BIOLOGICAL AND MEDICAL

PHYSICS
BIOMEDICAL ENGINEERING

Martin Beckerman

Molecular and
Cellular Signaling
With 227 Figures

Martin Beckerman
Y12 National Security Complex
Oak Ridge, TN 37831-7615
USA
[email protected]

Library of Congress Cataloging-in-Publication Data

Beckerman, Martin.
Molecular and cellular signaling/Martin Beckerman.
p. cm.(Biological and medical physics, biomedical engineering, ISSN 1618-7210)
Includes bibliographical references and index.
ISBN 0-387-22130-1 (alk. paper)
1. Cellular signal transduction. I. Title. II. Series.
QP517.C45B43 2005
571.74dc22
2004052556
ISBN-10: 0-387-22130-1
ISBN-13: 978-0387-22130-4

Printed on acid-free paper.

2005 Springer Science+Business Media, Inc.

AIP Press is an imprint of Springer Science+Business Media, Inc.
All rights reserved. This work may not be translated or copied in whole or in part without the
written permission of the publisher (Springer Science+Business Media, Inc., 233 Spring Street,
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Series Preface

The elds of biological and medical physics and biomedical engineering are
broad, multidisciplinary, and dynamic. They lie at the crossroads of frontier
research in physics, biology, chemistry, and medicine. The Biological and
Medical Physics/Biomedical Engineering series is intended to be comprehensive, covering a broad range of topics important to the study of the
physical, chemical, and biological sciences. Its goal is to provide scientists
and engineers with textbooks, monographs, and reference works to address
the growing need for information.
Books in the series emphasize established and emergent areas of science
including molecular, membrane, and mathematical biophysics; photosynthetic energy harvesting and conversion; information processing; physical
principles of genetics; sensory communications; and automata networks,
neural networks, and cellular automata. Equally important will be coverage
of applied aspects of biological and medical physics and biomedical engineering, such as molecular electronic components and devices, biosensors,
medicine, imaging, physical principles of renewable energy production,
advanced prostheses, and environmental control and engineering.
Oak Ridge, Tennessee

Elias Greenbaum
Series Editor-in-Chief

Preface

This text provides an introduction to molecular and cellular signaling in

biological systems. Cells partition their core cellular processes into a xed
infrastructure and a control layer. Proteins in the control layer, the subject
of this textbook, function as signals, as receptors of the signals, as transcription factors that turn genes on and off, and as signaling transducers and
intermediaries. The signaling and regulatory proteins and associated small
molecules make contact with the xed infrastructure responsible for metabolism, growth, replication, and reproduction at well-dened control points,
where the signals are converted into cellular responses.
The text is aimed at a broad audience of students and other individuals
interested in furthering their understanding of how cells regulate and coordinate their core activities. Malfunction in the control layer is responsible
for a host of human disorders ranging from neurological disorders to
cancers. Most drugs target components in the control layer, and difculties
in drug design are intimately related to the architecture of the control layer.
The text will assist students and individuals in medicine and pharmacology
interested in broadening their understanding of how the control layer
works. To further that goal, there are chapters on cancers and apoptosis,
and on bacteria and viruses. In those chapters not specically devoted to
pathogens, connections between diseases, drugs, and signaling are made.
The target audience for this text includes students in chemistry, physics,
and computer science who intend to work in biological and medical physics,
and bioinformatics and systems biology.To assist them, the textbook includes
a fair amount of background information on the main points of these areas.
The rst ve chapters of the book are mainly background and review
chapters. Signaling in the immune, endocrine (hormonal), and nervous
systems is covered, along with cancer, apoptosis, and gene regulation.
Biological systems are stunningly well engineered. Proof of this is all
around us. It can be seen in the sheer variety of life on Earth, all built pretty
much from the same building blocks and according to the same assembly
rules, but arranged in myriad different ways. It can be seen in the relatively
modest sizes of the genomes of even the most complex organisms, such as
vii

viii

Preface

ourselves. The genomes of worms, ies, mice, and humans are roughly
comparable, and only a factor of two or three larger than those of some
bacteria. The good engineering of biological systems is exemplied by the
above-mentioned partition of cellular processes into the xed infrastructure and the control layer. This makes possible machinery that always works
the same way in any cell at any time, and whose interactions can be exactly
known, while allowing for the machinerys regulation by the variable
control layer at well-dened control points.
Another example of good engineering design is that of modularity of
design. Proteins, especially signaling proteins, are modular in design and
their components can be transferred, arranged, and rearranged to make
many different proteins. The protein components interact with one another
through their interfaces. There are interfaces for interactions with other
proteins and with lipids DNA and RNA. Modularity is encountered not
only in the largely independent components, but also in the DNA regulatory sequences. These sequences serve as control points for the networks
that regulate gene expression. The DNA regulatory sequences can also
be rearranged in a multitude of ways along the chromosomes, and these
rearrangements, rather than the genes themselves, are largely responsible
for the richness of life on Earth. Two of the key objectives of the text are
to examine how modularity in design is used and how interfaces are
exploited. X-ray crystal structures and nuclear magnetic resonance (NMR)
solution structures provide insights at the atomic level of how the interfaces
between modules operate, and these will be looked at throughout the text.
One of the great conceptual breakthroughs in explorations of the control
layer was the idea that signaling proteins involved in cell-to-cell communication are organized into signaling pathways. In a signaling pathway, there
is a starting point, usually a receptor at the plasma membrane, and an endpoint (control point), more often than not a transcription regulatory site
in the nucleus, and there is a linear route leading from one to the other.
In spite of the enormous complexity of metazoans, there are only about a
dozen or so such pathways. These will be explored in the context of where
they are most strongly associated. For example, some pathways are prominent during development and are best understood in that context. Other
pathways are associated with stress responses and are best understood
within that framework, and still others are associated with immune
responses.
Signaling and the cellular responses to signals are complex.The responses
are controlled by a plethora of positive and negative feedback loops. The
presence of feedback complicates the simple picture of a linear pathway,
but this aspect is an essential part of the signaling process. Positive feedback ensures that once the appropriate thresholds are passed there will be
a rm commitment to a specic action and the system will not jump back
and forth between alternative responses. Negative feedback generates the
thresholds that ensure random excursions and perturbations do not unnec-

Preface

ix

essarily commit the cell to some irreversible response when it ought not to,
and it permits the cells to turn off the signaling once it has served its
purpose. These feedback loops will be examined along with the discussions
of the linear signaling pathways.
The goal of this textbook is to provide an introduction to the molecular
and cellular signaling processing comprising the control layer. The topic is
a vast one, and it is not possible to cover every possible aspect and still keep
the text concise and readable. To achieve the stated goal, material of a historical nature has been omitted, as have lengthy descriptions of all proteins
identied as being involved in the particular aspect of signaling being considered. In place of such an encyclopedic approach, selected processes are
presented step-by-step from start to end. These examples serve as simple
models of how the control process is carried out.
Oak Ridge, Tennessee

Martin Beckerman

Contents

Series Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Guide to Acronyms . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.

2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1
Prokaryotes and Eukaryotes . . . . . . . . . . . . . .
1.2
The Cytoskeleton and Extracellular Matrix . . . . .
1.3
Core Cellular Functions in Organelles . . . . . . . .
1.4
Metabolic Processes in Mitochondria and
Chloroplasts . . . . . . . . . . . . . . . . . . . . . . .
1.5
Cellular DNA to Chromatin . . . . . . . . . . . . . .
1.6
Protein Activities in the Endoplasmic Reticulum and
Golgi Apparatus . . . . . . . . . . . . . . . . . . . . .
1.7
Digestion and Recycling of Macromolecules . . . . .
1.8
Genomes of Bacteria Reveal Importance of
Signaling . . . . . . . . . . . . . . . . . . . . . . . . .
1.9
Organization and Signaling of Eukaryotic Cell . . .
1.10 Fixed Infrastructure and the Control Layer . . . . .
1.11 Eukaryotic Gene and Protein Regulation . . . . . .
1.12 Signaling Malfunction Central to Human
Disease . . . . . . . . . . . . . . . . . . . . . . . . . .
1.13
Organization of Text . . . . . . . . . . . . . . . . . .
The Control Layer . . . . . . . . . . . . . . . . . . . . . . .
2.1
Eukaryotic Chromosomes Are Built from
Nucleosomes . . . . . . . . . . . . . . . . . . . . . .
2.2
The Highly Organized Interphase Nucleus . . . . .
2.3
Covalent Bonds Dene the Primary Structure of a
Protein . . . . . . . . . . . . . . . . . . . . . . . . .
2.4
Hydrogen Bonds Shape the Secondary Structure .
2.5
Structural Motifs and Domain Folds:
Semi-Independent Protein Modules . . . . . . . .

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Contents

2.6
2.7
2.8
2.9
2.10
2.11
2.12
2.13
2.14
2.15
2.16

Arrangement of Protein Secondary Structure

Elements and Chain Topology . . . . . . . . . . .
Tertiary Structure of a Protein: Motifs and
Domains . . . . . . . . . . . . . . . . . . . . . . .
Quaternary Structure: The Arrangement of
Subunits . . . . . . . . . . . . . . . . . . . . . . .
Many Signaling Proteins Undergo Covalent
Modications . . . . . . . . . . . . . . . . . . . .
Anchors Enable Proteins to Attach to
Membranes . . . . . . . . . . . . . . . . . . . . .
Glycosylation Produces Mature Glycoproteins .
Proteolytic Processing Is Widely Used in
Signaling . . . . . . . . . . . . . . . . . . . . . . .
Reversible Addition and Removal of Phosphoryl
Groups . . . . . . . . . . . . . . . . . . . . . . . .
Reversible Addition and Removal of Methyl and
Acetyl Groups . . . . . . . . . . . . . . . . . . . .
Reversible Addition and Removal of SUMO
Groups . . . . . . . . . . . . . . . . . . . . . . . .
Post-Translational Modications to Histones . .

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3. Exploring Protein Structure and Function . . . . . . . . .

3.1
Interaction of Electromagnetic Radiation with
Matter . . . . . . . . . . . . . . . . . . . . . . . . .
3.2
Biomolecule Absorption and Emission Spectra . .
3.3
Protein Structure via X-Ray Crystallography . . .
3.4
Membrane Protein 3-D Structure via Electron and
Cryoelectron Crystallography . . . . . . . . . . . .
3.5
Determining Protein Structure Through NMR . .
3.6
Intrinsic Magnetic Dipole Moment of Protons and
Neutrons . . . . . . . . . . . . . . . . . . . . . . . .
3.7
Using Protein Fluorescence to Probe Control
Layer . . . . . . . . . . . . . . . . . . . . . . . . . .
3.8
Exploring Signaling with FRET . . . . . . . . . . .
3.9
Exploring Protein Structure with Circular
Dichroism . . . . . . . . . . . . . . . . . . . . . . .
3.10 Infrared and Raman Spectroscopy to Probe
Vibrational States . . . . . . . . . . . . . . . . . . .
3.11 A Genetic Method for Detecting Protein
Interactions . . . . . . . . . . . . . . . . . . . . . .
3.12 DNA and Oligonucleotide Arrays Provide
Information on Genes . . . . . . . . . . . . . . . .
3.13 Gel Electrophoresis of Proteins . . . . . . . . . . .
3.14
Mass Spectroscopy of Proteins . . . . . . . . . . . .

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Contents

4.

5.

Macromolecular Forces . . . . . . . . . . . . . . . . . . .
4.1
Amino Acids Vary in Size and Shape . . . . . . .
4.2
Amino Acids Behavior in Aqueous
Environments . . . . . . . . . . . . . . . . . . . .
4.3
Formation of H-Bonded Atom Networks . . . .
4.4
Forces that Stabilize Proteins . . . . . . . . . . .
4.5
Atomic Radii of Macromolecular Forces . . . . .
4.6
Osmophobic Forces Stabilize Stressed Cells . . .
4.7
Protein Interfaces Aid Intra- and Intermolecular
Communication . . . . . . . . . . . . . . . . . . .
4.8
Interfaces Utilize Shape and Electrostatic
Complementarity . . . . . . . . . . . . . . . . . .
4.9
Macromolecular Forces Hold Macromolecules
Together . . . . . . . . . . . . . . . . . . . . . . .
4.10 Motion Models of Covalently Bonded Atoms . .
4.11
Modeling van der Waals Forces . . . . . . . . . .
4.12 Molecular Dynamics in the Study of System
Evolution . . . . . . . . . . . . . . . . . . . . . . .
4.13 Importance of Water Molecules in Cellular
Function . . . . . . . . . . . . . . . . . . . . . . .
4.14 Essential Nature of Protein Dynamics . . . . . .

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Protein Folding and Binding . . . . . . . . . . . . . . . . .

5.1
The First Law of Thermodynamics: Energy Is
Conserved . . . . . . . . . . . . . . . . . . . . . . .
5.2
Heat Flows from a Hotter to a Cooler Body . . . .
5.3
Direction of Heat Flow: Second Law of
Thermodynamics . . . . . . . . . . . . . . . . . . .
5.4
Order-Creating Processes Occur Spontaneously as
Gibbs Free Energy Decreases . . . . . . . . . . . .
5.5
Spontaneous Folding of New Proteins . . . . . . .
5.6
The Folding Process: An Energy Landscape
Picture . . . . . . . . . . . . . . . . . . . . . . . . .
5.7
Misfolded Proteins Can Cause Disease . . . . . . .
5.8
Protein Problems and Alzheimers Disease . . . .
5.9
Amyloid Buildup in Neurological Disorders . . . .
5.10 Molecular Chaperones Assist in Protein Folding
in the Crowded Cell . . . . . . . . . . . . . . . . .
5.11 Role of Chaperonins in Protein Folding . . . . . .
5.12 Hsp 90 Chaperones Help Maintain Signal
Transduction Pathways . . . . . . . . . . . . . . . .
5.13 Proteins: Dynamic, Flexible, and Ready to
Change . . . . . . . . . . . . . . . . . . . . . . . . .

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xiv

Contents

6. Stress and Pheromone Responses in Yeast . . . . . . . . . .

6.1
How Signaling Begins . . . . . . . . . . . . . . . . .
6.2
Signaling Complexes Form in Response to
Receptor-Ligand Binding . . . . . . . . . . . . . . . .
6.3
Role of Protein Kinases, Phosphatases, and
GTPases . . . . . . . . . . . . . . . . . . . . . . . . .
6.4
Role of Proteolytic Enzymes . . . . . . . . . . . . . .
6.5
End Points Are Contact Points to Fixed
Infrastructure . . . . . . . . . . . . . . . . . . . . . .
6.6
Transcription Factors Combine to Alter Genes . . .
6.7
Protein Kinases Are Key Signal Transducers . . . . .
6.8
Kinases Often Require Second Messenger
Costimulation . . . . . . . . . . . . . . . . . . . . . .
6.9
Flanking Residues Direct Phosphorylation of
Target Residues . . . . . . . . . . . . . . . . . . . . .
6.10 Docking Sites and Substrate Specicity . . . . . . . .
6.11 Protein Phosphatases Are Prominent Components of
Signaling Pathways . . . . . . . . . . . . . . . . . . .
6.12 Scaffold and Anchor Protein Role in Signaling and
Specicity . . . . . . . . . . . . . . . . . . . . . . . .
6.13 GTPases Regulate Protein Trafcking in the Cell . .
6.14 Pheromone Response Pathway Is Activated by
Pheromones . . . . . . . . . . . . . . . . . . . . . . .
6.15 Osmotic Stresses Activate Glycerol Response
Pathway . . . . . . . . . . . . . . . . . . . . . . . . .
6.16 Yeasts Have a General Stress Response . . . . . . .
6.17 Target of Rapamycin (TOR) Adjusts Protein
Synthesis . . . . . . . . . . . . . . . . . . . . . . . . .
6.18 TOR Adjusts Gene Transcription . . . . . . . . . . .
6.19 Signaling Proteins Move by Diffusion . . . . . . . .

7.

Two-Component Signaling Systems . . . . . . . . . .

7.1
Prokaryotic Signaling Pathways . . . . . . . .
7.2
Catalytic Action by Histidine Kinases . . . .
7.3
The Catalytic Activity of HK Occurs at the
Active Site . . . . . . . . . . . . . . . . . . . .
7.4
The GHKL Superfamily . . . . . . . . . . . .
7.5
Activation of Response Regulators by
Phosphorylation . . . . . . . . . . . . . . . . .
7.6
Response Regulators Are Switches Thrown at
Transcriptional Control Points . . . . . . . . .
7.7
Structure and Domain Organization of
Bacterial Receptors . . . . . . . . . . . . . . .
7.8
Bacterial Receptors Form Signaling Clusters

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Contents

7.9
7.10
7.11
7.12
7.13
7.14

Bacteria with High Sensitivity and Mobility .

Feedback Loop in the Chemotactic Pathway
How Plants Sense and Respond to Hormones
Role of Growth Plasticity in Plants . . . . . .
Role of Phytochromes in Plant Cell Growth .
Cryptochromes Help Regulate Circadian
Rhythms . . . . . . . . . . . . . . . . . . . . .

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8. Organization of Signal Complexes by Lipids, Calcium, and

Cyclic AMP . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1
Composition of Biological Membranes . . . . . . . .
8.2
Microdomains and Caveolae in Membranes . . . . .
8.3
Lipid Kinases Phosphorylate Plasma Membrane
Phosphoglycerides . . . . . . . . . . . . . . . . . . . .
8.4
Generation of Lipid Second Messengers from
PIP2 . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.5
Regulation of Cellular Processes by PI3K . . . . . .
8.6
PIPs Regulate Lipid Signaling . . . . . . . . . . . . .
8.7
Role of Lipid-Binding Domains . . . . . . . . . . . .
8.8
Role of Intracellular Calcium Level Elevations . . .
8.9
Role of Calmodulin in Signaling . . . . . . . . . . . .
8.10 Adenylyl Cyclases and Phosphodiesterases Produce
and Regulate cAMP Second Messengers . . . . . . .
8.11 Second Messengers Activate Certain Serine/
Threonine Kinases . . . . . . . . . . . . . . . . . . .
8.12 Lipids and Upstream Kinases Activate PKB . . . . .
8.13 PKB Supplies a Signal Necessary for Cell
Survival . . . . . . . . . . . . . . . . . . . . . . . . . .
8.14 Phospholipids and Ca2+ Activate Protein
Kinase C . . . . . . . . . . . . . . . . . . . . . . . . .
8.15 Anchoring Proteins Help Localize PKA and PKC
Near Substrates . . . . . . . . . . . . . . . . . . . . .
8.16 PKC Regulates Response of Cardiac Cells to
Oxygen Deprivation . . . . . . . . . . . . . . . . . .
8.17 cAMP Activates PKA, Which Regulates Ion
Channel Activities . . . . . . . . . . . . . . . . . . . .
8.18 PKs Facilitate the Transfer of Phosphoryl Groups
from ATPs to Substrates . . . . . . . . . . . . . . . .
9. Signaling by Cells of the Immune System . . . .
9.1
Leukocytes Mediate Immune Responses
9.2
Leukocytes Signal One Another Using
Cytokines . . . . . . . . . . . . . . . . . .
9.3
APC and Nave T Cell Signals Guide
Differentiation into Helper T Cells . . .

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xvi

Contents

9.4
9.5
9.6
9.7
9.8
9.9
9.10
9.11
9.12
9.13
9.14
9.15
9.16
9.17
9.18
9.19
9.20

10.

Five Families of Cytokines and Cytokine

Receptors . . . . . . . . . . . . . . . . . . . . . .
Role of NF-kB/Rel in Adaptive Immune
Responses . . . . . . . . . . . . . . . . . . . . . .
Role of MAP Kinase Modules in Immune
Responses . . . . . . . . . . . . . . . . . . . . . .
Role of TRAF and DD Adapters . . . . . . . . .
Toll/IL-1R Pathway Mediates Innate Immune
Responses . . . . . . . . . . . . . . . . . . . . . .
TNF Family Mediates Homeostasis, Death, and
Survival . . . . . . . . . . . . . . . . . . . . . . . .
Role of Hematopoietin and Related Receptors .
Role of Human Growth Hormone Cytokine . . .
Signal-Transducing Jaks and STATs . . . . . . . .
Interferon System: First Line of Host Defense in
Mammals Against Virus Attacks . . . . . . . . . .
Chemokines Provide Navigational Cues for
Leukocytes . . . . . . . . . . . . . . . . . . . . . .
B and T Cell Receptors Recognize Antigens . .
MHCs Present Antigens on the Cell Surface . .
Antigen-Recognizing Receptors Form Signaling
Complexes with Coreceptors . . . . . . . . . . .
Costimulatory Signals Between APCs and
T Cells . . . . . . . . . . . . . . . . . . . . . . . .
Role of Lymphocyte-Signaling Molecules . . . .
Kinetic Proofreading and Serial Triggering of
TCRs . . . . . . . . . . . . . . . . . . . . . . . . .

Cell Adhesion and Motility . . . . . . . . . . . . . . . .

10.1 Cell Adhesion Receptors: Long Highly Modular
Glycoproteins . . . . . . . . . . . . . . . . . . .
10.2 Integrins as Bidirectional Signaling Receptors .
10.3 Role of Leukocyte-Specic Integrin . . . . . .
10.4 Most Integrins Bind to Proteins Belonging to
the ECM . . . . . . . . . . . . . . . . . . . . . .
10.5 Cadherins Are Present in Most Cells of the
Body . . . . . . . . . . . . . . . . . . . . . . . .
10.6 IgCAMs Mediate CellCell and CellECM
Adhesion . . . . . . . . . . . . . . . . . . . . . .
10.7 Selectins Are CAMs Involved in Leukocyte
Motility . . . . . . . . . . . . . . . . . . . . . . .
10.8 Leukocytes Roll, Adhere, and Crawl to Reach
the Site of an Infection . . . . . . . . . . . . . .
10.9 Bonds Form and Break During Leukocyte
Rolling . . . . . . . . . . . . . . . . . . . . . . .

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10.10 Bond Dissociation of Rolling Leukocyte as Seen in

Microscopy . . . . . . . . . . . . . . . . . . . . . . .
10.11 Slip and Catch Bonds Between Selectins and
Their Carbohydrate Ligands . . . . . . . . . . . . .
10.12 Development in Central Nervous System . . . . .
10.13 Diffusible, Anchored, and Membrane-Bound
Glycoproteins in Neurite Outgrowth . . . . . . . .
10.14 Growth Cone Navigation Mechanisms . . . . . . .
10.15 Molecular Marking by Concentration Gradients of
Netrins and Slits . . . . . . . . . . . . . . . . . . . .
10.16 How Semaphorins, Scatter Factors, and Their
Receptors Control Invasive Growth . . . . . . . .
10.17 Ephrins and Their Eph Receptors Mediate
Contact-Dependent Repulsion . . . . . . . . . . .
11.

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Signaling in the Endocrine System . . . . . . . . . . . . . . . .

11.1 Five Modes of Cell-to-Cell Signaling . . . . . . . . . .
11.2 Role of Growth Factors in Angiogenesis . . . . . . . .
11.3 Role of EGF Family in Wound Healing . . . . . . . .
11.4 Neurotrophins Control Neuron Growth,
Differentiation, and Survival . . . . . . . . . . . . . . .
11.5 Role of Receptor Tyrosine Kinases in Signal
Transduction . . . . . . . . . . . . . . . . . . . . . . . .
11.6 Phosphoprotein Recognition Modules Utilized Widely
in Signaling Pathways . . . . . . . . . . . . . . . . . . .
11.7 Modules that Recognize Proline-Rich Sequences
Utilized Widely in Signaling Pathways . . . . . . . . .
11.8 ProteinProtein Interaction Domains Utilized Widely
in Signaling Pathways . . . . . . . . . . . . . . . . . . .
11.9 Non-RTKs Central in Metazoan Signaling Processes
and Appear in Many Pathways . . . . . . . . . . . . .
11.10 Src Is a Representative NRTK . . . . . . . . . . . . .
11.11 Roles of Focal Adhesion Kinase Family of
NRTKs . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.12 GTPases Are Essential Regulators of Cellular
Functions . . . . . . . . . . . . . . . . . . . . . . . . . .
11.13 Signaling by Ras GTPases from Plasma Membrane
and Golgi . . . . . . . . . . . . . . . . . . . . . . . . . .
11.14 GTPases Cycle Between GTP- and GDP-Bound
States . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.15 Role of Rho, Rac, and Cdc42, and Their Isoforms . . .
11.16 Ran Family Coordinates Trafc In and Out of the
Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.17 Rab and ARF Families Mediate the Transport of
Cargo . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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12. Signaling in the Endocrine and Nervous Systems

Through GPCRs . . . . . . . . . . . . . . . . . . . . . . . .
12.1
GPCRs Classication Criteria . . . . . . . . . . . .
12.2 Study of Rhodopsin GPCR with Cryoelectron
Microscopy and X-Ray Crystallography . . . . . .
12.3 Subunits of Heterotrimeric G Proteins . . . . . . .
12.4 The Four Families of Ga Subunits . . . . . . . . . .
12.5 Adenylyl Cyclases and Phosphodiesterases Key to
Second Messenger Signaling . . . . . . . . . . . . .
12.6 Desensitization Strategy of G Proteins to Maintain
Responsiveness to Environment . . . . . . . . . . .
12.7 GPCRs Are Internalized, and Then Recycled or
Degraded . . . . . . . . . . . . . . . . . . . . . . . .
12.8 Hormone-Sending and Receiving Glands . . . . .
12.9 Functions of Signaling Molecules . . . . . . . . . .
12.10 Neuromodulators Inuence Emotions, Cognition,
Pain, and Feeling Well . . . . . . . . . . . . . . . .
12.11 Ill Effects of Improper Dopamine Levels . . . . .
12.12 Inadequate Serotonin Levels Underlie Mood
Disorders . . . . . . . . . . . . . . . . . . . . . . . .
12.13 GPCRs Role in the Somatosensory System
Responsible for Sense of Touch and
Nociception . . . . . . . . . . . . . . . . . . . . . .
12.14 Substances that Regulate Pain and Fever
Responses . . . . . . . . . . . . . . . . . . . . . . .
12.15 Composition of Rhodopsin Photoreceptor . . . . .
12.16 How G Proteins Regulate Ion Channels . . . . . .
12.17 GPCRs Transduce Signals Conveyed by
Odorants . . . . . . . . . . . . . . . . . . . . . . . .
12.18 GPCRs and Ion Channels Respond to
Tastants . . . . . . . . . . . . . . . . . . . . . . . . .
13.

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Cell Fate and Polarity . . . . . . . . . . . . . . . . . . . . . .

13.1 Notch Signaling Mediates Cell Fate Decision . . . .
13.2 How Cell Fate Decisions Are Mediated . . . . . . .
13.3 Proteolytic Processing of Key Signaling
Elements . . . . . . . . . . . . . . . . . . . . . . . . .
13.4 Three Components of TGF-b Signaling . . . . . . .
13.5 Smad Proteins Convey TGF-b Signals into the
Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . .
13.6 Multiple Wnt Signaling Pathways Guide Embryonic
Development . . . . . . . . . . . . . . . . . . . . . .
13.7 Role of Noncanonical Wnt Pathway . . . . . . . . .
13.8 Hedgehog Signaling Role During Development . . .
13.9
Gli Receives Hh Signals . . . . . . . . . . . . . . . .

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13.10 Stages of Embryonic Development Use

Morphogens . . . . . . . . . . . . . . . . . . . . . . .
13.11 Gene Family Hierarchy of Cell Fate Determinants in
Drosophila . . . . . . . . . . . . . . . . . . . . . . . .
13.12 Egg Development in D. Melanogaster . . . . . . . .
13.13 Gap Genes Help Partition the Body into
Bands . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.14 Pair-Rule Genes Partition the Body into
Segments . . . . . . . . . . . . . . . . . . . . . . . . .
13.15 Segment Polarity Genes Guide Parasegment
Development . . . . . . . . . . . . . . . . . . . . . .
13.16 Hox Genes Guide Patterning in Axially Symmetric
Animals . . . . . . . . . . . . . . . . . . . . . . . . .
14.

Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.1 Several Critical Mutations Generate a
Transformed Cell . . . . . . . . . . . . . . . . . . . .
14.2 Ras Switch Sticks to On Under Certain
Mutations . . . . . . . . . . . . . . . . . . . . . . . .
14.3 Crucial Regulatory Sequence Missing in Oncogenic
Forms of Src . . . . . . . . . . . . . . . . . . . . . . .
14.4 Overexpressed GFRs Spontaneously Dimerize in
Many Cancers . . . . . . . . . . . . . . . . . . . . . .
14.5 GFRs and Adhesion Molecules Cooperate to
Promote Tumor Growth . . . . . . . . . . . . . . . .
14.6 Role of Mutated Forms of Proteins in Cancer
Development . . . . . . . . . . . . . . . . . . . . . .
14.7 Translocated and Fused Genes Are Present in
Leukemias . . . . . . . . . . . . . . . . . . . . . . . .
14.8
Repair of DNA Damage . . . . . . . . . . . . . . . .
14.9 Double-Strand-Break Repair Machinery . . . . . . .
14.10 How Breast Cancer (BRCA) Proteins Interact with
DNA . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.11 PI3K Superfamily Members that Recognize
Double-Strand Breaks . . . . . . . . . . . . . . . . .
14.12 Checkpoints Regulate Transition Events in a
Network . . . . . . . . . . . . . . . . . . . . . . . . .
14.13 Cyclin-Dependent Kinases Form the Core of
Cell-Cycle Control System . . . . . . . . . . . . . . .
14.14 pRb Regulates Cell Cycle in Response to
Mitogenic Signals . . . . . . . . . . . . . . . . . . . .
14.15 p53 Halts Cell Cycle While DNA Repairs Are
Made . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.16 p53 and pRb Controllers Central to Metazoan
Cancer Prevention Program . . . . . . . . . . . . . .

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14.17 p53 Structure Supports Its Role as a Central

Controller . . . . . . . . . . . . . . . . . . . . . . . . .
14.18 Telomerase Production in Cancer Cells . . . . . . . . .
15.

16.

Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.1 Caspases and Bcl-2 Proteins Are Key Mediators of
Apoptosis . . . . . . . . . . . . . . . . . . . . . . . .
15.2 Caspases Are Proteolytic Enzymes Synthesized as
Inactive Zymogens . . . . . . . . . . . . . . . . . . .
15.3 Caspases Are Initiators and Executioners of
Apoptosis Programs . . . . . . . . . . . . . . . . . .
15.4 There Are Three Kinds of Bcl-2 Proteins . . . . . . .
15.5
How Caspases Are Activated . . . . . . . . . . . . .
15.6 Cell-to-Cell Signals Stimulate Formation of the
DISC . . . . . . . . . . . . . . . . . . . . . . . . . . .
15.7
Death Signals Are Conveyed by the Caspase 8
Pathway . . . . . . . . . . . . . . . . . . . . . . . . .
15.8 How Pro- and Antiapoptotic Signals Are
Relayed . . . . . . . . . . . . . . . . . . . . . . . . . .
15.9 Bcl-2 Proteins Regulate Mitochondrial Membrane
Permeability . . . . . . . . . . . . . . . . . . . . . . .
15.10 Mitochondria Release Cytochrome c in Response to
Oxidative Stresses . . . . . . . . . . . . . . . . . . . .
15.11 Mitochondria Release Apoptosis-Promoting
Agents . . . . . . . . . . . . . . . . . . . . . . . . . .
15.12 Role of Apoptosome in (Mitochondrial Pathway to)
Apoptosis . . . . . . . . . . . . . . . . . . . . . . . .
15.13 Inhibitors of Apoptosis Proteins Regulate Caspase
Activity . . . . . . . . . . . . . . . . . . . . . . . . . .
15.14 Smac/DIABLO and Omi/HtrA2 Regulate IAPs . .
15.15 Feedback Loops Coordinate Actions at Various
Control Points . . . . . . . . . . . . . . . . . . . . . .
15.16 Cells Can Produce Several Different Kinds of
Calcium Signals . . . . . . . . . . . . . . . . . . . . .
15.17 Excessive [Ca2+] in Mitochondria Can Trigger
Apoptosis . . . . . . . . . . . . . . . . . . . . . . . .
15.18 p53 Promotes Cell Death in Response to Irreparable
DNA Damage . . . . . . . . . . . . . . . . . . . . . .
15.19 Anti-Cancer Drugs Target the Cells Apoptosis
Machinery . . . . . . . . . . . . . . . . . . . . . . . .
Gene
16.1
16.2
16.3

Regulation in Eukaryotes . . . . . . . . . . . .
Organization of the Gene Regulatory Region
How Promoters Regulate Genes . . . . . . .
TFs Bind DNA Through Their DNA-Binding
Domains . . . . . . . . . . . . . . . . . . . . .

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16.4
16.5
16.6
16.7
16.8
16.9
16.10
16.11
16.12
16.13
16.14
16.15
16.16
16.17
17.

Transcriptional Activation Domains Initiate

Transcription . . . . . . . . . . . . . . . . . . . . .
Nuclear Hormone Receptors Are Transcription
Factors . . . . . . . . . . . . . . . . . . . . . . . .
Composition and Structure of the Basal
Transcription Machinery . . . . . . . . . . . . . .
RNAP II Is Core Module of the Transcription
Machinery . . . . . . . . . . . . . . . . . . . . . .
Regulation by Chromatin-Modifying
Enzymes . . . . . . . . . . . . . . . . . . . . . . .
Multiprotein Complex Use of Energy of ATP
Hydrolysis . . . . . . . . . . . . . . . . . . . . . .
Protein Complexes Act as Interfaces Between
TFs and RNAP II . . . . . . . . . . . . . . . . . .
Alternative Splicing to Generate Multiple
Proteins . . . . . . . . . . . . . . . . . . . . . . . .
Pre-Messenger RNA Molecules Contain Splice
Sites . . . . . . . . . . . . . . . . . . . . . . . . . .
Small Nuclear RNAs (snRNAs) . . . . . . . . . .
How Exon Splices Are Determined . . . . . . . .
Translation Initiation Factors Regulate Start of
Translation . . . . . . . . . . . . . . . . . . . . . .
eIF2 Interfaces Upstream Regulatory Signals and
the Ribosomal Machinery . . . . . . . . . . . . .
Critical Control Points for Protein Synthesis . . .

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Cell Regulation in Bacteria . . . . . . . . . . . . . . . . . .

17.1 Cell Regulation in Bacteria Occurs Primarily at
Transcription Level . . . . . . . . . . . . . . . . . .
17.2 Transcription Is Initiated by RNAP
Holoenzymes . . . . . . . . . . . . . . . . . . . . .
17.3 Sigma Factors Bind to Regulatory Sequences in
Promoters . . . . . . . . . . . . . . . . . . . . . . .
17.4 Bacteria Utilize Sigma Factors to Make Major
Changes in Gene Expression . . . . . . . . . . . .
17.5 Mechanism of Bacterial Transcription Factors . . .
17.6 Many TFs Function as Response Regulators . . . .
17.7 Organization of Protein-Encoding Regions and
Their Regulatory Sequences . . . . . . . . . . . . .
17.8 The Lac Operon Helps Control Metabolism in
E. coli . . . . . . . . . . . . . . . . . . . . . . . . . .
17.9 Flagellar Motors Are Erected in Several Stages . .
17.10 Under Starvation Conditions, B. subtilis Undergoes
Sporulation . . . . . . . . . . . . . . . . . . . . . . .
17.11 Cell-Cycle Progression and Differentiation in
C. crescentus . . . . . . . . . . . . . . . . . . . . . .

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17.12 Antigenic Variation Counters Adaptive Immune

Responses . . . . . . . . . . . . . . . . . . . . . . . .
17.13 Bacteria Organize into Communities When Nutrient
Conditions Are Favorable . . . . . . . . . . . . . . .
17.14 Quorum Sensing Plays a Key Role in Establishing a
Colony . . . . . . . . . . . . . . . . . . . . . . . . . .
17.15 Bacteria Form Associations with Other Bacteria on
Exposed Surfaces . . . . . . . . . . . . . . . . . . . .
17.16 Horizontal Gene Transfer (HGT) . . . . . . . . . . .
17.17 Pathogenic Species Possess Virulence Cassettes . . .
17.18 Bacterial Death Modules . . . . . . . . . . . . . . . .
17.19 Myxobacteria Exhibit Two Distinct Forms of
Social Behavior . . . . . . . . . . . . . . . . . . . . .
17.20 Structure Formation by Heterocystous
Cyanobacteria . . . . . . . . . . . . . . . . . . . . . .
17.21 Rhizobia Communicate and Form Symbiotic
Associations with Legumes . . . . . . . . . . . . . .
18.

Regulation by Viruses . . . . . . . . . . . . . . . . . . . . . .
18.1 How Viruses Enter Their Host Cells . . . . . . . . .
18.2 Viruses Enter and Exit the Nucleus in
Several Ways . . . . . . . . . . . . . . . . . . . . . . .
18.3
Ways that Viruses Exit a Cell . . . . . . . . . . . . .
18.4 Viruses Produce a Variety of Disorders in
Humans . . . . . . . . . . . . . . . . . . . . . . . . .
18.5 VirusHost Interactions Underlie Virus Survival and
Proliferation . . . . . . . . . . . . . . . . . . . . . . .
18.6 Multilayered Defenses Are Balanced by
Multilayered Attacks . . . . . . . . . . . . . . . . . .
18.7 Viruses Target TNF Family of Cytokines . . . . . . .
18.8 Hepatitis C Virus Disables Host Cells Interferon
System . . . . . . . . . . . . . . . . . . . . . . . . . .
18.9 Human T Lymphotropic Virus Type 1 Can Cause
Cancer . . . . . . . . . . . . . . . . . . . . . . . . . .
18.10 DNA and RNA Viruses that Can Cause Cancer . .
18.11 HIV Is a Retrovirus . . . . . . . . . . . . . . . . . . .
18.12 Role of gp120 Envelope Protein in HIV . . . . . . .
18.13 Early-Acting tat, rev, and nef Regulatory
Genes . . . . . . . . . . . . . . . . . . . . . . . . . . .
18.14 Late-Acting vpr, vif, vpu, and vpx Regulatory
Genes . . . . . . . . . . . . . . . . . . . . . . . . . . .
18.15 Bacteriophages Two Lifestyles: Lytic and
Lysogenic . . . . . . . . . . . . . . . . . . . . . . . . .
18.16 Deciding Between Lytic and Lysogenic Lifestyles . .
18.17 Encoding of Shiga Toxin in E. coli . . . . . . . . . .

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19.

20.

21.

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Ion Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19.1
How Membrane Potentials Arise . . . . . . . . . . . .
19.2 Membrane and Action Potentials Have Regenerative
Properties . . . . . . . . . . . . . . . . . . . . . . . . .
19.3 HodgkinHuxley Equations Describe How Action
Potentials Arise . . . . . . . . . . . . . . . . . . . . . .
19.4 Ion Channels Have Gates that Open and Close . . . .
19.5 Families of Ion Channels Expressed in Plasma
Membrane of Neurons . . . . . . . . . . . . . . . . . .
19.6
Assembly of Ion Channels . . . . . . . . . . . . . . . .
19.7 Design and Function of Ion Channels . . . . . . . . .
19.8 Gates and Filters in Potassium Channels . . . . . . . .
19.9 Voltage-Gated Chloride Channels Form a
Double-Barreled Pore . . . . . . . . . . . . . . . . . .
19.10 Nicotinic Acetylcholine Receptors Are Ligand-Gated
Ion Channels . . . . . . . . . . . . . . . . . . . . . . . .
19.11 Operation of Glutamate Receptor Ion Channels . . .

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Neural Rhythms . . . . . . . . . . . . . . . . . . . . . . . . . .
20.1 Heartbeat Is Generated by Pacemaker Cells . . . . .
20.2 HCN Channels Role in Pacemaker Activities . . . . .
20.3 Synchronous Activity in the Central Nervous
System . . . . . . . . . . . . . . . . . . . . . . . . . . .
20.4
Role of Low Voltage-Activated Calcium Channels . . . .
20.5 Neuromodulators Modify the Activities of
Voltage-Gated Ion Channels . . . . . . . . . . . . . . .
20.6 Gap Junctions Formed by Connexins Mediate
Rapid Signaling Between Cells . . . . . . . . . . . . .
20.7 Synchronization of Neural Firing . . . . . . . . . . . .
20.8 How Spindling Patterns Are Generated . . . . . . . .
20.9 Epileptic Seizures and Abnormal Brain Rhythms . . .
20.10 Swimming and Digestive Rhythms in Lower
Vertebrates . . . . . . . . . . . . . . . . . . . . . . . . .
20.11 CPGs Have a Number of Common Features . . . . .
20.12 Neural Circuits Are Connected to Other Circuits and
Form Systems . . . . . . . . . . . . . . . . . . . . . . .
20.13 A Variety of Neuromodulators Regulate Operation
of the Crustacean STG . . . . . . . . . . . . . . . . . .
20.14 Motor Systems Adapt to Their Environment and
Learn . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Learning and Memory . . . . . . . . . . . . . . . . . . . . . . .

21.1 Architecture of Brain Neurons by Function . . . . . .
21.2 Protein Complexes Structural and Signaling Bridges
Across Synaptic Cleft . . . . . . . . . . . . . . . . . . .

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21.3

The Presynaptic Terminal and the Secretion of

Signaling Molecules . . . . . . . . . . . . . . . . . . .
PSD Region Is Highly Enriched in Signaling
Molecules . . . . . . . . . . . . . . . . . . . . . . . .
The Several Different Forms of Learning and
Memory . . . . . . . . . . . . . . . . . . . . . . . . .
Signal Integration in Learning and Memory
Formation . . . . . . . . . . . . . . . . . . . . . . . .
Hippocampal LTP Is an Experimental Model of
Learning and Memory . . . . . . . . . . . . . . . . .
Initiation and Consolidation Phases of LTP . . . . .
CREB Is the Control Point at the Terminus of the
Learning Pathway . . . . . . . . . . . . . . . . . . . .
Synapses Respond to Use by Strengthening and
Weakening . . . . . . . . . . . . . . . . . . . . . . . .
Neurons Must Maintain Synaptic Homeostasis . . .
Fear Circuits Detect and Respond to Danger . . . .
Areas of the Brain Relating to Drug Addiction . . .
Drug-Reward Circuits Mediate Addictive
Responses . . . . . . . . . . . . . . . . . . . . . . . .
Drug Addiction May Be an Aberrant Form of
Synaptic Plasticity . . . . . . . . . . . . . . . . . . . .
In Reward-Seeking Behavior, the Organism Predicts
Future Events . . . . . . . . . . . . . . . . . . . . . .

515

518

520

521

.
.

523
524

525

.
.
.
.

526
528
529
529

531

532

533

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

539

Index

553

21.4
21.5
21.6
21.7
21.8
21.9
21.10
21.11
21.12
21.13
21.14
21.15
21.16

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Guide to Acronyms

This Guide to Acronyms contains a list arranged alphabetically of commonly

encountered acronyms all of which are discussed in the text. There are a
number of instances where the same acronym has more than one usage. In
some cases, the correct meaning can be discerned from the way the acronym
is denoted, but in other cases, the correct usage must be deduced from the
context. In the text, proteins are written starting with a capital letter, while
the genes encoding the proteins are written all in lowercase letters. Protein
names are, for the most part, not included in the list of acronyms. Proteins
appearing in the list with names ending in numerals such as Ste2 are entered
once; names of proteins of the same spelling with different numerals (e.g.,
Ste7, Ste11 in the case of Ste2) can be readily deduced.
5-HT

5-hydroxytryptamine (serotonin)

AA
AC
ACE
ACF
ACh
ACTH
ADAM
ADHD
ADP
AFM
AGC
AHL
AIDS
AIF
AIP
AKAP
ALK
ALS

arachidonic acid
adenylyl cyclase
angiotensin-converting enzyme
ATP-dependent chromatin assembly and remodeling factor
acetylcholine
adrenocorticotropic hormone
a disintegrin and metalloprotease
attention-decit hyperactivity disorder
adenosine diphosphate
atomic force microscopy
PKA, PKG, PKC family
acetyl homoserine lactase
acquired immunodeciency syndrome
apoptosis inducing factor
autoinducing peptides
A-kinase anchoring protein
activin receptor-like kinase
amytrophic lateral sclerosis
xxv

xxvi

Guide to Acronyms

AMP
AMPA
AMPK
ANT
APC
APC
APP
ARC-L
Arf
ARF
ARR
ATM
ATP
ATR
AVN
AVP

adenosine monophosphate
a-amino-3-hydroxyl-5-methyl-4-isoxazole propionate acid
AMP-dependent protein kinase
adenosine nucleotide translocator
adenomatous polyposis coli
antigen-presenting cell
amyloid b protein precursor
activation-recruited coactivator-large
ADP-ribosylation factor
alternative reading frame (of exon 2)
Arabidopsis response regulator
ataxia-telangeictasia mutated
adenosine triphosphate
ATM and Rad3-related
atrioventricular node
vasopressin

Bcl-2
BCR
BDNF
BER
BFGF
BIR
BLV
BMP
BRCA1
BRCT
BRE
bZIP

B cell leukemia 2
B-cell receptor
brain-derived neurotrophic factor
base excision repair
basic broblast growth factor
baculoviral IAP repeat
bovine leukemia virus
bone morphogenetic protein
breast cancer 1
BRCA1 C-terminal
TFIIB recognition element
basic region leucine zipper

C1
CAD
CaM
CaMKII
cAMP
CAP
CAPRI
CaR
CARD
CASK
CB
CBP
CBP
CD
Cdc25
Cdk

protein kinase C homology-1

caspase-activated deoxyribonuclease
calmodulin
calcium/calmodulin-dependent protein kinase II
cyclic AMP
catabolite activator protein
calcium-promoted Ras inactivator
extracellular calcium receptor
caspase recruitment domain
CaMK/SH3/guanylate kinase domain protein
Cajal body
complement binding protein
CREB binding protein
cluster of differentiation
cell division cycle (protein) 25
cyclin-dependent kinase

Guide to Acronyms

cDNA
CFP
CFTR
cGMP
CHRAC
Chromo
Ci
Ck2
ClC
Clk
CMGC
CNG
CNS
CNTF
CoA
COX
CPG
CR
CRD
CRE
CREB
CRF
CRH
CRSP
CSF
cSMAC
CST

complementary DNA
cyan uorescent protein
cystic brosis transmembrane conductance regulator
cyclic guanosine monophosphate
chromatin accessibility complex
chromatin organization modier
cubitus interruptus
casein kinase-2
chloride channel of the CLC family
cyclin-dependent kinase-like kinase
CDK, MAPK, GSK-3 CLK, CK2
cyclic nucleotide-gated
central nervous system
ciliary neurotrophic factor
acetyl coenzyme A
cyclo-oxygenase
central pattern generator
consensus repeat
cysteine-rich domain
cAMP response element
cAMP response element-binding protein
corticotropin-releasing factor
corticotropin-releasing hormone
coactivator required for Sp1 activation
cerebrospinal uid
central supramolecular activation cluster
cortistatin

DA
DAG
DAT
dATP
DC
DCC
DD
DED
DEP
DFF
Dhh
DIABLO
DISC
DIX
DLG
DNA
DNA-PK
DPE

dopamine
diacylglycerol
dopamine transporter
deoxyadenosine triphosphate
dendritic cell
deleted in colorectal cancer
death domain
death effector domain
disheveled, egl-10, and pleckstrin
DNA fragmentation factor
desert hedgehog
direct IAP binding protein with low pI
death-inducing signaling complex
disheveled and axin
discs large
deoxyribonucleic acid
DNA-dependent protein kinase
downstream promoter element

xxvii

xxviii

Guide to Acronyms

DR
DSB
DSL
dsRNA

death receptor
double-strand break
delta/serrate/lin
double-stranded RNA

E
ECF
ECM
EEG
EGF
EGFR
eIF
EPEC
ER
ERK
ESCRT
ESE
ESI
ESS
EVH1

epinephrine (adrenaline)
extracytoplasmic function
extracellular matrix
electroencephalographic
epidermal growth factor
epidermal growth factor receptor
eukaryotic initiation factors
enteropathogenic E. coli
endoplasmic reticulum
extracellular signal-regulated kinase
endosomal-sorting complexes required for transport
exonic splice enhancer
electrospray ionization
exonic splice silencer
enabled/vasodilator-stimulated phosphoprotein homology-1

FA
FADD
FAK
FAT
FH
FHA
FNIII
FRAP
FSH
FYVE

focal adhesion
Fas-associated death domain
focal adhesion kinase
focal adhesion targeting
forkhead
forkhead associated
bronectin type III
uorescence recovery following photobleaching
follicle-stimulating hormone
Fab1p, YOTB, Vac1p, Eea1

GABA
GAP
GAS
GAS
GDI
GDNF
GDP
GEF
GFP
GFR
GH
GHIH
GHRH

g-aminobutyric acid
GTPase-activating protein
group A streptococcus
interferon-gamma activated site
GDP dissociation inhibitors
glial-derived neurotrophic factor
guanosine diphosphate
guanine nucleotide exchange factor
green uorescent protein
growth factor receptor
growth hormone
growth hormone-inhibiting hormone
growth hormone-releasing hormone

Guide to Acronyms

GIRK
GKAP
GPCR
GPI
GRH
GRIP
GRK
GSK-3
GTP

G protein-linked inward rectied K+ channels

guanylate kinase-associated protein
G protein-coupled receptor
glycosyl phosphatidyl inositol
gonadotropin-releasing hormone
glutamate receptor interacting protein
G protein-coupled receptor kinase
glycogen synthase kinase-3
guanosine triphosphate

HA
HAT
HDAC
hGH
HGT
Hh
HHV
HIV
HK
HLH
HNC
hnRNP
HOG
HPt
HR
Hsp
HSV-1
hTERT
HTH
HTLV-1
hTR
HtrA2

histamine
histone acetyltransferase
histone deacetylase
human growth hormone
horizontal gene transfer
hedgehog
human herpesvirus
human immunodeciency virus
histidine kinase
helix-loop-helix
hyperpolarization-activated cyclic nucleotide gated
heterogeneous nuclear RNP
high osmolarity glycerol
histidine phosphotransfer
homologous recombination
heat shock protein
herpes simplex virus type 1
human telomerase reverse transcriptase
helix-turn-helix
human T lymphotropic virus type 1
human telomerase RNA
high temperature requirement factor A2

IAP
ICAD
ICAM
ICE
IEG
IFN
Ig
IGC
IgCAM
IGluR
IGluR
Ihh

inhibitor of apoptosis
inhibitor of CAD
intercellular cell adhesion molecule
interleukin-1b converting enzyme
immediate early gene
interferon
immunoglobulin
interchromatin granule clusters
immunoglobulin cell adhesion molecule
inhibitory glutamate receptor ion channel
ionotropic glutamate receptor
Indian hedgehog

xxix

xxx

Guide to Acronyms

IL
ILP
IN
Inr
InsP3R
IP
IPSP
IRAK
IRES
IRF
IS
IS
ISE
ISRE
ISS
ISWI
ITAM

interleukin
IAP-like protein
integrase
initiator
inositol (1,4,5) triphosphate receptor
Ischemic preconditioning
inhibitory postsynaptic potential
IL-1R-associated kinase
internal ribosomal entry site
interferon regulatory factor
immunological synapse
intracellular stores
intronic splice enhancer
interferon stimulated response element
intronic splice silencer
imitation SWI
immunoreceptor tyrosine-based activation motif

Jak
JNK

Janus kinase
c-Jun N-terminal kinase

KSHV

Kaposis sarcoma-associated herpesvirus

L
LAMP
LANA-1
LH
LNR
LNS
LPS
LRR
LTD
LTP
LTR
LZ

late (domain)
latency-associated membrane protein
latency-associated nuclear antigen type 1
luteinizing hormone
lin/notch repeat
laminin, neurexin, sex hormone-binding globulin
lipopolysaccharides
leucine-rich repeat
long-term depression
long-term potentiation
long terminal repeat
leucine zipper

MA
MAGE
MALDI
MAOI
MAP
MAPK
MCP
MD
MH1
MHC

matrix
melanoma-associated antigen
matrix-assisted laser desorption ionization
monoamine oxidase inhibitor
mitogen-activated protein
mitogen-activated protein kinase
methyl-accepting chemotaxis protein
molecular dynamics
mad homology-1
major histocompatibility complex

Guide to Acronyms

MIP
MM
MMP
MMR
MRI
mRNA
MSH
MVB

macrophage inammatory protein

molecular mechanics
matrix metalloproteinase
mismatch repair
magnetic resonance imaging
messenger RNA
melanocyte-stimulating hormone
multivesicular body

NAc
nAChR
NADE
NAIP
NBS
NC
NCAM
NE
NER
NES
NFAT
NF-kB
NGF
NH
NHEJ
NICD
NKA
NKB
NLS
NMDA
NMR
NPC
NRAGE
NRIF
NSAID
NSF
NURF

nucleus accumbens
nicotinic acetylcholine receptor
p75-associated cell death executioner
neuronal inhibitory apoptosis protein
Nijmegem breakage syndrome
nucleocapsid
neural cell adhesion molecule
norepinephrine (noradrenaline)
nucleotide excision repair
nuclear export signal (sequence)
nuclear factor of activated T cells
nuclear factor kappa B
nerve growth factor
amide (molecule)
nonhomologous end joining
notch intracellular domain
neurokinin A
neurokinin B
nuclear localization signal (sequence)
N-methyl-d-aspartate
nuclear magnetic resonance
nuclear pore complex
neurotrophin receptor-interacting MAGE homolog
neurotrophin receptor-interacting factor
nonsteroidal anti-inammatory drug
N-ethylmaleimide-sensitive fusion protein
nucleosome remodeling factor

OCT
OPR
OT

octopamine
octicopeptide repeat
oxytocin

PACAP
PAGE
PBP
PCP
PCR

pituitary adenylate cyclase-activating polypeptide

polyacrylamide gel electrophoresis
periplasmic binding protein
planar cell polarity
polymerase chain reaction

xxxi

xxxii

Guide to Acronyms

PDB
PDE
PDGF
PDK
PDZ
PGHS
PH
PIC
PIH
PIKK
PIP
PKA
PKB
PKC
PKG
PKR
PLA2
PLC
PMCA
PNS
POMC
PP-II
PRH
PRL
PS
PSD
PSD-95
pSMAC
PTB
PTH
PTHrH
PTPC
PYD

protein data bank

phosphodiesterase
platelet-derived growth factor
phosphoinositide-dependent protein kinase
PSD-95, DLG, ZO-1
endoperoxide H synthase
pleckstrin homology
pre-initiation complex
prolactin-inhibiting hormone
phosphoinositide 3-kinase related kinase
phosphatidylinositol phosphatase
protein kinase A
protein kinase B
protein kinase C
protein kinase G
protein kinase R
phospholipase A2
phospholipase C
plasma membrane calcium ATPase
peripheral nervous system
pro-opiomelanocortin
polyproline (helix)
prolactin-releasing hormone
prolactin
pseudosubstrate
postsynaptic density
postsynaptic density protein of 95 kDa
peripheral supramolecular activation cluster
phosphotyrosine binding
parathyroid hormone
parathyroid hormone related protein
permeability transition pore complex
pyrin domain

QM

quantum mechanics

RACK
RAIP
RE
REM
RF
RGS
RH
RHD
RIP

receptor for activated C-kinase

Arg-Ala-Ile-Pro (motif)
responsive (response) element
rapid eye movement
radiofrequency
regulator-of-G-protein signaling
RGS homology
rel homology domain
receptor-interacting protein

Guide to Acronyms

RNA
RNP
ROS
RPA
RR
RRE
RRM
rRNA
RSC
RT
RTK
RyR

ribonucleic acid
ribonucleoprotein
reactive oxygen species
replication protein A
response regulator
rev response region
RNA recognition motif
ribosomal RNA
remodels the structure of chromatin
reverse transcriptase
receptor tyrosine kinase
ryanodine receptor

S/T
S6K
SAGA
SAM
SAM
SAN
SARA
SC1
SCR
SDS
SE
SERCA
SH2
Shh
SIV
Ski
Smac
SMCC
SN
SNAP
SNARE
SNF
SnoN
snRNA
snRNP
SODI
Sos
SP
SSRI
SST
STAT
Ste2

serine/threonine
ribosomal S6 kinase
Spt-Ada-Gen5 acetyltransferase
S-adenosyl-l-methionine
sterile a motif
sinoatrial node
smad anchor for receptor activation
Schwann cell factor-1
short consensus repeat
sodium dodecyl sulfate
spongiform encephalopathies
sarco-endoplasmic reticulum calcium ATPase
Src homology-2
sonic hedgehog
simian immunodeciency virus
SloanKettering Institute proto-oncogene
second mitochondrial activator of caspases
SRD- and MED-containing cofactor complex
sunstantia nigra
soluble NSF-attachment protein
soluble NSF-attachment protein receptor
sucrose nonfermenting
ski-related novel gene N
small nuclear RNA
small nuclear ribonucleoprotein particle
superoxide dismutase
Son-of-sevenless
substance P
selective serotonin reuptake inhibitor
somatostatin
signal transducer and activator of transcription
sterile 2

xxxiii

xxxiv

Guide to Acronyms

STG
STRE
STTK
SUMO
SWI

stomatogastric ganglion
stress responsive element
serine/threonine and tyrosine kinase
small ubiquitin-related modier
(mating type) switch

TACE
TAF
TAR
TBP
TCA
TCR
TF
TGF-b
TGIF
TM
TNF
TOF
TOP
TOR
TOS
TRADD
TRAF
TRAIL
TRAP
TRF1
tRNA
TSH

tumor necrosis factor-a converting enzyme

TBP-associated factor
transactivating response (region)
TATA box binding protein
tricyclic antidepressants
T-cell receptor
transcription factor
transforming growth factor-b
TG3-interacting factor
transmembrane
tumor necrosis factor
time-of-ight
terminal oligopyrimidine
target of rapamycin
Phe-Glu-Met-Asp-Ile (motif)
TNF-R-associated death domain
TNF receptor-associated factor
TNF-related apoptosis-inducing ligand
thyroid hormone receptor-associated protein
telomeric repeat binding factor 1
transfer RNA
thyroid-stimulating hormone

UP
UPEC
UTR

upstream (sequence)
uropathogenic E. coli
untranslated region

VAMP
VDAC
VEGF
VIP
Vps
VTA

vesicle-associated membrane protein

voltage-dependent anion channels
vascular endothelial growth factor
vasoactive intestinal peptide
vascular protein sorting
ventral tegmental area

Wg

wingless

XIAP

X-chromosome-linked inhibitor of apoptosis

YFP

yellow uorescent protein

ZO-1

zona occludens 1

1
Introduction

Life on Earth is remarkably diverse and robust. There are organisms that
live in the deep sea and far underground, around hot midocean volcanic
vents and in cold arctic seas, and in salt brines and hot acidic springs. Some
of these creatures are methanogens that synthesize all their essential biomolecules out of H2, CO2 and salts; others are hyperthermophiles that use
H2S as a source of hydrogen and electrons, and still others are halophiles
that carry out a form of photosynthesis without chlorophyll. Some of these
extremeophiles are animallike, while others are plantlike or funguslike or
like none of these.
What is signicant about this diversity is that although the details vary
from organism to organism, all carry out the same core functions of metabolism, cell division and signaling in roughly the same manner. The underlying unity extends from tiny parasitic bacteria containing minimal
complements of genes to large differentiated multicellular plants and
animals. Each organism has a similar set of basic building blocks and
utilizes similar assembly principles. The myriad forms of life arise mostly
through rearrangements and expansions of a basic set of units rather than
different biochemistries or vastly different parts or assembly rules.

1.1 Prokaryotes and Eukaryotes

There are two basic forms of cellular organization, prokaryotic and eukaryotic. Prokaryotesbacteria and archaeonsare highly streamlined unicellular organisms. Prokaryotes such as bacteria are small, typically 1 to 10
microns in length and about 1 micron in diameter. They may be spherical
(coccus), or rod shaped (bacillus), or corkscrew shaped (spirochette).
Regardless of their shape, prokaryotic cells consist of a single compartment
surrounded by a plasma membrane that encloses the cytoplasm and separates outside from inside. The genetic material is contained in a small
number, usually one, of double-stranded, deoxyribonucleic acid (DNA)
molecules, the chromosomes that reside in the intracellular uid medium
1

1. Introduction

(cytosol). Bacterial chromosomes are typically circular and are compacted

into a nucleoid region of the cytosol. Many bacterial species contain
additional (extra-chromosomal) shorter, circular pieces of DNA called
plasmids.
The bacterial plasma membrane contains the molecular machinery
responsible for metabolism and the sensory apparatus needed to locate
nutrients. When nutrients are plentiful the bacterial cell organization is
ideally suited for rapid growth and proliferation. There are two kinds of
bacterial cell envelopes. The envelopes of gram-positive bacteria consist of
a thick outer cell wall and an inner plasma membrane. Those of gramnegative bacteria consist of an outer membrane and an inner plasma membrane. A thin cell wall and a periplasmic space are situated between the two
membranes.The plasma membrane is an important locus of activity. In addition to being sites for metabolism and signaling, the plasma membrane and
cell wall are sites of morphological structures extending out from the cell
surface of the bacteria. These include agellar motors and several different
kinds of secretion systems.
Eukaryotic cells are an order of magnitude larger in their linear dimensions than prokaryotic cells. Cells of eukaryotesprotists, plants, fungi, and
animalsdiffer from prokaryotes in two important ways. First, eukaryotic
cells have a cytoskeleton, a highly dynamic meshwork of protein girders that
crisscross these larger cells and lend them mechanical support. Second,
eukaryotic cells contain up to ten or more organelles, internal compartments, each surrounded by a distinct membrane and each containing their
own complement of enzymes. In contrast to prokaryotes, core cellular functions such as metabolism are sequestered in these compartments.

1.2 The Cytoskeleton and Extracellular Matrix

The cytoskeleton and extracellular matrix perform multiple functions. The
cytoskeleton provides structural support, and serves as a transportation
highway and communications backbone. Chromosomes, organelles, and
vacuoles are transported along actin laments and microtubules of the
cytoskeleton. Actin laments are used for short distance transport, while
microtubules serve as a rail system for delivering cargo over long distances.
Signal molecules are anchored at sites along the cytoskeleton, and the
cytoskeleton functions as a communications backbone linking signaling
molecules in the plasma membrane and extracellular matrix (ECM) to
signaling units in the cell nucleus.
The extracellular matrix consists of an extended network of polysaccharides and proteins secreted by cells. The ECM provides structural support
for cells forming organs and tissues in multicellular eukaryotes. In plants,
the ECM is referred to as the cell wall and serves a protective role. Cells of
animals secrete a variety of signaling molecules onto the extracellular

1.3 Core Cellular Functions in Organelles

matrix, and these molecules guide cellular migration and adhesion during
development. The ECM is not a simple passive medium. Instead, signaling
between ECM and the cytoskeleton is maintained throughout development
and into adult life.
The existence of a transport system in which large numbers of molecules can be moved along the cytoskeleton to and from the plasma membrane is important for signaling between cells in the body. In the immune
system, transport vacuoles move signal molecules called cytokines (antiinammatory agents such as histamines, and antimicrobial agents that
attack pathogens) to the cell surface where they are secreted from the cell.
In the nervous system, neurotransmitters are moved over long distances
down the axon and into the axon terminal via transport vesicles. In addition to outbound trafcking, there is inbound trafcking. Surface components are continually being recycled back to the internal organelles, where
they are then either reused or degraded.

1.3 Core Cellular Functions in Organelles

In prokaryotes, a single outer membrane is sufcient for membranedependent processes such as photosynthesis and oxidative phosphorylation
(respiration), and protein and lipid synthesis. However, a single membrane
is not adequate in eukaryotes because of the large, cubic increase in cell
volume. Natures solution to this design problem is a system of organelles
surrounded by membranes that perform membrane-specic cell functions
and sequester specic sets of enzymes. There are more than a half dozen
different kinds of organelles in a typical multicellular eukaryote. Organelles
present in typical multicellular eukaryotic cells are listed in Table 1.1, along
with their cellular functions.

Table 1.1. Organelles of the eukaryotic cell: The

principal functions of the proteins sequestered in these
organelles are listed in the second column.
Organelle
Mitochondria
Chloroplasts
Nucleus
Endoplasmic reticulum
Golgi apparatus
Lysosomes
Peroxisomes
Endosomes

Function
Respiration
Photosynthesis (plants)
Stores DNA; transcription and
splicing
Protein synthesis-translation
Processing, packaging, and shipping
Degradation and recycling
Degradation
Internalization of material

1. Introduction

Organelles are characterized by the mix of enzymes they contain and by

the assortment of proteins embedded in their membranes. Three kinds of
proteinspores, channels, and pumpsembedded in plasma and organelle
membranes allow material to enter and leave a cell or organelle.
Pores: Pore-forming proteins, or porins, are membrane-spanning proteins
found in the outer membrane of gram-negative bacteria, mitochondria
and chloroplasts. They form water-lled channels that enable hydrophilic
molecules smaller than about 600 Da to pass through the membrane in
and out of the cell or organelle. For example, bacterial porins allow nutrients to enter and waste products to exit the cell while inhibiting the
passage of toxins and other dangerous materials.
Ion channels: These are membrane-spanning proteins forming narrow
pores that enable specic inorganic ions, typically Na+, K+, Ca2+ or Cl-, to
pass through cell membranes. Ion channels are an essential component
of the plasma membranes of nerve cells, where they are responsible for
all electrical signaling. Ion channels regulate muscle contractions and
processes associated with them, such as respiration and heartbeat, and
regulate osmobalance and hormone release.
Pumps: Pumps are membrane-spanning proteins that transport ions and
molecules across cellular and intracellular membranes. While ion channels allow ions to passively diffuse in or out of cells along electrochemical
gradients, pumps actively transport ions and molecules.Thus, they are able
to act against electrochemical gradients, whereas ion channels cannot,
and maintain homeostatic balances within the cell. The transport involves
the performance of work and must be coupled to an energy source. A
variety of energy sources are utilized by pumps, including adenosine
triphosphate (ATP) hydrolysis, electron transfer, and light absorption.

1.4 Metabolic Processes in Mitochondria

and Chloroplasts
In all cells, energy is stored in the chemical bonds of adenosine triphosphate
(ATP) molecules. In metabolism, enzymes break down large biomolecules
into small basic components, synthesize new biomolecules out of those basic
components, and produce ATP. In catabolic processes such as glycolysis and
oxidative phosphorylation, large polymeric molecules are disassembled into
smaller monomeric units. The intermediates are then further broken down
into cellular building blocks such as CO2, ammonia, and citric acid. Key
goals of the catabolic processes are the production of ATP and reducing
power needed for the converse, anabolic processesthe assembly of cellular building blocks into small biomolecules, the synthesis of components,
and their subsequent assembly into organelles, cytoskeleton, and other cellular structures.

1.5 Cellular DNA to Chromatin

Glycolysis takes place in the cytoplasm while the citric acid cycle occurs
in the mitochondrial matrix. Five complexes embedded in the inner mitochondrial membrane carry out oxidative phosphorylation (respiration). The
constituents of the ve respiratory complexesenzymes of the electron
transport chainpump protons from the matrix to the cytosol of the mitochondria, then use the free energy released by these actions to produce ATP
from adenosine diphosphate (ADP). Their photosynthetic counterparts,
photosystems I and II, function in chroloplasts.
Chloroplasts and mitochondria are enclosed in double membranes. The
inner membrane of a mitochondrion is highly convoluted, forming structures called cristae. A similar design strategy is used in chroloplasts. The
inner membrane of a chloroplast encloses a series of folded and stacked
thylakoid structures. These designs give rise to organelles possessing large
surface areas for metabolic processes. The ATP molecules are used not only
for anabolism, but also in other core cellular processes, including signaling,
where work is done and ATP is needed.
The relocation of the machinery for metabolism from the plasma membrane to internal organelles is a momentous event from the viewpoint of
signaling. It not only provides for a far greater energy supply but also frees
up a large portion of the plasma membrane for signaling. In eukaryotic cells,
the plasma membrane is studded with large numbers of signaling proteins
that are either embedded in the plasma membrane, running from the
outside to the inside, or attached to one side or the other by means of a
tether.

1.5 Cellular DNA to Chromatin

Cellular DNA is sequestered in the nucleus where it is packaged into chromatin. As shown in Figure 1.1, all organisms on Earth today use DNA to
encode instructions for making proteins and use RNA as an intermediate
stage. This fundamental aspect of all of biology was rmly established by
Crick and Watson in their pioneering study in the mid-twentieth century.
In the rst steptranscriptionprotein machines copy selected portions
of a DNA molecule onto mRNA templates. In prokaryotes the ribosomal machinery operating concurrently with the transcription apparatus
translates the mRNA molecules into proteins. In eukaryotes there is an

Figure 1.1. Genes and proteins: Depicted is the two-step process in which DNA
nucleotide sequences, or genes, are rst transcribed onto messenger RNA (mRNA)
nucleotide sequences, and then these templates are used to translate the nucleotide
sequences into amino acid sequences.

1. Introduction

intermediate step: Protein machines known as spliceosomes edit the initial

RNA transcripts called pre-mRNA molecules, and produce as their output
mature mRNA molecules. The ribosomal machines then translate the
mature mRNAs into proteins.
In eukaryotes, cellular DNA is sequestered within the nucleus, and this
organelle is the site of transcription and splicing. The nucleus is enclosed in
a concentric double membrane studded with large numbers of aqueous
pores. The pores enable the two-way selective movement of material
between the nucleus and cytoplasm. Since proteins are synthesized in the
cytoplasm, nuclear proteinsproteins that carry out their tasks inside the
nucleusare imported from the cytoplasm to the nucleus, while messenger
RNAs and ribosomal subunits are exported. A variety of structural and regulatory proteins regularly shuttle back and forth between nucleus and cytoplasm. The pores, referred to as nuclear pore complexes, are composed of
about 100 proteins and are approximately 125 MDa in mass. All particles
entering or exiting the nucleus pass through these large pores. Small particles passively diffuse through the pores while large macromolecules are
actively transported in a regulated fashion.
The sequestering of the DNA within a nucleus is advantageous for
several reasons. It insulates the DNA against oxidative byproducts of
normal cellular processes taking place in the cytoplasm and from mechanical forces and stresses generated by the cytoskeleton. It separates the
transcription apparatus from the translation machinery, thereby allowing
independent control of both, and it makes possible the intermediate ribonucleic RNA editing (splicing) stage.
Eukaryotic DNA is wrapped in proteins called histones and tightly packaged into a number of chromosomes in the nucleus. As a result of sequestering and packaging, far more information can be stored in eukaryotic
DNA than in prokaryotic DNA. The wrapping up of the DNA to form chromatin enables the cells to regulate transcription of its genes in a particularly simple way that is not possible in prokaryotes. When the DNA is
wrapped tightly about the histones the DNA cannot be transcribed since
the sites that need to be accessible to the transcription machinery are
blocked. When the wrapping is loosened, these sites become available and
transcription can be carried out. A large number of eukaryotic regulators
of transcription operate on a chromatin-level of organization, making transcription easier or harder by manipulating chromatin.

1.6 Protein Activities in the Endoplasmic Reticulum

and Golgi Apparatus
The endoplasmic reticulum (ER) encompasses more than half the membrane surface of a eukaryotic cell and about 10% of its volume. It is
the primary site of protein synthesis (translation), fatty acid and lipid

1.6 Protein Activities in the Endoplasmic Reticulum and Golgi Apparatus

synthesis, and bilayer assembly. It is divided into a rough ER and a smooth

ER. The rough ER gets its name from the presence of numerous ribosomes
bound to its cytosolic side.The rough ER is the site where membrane-bound
proteins, secreted proteins, and proteins destined for the interior (lumen)
of organelles are synthesized. The smooth ER lacks ribosomes. It is the site
where lipids are synthesized and assembled and where fatty acids such as
steroids are synthesized. It stores intracellular Ca2+ and assists in carbohydrate metabolism and in drug and poison detoxication.
Not all ribosomes are bound to the endoplasmic reticulum. Instead, there
are two populations of ribosomes, bound and free. Bound ribosomes are
attached to the rough ER, but free ribosomes are distributed in the cytosol.
The free ribosomes are otherwise identical to their membrane-bound
counterparts, and they synthesize cytosolic proteins.
In order for a protein to carry out its physiological function it must fold
into and maintain its correct three-dimensional shape. Proteins are subject
to several different kinds of stresses. Abnormal conditions, such as elevated
or reduced temperatures and abnormal pH conditions, can result in the
denaturization (unfolding) or misfolding of proteins so that they no longer
have the correct shape and cannot function. Another type of condition that
can affect the shape of the protein is molecular crowding. A group of small
protein-folding machines called heat shock proteins or stress proteins or
molecular chaperones guide nascent polypeptide chains to the correct location and maintain the proteins in folded states that permit rapid activation
and assembly. They also refold partially unfolded proteins. In those cases
where the proteins cannot be returned to a proper state the misfolded proteins are tagged for destruction by another set of small protein machines
called proteases. These proteolytic machines enable a cell to degrade and
recycle proteins that are no longer needed, as well as those that are
damaged and cannot be refolded properly by the stress proteins.
Newly synthesized proteins are processed, subjected to quality control
with respect to their folding, and then shipped to their cellular destinations.
Prosthetic groupssugars and lipidsare added to proteins destined for
insertion in the membrane to enable them to attach to the membranes.
These modications are made subsequent to translation in several stages,
as the proteins are passed through the ER and Golgi apparatus. The overall
process resembles an assembly line that builds up the proteins, folds them,
inserts them into membranes, sorts them, labels them with targeting
sequences, and ships them out to their cellular destinations (Figure 1.2).
The Golgi apparatus consists of a stacked system of membrane-enclosed
sacs called cisternae. Some of the polysaccharide modications needed to
make glycoproteins are either made or started in the rough ER. Proteins,
especially signaling proteins destined for export (secretion) from the cell
or for insertion into the plasma membrane, are sent from the rough ER to
the smooth ER where they are encapsulated into transport vesicles pinched
off from the smooth ER. The transport vesicles are then sent to the Golgi

1. Introduction

Figure 1.2. Movement of proteins through the endoplasmic reticulum and Golgi
apparatus: Proteins synthesis and processing start with the export of mRNAs from
the nucleus to the ribosome-studded rough endoplasmic reticulum. Nascent proteins synthesized in ribosomes are processed and then shipped in transport vesicles
to the Golgi. They pass through the cis (nearest the ER) and trans (furthest from
the ER) Golgi, and the nished products are then shipped out to their lysosomal
and the plasma membrane destinations.

for further processing and eventual shipping to their cellular destinations.

The Golgi apparatus takes the carbohydrates and attaches then as oligosaccharide side chains to some of these proteins to form glycoproteins and to
complete modications started in the rough ER. Both proteins and lipids
are modied in the Golgi. Other proteins, synthesized as inactive precursor
molecules, are processed to produce activated forms in the Golgi. Modied
proteins are enclosed in transport vesicles, pinched off from the Golgi, and
shipped to destinations such as the plasma membrane and the extracellular matrix (Figure 1.2).

1.7 Digestion and Recycling of Macromolecules

Digestion and the recycling of macromolecules take place in a network of
transport and digestive organelles. The last three organelles listed in Table
1.1 are involved in digestion. Peroxisomes and lysosomes contain sets of
enzymes used for digestion of macromolecules. In these highly acidic environments, macromolecules are broken down into smaller molecules. By
sequestering enzymes in these compartments the rest of the cell is protected
from the digestive properties of the enzymes. Lysosomes are small
organelles that degrade ingested bacteria and nonfunctional organelles.

1.8 Genomes of Bacteria Reveal Importance of Signaling

Perixosomes are utilized for the sequestering of oxidative enzymes. Their

digestive enzymes degrade fatty acids to small biomolecules. Peroxisomes
are a diverse collection of organelles, each with its own mix of enzymes.
Some peroxisomes detoxify harmful substances. Others, in plants, convert
fatty acids to sugars and carry out photorespiration.
Endosomes, the nal set of eukaryotic organelles listed in Table 1.1, facilitate the transport of extracellular material and membrane proteins from
the plasma membrane to lysosomes for degradation. Several kinds of
organellesearly endosomes, carrier vesicles, and late endosomesform a
transport and sorting system that moves ingested foodstuffs, captured
pathogens, dead material, and ligand-bound receptors and lipid plasma
membrane components to the lysosomes and other cellular compartments
for either degradation or reuse.
In summary, cells are highly dynamic entities; materials are continually
being brought in and out of the cell, and moved back and forth to the
surface. There is a continual ow of outbound trafc of cargo from the ER
and Golgi to organelles and the cell surface, and there is a continual ow
of inbound trafc from the cell surface to lysosomal and peroxisomal compartments. Signal proteins destined for the plasma membrane and for secretion are packaged into vacuoles. These transient structures are formed by
pinching off portions of membrane. The vacuoles are moved over the rail
system and fused with membranes at the destination (exocytosis). Similarly,
materials from the cell surface are captured, packaged into vesicles, and
shipped to digestive compartments for processing (endocytosis).

1.8 Genomes of Bacteria Reveal Importance

of Signaling
Insights into the importance of signaling can be obtained from analyses of
the composition of the genomes of bacteria. Prokaryotes are tiny organisms
that tell us a lot about signaling. Prokaryotic genomes range in size from
0.5 MBp to more than 12 MBp. The Mycoplasmas sit at the bottom of this
range; they are minimal organisms. They occupy very limited ecological
niches, are restricted in their metabolic capabilities, and have the smallest
genomes of any organisms. Genes encoding signal proteins are largely nonexistent, taking up no more than about 1% of their genomes. Prokaryotes
with somewhat larger genomes include the archaeal extremophiles mentioned at the beginning of the chapter, and many obligate bacterial parasites that are the causal agents of diseases in humans. These organisms
live in fairly constant and unvarying environments, and as a result their
requirements on signaling and control are modest. Their signaling proteins
account for no more than a few percent of their genomes.
Prokaryotes that can alter their metabolic and reproductive strategies to
match their changing environmental conditions have larger genomes than

10

1. Introduction

those that live under constant conditions. Because of their ability to adapt
their physiology to their environment, these bacteria may be referred to as
environmentalists. Escherichia coli and Psuedomonas aeruginosa are typical
environmentalists. Their genomes are ve to ten times larger than the
Mycoplasmas and encode ensembles of signaling and regulatory proteins
that are 50 to 100 times larger than those of the Mycoplasmas. As a result,
they are able to thrive in a variety of environmentssoil, water, airand
they deal with many different stresses. Thus, not only are the genomes
becoming larger as the bacteria become more versatile and adaptive, but
the fractions of the genomes devoted to regulatory functions are increasing as well.
Colony-forming bacteria have even larger genomes. These bacteria can
not only cope with environmental changes and stresses, but also they can
assemble into colonies and exhibit a limited form of differentiation. Their
genomes are the largest of all the prokaryotes, and the fraction of their
genomes devoted to signaling and control approaches or exceeds 10%. An
example of a prokaryote that exhibits environmental diversity and colonial
behavior is Streptomyces coelicolor. This versatile soil bacterium is used
for the production of antibiotics such as tetracycline and erythromycin. Its
signaling component accounts for 12% of its genome.
As might be expected, the genomes for multicellular plants and animals
are larger than those for even the most sophisticated prokaryotes, but not
by as much as one might expect. The rst estimates from the complete
sequencings of the human genome are in the range of 26,000 genes, of which
roughly one quarter is devoted to signaling. There are still some genes that
remain to be identied, and when further analyses are completed there may
be as many as 32,000 to 40,000 genes. This number appears to be astonishingly low. It is scarcely a factor of three larger than the genomes for some
of the bacteria. Furthermore, the genome for the bacterium S. coelicolor is
nearly as large as that for the highly differentiated, multicellular fruit y
Drosophila melanogaster.

1.9 Organization and Signaling of Eukaryotic Cell

Eukaryotic cell organization and expanded signaling capabilities make
multicellularity possible.The signicance of these observations is that something more than a simple increase in genome size produced the greatly
increased complexity associated with multicellular plants and animals. The
answer to the question of what is happening has several parts. It involves
the way eukaryotic cells are organized and the way the expanded repertoire of signaling proteins is organized and used. There are four broad
categories of environmental and regulatory signals. These are as follows:
Physical and chemical sensations indicative of external conditions.
Contact signals indicative of ECM-to-cell and cell-to-cell adhesion.

1.9 Organization and Signaling of Eukaryotic Cell

11

Signals sent from one cell to another that allow the sender to regulate
gene expression and other cellular responses in the recipient.
Signals and sensations indicative of internal stresses and balances.
The rst category includes a diverse set of physical and chemical signals.
Most organisms can neither alter their environments nor move over large
distances. Instead, they must continually adapt their metabolism and growth
strategies to match the environmental conditions in which they nd themselves. In this grouping of signals are environmental cues important to unicellular organisms, such as light, temperature, osmolarity, pH, and nutrients.
Also included in this category are signals such as odorants and tastants
detected by sensory organs in multicellular animals, or metazoans.
The next category encompasses contact signals between surfaces and is
specic to multicellular organisms. Proteins embedded in the plasma membrane and in the ECM convey contact signals. These signals allow cells to
establish and maintain physical contact with supporting structures within
the body, and mediate two-way communication between cells in physical
contact. This category is greatly expanded in vertebrates, and includes elements of the immune system such as antibodies that have evolved from
adhesion molecules.
The third category of signals consists of the cell-to-cell messages. This
category includes pheromones, chemical signals that promote mating and
colonial behavior in unicellular organisms, and it includes the signals in
multicellular plants and animals that allow cells in tissues and organs to
work together. In humans, there are several systems of tissues, organs, and
glands that continually send and receive chemical messages. Cells of the
immune systems send and receive cytokines; cells residing in glands of the
endocrine system secrete hormones; and neurons in the nervous system
communicate using neurotransmitters and neuromodulators. Embryonic
development is controlled from cell division to cell division by programs of
gene expression. The category of cell-to-cell signals encompasses the signals
that help establish cell fate and polarity during development, and the
growth factor and hormonal signals that shape and guide the programs of
cell growth and differentiation.
The last category consists of signals generated within the cell that help
maintain proper internal balances, or homeostasis. This category includes
signals indicative of internal stresses such as improper pH conditions, excessive temperatures, and water imbalances. Macromolecules such as DNA
and proteins are marginally stable under physiological conditions. Cellular
DNA can be damaged by ultraviolet radiation, by ionizing radiation, and
by oxidative byproducts of normal cellular processes. In addition, DNA
strand breaks can occur during the DNA replication stage that precedes
mitosis and meiosis. All cells, prokaryotic and eukaryotic, possess DNA
repair systems that continually sense and repair single and double strand
breaks. In multicellular organisms, whenever DNA damage is detected and

12

1. Introduction

found to be irreparable, the cell is targeted for destruction. The process of

eliminating a cell that is damaged, or infected, or deemed to be no longer
needed is called apoptosis. The apoptosis, or cell suicide, machinery cuts up
(cleaves) and disassembles large cellular componentsDNA and membranesand packages the cellular contents in such a way that they do no
harm to neighboring cells. The last category of signals includes those that
integrate growth and survival signals with repair and apoptosis signals,
determining whether a cell grows and proliferates, repairs itself, or dies.

1.10 Fixed Infrastructure and the Control Layer

The proteins responsible for signaling and control form a control layer. This
layer sits on top of a lower layer, the xed infrastructure, which is responsible for core cellular functions such as metabolism and replication. Proteins belonging to two layers carry out their cellular roles synergistically.
Proteins belonging to the control layer make contact with elements of the
xed infrastructure at well-dened loci, or control points, where they exert
their regulatory functions, but dont otherwise interfere with the machinelike operations of those proteins. In turn, eukaryotic architecture, with its
organelles and cytoskeleton, is especially well suited for signaling and is
extensively exploited for that purpose.
Unlike the proteins of the xed infrastructure, the proteins belonging
to the control layer are not sequestered within a single compartment or
organelle. Instead, they form a meshwork of signaling pathways that extend
throughout the cytoplasm and into organelles, most notably the nucleus,
but other as well. Each signaling pathway has a start point and an end
point. The start points are typically proteins that function as sensors and
as receivers of signals from other cells. These proteins are often associated
with the plasma membrane, where outside meets inside, and are referred to
as receptors. The receptors not only detect the signals but also convert them
into forms that can be understood and processed further within the cell.
This conversion process is called signal transduction.The signaling pathways
terminate at sites where the elements of the control layer come into contact
with the components of the xed infrastructure.These are the control points
where the environmental and regulatory signals are converted into cellular
responses.
The cell nucleus contains large numbers of control points, and when these
sites are the end points the signaling process is termed gene regulation.
Alternatively, and more generally, the control processes carried out by
signaling proteins is referred to as cell regulation. One of the key factors
making possible the emergence of complex multicellular organisms is the
formation of gene regulatory networks composed of transcription regulating proteins, or transcription factors, and the DNA sequences they bind.

1.11 Eukaryotic Gene and Protein Regulation

13

Table 1.2. Comparison of proteins in the xed infrastructure and control layer.
Property
Location
Mobility
Lifetime
Structure
Function

Fixed infrastructure
Machines/factories in organelles
Little
Longer
Fixed
Unifunctional

Control layer
Complexes in subcompartments
Considerable
Shorter
Variable
Multifunctional

Changes in how these networks are built rather than in the genes they
control underlie the increased complexity of multicellular life.
Signal proteins are not only the messengers but also the messages. Since
the messages must be conveyed from one place to another, mobility is a
key property of the proteins. Mobility is less important for functions such
as metabolism carried out as part of large molecular machines in the
organelles. Another way signal proteins differ from the other proteins is in
the presence of post-translational modications. Signal proteins are subjected to a host of post-translational modications. Some are made in the
ER and Golgi as part of the nishing process, but others, to be discussed in
the next chapter, are part of the signaling process itself. These alterations
endow the proteins with switchlike response properties, turning them on
and off.This property is absolutely essential for a signaling element, keeping
it available for conveying a message, but in an off-conguration until an
activating signal arrives.
There are several other ways that proteins belonging to the control layer
and xed infrastructure differ from one another (Table 1.2). The function
of a protein, that is, what it does, is determined by its associations, and by
where and when it establishes them. Proteins that function as part of the
transcription machinery in the nucleus or as part of the electron transport
chain in the mitochondria, usually have a single purpose, and they carry
out this task over and over. The signaling proteins form complexes too.
However, the protein complexes are smaller than the large machines used
for metabolism and replication, and the associations and interactions are
more variable. They can be different in different cell types and will even
vary somewhat over time in the same cell type. Because of this exibility,
signaling proteins are multifunctional, or pleiotropic, in their actions.

1.11 Eukaryotic Gene and Protein Regulation

Eukaryotic genes and proteins can be regulated in several ways. One of the
most important consequences of switch from prokaryotic to eukaryotic cell
organization is the creation of a large number of ways of controlling the

14

1. Introduction

mix of proteins being expressed at any given time in a cell. In place of DNA
regulation of prokaryotic gene and protein expression there is now a multiplicity of eukaryotic control mechanisms. Gene and protein expression can
be controlled through

DNA regulation
histone modication
splicing regulation
translation regulation
nuclear import/export

Histone modication has already been discussed. Nuclear import and

export refers to a strikingly simple and widely used form of regulation.
Many transcription factors are parked in convenient locations in the
cytoplasm awaiting activating signals. When activated they diffuse to
the nucleus, where they carry out their transcriptional activities. Exporting
the proteins back out of the nucleus into the cytoplasm is an equally simple
way of terminating their activities.
Translational regulation allows for the placement of messenger RNAs
(mRNAs) in sites where the proteins they encode might be needed at a
later time. Asymmetric distributions of mRNAs and proteins in cells early
in development lead to offspring that are dissimilar. Localized populations
of mRNAs are utilized as part of adult physiology. They are used, for
example, in nerve cells to avoid long time delays arising when signals must
be sent over long distances to the nucleus and the resulting proteins shipped
back out to the distant extrema.
The observation that for every one unique gene there is one unique
protein is correct as far as it goes, but in eukaryotes one must append the
equally true statement that the unique proteins come in several avors.
Alternative splicing permits adjustments to be made to the proteins
expressed in different cell types. Eukaryotic genes are larger than their
prokaryotic counterparts and contain greater numbers of units called
domains (these are discussed in the next chapter). In vertebrates there are,
on the average, about three alternative spliced forms, or avors, for each
protein. Signals sent to the control points in the splicing machinery help
determine which spliced variant, or isoform, gets made at that particular
time in that cell type.
A piece of the mystery of the relatively small size of the eukaryotic
genome is resolved by the observation that alternative splicing creates
many variants from a single gene, alleviating the need to store the instructions for making each variant in the DNA. Alternative splicing and posttranslational modications are extensively utilized in the control layer. If
all of these avors are counted as distinct items, the resulting signaling
protein numbers would comprise most of the genome, and the eukaryotic
totals would be far more impressive. One of the consequences of this multiplicity is that study of the control layer is more difcult than it would be

1.12 Signaling Malfunction Central to Human Disease

15

otherwise. Because of the large numbers of structural variations, and also

because of the multifunctionality, mobility, low copy numbers, and short
lifetimes of the signaling proteins (Table 1.2), understanding of the control
layer is not as advanced as that of the proteins in the xed infrastructure
involved in, for example, metabolism.
Lying at the heart of biology is the following observation, succinctly
stated by Francois Jacob in a 1977 article in Science: What distinguishes a
buttery from a lion, a hen from a y, and a worm from a whale is much
less about differences in chemical constituents than in the organization
and distribution of their components. In other words, all creatures, great
and small, carry out metabolism, replication, multiplication, and division in
much the same way. They differ from one another mainly in the way their
parts are arranged. Multicellular eukaryotes such as ies and worms are
clearly far more complex than bacteria. Yet it only takes a factor of two or
three more genes to get from one to the other. The answer to how this can
be possible is not in the numbers of genes or in a new type of biochemistry,
but rather in the way that the genes and their products, the proteins, are
used, and in the way eukaryotic cells are organized.

1.12 Signaling Malfunction Central to Human Disease

Malfunctions in molecular and cellular signaling lie at the heart of human
diseases. Proteins belonging to the control layer are involved in a host of
human disorders. They are key elements in cancers and in neurodegenerative disorders in the elderly and mood disorders in the young. Signaling
processes make complex organisms like humans possible, but when there
are malfunctions, the signaling processes give rise to diseases in those very
same organisms.
Improper expression levels and malfunctions of signaling proteins are
responsible for a host of human cancers. The underlying causes of cancers
are mutations and other alterations in DNA. These aberrations produce
malfunctions and inappropriate expression levels of genes encoding
proteins that either promote growth or restrain it, or direct the apoptosis
machinery, or are responsible for DNA damage repair and signaling, and
chromatin remodeling. Erroneous signaling conveys inappropriate growth
signals, fails to turn on the bodys cell suicide program when it is needed, and
fails to repair DNA damage when it occurs.
The brain is the most complex organ in the body. A substantial portion
of the human genome is taken up with encoding brain-specic signaling
proteins. Some of these, such as the ion channels, endow the neurons
with the ability to generate action potentials, which are used to signal other
neurons and control muscle cells. Improper and excessive rhythms resulting from imbalances between the excitation and inhibition of neurons are
responsible for epileptic seizures and a host of attention, learning, and

16

1. Introduction

mood disorders. Proteolytic processing is a prominent part of the signaling

routes activated during embryonic development. But some of the same processing elements that are crucial for embryonic development early in life
contribute to neurological disorders such as Alzheimers disease late in life.
Receptors, the proteins that reside in the plasma membranes of cells and
receive signals from other cells are key targets of therapeutic drugs. These
drugs act as ligands for the receptors, and are intended to elicit one of two
kinds of actions: (1) By binding the receptor, the drug may activate the signaling pathway into the cell, stimulating processes that are otherwise not
properly working; (2) or alternatively, the drug may serve as a null ligand
one that can bind the receptor but not stimulate signaling when doing so.
This second type of action is that of a blocker, since it ties up the receptor,
preventing other ligands from binding it and activating the signaling
pathway.
The preeminent family of receptors in humans is the G protein-coupled
receptor family. They are responsive to hormones, neuromodulators, and
neurotransmitters. Some 40 to 60% of all drugs target G protein-coupled
receptors. Some of the best known of these are the serotonin and adrenergic receptor-targeted drugs that treat depression, and the dopamine
receptor-directed drugs that treat schizophenia. Other examples are the
vasopressin receptor-mediated drugs that act as antidiuretics and the
angiotensin receptor-targeted drugs that treat hypertension. Three more
examples are: the histamine receptor-targeted drugs that alleviate allergic
symptoms, the opioid receptor-targeted drugs that alter mood, and the
neurokinin receptor-targeted drugs that alleviate pain.

1.13 Organization of Text

The textbook is organized into several parts. Chapters 2 through 5 serve as
an introduction, providing background information helpful for an understanding of signaling. Chapter 2 gives an overview of the control layer and
its relationship to cellular, nuclear, DNA, and protein organization. Chapter
3 examines the principal experimental methods used to probe the structure
of signaling proteins and their interactions with one another. One of the
most interesting and intensively studied processes in all of science is the
folding of newly synthesized proteins into their physiologically functional
three-dimensional shape. Chapter 4 has as its focus energy considerations.
It covers how energy considerations drive protein folding and binding, and
how proteins fold in the cell with the assistance of molecular chaperones.
Chapter 5 deals with the macromolecular forces that underlie not only how
proteins assume their functional forms but also how they interact with each
other.
The next three chapters serve as an introduction to signaling. A good
starting point for any discussion of signaling is the plasma membrane; it is

1.13 Organization of Text

17

the place where environmental conditions are sensed and most cell-to-cell
signals are received. The yeast stress and pheromone signaling systems, the
focus of Chapter 6, and the bacterial chemotaxis system, the main subject
of Chapter 7, are archetypical signaling systems. Yeasts must constantly
adapt to changing conditions in their environment, and, similarly, bacteria
must locate nutrients and sense and respond to changes in their external
environments. These systems exhibit many of the properties and principles
that characterize signaling in more complex organisms. For many years the
plasma membrane was regarded as a fairly homogeneous and passively uid
substrate into which signaling proteins were embedded. That view has
undergone considerable change over the past few years with the realization
that the plasma membrane is organized into distinct signaling domains, and
that several kinds of lipids serve as signaling intermediaries. This new
picture of the plasma membrane, along with the role of small molecules
lipid and nonlipidin signaling is explored in Chapter 8.
The immune system provides several layers of defense against viral,
bacterial, and eukaryotic pathogens. These defenses are coordinated and
regulated by networks of signaling molecules. These signaling molecules are
the subjects of Chapter 9. In response to certain signals, leukocytes, or white
blood cells, converge upon sites of an infection and kill pathogens. Chapter
10 explores the role of signaling events taking place at the cell surface that
make possible the directed movement of leukocytes into an infection site.
Leukocytes are not the only cells requiring motility. During development
cells must move about and aggregate into tissues, and nascent nerve cells
must send out growth messages to connect with other nerve cells. These
signaling processes are examined, too, in Chapter 10.
Cells secrete growth factors and hormones in order to coordinate cellular growth and proliferation. The mechanisms whereby polypeptide growth
factors are received and transduced into cellular responses are discussed in
Chapter 11. G protein-coupled receptors transduce a remarkably diverse
spectrum of messages into the cell. Among these are light, gustatory,
odorant, pheremone, pain, immunological, endocrine, and neural signals.
Signaling through these receptors is covered in Chapter 12.
During embryonic development cell-to-cell signals coordinate the genetic
programs of growth, differentiation, and proliferation. These signaling
events, along with those that regulate motility, guide the formation of organs
and tissues with well-dened boundaries composed of functionally specialized cells derived from less specialized progenitors. The determination of
which tissue or organ a particular cell becomes part of, or cell fate, is the
focus of Chapter 13.
As noted in the last section, cancer and signaling are intimately connected.
Cancer can be regarded as a collection of diseases associated with malfunctions of key elements of the control layer and DNA repair machinery.
The relationships between cancer and aberrant signaling are explored in
Chapter 14. During the past few years the subject of programmed cell death,

18

1. Introduction

or apoptosis, has moved to the forefront of cancer research. The goal is to

create drugs that kill cancers by forcing the cancerous cells to undergo apoptosis. This topic is explored in Chapter 15.
The main way that cells respond to environmental and cellular signals is
to alter gene expression, turning some genes on and others off. In eukaryotes, the primary terminus of the signaling pathways leading from the cell
surface into the cell interior is the transcription and the splicing machinery
located in the cell nucleus. The mechanisms involved in turning on and off
specic genes are the focus of Chapter 16.
The next two chapters, 17 and 18, deal with gene regulation in bacteria
and by viruses. Bacterial cell-to-cell communication and gene regulation are
discussed in Chapter 17. Viruses suborn host defenses in order to gain entry
into a cell and once inside the cell create environments conducive to their
replication. Several examples of how elements of the host and viral control
layers interact are presented in Chapter 18.
The last three chapters of the textbook are devoted to signaling in the
nervous system. Nerve cells, or neurons, send signals to other neurons and
to muscle cells. The main goal of Chapter 19 is to explore how ion channels
open and close and work together to generate action potentials, the hallmark of nerve cell signaling. The next chapter (Chapter 20) describes how
nerve cells working together generate rhythmic activities, some associated
with sleep and other with awakening, some with rhythmic motor activities
such as walking and chewing, and others with heartbeat and breathing. The
last chapter (Chapter 21) deals with how complex organisms ranging from
worms to ies to humans learn and remember and forget and learn again.

General References
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, and Walker P [2002]. Molecular
Biology of the Cell (4th ed). New York: Garland Publishers.
Jacob F [1977]. Evolution and tinkering. Science, 196: 11611166.
Matthews CK, van Holde KE, and Ahem KG [2000]. Biochemistry, 3rd ed. San
Francisco: Pearson Benjamin Cummings.
Stryer L [1995]. Biochemistry, 4th edition. New York: W.H. Freeman and Company.

References and Further Reading

Molecular Machines
Cramer P, et al. [2000]. Architecture of RNA polymerase II and implications for the
transcription mechanism. Science, 288: 640649.
Hirokawa N [1998]. Kinesin and dynein superfamily proteins and the mechanism of
organelle transport. Science, 279: 519526.
Rout, MP, et al. [2000]. The yeast nuclear pore complex: Composition, architecture,
and transport mechanism. J. Cell Biol., 148: 635651.

References and Further Reading

19

Saraste M [1999]. Oxidative phosphorylation at the n de siecle. Science, 283:

14881493.
Vale RD, and Milligan RA [2000]. The way things move: Looking under the hood
of molecular motor proteins. Science, 288: 8895.

Ubiquitin-Preotosome Complex
Ciechanover A [1998]. The ubiquitin-proteosome pathway: On protein death and
cell life. EMBO J., 17: 71517160.

Organelle Function and Trafcking

Mellman I, and Warren G [2000]. The road taken: Past and future foundations of
membrane trafc. Cell, 100: 99112.
Presley JF, et al. [1997]. ER-to-Golgi transport visualized in living cells. Nature, 389:
8185.

Quality Control
Ellgaard L, Molinari M, and Helenius A [1999]. Setting the standards: Quality
control in the secretory pathway. Science, 286: 18821888.
Lindahl T, and Wood RD [1999]. Quality control by DNA repair. Science, 286:
18971905.
Wickner S, Maurizi MR, and Gottesman S [1999]. Posttranslational quality control:
Folding, refolding and degrading proteins. Science, 286: 18881893.

2
The Control Layer

Eukaryotic cell organization impacts signaling by the control layer in

several ways. One of the most important of these is to provide exibility by
increasing the number of control points. The expression of a gene can be
regulated through interactions with the DNA and the transcription machinery, with the histones, and with the splicing machinery. This property of the
eukaryotic control layer is explored further in the rst part of this chapter.
The nucleosome, the basic repeating unit of chromatin, will be discussed
rst and then nuclear architecture will be examined. Chromosomes are not
randomly distributed through the nucleus. Rather, there is a considerable
amount of nuclear order in support of transcription and splicing. The
arrangement of chromosomes into distinct structures and compartments
will be looked at.
Signaling proteins are highly modular, and most signaling proteins appear
to have been built by the forming of different arrangements of a set of
building block domains. Modularity is a good example of efcient coding
if proteins are constructed from almost independent pieces, their parts can
be reused to make other proteins. This method of design facilitates genetic
rearrangements in which new proteins can be created without the body
having to rst design new building blocks. There is a hierarchy of protein
structures from primary to quaternary. This organization will be the presented in the middle part of the chapter.
Proteins belonging to the control layer are themselves controlled through
the addition and removal of small groups of atoms. These changes are
reversible.They are referred to as post-translational modications since they
occur subsequent to translation. They serve several signaling purposes. The
modications inuence the location of the signaling proteins within the cell;
they regulate their signaling activities by turning catalytic activities on and
off, and exposing and hiding interfaces, and they alter the messages they
convey. The main types of posttranslational modications will be presented
in the last part of the chapter.

21

22

2. The Control Layer

2.1 Eukaryotic Chromosomes Are Built

from Nucleosomes
The double-stranded DNA molecules of the eukaryotic cell nucleus form
associations with proteins called histones. The resulting DNA-histone material is known as chromatin. It was initially thought that DNA molecules
were uniformly wrapped in histones. The main function of the largely undifferentiated histones was in support of supercoiling, which allowed the
chromatin to pack tightly into the nucleus. The current picture differs radically from the earlier one. In the new picture, histones are not distributed
uniformly. Instead, there is a fundamental repeating unit of DNA plus
histone called the nucleosome, and these nucleosomes are strung together
much like individual beads on the string. The histones have a quite specic
stoichiometry and act in a dynamic manner to regulate gene transcription.
They are not merely a passive set of girders for the DNA.
Nucleosomes are composed of 146 base pairs of DNA wrapped about a
histone octamer. The duplex DNA is wrapped about a histone core composed of H2A, H2B, H3, and H4 histone pairs. The core nucleosome unit
(shown in Figure 2.4) is connected by a linker segment (called H1) of chromatin to the next nucleosome core unit. Each nucleosome has the DNA
wrapped about 1.75 times about the histone core forming a bead-like structure roughly 11 nm in diameter. The nucleosomes are themselves organized
into 30-nm diameter solenoidal structures, and these next larger structural
units are further wound into loops, and then into minibands, and nally into
tightly coiled chromosomes. By this means a meter or so of DNA is organized into a compact unit that ts into a region a few microns in each linear
dimension.
Growing crystals suitable for use in X-ray crystallography is often the
most difcult step in applying the technique to biomolecules, especially
large ones. In the case of the nucleosome, the challenge was in getting the
phases aligned. The exact positioning of the nucleosome along a length of
DNA is known as the phase. In order to create a crystal suitable for highresolution X-ray crystallography the DNA and histone core of each nucleosome must have the same phase. This problem was solved through the
development of a bacterially derived palindrome (reading the same forwards and backwards) DNA sequence that is able to attach to the histone
core in a repeatable (same phase) way. Multiple copies of these sequences
were inserted into bare histone cores taken from chicken red blood cell
nuclei. The resulting nucleosome and histone core structures are displayed
in Figures 2.1 and 2.2.
The transcription machinery and regulatory elements must have access to
DNA in order for transcription to occur. In its fully wound form, most of the
DNA binding sites are blocked, and are inaccessible. The DNA material
that is to be transcribed or replicated must be unwound and separated

2.2 The Highly Organized Interphase Nucleus

23

Figure 2.1. Structure of the nucleosome revealed by X-ray crystallography at

2.5 resolution: Shown in the gure is the double-stranded DNA wrapped around
a histone core. The DNA molecule is depicted in a stick model highlighting the base
pairings between complementary strands, and the histones are shown as ribbons and
strings. [From Harp et al. [2000]. Acta Cryst. D, 56: 15131534. Reprinted with permission from the authors.]

sufciently to expose the binding sites. Steric blockages are relieved, or alternatively enhanced, by proteins that interact with and modify DNA structure,
and also by proteins that alter histone structure. In eukaryotes, the ability to
inuence gene expression by modifying histone structure adds a further
layer of control to that available through the protein-DNA binding.
Multiple control points are situated on histone tails that extend out from the
histone core beyond the DNA. As can be seen in Figure 2.2, these sites are
well exposed, thereby providing regulatory elements with ease of access.

2.2 The Highly Organized Interphase Nucleus

The interphase nucleus is a highly organized structure. The cycle of cell
growth and division passes through four stages. The rst of these stages (G1)
is a cellular growth stage that prepares the way either for entry into a cell

24

2. The Control Layer

Figure 2.2. Structure of the histone octamer revealed by X-ray crystallography at

2.5 resolution: Depicted are the H2A, H2B, H3, and H4 core histones without the
surrounding DNA. Tails from the H2A, H2B, H3, and H4 histones are represented
by strings. These structures extend out from the core and interact with DNA and
with neighboring nucleosomes. [From Harp et al. [2000]. Acta Cryst. D 56:
15131534. Reprinted with permission from the authors.]

division series of stages (S, G2, and M), to growth arrest (G0), or to apoptosis (cell death). Cells that do not undergo growth arrest or apoptosis enter
a synthesis (S) stage where the DNA is replicated in preparation for mitosis.
This stage is followed by a further mitosis preparatory stage (G2) where
RNAs and proteins required for mitosis are synthesized. Finally, the cell
enters into mitosis (M) where the cell divides to produce two offspring. The
three stages preceding mitosis are collectively referred to as interphase.
Chromosomes are not randomly distributed in the nucleus during interphase. Instead, they occupy distinct chromatin territories and are positioned
in a way that reects their replication status. The chromatin territories
are partitioned into ~1Mbp-chromatin domains. Interchromatin spaces
separate the chromosome territories from one another. Gene-rich early
replicating chromatin is sequestered from gene-poor chromatin, and
from mid-to-late replicating chromatin. Early replicating chromosomes are
located in the nuclear interior, while inactive and late-replicating chromo-

2.2 The Highly Organized Interphase Nucleus

25

somes are situated at the periphery of the nucleus near or in contact with
the nuclear envelope.
The nuclear architecture facilitates transcription and splicing. Highly condensed chromatin serves to repress transcription while an open formation
allows access of transcription and splicing factors to the sites of transcription. Chromosomes containing active regions of transcription are more
open in their organization than those not actively undergoing transcription.
The active regions typically lie on the outer portion of the chromosome territories. They extend into the interchromatin spaces to permit a maximal
exposure of the surface of active genes to transcription and splicing factors;
the two activities, transcription and splicing, are synchronized with one
another.
The nucleus has several other kinds of structures in addition to chromatin
territories. The additional structuresnucleoli, coiled (Cajal) bodies, and
interchromatin granule clustersare nonmembrane-associated organelles.
They are used for sequestering and storage, and for preparation and assembly of materials used for gene transcription, pre-mRNA splicing, and protein synthesis. They are highly dynamic structures that form and reform
about local concentrations of materials that process RNA molecules. In more
detail:
The nucleolus is the site of ribosome assembly, and forms in response to
transcription of ribosomal DNA (rDNA), the genes that encode ribosomal RNA. Preribosomal RNA (pre-rRNA) units associate with the small
nucleolar RNAs (snoRNAs), and are processed to form mature rRNAs.
Pretransfer RNAs (pre-tRNAs) are imported into the nucleolus and
processed there.The mature rRNAs associate with the tRNAs and a large
ensemble of ribosomal proteins to form ribosomes. Once assembled the
ribosomes are exported to the cytoplasm.
Cajal bodies (CBs) are the sites of assembly of transcription complexes,
and contain high concentrations of RNA Pol I, Pol II, and Pol III. Recall
that there are three kinds of eukaryotic polymerases. RNAP Pol II transcribes pre-mRNAs and most splicing RNAs. The other RNA polymerases transcribe rRNA subunits, pre-tRNAs, and the U6 splicing
RNAs. A cell may contain up to ten Cajal bodies. Once assembled within
a CB, pol I complexes translocate to the nucleoli, where they transcribe
rRNA genes. Pol III assemblages diffuse to the U6 small nuclear RNA
(snRNA) involved in splicing, 5S rRNA and tRNA transcription sites,
and Pol II complexes diffuse out of the CBs and over to mRNA and
snRNA transcription sites.
Interchromatin granule clusters (IGCs), or speckles, are distributed
throughout the inter-chromatin spaces. These organelles are enriched in
U1, U2, U4/U6, and U5 small nucleolar ribonuclear particles (snRNPs)
and other components of the splicing machinery. (These will be discussed
in detail in Chapter 15.) The localization of splicing factors within the

26

2. The Control Layer

IGCs is not static, but instead changes in time in a way that reects the
transcriptional activity underway. Splicing factors diffuse in and out of
the granules to and from sites of transcription activity.

2.3 Covalent Bonds Dene the Primary Structure

of a Protein
Proteins have a primary structure, a secondary structure, and a tertiary
structure. Proteins constructed from more than one polypeptide chain have
a quaternary structure, as well. A protein is a linear chain of amino acids,
connected to one another by means of peptide bonds. Twenty different
amino acids contribute to the formation of proteins. All have the same basic
architecture shown in Figure 2.3. There is a central carbon atom, called an
alpha carbon. Tied to it are an amino group, a carboxyl group, and a hydrogen atom. The fourth bond is with atoms belonging to the side chain (R).
Two forms, uncharged and charged, are depicted in Figure 2.3.
The starting point in determining a proteins structure is the linear
sequence of amino acids. These sequences are unique for each type of
protein. In a protein, each amino acid residue in a sequence is linked to the
next amino acid residue by means of a covalent peptide bond, and for that
reason proteins are commonly referred to as polypeptides. The primary
structure of a protein macromolecule is its covalent structure, including all
covalent disulde bonds that form during folding. The core of the amino
acid is the repeating NCaC unit. These units, covalently linked to one
another by peptide bonds, form the main chain, or backbone, of the protein
in which a carboxyl group of one amino acid and an amino group of the
next amino acid in the sequence are linked together by a peptide bond. The
result of joining amino acids by means of peptide bonds is depicted in
Figure 2.4. By comparing Figures 2.3 and 2.4 one can see that two hydrogen atoms and an oxygen atom are removed during peptide bond formation. These are restored during hydrolysis of the peptide bond.

Figure 2.3. Amino acid structure: In amino acids, the bound organic groups,
denoted by the R symbols, are termed side chains. (a) Form of an uncharged
amino acid. (b) Form of a dipolar, or zwitterion, amino acid.

2.4 Hydrogen Bonds Shape the Secondary Structure

27

Figure 2.4. Two amino acids joined together by

means of a peptide bond.

Table 2.1. Properties of common secondary structure:

The second column lists the psi (y) values and the third
column the phi (j) values for the ideal structural
elements. The last column list the number of residues
per helical or strand turn.
Secondary structure
Alpha helix
3.10 helix
Pi helix
Beta strand

psi

phi

-57.8
-74.0
-57.1
-139.0

-47.0
-4.0
-69.7
+135.0

3.6
3.0
4.4
2.0

The peptide CN link is partially double bond in character. Consequently,

the atoms cannot rotate freely about the CN bond axis. Instead, the four
atoms highlighted in Figure 2.4 form a rigid planar peptide unit. The single
bonds on each side of the peptide bond, that is, the CaC and the NCa bonds,
are quite exible with respect to rotations (torsions) about the bond axes.
The angle of rotation about the CaC axis is usually denoted as psi (Y) and
that about the NCa axis as phi (j).The identication of (Y, j) for each amino
acid residue completes the specication of the main chain conformation. Not
all values of Y and j are allowed, due to steric constraints associated with
the van der Waals radii. Instead, a Ramachandran plot of the (Y, j) values
for a given protein will have areas of high density of points indicative of that
proteins b sheet and a helix secondary structural content. Large regions of
the Ramachandran plot will be empty corresponding to (Y, j) values that
are sterically forbidden. Characteristic psi and phi values for several kinds
of helical structures, and for the beta sheet, are presented in Table 2.1. The
tabulated values differ considerably from one another, and data points
falling about these values will be well separated in a Ramachandran plot.

2.4 Hydrogen Bonds Shape the Secondary Structure

Portions of the polypeptide chain that occur near one another tend to form
geometrically regular, repeating structures during the process of folding
into a three-dimensional functional form. The most commonly encountered
regular structures are alpha helices (a helices), beta sheets (b sheets) and

28

2. The Control Layer

turns. Sequences that do not form one of these kinds of regular structures
are grouped together in a general category called random coils. The
arrangement of these features within the protein constitutes the proteins
secondary structure.
Alpha helices and beta sheets are stabilized by hydrogen bonds between
amide (NH) and carbonyl (CO) groups. In alpha helices, the hydrogen
bonds form between a carbonyl group on the ith residue and the amide
group on the (i + 4)th residue lying below it. In beta sheets, the hydrogen
bonds form between the carbonyl group lying on one strand and the amide
group situated immediately adjacent to it on the other strand. The beta
strands can be oriented in either a parallel or an antiparallel manner. Some
proteins are mostly alpha helix; other mostly beta sheet, and some are a
mixture of the two. These are referred to as a/b if the two types of structural element are mixed and a + b if they remain distinct.
Alpha helices are about 10 residues in length and these structures
account for about a third of the amino acid residues in a typical protein.
Beta sheets are typically 6 amino acid residues in length and they account
for a quarter of the residues.Turns allow the chain to reverse direction.They
along with loops are usually located on the protein surface and thus contain
polar and charged residues. Protein structures tend to be compact with little
space left open in the interior. Hydrogen bonds formed in the protein interior neutralize buried polar groups. Structural compactness and the use of
hydrogen bonds to neutralize interior polarity drive the formation of the
alpha helices and beta sheets.

Figure 2.5. Ramachandran plot: Shown is a stereotypic contour plot of the distribution of main chain torsion angles j and Y determined from high-resolution Xray crystallographic data. Darker shading denotes more highly favored torsion angle
combinations. Blank (white) regions represent conformations that are sterically
forbidden. The most favorable conformations for alpha helices and beta sheets are
concentrated into the three main regions. Beta sheet torsion angles appear as a
double-peaked distribution in the upper left quadrant of the plot. The distribution
of torsion angles for right handed alpha helices peaks in the lower left quadrant,
and that for left handed alpha helices peaks in the upper right quadrant. Each of
the 20 amino acids populates similar, but slightly different, portions of the
Ramachandran plot.

2.6 Protein Secondary Structure Elements and Chain Topology

29

Figure 2.6. Topology of two common structural motifs: (a) The four-helix bundle,
and (b) the Greek key. In the plots, cylinders represent alpha helices, and arrows
oriented in the amino-to carboxyl-terminal direction denote beta strands. The
helices and strands are connected by short turns and longer loops. The four-helix
bundle can be regarded as a pair of helix-turn-helix structures. The Greek key is
assembled from two pairs of antiparallel beta strands.

2.5 Structural Motifs and Domain Folds:

Semi-Independent Protein Modules
In the alpha helix, the backbone forms the inner portion of structure while
the side chains rotate outward. Several different arrangements of helices can
be formed. One arrangement is as a stand-alone helix.Another arrangement
is as coiled coils in which two or more helices are twisted about one another
to make a particular stable structure.This type of structural motif is common
in muscle and hair. Yet another kind of structure is formed when two helices
are connected by a exible loop. This structure, a helix-loop-helix, is commonly encountered in DNA-binding proteins. More generally, when secondary structure elements associate with other secondary structures they form
supersecondary structures. In this process, strands, loops, and turns connect
the alpha helices and beta sheets, and form different kinds of stereotypic
compact structures. When these structures involve sequential arrangements
of two or three elements they are referred to as structural motifs.
Domains are structural units formed by sequential combinations of more
than three secondary structures, and by stable associations of two or more
structural motifs. Typical examples of a domain is the four-helix bundle
formed by two pairs of alpha helices connected by a loop; another typical
domain is the ve-element, alternating arrangement of beta sheets and
alpha helices known as a Rossman fold, and still another is a two pairs of
beta sheets known as a Greek key. Domains are semi-independent folding
units, and for that reason they are often referred to as domain folds.

2.6 Arrangement of Protein Secondary Structure

Elements and Chain Topology
A number of empirical rules can be formulated that summarize the packing
of amino acid sequences into three-dimensional folding domains. These
rules are quite general and summarize the observation that secondary struc-

30

2. The Control Layer

tures pack into one of a small number of geometries and the chains assume
one of an equally small number of topologies. For example, beta sheets form
layered structures with either alpha helices or other beta sheets on their
faces. Alpha helices either distribute themselves about a core or form
layered structures. The packing of helices about a core can be described by
regular polyhedra. Three helices pack into an octahedron, four helices
describe a dodecahedron, ve helices form a hexadecahedron, and six
helices pack into a icosahedron. The packing of sheets is more variable.
Most beta sheets pack into two-layered structures, with the sheets either
aligned or orthogonal. Some beta sheets, notably in a/b proteins, form
barrels. In barrel patterns the b sheets coil around to form a cylinder and
the a helices are arranged on the cylinder surface.
The rules for chain topology state that knots in the polypeptide chain do
not form nor do secondary structure elements cross through one another.
Instead, the chain topology is minimally convoluted. Secondary structure
elements that are adjacent in the polypeptide sequence prefer to pack in
an antiparallel manner, and groupings of the form b-X-b, where X is either
an a-helix or a b-strand in an adjacent sheet, are right handed. The helices
pass through the folding domain and so the connections between helices
form on the outside along the ribs of the polyhedra. Beta sheet topology
forms hairpin, Greek key, or jellyrolls, depending on the number of strands
(respectively, two, four, six).

2.7 Tertiary Structure of a Protein: Motifs and Domains

Proteins involved in signaling tend to be fairly large. They are constructed
from a number of structural motifs and domains connected by loops that
serve as exible linkers. The Src protein shown in Figure 2.7 serves as an
example of domain organization. Domains operate as functional units, and
when proteins are formed with a specic set of domains they inherit those
functions. Some signaling proteins contain just a few domains, while others
contain large numbers of domains and because of that are termed mosaic
proteins. Domain folds typically contain from 100 to 250 amino acids, but
can be as small as 25 to 30 amino acids. In mosaic proteins, small domains
along with larger ones often appear as tandem repeated sequences.
Prokaryotic genes average 850 to 1000 bp in length while eukaryotic genes
average 1400 to 1450 base pairs (bp) in size, and thus contain a greater
number of these semiautonomous folding units.
The tertiary structure of a protein refers to the way the constituent
domains and supersecondary structure elements come together to form the
folded and physiologically functional protein. For most proteins this is the
nal layer of protein organization, and proteins are classied into structural
families based on their domain composition. Listed in Table 2.2 are some
of the more common families of proteins encoded in the human genome as

2.7 Tertiary Structure of a Protein: Motifs and Domains

31

Figure 2.7. Domain organization of the Src protein, a nonreceptor tyrosine kinase:
It consists of four domainsan N-terminal SH3 domain, a C-terminal SH2 domain,
and two catalytic domains, connected by linker segments. Amino acid residues that
form well-dened secondary structure elements are drawn as (dark) coiled ribbons
and as (grey) planar ribbons or arrows. The coiled ribbons, like the cylinders used
in the previous gure, denote alpha helices. The planar ribbons or arrows form
layers that denote beta sheet secondary structures, either parallel or antiparallel.
The SH2 domain is of the form a + b; its secondary structure resembles a twolayered sandwich with two short alpha helices plus a central beta sheet. The SH3
domain contains a pair of antiparallel beta sheets. Tyrosine kinases catalyze the
transfer of phosphoryl groups to selected tyrosine residues. As can be seen in the
gure the catalytic domains are mostly alpha helical, except for the N-lobe, where
a prominent beta sheet component is present. Tyrosine kinases will be discussed in
Chapter 11. The gure was generated using Protein Explorer with the Brookhaven
Protein Data Bank (PDB) entry (accession number) 2Src containing the atomic
coordinates of Src determined by x-ray crystallography (to be discussed in
Chapter 3).

dened by the presence of one or more of the domains listed in the rst
column. The sizes vary from as few as 25 to 33 amino acid residues for the
minidomains to 120 amino acid residues for the PH domain. The domains
are not mutually exclusive, and a single protein will usually contain one or
more of several different kinds of domains. Minidomains are often arranged
in the proteins as tandemly repeated elements. Including the term repeat
as part of the name reects this propensity.
Several kinds of changes have taken place in the human genome. New
families of vertebrate genes appear that encode proteins belonging to the
immune and nervous systems, and there is a new family (KRAB) of zinc
nger transcription factors. A more widespread kind of change is the creation of new architectures, that is, of new arrangements of domains that can
subsume new functions. A third type of change is the large expansion of
existing families. Listed in Table 2.2 are representative examples of each of

32

2. The Control Layer

Table 2.2. Commonly encountered domains in the human: Sizes of the domains
are given in terms of the numbers of amino acid residues. The fourth column lists
the number of genes encoding the domains. All of these domains have signaling
roles. The last column indicates the most prominent activities associated with proteins containing these domains. Im: immune system function; Extra: Extracellular
adhesion; Trans: transcription regulation.
Domain
Immunoglobulin
EGF-like
Fibronectin Type III
EF hand
Ankyrin repeat
Leucine-rich repeat
Cadherin
WD-40 repeat
Pleckstrin homology
Src homology 3
Src homology 2
PDZ
C2H2 zinc nger
Homeobox
RING nger
Krueppel-associated box

Symbol

Size

Genes*

Function

Ig
EGF
Fn3
EF
Ankyrin
LRR
Cadherin
WD40
PH
SH3
SH2
PDZ
C2H2
Homeobox
RING
KRAB

100
35
90
40
33
25
110
50
120
60
100
80
30
60
50
75

765
222
165
242
276
188
114
277
193
143
119
162
706
267
210
204

Im, Extra
Im, Extra
Im, Extra
Im, Extra
Im, Extra, Signal
Im, Extra, Signal
Im, Extra, Signal
Signal
Signal
Signal
Signal
Signal
Trans
Trans
Trans
Trans

* Data from International Human Genome Sequencing Consortium [2001]. Nature 409:
860921; Venter JC et al. [2001]. Science, 291: 13041351.

these categories. The table includes domain families prominently associated

with immune and extracellular functions such as adhesion to the extracurricular matrix, the ECM, and also large numbers of domains that mediate
interactions between control layer proteins and between proteins and
DNA. These domains and the signaling proteins that contain them will be
examined in detail in later chapters.

2.8 Quaternary Structure: The Arrangement of Subunits

Signaling proteins are frequently assembled from distinct polypeptide
chains. In these situations, the protein is said to have a quaternary structure.
Quaternary structure describes how the individual chains, or subunits,
are arranged in the protein. Several kinds of multichain proteins play
important roles in the control layer. One type of multichain protein is the
ion channel, where several subunits form a membrane-spanning pore that
permits passage of ions and small molecules across the membrane. In these
proteins, the subunits are tightly bound to one another. Other classes of
proteins embedded in the plasma membrane, and serving as receivers of
cell-to-cell signals, are assembled from multiple subunits that are much
more loosely bound to one another. This kind of protein organization is

2.9 Many Signaling Proteins Undergo Covalent Modications

33

widely encountered in the immune system, and in these cases, unlike the
ion channels, the different chains may perform distinct functions.
Some of the large signaling proteins that reside in the cytosol and serve
as central organizers of the signal pathways are constructed from multiple
subunits. Specic functions are sequestered in the different subunits of the
proteins, and the ability of the proteins subunits to associate and dissociate from one another in a key part of how the signaling system works. Alternative splicing is often used to generate different forms of specic subunits.
This makes possible the creation of many different combinations of subunits; each one specialized for a specic cell or tissue type. By this means
many proteins of a similar kind, but each performing a slightly different
task, can be constructed from a small instruction set.

2.9 Many Signaling Proteins Undergo

Covalent Modications
Proteins can associate with the plasma membrane in several ways.If they pass
completely through the plasma membrane they are able to convey a signal
from one side to the other. Most receptors work this way relaying signals
from outside the cell to the inside. Alternatively, the proteins may attach to
either the extracellular side or the intracellular side by means of the tether.
The tether, a type of post-translational modication, passes into one of the
leaets but does not pass all the way through to the other side of the plasma
membrane. The proteins so anchored tend to form clusters of signaling
elements, which may relay signals to nearby proteins that pass through the
plasma membrane or not, depending on the composition of the cluster.
One of the most striking features of the control layer is its widespread
use of post-translational modications. Some of the modications are made
during protein processing and nishing as discussed in the last chapter. This
type of modication is common in proteins that function in the plasma
membrane. But many others are made later and are part of the signaling
process itself. There are several different kinds of modications all involving either the covalent addition of a group or structure, or the cleavage of
the protein or a part thereof.
The main modications are:
Covalent attachment of anchors that tether signal proteins to one side or
the other of the plasma membrane.
Addition of sugar groups to the extracellular region of transmembrane
signaling proteins.
Proteolytic cleavage of anchors and proteins to free up the proteins for
movement and conveyance of a message, or, alternatively, to keep them
inactive and degrade them.
Covalent addition and removal of phosphoryl, methyl, and acyl groups
to cytosolic proteins.

34

2. The Control Layer

2.10 Anchors Enable Proteins to Attach to Membranes

The region at and just below the plasma membrane is an important
locus of signaling molecules. In response to the onset of signaling, many
cytosolic proteins translocate to the plasma membrane where they are
anchored to the cytosolic face and become activated. There are four types
of modications to cytosolic proteins that enable them to anchor to the
plasma membrane and to the membranes of organelles: myristoylation,
palmitoylation, farnesylation, and geranylgeranylation (Table 2.3, and
Figures 2.8 and 2.9).
A crucial feature of acyl and prenyl anchors is that they are weak. Electrostatic interactions between N-terminal basic (+) residues and acidic ()
lipids contribute along with the anchor to the attachment. Because of the
weak attachment the proteins can be detached easily by altering the electrostatic environment. This is usually done by phosphorylation, the covalent attachment of a phosphoryl group (with two negative charges). The
modication to the basic residues weakens the electrostatic forces sufciently to enable the protein to detach from the membrane and translocate
to the cytoplasm to carry out its signaling function.This allows for reversible
attachment and operation as an electrostatic switch.
The addition of the aforementioned fatty groups to proteins makes
possible the proteins anchoring to the inner, or cytoplasmic, leaet of the
plasma membrane. A different kind lipid modication is made to proteins
to allow them to be anchored to the outer, or exoplasmic, leaet. These
Table 2.3. Membrane anchors: Abbreviationscysteine (C); alipathic residue
(residues with long hydrocarbon side chains such as leucine and isoleucine) (a);
X = serine (S), methionine (M), alanine (A), or glutamine (Q); leucine (L).
Location and type of anchor
Inner Leaet
Acyl type
Myristoyl
Palmitoyl
Prenyl type

Farnesyl
Geranylgeranyl
Outer Leaet
Glycosylphosphatidylinositol
(GPI)

Attachment

Preferentially attaches to glycine residues at the

N-terminus
Preferentially attaches to cysteine residues variably
located through a thioester link
Covalently attaches through a thioester link to a cysteine
residue located four residues from the C-terminus. The
last three residues are removed and the new C-terminus
group is methylated.
C-terminal sequence CaaX
C-terminal sequence CaaL

Attaches to the C-terminal amino acid; directed by a

signal peptide that is removed and replaced by the
anchor

2.10 Anchors Enable Proteins to Attach to Membranes

35

Figure 2.8. Acyl anchors covalently attached to membrane proteins: (a) A 16-C
palmitoyl anchor is attached to a cysteine residue. (b) A 14-C myristoyl anchor is
covalently attached to a glycine residue.

Figure 2.9. Prenyl anchors covalently attached to membrane proteins: (a) A 15-C
farnesyl anchor is attached to a cysteine residue. (b) A 20-C geranylgeranyl anchor
is covalently attached to a cysteine residue.

36

2. The Control Layer

anchors are made from a complex sugar plus a phosphatidylinositol

grouping, and are called GPI (glycosyl phosphatidyl inositol) anchors.
The GPI anchors are preformed in the endoplasmic reticulum (ER) and
attached to the newly synthesized proteins. Proteins bearing these anchors
are localized on the cell exterior, where they can be easily freed up by
proteolytic proteins called sheddases. These proteolytic proteins are given
that particular name because they cleave, or shed, the exoplasmic domains,
or ectodomains of their targets. The proteins, once freed of their anchors,
function as soluble proteins. Dual form proteins, membrane-associated and
soluble, are frequently encountered in immune and endocrine (hormonal)
signaling.

2.11 Glycosylation Produces Mature Glycoproteins

Most proteins destined for insertion in the plasma membrane contain covalently linked oligosaccharides that extend out from their extracellular side.
These proteins are referred to as glycoproteins. Their post-translational
modications are started in the ER and nished in the Golgi apparatus.
There are two forms of modication, N-linked and O-linked. In N-linked
glycoproteins, a carbohydrate is added to a side chain NH2 group of an
asparagine amino acid residue. In O-linked glycoproteins, the oligosaccharide chain is appended to a side chain hydroxyl group of a serine or threonine amino acid residue.
These modications alter the binding properties of the signaling proteins.
By adding or removing the carbohydrate groups, the afnity for a particular ligand can be either increased or decreased, and can even be shifted to
favor one ligand over another. These properties have been explored for a
prominent signaling protein called Notch that is involved in embryonic
development. Notch signaling and other developmentally important processes will be explored in Chapter 13. What is of interest here is that rather
than genetically encoding a variety of Notchlike proteins, each with slightly
different ligand-binding properties, a small number of Notch proteins are
expressed and their properties are then altered post-translationally as the
need arises.

2.12 Proteolytic Processing Is Widely Used in Signaling

Notch undergoes additional modications subsequent to ligand binding.
It is proteolytically processed to form a diffusible messenger protein that
translocates to the nucleus where it functions as a transcription factor. Proteolytic processing of membrane proteins to create mobile messengers is
not limited to Notch, but instead is used in several signaling pathways. In

2.13 Reversible Addition and Removal of Phosphoryl Groups

37

this way, a single protein performs multiple tasks within a single signaling
pathway. Because several signaling intermediates are eliminated, the signaling process is a rapid one requiring few steps to go from the initiating
sensing stage to a terminating control point.
Proteolytic processing is not restricted to membrane proteins. It is utilized heavily to change the properties of cytosolic proteins from immobilized forms to mobile ones. Proteolytic processing is a key component of
signaling pathways involved in regulating the cell cycle, embryonic development, and immune function. The mechanism is similar in all of the
pathways. A crucial signaling element is parked in a specic location in the
cell through its binding to an inhibitory protein. In response to activating
signals, proteolytic enzymes (the 26S proteosome) chop up the inhibitory
proteins, thereby freeing the signaling proteins for movement into the
nucleus where they promote gene transcription. These mechanisms will be
discussed in more detail when the specic pathways in which they appear
are examined.

2.13 Reversible Addition and Removal of

Phosphoryl Groups
Reversible protein phosphorylation is the preeminent mechanism used by
all cells to convey a signal. It is used to direct cellular responses to the
binding of hormones, growth factors, and neurotransmitters to receptors
and ion channels at the cell surface. It is used to regulate metabolism,
growth and differentiation, and learning and memory. In this process, a
phosphoryl group is covalently attached to a specic residue on a target
protein thereby modifying that proteins activity. ATP serves as the donor
of the phosphoryl group. Protein kinases are enzymes that catalyze the
transfer of a phosphoryl groups from the ATP molecules to protein targets.
Another class of signaling molecules, protein phosphatases, does the opposite. Protein phosphatases catalyze the removal of phosphoryl groups, that
is, they catalyze their hydrolysis.
Different amino acid residues serve as the primary recipients of phosphoryl groups in bacteria and eukaryotes. In bacteria, phosphoryl groups
are transferred to aspartate and histidine residues forming His-Asp phosphorelays. In eukaryotes, there are two classes of protein kinases. One
group catalyzes the covalent attachment of phosphoryl groups to serine and
threonine residues and the other promotes the attachment to tyrosine
residues (Figure 2.10). Protein kinases are central elements in most signal
pathways and are present in large numbers of metazoan and plant genomes.
The human genome encodes close to 900 eukaryotic protein kinases. These
signaling elements will be introduced again in Chapters 6 (bacteria) and 7
(yeasts and other eukaryotes).

38

2. The Control Layer

Figure 2.10. Phosphorylation of serine, threonine, and tyrosine side chains:

Hydroxyl (OH) groups located at the ends of the side chains provide sites for covalent attachment of phosphoryl groups.

Figure 2.11. Covalent attachment of methyl and acetyl groups to arginine and
lysine side chains: Amino groups located at the ends of arginine and lysine side
chains provide sites for attachment of one or more methyl or acetyl groups.

2.14 Reversible Addition and Removal of Methyl and

Acetyl Groups
Phosphoryl groups are not the only groups that are added and removed
from signaling proteins as part of their cellular function. Methyl and acetyl
groups are added and removed, as well. Whereas side chain hydroxyls
provide binding sites for the phosphoryl groups, side chain nitrogens do the
same for the methyl and acetyl groups. The transfer targets are the amino
groups lying at the ends of the side chains of lysine and arginine residues.
The amino groups provide multiple attachment sites for methyl groups.
Several examples of methylation are presented in Figure 2.11. Two

2.15 Reversible Addition and Removal of SUMO Groups

39

nitrogens are present at the ends of arginine side chains, allowing for a
symmetric distribution of methyl groups between the two nitrogens, or,
alternatively, for one or the other of the nitrogens to have most if not all of
the methyl groups.
The enzymes that catalyze the transfer of methyl groups to arginine
and lysine residues are referred to as arginine methyltransferases and as
lysine methyltransferases, respectively. These enzymes use an endogenous
cellular molecule called S-adenosyl-l-methionine, or SAM, as the methyl
group donor. SAM is synthesized in cells of the body from methionine
and ATP, and readily donates its methyl group to the guanidine groups
on the arginine side chains and to the amino groups on the lysine side
chains.
The amino groups at the end of lysine side chains are the main
attachment sites for acetyl groups. The enzymes responsible for catalyzing
the transfer of acetyl groups to lysines are known as acetyltransferases.
These enzymes use acetyl coenzyme A (acetyl CoA) as the acetyl group
donor. The acetyl group is attached by a sulfur atom to CoA forming a
high-energy thioster bond, making it easy to transfer to acceptors such as
lysine.

2.15 Reversible Addition and Removal of

SUMO Groups
Small ubiquitin-related modier (SUMO) is a member of the ubiquitin
family of regulatory proteins. Like ubiquitin, it is covalently attached to
a variety of proteins through the sequential actions of three sets of
enzymes. Recall that ubiquitin is rst activated in an ATP-dependent
way by E1 enzymes. A thioester bond is formed between the C-terminal
of the ubiquitin protein and the E1 ubiquitin-activating enzyme. The
ubiquitin protein is then transferred to an E2 ubiquitin-conjugating
enzyme, and then to the E3 ubiquitin protein ligase, which then transfers
it to either the substrate or to multiubiquitin chains formed there. The
substrate so tagged by one or more ubiquitin molecules is then degraded
by the 26S proteosome.
The SUMO system of enzymes operates in a somewhat similar fashion.
There is an E1 SUMO-activating enzyme, which consists of a heterodimer
in place of the single polypeptide chain found for the ubiquitin E1. There
is an E2 SUMO-conjugating enzyme, and there is an E3 SUMO protein
ligase, which accelerates the direct transfer of SUMO from the E2 to the
substrate. These attachments are illustrated in Figure 2.12.
Both ubiquitination and sumoylation have regulatory roles. Whereas
ubiquitination of a protein generally tags that protein for destruction by the
26S proteosome, attachment of a SUMO group has a different role. It is a
reversible process. It helps stabilize proteins and their interactions with

40

2. The Control Layer

Figure 2.12. Ubiquitination and sumoylation: Conjugates are formed between

lysine NH group and carbonyl groups at the C-terminal of ubiquitins and SUMO.

other proteins, and assists in localizing proteins to specic compartments

within the cell.

2.16 Post-Translational Modications to Histones

Post-translational modications to histones are an important regulatory
mechanism. Chromatin structure plays an important role in determining
which genes are to be transcribed at any given moment in time. There are
two forms of chromatin. Transcriptionally inactive chromatin, called heterochromatin, is tightly compacted. In heterochromatin, sites where transcription initiation and regulation take place are largely inaccessible to the
proteins responsible for these actions. In contrast, transcriptionally active
chromatin, or euchromatin, has a far more open shape. In euchromatin, sites
where transcription factors and the transcription machinery bind are accessible to the responsible proteins.
As discussed earlier in this chapter, nucleosomes contain two each of
several core histones. The amino terminals of these core histones extend
out from the nucleosomes to form histone tails. These tails provide a means
for other proteins to inuence transcription. The histone tails provide sites
for attachment of methyl groups, acetyl groups, and SUMO groups. The predominant targets of the post-translational modications are the amino
groups lying at the end of lysine side chains. The lysine residues, along with
arginine residues, can be methylated, acetylated, sumoylated, or ubiquitinated. As shown in Figure 2.13, the tails of the histones are enriched in these
residues, and thus supply multiple sites for attachment of these groups.
Attachment of acetyl and other groups neutralizes the net positive charge
on the tail regions, and this reduction weakens the attraction between the
tails and the DNA. As a consequence the histone-DNA interactions are

2.16 Post-Translational Modications to Histones

41

Figure 2.13. Covalent modications of histone tails: Shown are regulatory sites
modied by acetylation (A), methylation (M), phosphorylation, or ubiquitination
(U). As is customary the sites are numbered in ascending order starting from the
N-terminal. One-letter codes for the amino acids are arginine (R), lysine (K), and
serine (S). (Three- and one-letter abbreviations for the amino acids are listed in
Table 4.1.)

lessened allowing for a greater access of the DNA to transcription regulators. Similarly, addition of net negative charge through phosphorylation on
serine residues also serves to decondense chromatin.
Many of the proteins that act in a supporting role to help activate or
suppress transcription interact directly with the histones rather than with the
DNA. Some of these enzymes function as histone acetyltransferases (HATs)
or alternatively as histone deacetylases (HDACs), adding or removing
acetyl groups from histone tails, using acetyl coenzyme A (acetyl-CoA) as
the donor. Other coregulatory enzymes operate in a similar manner, adding
or removing methyl groups or phosphoryl groups or SUMO groups or ubiquitin groups.

42

2. The Control Layer

General References
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, and Walker P [2002]. Molecular
Biology of the Cell, 4th edition. New York: Garland Publishers.
Matthews CK, van Holde KE, and Ahem KG [2000]. Biochemistry, 3rd edition. San
Francisco: Pearson Benjamin Cummings.
Stryer L [1995]. Biochemistry, 4th edition. New York: W.H. Freeman and Company.

References and Further Reading

Chromatin and the Nucleosome
Harp JM, et al. [2000]. Asymmetries in the nucleosome core particle at 2.5 resolution. Acta Cryst., D56: 15131534.
Kornberg RD, and Lorch YL [1999]. Twenty-ve years of the nucleosome, fundamental particle of the eukaryote chromosome. Cell, 98: 285294.

Nuclear Organization
Lamond AI, and Earnshaw WC [1998]. Structure and function in the nucleus.
Science, 280: 547553.
Lewis JD, and Tollervey D [2000]. Like attracts like: Getting RNA processing
together in the nucleus. Science, 288: 13851389.

Chromosome Organization
Cremer T, and Cremer C [2001]. Chromosome territories, nuclear architecture and
gene regulation in mammalian cells. Nat. Rev. Genet., 2: 292301.
Manuelidis L [1990]. A view of interphase chromosomes. Science, 250: 15331540.

Protein Organization
Hovmller S, Zhou T, and Ohlson T [2002]. Conformations of amino acids in proteins. Acta Cryst., D58: 768776.
Kleywegt GJ, and Jones TA [1996]. Phi/Psi-chology: Ramachandran revisited. Structure, 4: 13951400.

Post-Translational Modications
Fortini ME [2000]. Fringe benets to carbohydrates. Nature, 406: 357358, and references cited therein.
McLaughlin S, and Aderem A [1995]. The myristoyl-electrostatic switch: A modulator of reversible protein-membrane interactions. Trends Biochem. Sci., 20:
272276.
Milligan G, Parenti M, and Magee AI [1995]. The dynamical role of palmitoylation
in signal transduction. Trends Biochem. Sci., 20: 181186.
Peschon JJ, et al. [1998]. An essential role for ectodomain shedding in mammalian
development. Science, 282: 12811284.
Resh MD [1999]. Fatty acylation of proteins: New insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta, 1451:
116.

Problems

43

Regulated Proteolysis
Brown MS, et al. [2000]. Regulated intramembrane proteolysis: A control mechanism conserved from bacteria to humans. Cell, 100: 391398.
Maniatis T [1999]. A ubiquitin ligase complex essential for the NF-B, Wnt/Wingless,
and Hedgehog signaling pathways. Genes Dev., 13: 505510.
Townsley FM, and Ruderman JV [1998]. Proteolytic ratchets that control progression through mitosis. Trends Cell Biol., 8: 238244.

Sumoylation
Mller S, et al. [2001]. SUMO, ubiquitins mysterious cousin. Nature Rev. Mol. Cell
Biol., 2: 202210.

Histone Modications
Grunstein M [1997]. Histone acetylation in chromatin structure and transcription.
Nature, 389: 349352.
Jenuwein T, and Allis CD [2001]. Translating the histone code. Science, 293: 1074
1080.

Problems
These problems require use of a PC to run the Protein Explorer, the free
software used to model the Src protein as shown in Figure 2.7. The rst step
is to install Chime to enable your browser to talk to the Protein Explorer.
Then bring up the Protein Explorer. (You may want to save the URL under
your Favorites pull-down.)
2.1 Bring up the atomic coordinates for the Src protein displayed in Figure
2.7. Its Protein Data Bank (PDB) accession number is 2src. Next
remove the water molecules and ligands leaving just Src. Then display
the image in the form shown in Figure 2.7. Hint: Go to Quick Views
and use the Display feature to bring up the cartoon depiction of
the secondary structure. Then drag on the image to rotate into the orientation presented in Figure 2.7.
2.2 Click on Mol Info and then the Header button to bring up additional information about the protein Src. Note the list of amino acids
comprising the primary structure.
2.3 Returning to the image of the Src protein. Bring up a backbone
display of the protein. What does this image show?
2.4 Now, bring up a ball-and-stick model of Src. What do the balls represent and what do the sticks denote? Click on some of the balls shown
in the image. What happens when this is done?
2.5 Change the display to a space-ll model. What do the spheres now
mean; that is, what do their radii represent? How is hydrogen treated?

3
Exploring Protein Structure
and Function

A variety of methods have been developed that enable researchers to study

the structure and function of proteins at the atomic, molecular, intermolecular and cellular levels. Some methods exploit differences in charge and
mass of the proteins in order to distinguish one kind of protein from
another. Other methods exploit the many kinds of interactions occurring
between electromagnetic radiation and biomolecules. Depending on the
wavelength and the properties of the target material, electromagnetic
radiation will be scattered and diffracted, absorbed and emitted. In all, an
ensemble of methods is used in the laboratory not only to explore protein
structure, but also to investigate posttranslational modications, intermolecular interactions, cellular localization, and pleiotropy.
The preeminent methods for exploring the shape and internal structure
of proteins at atomic level detail are X-ray crystallography and nuclear
magnetic resonance (NMR). These techniques provide detailed threedimensional information on how the proteins are organized into their functionally distinct domains and motifs, and what happens when one protein
binds to another. They identify which amino acid residues are critical for
protein-protein and protein-DNA binding, and they reveal the functional
consequences of mutations of specic amino acid residues. As will be seen
in later chapters, proteins involved in intracellular signaling often possess
catalytic domains that stimulate the transfer of phosphoryl groups from one
protein to another. X-ray crystallography and NMR show how these catalysts work, and how their activities are regulated.
The goal of techniques such as gel electrophoresis and DNA microarrays
is to examine which proteins are expressed at higher levels and which ones
at lower levels in response to specic kinds of signaling events and conditions. That is, they provide intermolecular and cellular level details. The
mass spectrograph, often used in conjunction with these methods, permits
the researcher to determine with high resolution the masses of proteins that
have been isolated by gel electrophoresis and the microarrays. Another
method, the yeast two-hybrid method, has become the leading method for
determining which sets of proteins interact with one another to form the
45

46

3. Exploring Protein Structure and Function

Table 3.1. Methods using electromagnetic interactions to explore protein structure

and interactions.
Experimental method

Level of detail

X-ray crystallography
Circular dichroism

Atomic
Molecular

Fluorescence resonance energy transfer

Intermolecular

IR and Raman spectroscopy

Molecular

NMR spectroscopy

Atomic

Process
X-ray diffraction
Absorption of polarized
UV light
Visible light absorption
and emission
Absorption (IR) and
scattering (Raman) of IR
light
Nuclear spin ips

Table 3.2. Physical methods used to explore protein structure and interactions.
Experimental method
Yeast two-hybrid
Gel electrophoresis
Mass spectrograph
DNA microarrays

Level of detail
Intermolecular
Molecular, Intermolecular
Molecular
Cellular

Process
Protein-protein interactions
Mass/charge separation
Mass/charge separation
Complementary base-pairing

signaling pathways in the cell. And another method, uorescence resonance

energy transfer, is used to explore protein interactions and view the movements of the signaling proteins in the cell. All of the methods listed in Tables
3.1 and 3.2 will be discussed in this chapter, some to a greater extent than
others. The discussion will start with a review of the electromagnetic spectrum and how electromagnetic waves interact with matter. X-ray crystallography, NMR, and FRET will be explored next, followed by discussions
of the physical methods.

3.1 Interaction of Electromagnetic Radiation

with Matter
Electromagnetic radiation interacts with matter in a variety of ways. Recall
that the wavelength and frequency of an electromagnetic wave are inversely
proportional to one another, and the speed of light is the constant of proportionality. That is,
n=

c
,
l

(3.1)

where l (cm) is the wavelength, n (cycles/s) denotes the frequency, and c

represents the speed of light, equal to 2.99 1010 cm/s. Electromagnetic radi-

3.1 Interaction of Electromagnetic Radiation with Matter

47

ation is quantized into discrete packets called photons.The energy E of each

packet is given by Plancks formula:
E = hn,

(3.2)

-27

where h = 6.626 10 erg-sec is Plancks constant. According to Eqs. (3.1)

and (3.2), as the frequency increases, or equivalently the wavelength
decreases, the photon energy goes up.
Electromagnetic radiation is not limited to a narrow range of wavelengths, but rather spans a broad range of wavelengths from less than a
nanometer to more than a meter. The shortest waves are the gamma rays
(g-rays) emitted by atomic nuclei, and the longest waves are radio waves
emitted by charged particles as they move back and forth in, for example,
interstellar gases. The continuum of different kinds of radiation, each characterized by a unique wavelength, is known as the electromagnetic spectrum.
The middle portion of the electromagnetic spectrum contains the infrared
and ultraviolet regions with the visible range sandwiched in between. These
three regimes are the most important portion of the spectrum with respect
to biological systems.
According to the formulas just presented, as wavelengths decrease,
photon energies increase. The energies corresponding to the different
wavelength regimes are presented in Figure 3.1 in units of kcal/mol to
facilitate comparison to familiar bonding energies, which are usually
given in these unitsthe energies of covalent bonding are on the order of
100 kcal/mol. Noncovalent interactions such as hydrogen bonds have
energies in the range 1 to 10 kcal/mol, and thermal energies are roughly
0.6 kcal/mol.

Figure 3.1. Electromagnetic spectrum: Shown in the upper portion of the gure are
the types of radiation emitted. The kinds of transitions, or motions, that absorb and
emit the radiation are shown in the lower part of the gure. Vertical dashed lines
delineate the boundaries between the different regimes. The actual boundaries
between radio waves and microwaves, and between ultraviolet and X-rays, are not
sharp but instead the regimes merge into one another.

48

3. Exploring Protein Structure and Function

Molecules are not static entities but instead are in constant thermal
motion, gaining and losing energy through random collisions with other
molecules. The kinetic energy absorbed in the collisions is converted into
vibrations and rotations about bond angles. These so-called collective modes
can also be excited when the atoms and molecules absorb radiation in the
appropriate wavelength range. The correspondence between wavelength
regimes of electromagnetic radiation and protein motions (transitions) that
produce the radiation is presented in the lower portion of Figure 3.1.
Recall that electrons move in specic atomic and molecular orbits, each
with a well-dened energy. The lowest energy state of the electrons in their
various orbits around a nucleus is called the ground state, and all others are
referred to as excited states. Besides inducing vibrational and rotational
activity, electrons can transition into excited states when radiation of the
correct wavelength is absorbed. Electromagnetic radiation of well-dened
energies is involved whenever electrons, atoms, and molecules undergo
transitions from one state to another, either higher or lower. The energy of
the absorbed and emitted radiation is equal to the difference in energies
between the two energy levels (states), and its frequency (wavelength) is
given by the following form of Plancks formula:
E2 - E1 = DE = hn.

(3.3)

The absorption and emission of photons is depicted in Figure 3.2.

Figure 3.2. Absorption and emission of electromagnetic radiation: Horizontal lines

represent energy levels of a simplied molecule possessing just a single ground state
and a single excited state. From left to right: (a) AbsorptionThe molecule, initially
in its ground state, absorbs a photon and undergoes a transition to an excited state.
(b) EmissionThe molecule, initially in its excited state, undergoes a transition to
the ground state and emits a photon in the process.

3.3 Protein Structure via X-Ray Crystallography

49

3.2 Biomolecule Absorption and Emission Spectra

Biomolecules such as amino acids, peptides, and proteins absorb and scatter
light in the ultraviolet and visible portions of the electromagnetic spectrum.
Many of these biomoleucles selectively absorb light of particular wavelengths while scattering light at other wavelengths. These molecules act as
pigments, imparting color to the materials in which they reside. The specic
groups of atoms responsible for these absorption properties are known as
chromophores, or color bringers. The chlorophylls are prominent examples of chromophore-bearing molecules. They selectively absorb light in the
blue and red portions of the spectrum, and scatter light in the green range.
The result is the pronounced green color of plants.
Another common example of a biomolecule with striking chromatic properties is hemoglobin. Its absorption/scattering properties impart a red color
to fully oxygenated blood while conveying a bluish tint to deoxygenated
blood.Those electrons in a chromophore responsible for the light absorption
are sensitive to their local environments, especially the presence of nearby
charged groups. In the case of hemoglobin, the heme group forming the
chromophore is sensitive to the presence or absence of bound oxygen atoms.
Photoreceptors form another class of chromophore-bearing proteins.
The class includes ospins found in the mammalian retina along with phytochromes and cryptochromes, which enable bacteria, plants, and animals to
adapt their cellular responses to local lighting conditions and undergo circadian rhythms. Opsins will be discussed in Chapter 12. Phytochromes and
cryptochromes will be explored in Chapter 7. Protein chromophores can be
built about the peptide bond, or about side chains, or upon prosthetic
groups covalently attached chains that are nominally not part of the protein.
Examples of prosthetic groups operating as chromophores are the heme
groups and opsins.
When atomic groups absorb light, electrons are promoted into excited
states (orbits). One of three things can then happen. The electrons may
deexcite to the ground state without emitting radiation in a process called
internal conversion. Alternatively, the excess energy may be lost through
emission of photons having energies less than those of the absorbed light,with
the remaining energy lost in a radiationless manner. If this happens rapidly, in
a time scale of nanoseconds, the process is referred to as uorescence (Figure
3.3), and the chromophores are called uorophores. If, on the other hand, the
lifetime of the excited state is long-lived, in the millisecond to second range,
the process of absorbing and then emitting light is called phosphorescence.

3.3 Protein Structure via X-Ray Crystallography

Because of their short wavelength, X-rays can be used to explore the
arrangements of individual atoms in proteins and other biomolecules. Xrays are produced whenever swiftly moving electrons strike a solid target.

50

3. Exploring Protein Structure and Function

Figure 3.3. Schematic depiction of uorescence: Shown are sets of related states,
or bands, along with vertical arrows denoting transitions between states. From left
to right, a packet of electromagnetic radiation is absorbed by a uorophore, producing a transition from the ground state to an excited state. Shortly thereafter, the
uorophore deexcites back to the ground state, emitting a photon with a slightly
lower energy and longer wavelength. Energy not carried off by the emitted photon
is lost through radiationless transitions between vibrational states within the excited
state band and within the ground state band.

In an X-ray tube, a beam of electrons is generated that strikes a metallic

anode (typically copper) to produce an X-ray beam. Two types of X-rays
are producedcharacteristic X-rays that generate a line spectrum, containing a set of narrow strong peaks, and a smooth spectrum of continuum
X-rays produced by coulombic interactions between the electron beam and
the positive-charged nuclei (called bremsstrahlung, or braking radiation). In
X-ray studies, characteristic Ka X-rays produced by transitions among inner
shell electrons are typically used. These have a well-dened wavelength of
about 1.5 , comparable to atom-atom bond lengths.
X-rays are scattered by the electron clouds of atoms, particularly by
tightly bound electrons near the center of atoms, with no change in wavelength and no change in phase. Light scattered from single atoms is too
weak to observe, but the amount of light can be amplied using puried
crystals. In a crystal, large numbers of identical molecules are arranged in
a regular lattice. Light passing through a crystal will be scattered in a variety
of directions. Spherical wavelets scattered by different atoms will interfere,
some constructively and some destructively. For certain wavelengths and
scattering directions, the wavelets will be in phase to produce strong constructive interference. Constructive interference taking place between light
waves as they are scattered off of the atoms serves to amplify the light, producing a characteristic pattern of light spots and dark areas, a diffraction
pattern, that can be seen and analyzed to yield information on how the
atoms are arranged.

3.3 Protein Structure via X-Ray Crystallography

51

Figure 3.4. Scattering of X-rays from a series of planes in a crystal: A beam of

X-rays impinges on a series of crystal planes. Some of the light is scattered back
(reected) from each of the planes while the remainder of the light is transmitted.
The optimal situation where the angle of incidence equals the angle of reection is
depicted in the gure.

W.H. Bragg and his son W.L. Bragg formulated the basic theory of relating diffraction patterns to atomic positions in a crystal in 19121913. They
noted that a three-dimensional crystal could be viewed as a set of equidistant parallel planes. The conditions for maximal constructive interference
are twofold. First, the scattering from each plane must be a specular, or
mirror, reection in which the angle of incidence equals the angle of reection. This situation is depicted in Figure 3.4. Second, the X-ray wavelength
l, distance between parallel planes d, and angle of reection q, obey the
relationship known as Braggs law:
2d sin q = nl.

(3.4)

In Eq. (3.4), n is an integer that can take on the values 1, 2, 3, and so on.
When n = 1, the spots of light are known as rst order reections, and when
n = 2, they are called second order reections. First order reections are
more intense than second order reections, and similarly for third and
higher order contributions.
The light spots are produced when the light waves arrive at the detector
in phase with one another and thus constructively interfere. In an X-ray
diffraction experiment, the intensities and positions of the light spots are
recorded. The diffraction patterns are converted into electron density maps
through application of a mathematical operation known as a Fourier transform. Several tens of thousands of reections are collected in a typical
X-ray diffraction experiment. Computer programs, taking as input the
resulting electron density map and knowledge of the primary sequence, are

52

3. Exploring Protein Structure and Function

Figure 3.5. X-ray crystallography: A unit cell is shown containing the molecule to
be studied. It is replicated many times in the crystal sample. X-rays scattered from
the atoms in the crystal produce a complex three-dimensional pattern of light and
dark spots in the detector called reections. The patterns of light and dark spots are
a consequence of the way the waves from different atoms in different locations in
the unit cell interfere with one another. A typical diffraction pattern produced in
one-dimension from a pair of atoms is depicted in the gure. As can be seen, there
is a series of peaks where the waves constructively interfere, and each peak is separated from the next by a trough where the waves from the two atoms destructively
interfere. These reections are then converted into an electron density map. A
typical portion of an electron density map is depicted. The map, a contour plot,
shows peaks where the electron density is high and valleys or open areas where the
electron density is low. In the model-building portion of the data analysis, the threedimensional structure of the molecule is deduced using computer programs.

used deduce the three-dimensional arrangement of atoms in the protein

(Figure 3.5).
X-ray crystallographic data have been used to deduce the atomic structure of more than 16,000 proteins to date. In 1953, Crick and Watson
deduced the double helix structure of DNA using the X-ray crystallographic data of Franklin and Wilkins. This discovery was followed by those
of Perutz and Kendrew, who used X-ray crystallography to deduce the
atomic structure of hemoglobin and myoglobin in the period from 1953 to
1960. These were the rst two proteins to be so described in atomic detail.
Since that time a rapidly increasing number of proteins have had their
atomic structure solved.The three-dimensional coordinates (x, y, z) for each
atom in the protein are deposited in the Protein Data Bank (PDB) and
made available to the research community. The PDB repository was estab-

3.5 Determining Protein Structure Through NMR

53

lished at Brookhaven National Laboratory in Long Island, and at several

mirror sites throughout the world. Of the more than 16,000 structures for
proteins and peptides that have been deposited in the PDB to date, 14,000
were determined using X-ray crystallography and 2000 using nuclear magnetic resonance (NMR) spectroscopy.

3.4 Membrane Protein 3-D Structure via Electron and

Cryoelectron Crystallography
The overall limitation of X-ray crystallography is the growing of puried
crystals with sufcient numbers of molecules. This outcome is exceptionally
difcult to achieve in the case of large and complex protein molecules such
as those embedded in membranes that pass back and forth through the lipid
bilayer several times. These proteins have a hydrophobic band that tends
to destabilize them when they are removed from the lipid bilayer and solubilized. In electron crystallography, both the native environment and the
biological activity of the membrane proteins are preserved.
The central idea in electron crystallography is similar to that which drives
X-ray crystallography. By growing a crystal containing a large number of
the molecules of interest one can greatly amplify the crystals weak signals.
In this approach, two-dimensional membrane crystals are prepared. Electron diffraction patterns are then acquired using high-resolution cryoelectron microscopes. This method has been used to study light-harvesting
complexes that function as antennae of solar energy in plants, and to investigate the atomic structure of porins of gram-negative bacteria. The method
was rst applied to the study of bacteriorhodopsin, the light-driven proton
pump located in the purple membrane of Halobacteria and, more recently,
to the analysis of the three-dimensional structure of the acetylcholine
receptor, a neurotransmitter-gated ion channel located in nerve-muscle
synaptic membranes.

3.5 Determining Protein Structure Through NMR

Nuclear magnetic resonance (NMR) spectroscopy is the second major
experimental method used to determine the three-dimensional atomic
structure of proteins. In this method, one utilizes pulses of electromagnetic
radiation in the radiofrequency (RF) range of the EM spectrum. NMR
pulse frequencies are typically in the range 300 to 600 MHz. These frequencies correspond to wavelengths of 100 to 50 cm, and the corresponding
RF energies are 10 orders of magnitude weaker that the X-ray energies.
Photons of these energies are used to induce transitions between nuclear
(proton and neutron) spin states. The subsequent relaxation of the nuclear
spin distributions back to their equilibrium distribution is sensitive both to

54

3. Exploring Protein Structure and Function

the electron distribution surrounding the nucleus and to neighboring

nuclear spins. As a consequence, one can deduce atomic structure of a
protein from an analysis of its NMR spectra.
Electrons, protons, and neutrons have an intrinsic angular momentum, or
spin. Because they have a spin angular momentum, they have a magnetic
dipole moment and can interact with an external magnetic eld as depicted
in Figure 3.6. Like electrons, protons and neutrons have a spin of 1/2. Nuclei
with an odd number of neutrons and an even number of protons, or alternatively, an odd number of protons and an even number of neutrons will
have a net spin of 1/2. Spin 1/2 nuclei encountered in biomolecules are 1H,
13
C, 15N, 19F, and 31P. This is not the only nonzero spin value possible. Nuclei
with an odd number of proteins and also an odd number of neutrons, like
2
H, have a spin of 1. In Figure 3.6, spin is indicated by Iz.

Figure 3.6. Splitting of energy levels of a spin 1/2 particle by an external magnetic
eld: (a) In the absence of a magnetic eld, the energies of the spin up and spin
down states of the spin 1/2 particle are the same. The energy levels are no longer the
same in the presence of a magnetic eld. The energy of the particle whose spin is
aligned parallel to the magnetic eld, the spin +1/2 particle, is lower than the particle whose spin is aligned against the external magnetic eld, that is, the spin -1/2
particle. The energy levels are said to be split by the magnetic eld. (b) If the spin
1/2 particles are exposed to a source of electromagnetic energy, while in the magnetic eld, there will be a sharp absorption peak when the frequency of the electromagnetic wave exactly matches the frequency of the photon needed to trigger a
transition from the lower to the higher energy level.

3.5 Determining Protein Structure Through NMR

55

The negatively charged electron clouds surrounding nuclei reduce the

magnitude of the magnetic eld experienced by the spin 1/2 protons and
neutrons residing in the atomic nucleus. This effect is called shielding. Each
atomic species has a uniquely different electron cloud, and thus different
atoms will undergo different line splittings in the presence of the same
external magnetic eld. Nearby atoms will inuence the energy splitting
through the shielding effects, as well. Atoms such as oxygen that are
strongly electronegative will have a far greater inuence on neighboring
atoms than will, for example, carbon atoms.
Shielding by the atoms electron cloud and those of its neighbors give rise
to several different observable effects. The electron clouds will alter the
locations of the peaks; they will create clusters of peaks in place of single
isolated peaks and produce variations in the peak heights. The creation of
clusters of peaks arises through interactions of the spins (magnetic elds)
of the atoms in one group with the spins (magnetic elds) of atoms in neighboring groups. In more detail, interactions between spins split the energy
levels, and the transitions between these energy levels appear as the distinct peaks in the plot of chemical shifts. An example of this, for the case of
interactions between a methyl group and a methylene group, is presented
in Figure 3.7. If the methyl group were isolated from its neighbors, there
would be a single hydrogen peak, since each of the three hydrogen atoms
sees the same environment. The presence of a nearby CH2 group induces
the splitting of the single peak into three peaks, and similarly, the presence

Figure 3.7. Chemical shifts of hydrogen atoms in a CH3CH2 molecular environment: The arrangement of atoms in the two groups is depicted in the insert. The
chemical shifts are plotted with respect to a reference shift, taken as the zero of the
axis, and are given in units of parts per million (ppm).

56

3. Exploring Protein Structure and Function

of the nearby CH3 group induces the splitting of the single peak for CH2
into four peaks. Variation in peak heights is not arbitrary, but instead is a
direct consequence of the relative contribution to each peak from different
spin-spin interactions (see Problem 3.4).

3.6 Intrinsic Magnetic Dipole Moment of Protons

and Neutrons
Protons and neutrons, like electrons, have an intrinsic magnetic dipole
moment. The magnitude of the magnetic moment for a proton is
m p = 2.79 m N ,
and for a neutron it is
m n = -1.91 m N .
The quantity mN is known as the nuclear magneton. It is dened in a manner
analogous to the electron dipole moment as
mN =

eh
,
2 Mp

(3.5)

- is Plancks constant divided by 2p, Mp is the

where e is the unit charge, h
proton rest mass, and c is the speed of light. Since the mass of a proton is
1837 times the mass of an electron, the magnetic dipole moment of a proton
or neutron is 1/1000 that of an electron.
A free proton at rest in a uniform static magnetic eld B0 oriented along
the z-axis can occupy one of two spin states. It can either be in a spin up
state (Iz = +1/2) or a spin down state (Iz = -1/2). The energy associated with
lower energy spin up state is
E+ = -m p B0 ,
and that of the higher energy spin down state is
E- = m p B0 .
If an RF pulse of electromagnetic energy is applied that matches the transition energy, or energy difference, between these two states, the spin can
be ipped from the lower energy state to the higher. The proton will then
relax back to its lower energy state. The resonant frequency of the RF pulse
that triggers the spin transition is
n0 =

2m p B0
.
h

(3.6)

Nuclear magnetic resonance of protons in bulk matter was rst demonstrated in 1946 by Felix Bloch and, independently, by Edward M. Purcell.

3.7 Using Protein Fluorescence to Probe Control Layer

57

Their efforts followed earlier work by Isidor I. Rabi, who discovered the
NMR effect in molecular beam experiments in 1937. Norman F. Ramsey,
working in Rabis laboratory, acquired the rst radiofrequency (RF) spectra
the following year, and developed the chemical shift theory in 1949. These
initial studies have evolved into a powerful technique used to study biomolecules in solution, and to a diagnostic tool, magnetic resonance imaging
(MRI), used in medicine.

3.7 Using Protein Fluorescence to Probe Control Layer

Fluorescent proteins can be used as sensitive probes of the movements and
interactions of control layer elements. Proteins that uoresce emit light at
a characteristic emission wavelength, lem, shortly after absorbing light at
their characteristic excitation wavelength, lex. The wavelengths at which
both absorption and emission occur fall in the short to middle portion of
the visual spectrum. An energy level diagram, a schematic depiction of the
energies of the low-lying electron orbitals of the proteins uorophore, is
presented in Figure 3.3. As shown in the gure, there is a ground state band
consisting of number of closely spaced energy levels, and an excited state
band, also consisting of a cluster of energy levels. Levels within the excited
state band are populated when light is absorbed. The emission of light corresponds to electronic transitions from the excited state band back down
to the ground state band. The energies of absorbed and emitted photons
differ slightly. The emitted photon has a greater wavelength, reecting its
slightly lower energy.
Green uorescent protein (GFP) and other naturally uorescent molecules are found in organisms ranging from the bioluminescent jellysh
Aequorea victoria to the non-bioluminescent coral Discosoma striata. The
ability of GFP to uoresce is due to the presence of a uorophore consisting of a sequence of three amino acid residuesSer 65-Tyr 66-Gly 67. This
internal sequence is post-translationally modied to a ring structure and the
tyrosine is oxidized. These two changes convert the trio of amino acid
residues into a uorescing center. The natural form, or wild type, of GFP
has an excitation maximum at 395 nm, a secondary excitation maximum at
470 nm, and an emission peak at 509 nm. These peaks correspond to green
light. The protein extracted from Discosoma striata uoresces in the red
range, and is named dsRed. In addition to these naturally occurring uorescent proteins a number of variants have been created with altered properties that improve their utility as markers. The excitation and emission
peaks for several uorescent protein variants are presented in Table 3.3.
The DNA sequence for the 28kDa green uorescent protein has been
determined. To make a fusion protein, the complementary DNA, or cDNA,
of the protein of interest is inserted into a vector along with the DNA for
the GFP. When the combined gene is expressed in a transfected cell (the

58

3. Exploring Protein Structure and Function

Table 3.3. Spectral properties of green uorescent
protein variants.
GFP variant

Color

lex (nm)

lem (nm)

eBFP
eCFP
eGFP
eYFP
dsRed

blue
cyan
green
yellow
red

380
434
488
514
558

440
476
509
527
583

cell into which the vector was inserted) the resulting fusion protein will
contain the GFP covalently attached to the study protein, in a way that does
not interfere with the normal operation of that protein. The fusion protein
functions as a uorescent reporter. When excited either by a laser or by an
incandescent lamp, the protein will emit light and thus report its presence.
Fusion proteins made in this manner have been used to study how signaling proteins move about the living cell. They have been employed to visualize the subcellular compartmentalization of the signaling molecules, and
to view how the proteins move from one locale to another, in and out of
organelles, and back and forth to the plasma membrane.
In Aequorea victoria, GFP operates in close association with another
protein, aquorin, a naturally luminescent protein that emits blue light.
Energy is transferred in a radiationless manner from the uorophore of
aquorin to the uorophore of GFP. The GFP absorbs the blue light and in
turn emits green light. The process of radiationless energy transfer from one
uorophore to another is called uorescence resonance energy transfer
(FRET). This process can occur when the energy level differences involved
in emission from one uorophore overlap the energy level differences
involved in excitation of the second uorophore. That is, FRET can occur
if the donor emission spectrum overlaps the acceptor excitation spectrum.

3.8 Exploring Signaling with FRET

Fluorescence resonance energy transfer can be used to explore signaling
within the living cell. Fluorescence resonance energy transfer and related
techniques can be used to study protein-protein interactions. In this approach, one uorophore is fused to the rst of two proteins and the other
to the second protein. When the rst protein is well separated from the
second, the rst protein will emit light at its characteristic emission wavelength upon stimulation. If the two uorophores satisfy the conditions for
FRET, there will be a shift in wavelength of the emitted light from that of

3.8 Exploring Signaling with FRET

59

the rst protein (the donor) to that of the second (the acceptor) when the
two proteins some into contact. These processes can then be studied with
light microscopy. The nature of the emitted light is sensitive to relative
orientation of the two uorophores and to their spatial separation. These
dependencies can be exploited in the design of the fusion proteins, so that
the emission spectra reect conformational changes and properties of the
protein-protein interactions.
The principle behind the use of FRET for studying protein-protein
interactions can be applied in a variety of ways to the study of signaling.
As was the case for protein-protein interactions, two particles are brought
into close proximity, one fused to a uorescent donor and the other to a
uorescent acceptor. The sought after signaling interactions are then
studied and quantied by measuring the changes in FRET efciency. When
the two uorophores are far apart the efciency is low, but this changes in
the presence of the interaction of interest. This is the basis for studying
post-translational modications such as proteolytic cleavage and phosphorylation; the movement of small signaling intermediaries such as cyclic
adenosine monophosphate (cAMP), Ca2+, and cytochrome c; and it has
been used to study the trafcking of proteins as they are secreted from the
cell.
Several examples illustrating how the FRET principle is applied are
presented in Figure 3.8. In the rst example, that of phosphorylation by a
protein kinase, a phosphorylation reporter is created. This (the reporter)
protein contains an amino acid sequence that is typically phosphorylated
by the kinase of interest. When the sequence is phosphorylated by the
kinase a second domain in the reporter protein recognizes and binds to the
phosphorylated sequence. The second domain contains a uorescent
protein that acts as the acceptor, while the rst domain contains the donor.
When the protein is phosphorylated by the kinase and the second domain
binds it, the uorescent acceptor is brought closer to the donor and the
FRET efciency increases.
A similar procedure is followed for proteins called cameleons, which
report the presence of calcium, an exceptionally important small signaling
molecule. In this case, calmodulin (CaM), a calcium-binding protein, is used
in constructing the reporter. Again a donor and acceptor are appended; the
donor is appended to CaM and the acceptor to a peptide known as M13
that binds tightly to CaM what the latter binds calcium. The third example
presented in Figure 3.8 represents the converse situation, that of proteolysis. To study proteolysis, a protein is created with donor and acceptor uorescent proteins. The two regions are connected to one another by an amino
acid sequence that is characteristically cleaved by the proteolytic enzyme
(protease) of interest. When the reporter protein is cleaved by the proteolytic protein, FRET ceases as the two uorescent proteins drift apart and
no longer interact.

60

3. Exploring Protein Structure and Function

Figure 3.8. Fluorescence resonance energy transfer (FRET) used to explore intracellular signaling: (a) PhosphorylationA laser emits radiation at 440 nm, triggering emission from a CFP at 480 nm in the left hand panel. The phosphorylation
recognition domain connected to the rst domain by a exible linker binds the phosphorylated protein following kinase activity in the right hand panel. These changes
result in FRET and an increased emission from the YFP at 535 nm. (b) Calcium
bindingIn the absence of calcium, a calcium-binding protein, calmodulin, is in a
conrmation that loosely tethers an M13 peptide. When it binds four calcium ions,
it assumes a dumbbell-like shape that induces a tight binding of the M13 peptide
resulting in FRET and increased emission at 535 nm. (c) ProteolysisIn the absence
of the protease, the two domains remain in close proximity and emit radiation at
535 nm. When the protease cleaves the linker, the two portions of the protein move
apart and no longer exhibit FRET.

3.9 Exploring Protein Structure with Circular Dichroism

Another important property of biomolecules is their chirality, or handedness. All amino acids used to make proteins are left handed (L-amino acids)
and all sugars used in DNA and RNA are right handed (D-sugars). This is
not accidental. A consistent handedness on the part of protein-forming
amino acids is essential for the proper folding of the polypeptide chains into
compact three-dimensional shapes. Not all molecules possess a handedness.
For this to occur a central atom surrounded by four different atoms or
atomic groups must be present. The alpha carbon in the amino acids serves
as the chiral center, and all amino acids with the exception of glycine satisfy

3.11 A Genetic Method for Detecting Protein Interactions

61

the chirality condition. The left and right handed forms of chiral molecules
are collectively referred to as optical isomers or as enantiomers
nonsuperimposable mirror images of one another.
Chiral biological materials are sensitive to the chiral behavior of left and
right circularly polarized light and absorb the two light forms in different
ways. In circular dichroism (CD), differences in absorption between left and
right circularly polarized light are measured as a function of wavelength.
These measurements provide information on the secondary structure
content of the proteins.The measurements can be used to determine whether
the protein is mostly alpha helical, or whether it is made up mostly of antiparallel beta sheets and turns.Circular dichroism utilizes the ultraviolet (UV)
portion of the electromagnetic spectrum, typically in the range of 200 nm.

3.10 Infrared and Raman Spectroscopy to Probe

Vibrational States
Transitions between rotational states in proteins are of fairly low energy and
correspond to absorption of EM radiation in the microwave portion of the
spectrum. Vibrational transitions are somewhat higher in energy. There are
three kinds (modes) of vibrationssymmetric stretching, asymmetric
stretching, and bending. In symmetric stretching, the stretching and contractions in bond length of a pair of bonds connecting a central atom to its two
neighbors are of the same magnitude. In asymmetric stretching, the stretches
and contractions are of unequal magnitude. The energies of the transitions
between vibrational states lie in the infrared portion of the EM spectrum and
can be explored using infrared (IR) spectroscopy and Raman spectroscopy.
In infrared spectroscopy, measurements are made of the frequencies of
IR light that are being absorbed by the sample of interest. The IR wavelengths of most interest in explorations of biomolecules span the range from
2.5 to 15 microns, which is in the middle IR regime. The absorption spectra
measured using IR spectrometers contain dips at wavelengths where the
light has the correct energy to induce transitions between vibrational states
and be absorbed. In Raman spectroscopy, inelastically scattered light is
examined. The difference in energy between absorbed and emitted light is
taken up by other vibrational transitions, and these transitions appear as
spikes in the spectra.

3.11 A Genetic Method for Detecting

Protein Interactions
The yeast two-hybrid system is a genetic method for detecting proteinprotein interactions. There are a number of methods for detecting interactions between proteins. One of these, rst introduced in 1989 and widely

62

3. Exploring Protein Structure and Function

used since then, is the yeast two-hybrid system. This method involves the use
of designed transcription factors (the hybrids) and reporter genes. Reporter
genes are genes whose protein products have easily detected activities.Transcription factors are proteins that stimulate transcription. The transcription
factors used in the yeast two-hybrid approach activate the reporters.The rst
of these to be used is the Ga l4 protein in the yeast S. cerevisiae. This protein
stimulates transcription of the lacZ reporter gene that encodes the enzyme
b-galactosidase, whose activity can be easily measured (assayed).
Ga l4, like many transcription factors, contains a DNA-binding domain
and a transcription activation domain. (Transcription factors will be examined in detail in Chapter 16.) The modular organization of the Ga l4 protein
is exploited to create a pair of hybrid proteins. One hybrid consists of the
DNA-binding domain of Ga l4 fused to protein X. The other contains the
activation domain of Ga l4 fused to protein Y. The two hybrids are reintroduced into the yeast cell. If protein X (the bait) interacts with protein Y
(the prey) the Ga l4 DNA and activation domains will be brought into close
proximity and the pair will be able to stimulate transcription of the reporter
gene.
In the yeast two-hybrid method, different combinations of bait and prey
are tested to identify interacting proteins. A common approach to carrying
out large scale testing is to utilize two haploid yeast strains. One strain
contains the bait and the other the prey. The strains are then mated and the
interactions are determined by assaying the activity of the reporter gene in
the diploid strain.This variant of the two-hybrid method is called interaction
mating or mating assaying. It has been applied to several organisms whose
genomes have been sequenced. For example, several hundred proteinprotein interactions have been identied in this manner in S. cerevisiae.

3.12 DNA and Oligonucleotide Arrays Provide

Information on Genes
One consequence of the work on complete genome sequences is the
existence of libraries of genomes for many organisms. These libraries of
DNA sequences form the basis for the newly developed microarray
technologies. The physical basis for the microarray approaches is the complementary binding propensity of single-stranded nucleic acid sequences
nucleic acid bases preferentially bind to their complementary bases. A
microarray is a glass slide containing single-stranded genetic material
afxed in a regular grid. Each grid spot contains a single gene product that
has been amplied (i.e., many copies made) in a polymerase chain reaction
(PCR). A sample set of mRNAs from a cell of interest is then washed over
the array. These nucleic acids will bind (hybridize) to the cDNA counterparts immobilized in the array yielding a prole of the particular gene being
expressed in the cell.

3.13 Gel Electrophoresis of Proteins

63

In order to determine relative abundances the mRNA samples are

labeled with a uorescence dye. Typically, the sample of interest is labeled
with a red uorescent dye and there is a control sample that is labeled
with a green uorescent dye. By illuminating the spots with a laser and
observing the color of the spot the relative abundances can be determined.
Red would indicate a high abundance of the sample relative to the
control, and green the opposite situation. Yellow would indicate equal
abundances, and black a lack of hybridization by either sample or control
at that spot.
DNA microarrays generate enormous amounts of data. They provide
information on which sets of genes are expressed (and how strongly) at different times in the cell cycle and in response to which kinds of environmental stimuli. When interpreted and assembled into a regulatory model
these kinds of data provide a molecular level explanation of how the cell
or organism under study behaves.

3.13 Gel Electrophoresis of Proteins

Size and composition of protein complexes can be determined using oneand two-dimensional gel electrophoresis. In polyacrylamide gel electrophoresis, or SDS-PAGE, a polyacrylamide gel serves as an inert matrix
over which proteins in a detergent solution migrate. Sodium dodecyl sulfate
(SDS), the detergent, binds to hydrophobic portions of proteins causing
then to unfold (denature) into extended chains, to dissociate from other
proteins and lipid molecules, and for their subunits to unbind to each other.
The large number of bound, negatively charged detergent molecules more
than cancels any net positive charge on the protein. The overall negatively
charged proteins migrate towards the positive electrode when a voltage is
applied. The distance the protein moves is dependent upon the mass (size)
of the protein, and so the proteins separate into bands arranged according
to mass. The mass bands can then be analyzed to yield the protein masses
and subunit composition.
In 2D gel electrophoresis, proteins are separated by both mass and
charge. The proteins in the sample are again separated by a detergent, but
this time the detergent is uncharged. The net charge on the now separated
proteins is left unchanged. Recall that altering the pH of the solute will
change the net charge on a protein, and proteins have an isoelectric point
the pH value at which the protein net change is zero. When electrophoresed
in a gel in which a pH gradient has been applied the proteins will migrate
to their individual isoelectric points and stop because at this point there is
no force due to the electric eld acting on the protein. When this procedure
is followed with an SDS-PAGE procedure carried in a second direction
orthogonal to that of the isoelectric focusing, the result is a set of proteins
immobilized in a 2D gel separated by charge and mass.

64

3. Exploring Protein Structure and Function

3.14 Mass Spectroscopy of Proteins

Mass spectroscopy can be used to measure the masses of proteins, and to
determine post-translational modications. J.J. Thomson developed the
mass spectrograph in the period from 1906 to 1913. He initially used his
devices to study canal rays, positively charged ions produced in a cathode
ray tube. He later used the mass spectrograph to measure the masses of a
variety of atomic species, establishing their discrete character, and then
by this means established the existence of isotopes of stable elements.
A number of researchers, most notably Aston, Dempster, Bainbridge,
Mattuach, and Nier in the 1920s and 1930s further developed the discipline
of mass spectrometry in which combinations of electric and/or magnetic
elds are used to lter, disperse, and separate charged particles according
to their charge (z) to mass (M) ratios.
All mass spectrometers have three main components: an ion source, a
mass analyzer, and an ion detector. The source is responsible for producing
a beam of ionized particles of the material to be mass analyzed. The analyzer separates the beam ions according to their M/z values, and the detector is responsible for their detection. The main breakthrough that lead to
the use of mass spectrometry in the study of proteins was the development
of techniques for producing beams of charged gaseous proteins. Two
techniques are widely used. The rst is electrospray ionization (ESI) and the
second is matrix-assisted laser desorption ionization (MALDI). These
methods were introduced during the 1980s, and can be employed for
biomolecules with masses up to 50 kDa (ESI) and more than 300 kDa
(MALDI).
In a mass spectrometer, a beam of particles with charge z moves with
velocity u. The ions are rst subjected to an accelerating voltage V in an
ionization chamber, and, as a result, attain kinetic energies equal to zV,
where z is the charge of the ion. They are then sent through a magnetic eld
that is applied in a direction perpendicular to the direction of motion of the
particles. The particles experience a force equal to zuB, where B is the magnetic eld strength, which is balanced by the centrifugal force. By subjecting the beam of charged biomolecules to a magnetic eld, various M/z
values are scanned. The basic expression is
M z = r 2 B2 2V .

(3.7)

The voltage V is usually kept constant; the radius of curvature r is determined by the geometric properties of the magnet, and the user varies B to
select different M/z ratios. In a further renement of the technique, a pair
of parallel electrodes can be added to the magnet. By adjusting the electric
and magnetic elds according to the expression r = 2V/E, the velocity
dependence can be removed so that all ionized proteins of a given M/z
value fall on the same point in the focal plane of the detector.

References and Further Reading

65

In still another approach, a time-of-ight (TOF) instrument can be

devised in which differences in arrival times are used to measure masses.
In a TOF system all ions are accelerated to the same kinetic energy, and
the time of ight t is measured over the ight length L. The M/z value is
then determined according to the formula:
2

M z = 2V (t L) .

(3.8)

Books on Protein Structure, X-Ray Crystallography,

and NMR
Brandon C, and Tooze J [1999]. Introduction to Protein Structure, 2nd edition. New
York: Garland Science Publishing.
Drenth J [1998]. Principles of Protein X-ray Crystallography. New York: SpringerVerlag.
Hore PJ [1995]. Nuclear Magnetic Resonance. Oxford: Oxford University Press.
Levitt MH [2001]. Spin Dynamics: Basics of Nuclear Magnetic Resonance. New
York: John Wiley and Sons.
Rhodes G [2000]. Crystallography Made Crystal Clear: A Guide for Users of
Macromolecular Models, 2nd edition. San Diego: Academic Press.
Woolfson MM [2001]. An Introduction to X-ray Crystallography. Cambridge:
Cambridge University Press.

References and Further Reading

X-Ray Crystallography, Electron Crystallography, and NMR
Campbell ID, and Downing AK [1998]. NMR of modular proteins. Nat. Struct. Biol.
(Suppl.), 5: 496499.
Khlbrandt W, and Wang DN [1991]. Three-dimensional structure of plant light-harvesting complex determined by electron crystallography. Nature, 350: 130134.
Unwin N [1993]. Nicotinic acetylcholine receptor at 9 resolution. J. Mol. Biol., 229:
11011124.
Unwin N [1995]. Acetylcholine receptor channel imaged in the open state. Nature,
373: 3743.
Wilson KS [1998]. Illuminating crystallography. Nat. Struct. Biol. (Suppl.), 5:
627630.

Fluorescence Resonance Energy Transfer

Bastiaens PIH, and Pepperkok R [2000]. Observing proteins in their natural habitat:
The living cell. Trends Biochem. Sci., 25: 631637.
Matz MV, et al. [1999]. Fluorescent proteins from nonbioluminescent Anthozoa
species. Nat. Biotechnol., 17: 969973.
Pollok BA, and Heim R [1999]. Using GFP in FRET-based applications. Trends in
Cell Biol., 9: 5760.
Weiss S [1999]. Fluorescence spectroscopy of single biomolecules. Science, 283:
16761683.

66

3. Exploring Protein Structure and Function

Circular Dichroism
Johnson WC [1990]. Protein secondary structure and circular dichroism: A practical guide. Proteins, 7: 205214.
Woody RW [1995]. Circular dichroism. Methods in Enzymology, 246: 3471.

Yeast Two-Hybrid System

Chien CT, et al. [1991]. The two-hybrid system: A method to identify and clone genes
for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA, 88:
95789582.
Finley RL, Jr., and Brent R [1994]. Interaction mating reveals binary and ternary
connections between Drosophila cell cycle regulators. Proc. Natl. Acad. Sci. USA,
91: 1298012984.
Ito T, et al. [2001]. A comprehensive two-hybrid analysis to explore the yeast protein
interactome. Proc. Natl. Acad. Sci. USA, 98: 45694574.
Uetz P, et al. [2000]. A comprehensive analysis of protein-protein interactions in
Saccharomyces cerevisiae. Nature, 403: 623627.
Walhout AJM, et al. [2000]. Protein interaction mapping in C. elegans using proteins
involved in vulval development. Science, 287: 116122.

2D Gel Electrophoresis
Gygi SP, et al. [1999]. Quantitative analysis of complex protein mixtures using
isotope-coded afnity tags. Nat. Biotechnol., 17: 994999.

Mass Spectrometry
Link AJ, et al. [1999]. Direct analysis of protein complexes using mass spectrometry, Nat. Biotechnol., 17: 676682.
Yates JR, 3rd [1998]. Mass spectrometry and the age of the proteome. J. Mass
Spectrom., 33: 119.

Gene Fusion, Gene Expression Proles, and Combined Methods

Enright AJ, et al. [1999]. Protein interaction maps for complete genomes based on
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Lockhart DJ, and Winzeler EA [2000]. Genomics, gene expression and DNA arrays.
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Marcotte EM, et al. [1999a]. Detecting protein function and protein-protein interactions from genome sequences. Science, 285: 751753.
Marcotte EM, et al. [1999b]. A combined algorithm for genome-wide prediction of
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Young RA [2000]. Biomedical discovery with DNA arrays. Cell, 102: 915.

Problems

67

Problems
3.1 (a) The emission of laser light at 440 nm for use in FRET was discussed
in Section 3.10. What is the energy of these photons in (i) eV, and
(ii) kcal/mol? (b) What is the energy of a 1.5 X-ray in these two sets
of units? (c) What are the corresponding frequencies of the photons at
440 nm and 1.5 .
The following list of physical constants and conversion factors may be
helpful:
Physical constants and conversion factors
Avogardos number N = 6.022 1023/mol
Velocity of light c = 2.9979 1010 cm/s
Plancks constant h = 6.626 10-27 erg sec
1 eV = 1.6 10-12 erg
1 cal = 4.184 joules .004 4.184 107 ergs

3.2 Using the gure shown below, derive Braggs law, Eq. (3.4).

Start with the relationship that the increase in path length between the
two scattered rays shown in gure must be an integral multiple of the
wavelength, that is, the waves must be in phase with one another in
order to constructively interfere. In terms of the notation of the gure
this condition is
r + x = nl .
Hint: Make use of the trigonometric identity:
2 sin 2 q - 1 = cos (180 -2q) .
3.3 Show that the nuclear magneton
m N = 5.05 10 -27 J T .

68

3. Exploring Protein Structure and Function

In the unit J/T, J denotes Joules and the symbol T denotes Tesla, the SI
unit for B-elds. This quantity has dimensions of kilograms per second
per second per ampere; that is 1 T = 1 kg s-2 amp-1. Use values for physical constants from the table in Problem 3.1 and the one shown below.
(Note: The ampere is a unit of current; that is, 1 amp = 1 coulomb per
second).
Physical constants and conversion factors
Charge on an electron e = 1.6 10-19 coulombs
Mass of a proton Mp = 1.672 10-27 kg

3.4 Recall from Figure 3.7 that the CH2 group splits the single peak for the
hydrogen atoms in CH3 into three peaks, and the CH3 group splits the
single hydrogen peak for CH2 into four peaks. In the gure shown
below, the spin-spin effect of the CH2 group on the CH3 group is shown.
The two hydrogen atoms in CH2 can each have their spins either
aligned with the external eld (+) or have their spins aligned opposite
(-). When opposite they shield the magnetic eld and reduce the splitting, while aligned they add to the eld and increase the splitting. There
are three different spin-spin combinations: both negative, one positive
and one negative, and both positive. This gives rise to the three peaks.
Since there are two ways of arriving at a [+, -] combination and once
each for the others the peak intensities are in a 1 : 2 : 1 ratio as illustrated
in Figure 3.7.

(a) In this problem, write down the possible spin combinations for the
CH3 group that splits the peak for CH2 into four peaks shown in Figure
3.7, and give the ratios of the peak heights. (b) From the results for two
and three hydrogens, infer the splittings of a hydrogen peak produced
by four nearby hydrogen atoms. How would their peak heights vary?

Problems

69

3.5 Recall from the discussion on mass spectrometers that the beams of
charged particles are subjected to rst an accelerating voltage allowing
the particle to attain a kinetic energy equal to
zV = mu 2 2.
When the particles feel the magnetic eld the force zuB is exactly balanced by the centrifugal force:
zuB = mu 2 r .
(a) From these two relationships derive Eq. (3.7). (b) Then derive the
time-of-ight expression given by Eq. (3.8).

4
Macromolecular Forces

Ordinary polymers such as rubber are constructed as repetitive sequences

of a small number of basic units. In contrast, proteins are synthesized as nonrepetitive sequences of the 20 different amino acids. Each kind of amino acid
has a different side chain, and the variations in side chains endow each
amino acid with a distinct set of physical and chemical properties.The amino
acid compositions of proteins are not randomly selected. Instead, amino acid
sequences are selected to allow the proteins to fold into compact threedimensional forms in physiologically meaningful time periods, with specic
binding properties that enable the proteins to carry out their cellular
tasks.
Macromolecular forcesmixtures of covalent and noncovalent forces
generated by the electron clouds surrounding the atomic nucleihold
protein together. The starting point in understanding how proteins signal
one another is to understand the physical properties of the amino acids and
the macromolecular forces that hold biomolecules together. One question
that one would like to answer is How do amino acids pack together?
Another is How do the macromolecular forces and amino acid geometry
shape the proteins? How do they guide their interactions with one another,
and with lipids, carbohydrates, DNA, and RNA? The goal of this chapter is
to begin to answer these questions and others like them. Amino acid composition and organization in proteins will be looked at along with macromolecular forces holding the proteins together and mediating their
interactions with one another through their interfaces. This exploration will
continue into the next chapter, where the problem of how a protein folds,
one of the most enduring and distinguished problems in all of science, will
be explored.

4.1 Amino Acids Vary in Size and Shape

Amino acid side chains vary considerably in length and mass. The smallest
amino acid is glycine. Its side chain consists of a single hydrogen atom. The
next smallest amino acid is alanine with a CH3 group as its side chain. The
71

72

4. Macromolecular Forces
Table 4.1. Physical properties of amino acid residues:
Three- and one-letter abbreviations (codes) are listed in
column 2. Masses are given in column 3 in Daltons. The
volumes presented in column 4 are in cubic angstroms.
Amino acid

Code

Mass (Da)

Volume (3)

Alanine
Arginine
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine

Ala-A
Arg-R
Asn-N
Asp-D
Cys-C
Glu-E
Gln-Q
Gly-G
His-H
Ile-I
Leu-L
Lys-K
Met-M
Phe-F
Pro-P
Ser-S
The-T
Trp-W
Tyr-Y
Val-V

71.08
156.2
114.1
115.1
103.1
129.1
128.1
57.05
137.1
113.2
113.2
128.2
131.2
147.2
97.12
87.08
101.1
186.2
163.2
99.13

89.3
190.3
122.4
114.4
102.5
138.8
146.9
63.8
157.5
163.0
163.1
165.1
165.8
190.8
121.3
93.5
119.6
226.4
194.6
138.2

longest side chains belong to arginine and lysine, while the most massive
are arginine, tyrosine and tryptophan. The lengths of the side chains vary
by an order of magnitude and the masses of the amino acids, which are
listed in Table 4.1, differ by more than a factor of three.
The amino acids used to make proteins vary not only in size, but also in
shape. Some amino acids have open side chains (alipathic); others contain
closed rings (aromatic); and one amino acid, proline, has a side chain that
closes back onto the main chain nitrogen. That the amino acids vary in size
and shape is important for their packing. These variations make possible
the close packing of amino acids in the core of the folded protein. The
resulting interior packing densities approach those of organic solids.

4.2 Amino Acids Behavior in Aqueous Environments

Amino acids differ in their charge properties. Some are polar, charged, and
either acidic or basic; others are polar and uncharged; and still others are
nonpolar. Several side chains contain hydroxyl groups and are highly reactive, and two contain a sulfur atom. The physical properties of the amino
acidssize, mass, shape, charge distribution, and bonding propensityplay
major roles in determining how a protein folds, how a protein interacts

4.2 Amino Acids Behavior in Aqueous Environments

73

Table 4.2. Electrostatics and bonding propensities of the amino acids: The polar
amino acids listed in column 3 are of three kinds. Some are basic (arginine, histidine,
and lysine); others are acidic (aspartic and glutamic acids); and the remainder are
uncharged polar molecules. Histidine is only weakly basic and is often considered
a nonpolar amino acid, along with proline.
Amino acid
Alanine
Arginine
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine

Nonpolar

Polar

H-bond

Salt Bridge

*
*
*

S Bridge

*
*
*

*
*
*
*
*
*
*
*

*
*

*
*

*
*

with other biomolecules, and how proteins interact with their aqueous
environments.
The results of grouping the 20 amino acids according to their charge and
bonding properties are presented in Table 4.2. Columns 2 and 3 in the table
group the amino acids according to whether they are polar or nonpolar. The
signicance of this partitioning is that amino acids with nonpolar side chains
are hydrophobic, and those with polar side chains are hydroplilic. The
distinction between the two kinds of interaction arises from the binding
preference of water for the amino acid. In the case of a hydrophilic (waterloving) amino acid, a water molecule would rather bind to the amino acid
than to another water molecule. In the case of a hydrophobic amino acid,
a water molecule prefers another water molecule to the amino acid.
Nonpolar groups tend to come together in water, not because of an
attraction for each other but rather because their clustering enables water
molecules to make the maximum number of contacts with each another.
The term hydrophobic interactions denotes the process whereby water
molecules come together so that small nonpolar molecules and nonpolar
portions of large molecules minimize their contacts with water. The
propensity for water molecules to come together is a reection of their

74

4. Macromolecular Forces

hydrogen-bonding capabilities resulting in the formation of networks of

hydrogen-bonded molecules.

4.3 Formation of H-Bonded Atom Networks

Extensive networks of hydrogen-bonded atoms are formed. In small
hydrogen-bearing molecules such as water there is little screening of the
positively charged hydrogen nuclei by electron clouds. The hydrogen atoms
of these molecules easily form bonds with unshared electrons of electronegative atoms of nearby molecules. These hydrogen bonds form most
often between covalently bonded O-H, N-H, and F-H groups and other OH, N-H, and F-H groups. The tightness of the hydrogen bonding is limited
by the mutual repulsion of the electron clouds of the two electronegative
atoms. Typical bond lengths, representing the distances between centers of
the two electronegative binding partners, range from 2.6 to 3.2 angstroms.
Hydrogen bonding is dipole-dipole in character, and is therefore highly
directional. The bond is strongest when the three atoms are colinear and
rapidly diminishes with increasing bond angle.Typical maximum bond energies are 3 to 7 kcal/mol.
Water is a particularly important example of a molecule that forms
hydrogen bonds with other molecules of the same kind. Each water molecule
typically forms three or four hydrogen bonds with nearby water molecules.
The result is a three-dimensional latticework of H2O molecules. The ability
of water molecules to form hydrogen bonds with other water molecules is
responsible for waters cohesiveness. Most biomolecules are soluble in water
and can move freely from one location to another without clumping together.
But they are not so soluble that they are unable to expel intervening water
molecules when coming together to form complexes and machines.
Hydrogen bonding is responsible for the formation and stability of alpha
helices and beta sheets. The tendency is for the interior of proteins to be
predominately hydrophobic, and in some studies of how proteins fold,
hydrophobic and hydrophilic interactions of residues are found to have a
dominating inuence. However, polar amino acid residues do populate the
interior of proteins, and hydrophobic residues populate the surface. In those
instances where polar residues are located in the interior, hydrogen bonds
alleviate the disruptive inuences of charged groups on the stability of the
protein.

4.4 Forces that Stabilize Proteins

Salt bridges, van der Waals forces, and disulde bridges help stabilize proteins and their surface contacts. Salt bridges are formed by coulombic
attractions between positively and negatively charged atoms. In the context

4.5 Atomic Radii of Macromolecular Forces

75

of the amino acids, salt bridges are generated by electrostatic (coulombic)

attractions between positively charged lysine or arginine residues and negatively charged aspartic acid or glutamic acid residues. These bonds are
most often encountered on the surface of the protein.
Those atoms that do not form hydrogen bonds or salt bridges still attract
one another through van der Waals attractions. The van der Waals attractions are dipole forces. They are shorter ranged than point coulomb forces
and are weaker, as well. In a dipole-dipole interaction the region of negative charge of one molecule attracts the region of positive charge of another
molecule.The strongest dipole-dipole interactions occur between molecules
possessing permanent dipole moments. London (or London dispersion)
forces arise from the motion of the electron clouds that surround the atomic
nuclei. The electrons in any molecule are constantly in motion and undergo
spontaneous distortions to form instantaneous dipoles where one portion
of the molecule has a net positive charge and the other has a net negative
one. These instantaneous dipoles induce matching dipoles on neighboring
molecules. The result is a nonspecic and nondirectional attractive force
between the two molecules. London forces are most pronounced when the
molecules have large electron clouds that are easily polarized. It is the
various dipole forces involving induced and permanent dipoles that are collectively referred to as van der Waals forces.
The last column in Table 4.2 lists two amino acids whose side chains
contain sulfur atoms. Disulde bonds depart from the aforementioned rule
of weak bonding. The disulde bonds are covalent in character. They are
formed between sulfur atoms on cysteine side chains. These bonds make
important contributions to the overall stability of the proteins.

4.5 Atomic Radii of Macromolecular Forces

Macromolecular forces have characteristic strength and distinct atomic
radii. The different macromolecular forces vary considerably in strength
from covalent bonds (the strongest) to van der Waals interactions (the
weakest). Because the different kinds of interactions vary in their strengths,
the amount of interpenetration of the electron clouds will vary depending
on which forces are being experienced. This means that the radii of atoms
bound inside proteins and other macromolecules will depend on the forces
being experienced. This type of systematic behavior is quite useful, allowing investigators to deduce forces from observed bond lengths. Listed
in Table 4.3 are a set of atomic radii for nitrogen and oxygen under the
inuence of different forces.
The average bond lengths and atomic radii are related to one another in
a simple fashion. A bond length is just the sum of the two atomic radii for
the bond partners. For bonds between like atoms, this means that the bond
length is just twice the atomic radius for the interaction of interest. The

76

4. Macromolecular Forces
Table 4.3. Atomic radii: Listed are average values for
atomic radii deduced from X-ray crystallography data
for a variety of atomic groups in proteins. All atomic
radii are in angstroms (). The radii listed in columns
2 through 5 are for covalent, coulombic, hydrogenbonded and van der Waals interactions, respectively.
Atom

rcov

rcoul

rH

rvdW

Nitrogen
Oxygen

0.70
0.65

1.45
1.40

1.55
1.40

1.70
1.50

atomic radii and bond energies behave in a systematic way with respect to
one another. The stronger the bond the closer together the two atoms will
be. Covalent bonding strengths can be as high as 100 kcal/mol or more. This
is at least an order of magnitude greater than any of the noncovalent bond
forms. As a result the bond lengths are about a factor of two shorter than
those of any of the weaker bonding forms. The strongest of the noncovalent forces are the coulombic interactions. These can be as much as 5 kcal/
mol. The hydrogen bonds are somewhat weaker yet, broadly distributed in
the range of 2 kcal/mol. The weakest of the forces are the van der Waals
interactions. These are not more than about 1 kcal/mol, barely above typical
thermal energies of 0.6 kcal/mol.
The three kinds of interactions just discussed, hydrogen bonds, coulombic attractions and repulsions, and van der Waals forces, are all far weaker
than the covalent bonds that underlie the protein backbone. Rather than
forming a few strong covalent bonds proteins interact with one another by
forming multiple weak bonds that can be easily broken and reestablished
in the same or in different ways.

4.6 Osmophobic Forces Stabilize Stressed Cells

Osmophobic forces are an important stabilizing element when cells are
stressed. There is one more class of forces that has an important bearing on
protein-folding and stability in the cell. Cells are exposed to a variety of
stressful conditions. Among these are thermal, osmotic, and salt stresses.
Cells have developed a number of strategies that enable them to cope with
stresses when they arise. One of these adaptive mechanisms is to build up
intracellular concentrations of small organic metabolites called osmolytes.
Free amino acids and amino acid derivatives are common osmolytes. These
organic molecules protect the cell against denaturing effects on the proteins
of abnormal cellular conditions. They help maintain the proteins in their
folded conformations without interfering with the normal functioning of
the proteins.

4.7 Protein Interfaces Aid Intra- and Intermolecular Communication

77

By analogy with the characterization of disfavored water-protein interactions as a hydrophobic effect, disfavored osmolyte-protein interactions
are called osmophobic interactions. This effect manifests itself as a burial of
the protein backbone to shield it from the osmolytes. The gain in stability
from avoiding contacts between the backbone atoms and the osmolytes
more than compensates for the attractive effects between the side chains
and the osmolytes. By increasing the concentration of osmolytes when
stressed, a cell supplies a driving force that promotes the folded state over
the unfolded one.

4.7 Protein Interfaces Aid Intra- and

Intermolecular Communication
Proteins possess interfaces that enable them to communicate with proteins,
nucleotides, lipids, and carbohydrates.The region of contact between molecular surfaces is known as the interface. Interfaces belong to the domain level
of protein organization: When a protein acquires a particular domain it
inherits that domains binding properties by possessing its interfaces.
Protein interfaces recognize and bind other cellular components through
the latters interfaces. There are several different kinds of interfaces, listed
in Table 4.4. The rst entry is that of interfaces between adjacent domains
located within a single protein. Communication across these interfaces
allows the functionally separated parts of the protein to work together. The
next two entries, subunit interfaces, also pertain to communication within a
protein, but operate at the quaternary level of organization. One kind of
subunit interface (tight) enables protein subunits that stay together for
appreciable periods of time in membrane proteins to coordinate their activities. The other (loose) permits subunits of cytosolic proteins that only come
together for brief periods of time then immediately separate again to regulate one anothers actions.
Table 4.4. Classication of protein interfaces.
Type
Domain-domain
Subunit-subunit
(a) Tight
(b) Loose
Protein-protein
Protein-DNA
Protein-RNA
Protein-lipid
Protein-carbohydrate

Function
Coordinates activities of adjacent domains within a protein
Enables subunits on pore- and channel-forming proteins to work as
a single unit
Coordinates activities of cytosolic protein subunits that transiently
associate
Communication between proteins
Communication between proteins and regulatory sequences in
DNA molecules
Communication between proteins and RNA molecules
Communication between proteins and membrane lipids
Recognition of cellular and ECM carbohydrates

78

4. Macromolecular Forces

The other categories of interfaces operate between proteins and the different kinds of cellular biomoleculesproteins, DNA, RNA, lipids, and carbohydrates. These interfaces make possible the binding of proteins to their
ligands, transcription factors to DNA, and splicing factors to RNA. They
mediate interactions with the lipid membrane bilayer and are responsible
for the coordinated activities of signal complexes. From the viewpoint of
signaling the most widely encountered and studied interfaces are the
protein-protein and protein-DNA interfaces. Carbohydrate interfaces are
widely encountered in cells of the immune system, and these will be discussed in Chapter 10, which is devoted to cell adhesion and motility. Lipid
interfaces are crucial for a variety of cellular processes, including signaling.
These interfaces will be examined in detail in Chapter 8, where membrane
lipid composition and lipid signaling are explored.
Recall from Chapter 2 that proteins involved in signaling are frequently
post-translationally modied. Some amino acid residues acquire phosphoryl
groups, while others gain acetyl groups or methyl groups. Some protein
interfaces are sufciently precise in their binding afnities that they can
distinguish whether these groups are present or absent. As a result, an
upstream signaling event, where a group is added, can be followed by a
downstream event, which involves the recognition of that binding site.

4.8 Interfaces Utilize Shape and Electrostatic

Complementarity
Surfaces establish contact with one another though their interfaces. The
areas of surface contact may be planar, but most often they are irregular in
shape. Interfaces utilize shape and electrostatic complementarity for recognition and binding. Shape complementarity denotes the propensity of the
surfaces of two molecules to geometrically t together so that multiple contacts can be established. Electrostatic complementarity denotes the matching of hydrophobic patches, the complementary pairing of hydrogen bond
donors and acceptors, and the matching of positive and negative charges of
basic and acidic polar residues from one surface to the other.
The amino acid residues that form the interfaces on the two complementary surfaces do not each contribute equally to the binding energy and
specicity. Rather, some 5 to 10 amino acid residues in each complementary surface form energetic hot spots. These hot spots are responsible for
most of the binding afnity of one surface for the other. This number may
be compared to the 10 to 30 residues on each protein that form the interface. Interfaces are in general hydrophobic, but not overwhelmingly so.
Hydrophilic residues assume a greater role in binding than in folding (to
be discussed in the next chapter). For some interfaces electrostatic interactions help steer the ligand onto the correct docking orientation/location.
Hot spots tend to be localized in the center of the interface, surrounded by

4.10 Motion Models of Covalently Bonded Atoms

79

hydrophobic rings containing energetically less important residues that

shield the hot spot residues from the bulk solvent.

4.9 Macromolecular Forces Hold

Macromolecules Together
The macromolecular forces that hold macromolecules together are of two
kinds. One kind of force, the covalent bond, is generated when two atoms
share electrons. The other kind of force operates between atoms that do
not share electrons and are not covalently bonded. The interactions that
take place in these situations are electrostatic in character consisting of
combinations of point charge and dipole forces, and, as already discussed,
are referred to as coulombic (salt bridges) and van der Waals interactions.
Hydrogen bonds are included in this category. Unlike the electron-sharing
mechanism underlying covalent-bonding interactions, hydrogen bonds are
primarily electrostatic in nature. They arise from a balance between attractive and repulsive forces between partial charges.
Covalent-bonding forces can be thought of as operating in an elastic
springlike manner. In an elastic spring, there is an equilibrium length where
the spring is at rest. If compressed or stretched away from the equilibrium
length, potential energy builds up, or is stored, in the spring. Once the perturbing force is removed from the spring, an elastic spring force, called a
Hookes law force, restores the spring to its equilibrium rest length. Hooke
law expresses the relationship between the displacement d from equilibrium position, the spring constant Kd, and the restoring force F:
F = - Kd d.

(4.1)

Several different kinds of motions of atoms about their bonds are possible.
As depicted in Figure 4.1, there are stretching motions, bending motions,
and torsions about the bond axis.

4.10 Motion Models of Covalently Bonded Atoms

Hookes law and periodic potentials are often used to model how covalently
bonded atoms move. A variety of conceptual approaches have been developed that enable researchers to study how proteins and other macromolecules move under the inuence of bonding and nonbonding forces. These
methods have been used singly and in combination. The most popular
methods in use are:
Continuum electrostatics formalism
Molecular mechanics formalism
Molecular dynamics method

80

4. Macromolecular Forces

Figure 4.1. Motions about groups of two, three, and four covalently bonded atoms:
Displayed in the upper portion of the gure are bond-stretching motions of a pair
of covalently bonded atoms along their bond axis. Shown in the middle panel are
bending motions involving three bonded atoms forming an angle q. Displayed at
the bottom are groups of four atoms, where the leftmost atom has rotated about
the middle bond axis.

Brownian dynamics method

Quantum mechanics formalism
In continuum electrostatics, one replaces detailed models of interactions of
the macromolecule atoms with their surrounding waters with a simplied
treatment, one in which the molecule-solvent interactions are treated in an
average way. The water is modeled at a macroscopic level while the atoms
of the molecule of interest are still studied at an atomic level of detail. In
this approach, a macroscopic expression is solved to give the electrostatic
potential extending outward from the surface of the protein. When visualized, the potential highlights interesting regions of positive and negative
charge and provides insight into the interface and binding properties of the
protein.
The atomic level of detail is handled by the molecular mechanics (MM)
formalism. In the MM formalism, a classical force eld U is introduced consisting of a sum of bonding and nonbonding interactions. The three kinds
of covalent bonds illustrated in Figure 4.1 are modeled using Hookes law
and periodic potentials. As indicated in expression (4.2), Hookes law terms
represent bond and angle interactions, and a periodic term represents the
dihedrals, also referred to as torsions:
2

K (b - b ) + K (q - q
= + K (1 - cos (nf + d)).

U bonded =

bonds

eq

angles

torsions

eq

(4.2)

4.11 Modeling van der Waals Forces

81

Figure 4.2. Bond stretching: Plotted are the potential energies associated with bond
stretching. The harmonic potential has a minimum at the equilibrium bond length.
It increases as the bonded atoms are pushed along the bond axis towards one
another, so the bond is compressed, and as they are pulled apart, so the bond is
stretched out. For the stretch spring constant, Kb = 400 kcal/(mol 2), and for the
equilibrium bond length, beq = 1.3 .

The total potential energy of the macromolecule Ubonded is equal to the

sum (S) of the contributions from the individual bonds. All of the bondstretching interactions are included along with the bond-rotations and
bond-torsions. As the atoms forming these bonds move away from and
towards their equilibrium congurations, the potential energies rise and fall.
The shape of the potential energy curve for each of the covalent bond types
is illustrated in Figures 4.2 to 4.4.

4.11 Modeling van der Waals Forces

The noncovalentbonded interactions are of two typesvan der Waals
forces and a coulombic term representing salt bridges and other point
charge interactions. Because of the 1/r radius dependence the coulombic
attractions and repulsions fall off with increasing separation of the interacting atoms very slowly, far more so than the contributions from the other
terms in the nonbonded potential shown in Eq. (4.3).
Van der Waals forces are frequently modeled in terms of a Lennard
Jones, 612 potential. There are two terms in this potential. The term containing the sixth power of the radius is an attractive one while the term

82

4. Macromolecular Forces

Figure 4.3. Bond bending: Plotted are the potential energies associated with rotations of the angle made by three atoms covalently bonded to one another. In a
manner analogous to bond stretching, there is an equilibrium bond angle where the
potential energy is at a minimum. As the rotation angles are either widened out
or squeezed in, the potential energy rapidly increases. The bending spring constant
Kq = 40 kcal/(mol rad2), and equilibrium angle qeq = 120 deg.

Figure 4.4. Bond torsions: Plotted are the periodic contribution to the potential
energy from torsion (dihedrals) rotations of covalently bonded sets of four atoms.
Calculations were done using a Kj with four paths so that 3.625 kcal/mol is the constant in front of the expression. Parameters values used were n = 2 and d = 180 deg.

involving the twelfth power of the radius is a repulsive one. As can be seen
in the gure there are two distances of interest. One of these is the equilibrium radius r0, the location where the potential has its minimum and
where the forcethe derivative of the potentialis zero. The other key dis-

4.12 Moleculer Dynamics in the Study of System Evolution

83

Figure 4.5. Van der Waals interactions: Plotted are the van der Waals potential
energies in kcal/mol using a well depth e = 0.19 kcal/mol and a radius r0 = 3.59 ,
corresponding to nitrogen and oxygen contact radii of 1.7 + 1.5 = 3.2 .

tance is the radius at which the repulsion exactly cancels out the attraction
so that the net potential is zero. This repulsion is generated by the interpenetrating electron clouds. This contact distance is the van der Waals
radius. As can be seen in the plot, any further interpenetration is strongly
resisted by the rapidly increasing repulsive force arising from the Pauli
exclusion.
6
r0 12
r0 qi q j
+
U nonbonded = e
-2

r 4pe 0 r
i j r

(4.3)

Hydrogen-bonding contributions to the total potential are treated in

an approximate way in the MM formalism. They are modeled using
LennardJones and coulombic like terms suitably modied to match the
characteristics of the hydrogen bonds.

4.12 Moleculer Dynamics in the Study of

System Evolution
The evolution of macromolecular systems over time can be studied using
the method of moleculer dynamics (MD). In molecular dynamics, the
classical (Newtonian) equations of motion are solved through numerical

84

4. Macromolecular Forces

integration. The forces F are the spatial derivatives of the potential energies U presented in Eqs. (4.2) and (4.3). These quantities are then converted
to accelerations using Newtons second law, F = ma, where m is mass and a
is acceleration. Once this is done the equations of motion are solved to give
a picture of how the system evolves over time. Numerical methods are used
to convert the equations of motion into a form suitable for numerical integration on a computer. A number of computer programs are in use that
enable the researcher to carry out MD simulations.
The last entry in the list of methods is quantum mechanics (QM). The
covalent bonds are only handled in an approximate way in the MM formalism. In a quantum mechanical approach, the empirical treatment of the
covalent bonds through the use of a Hookes law is replaced by an exact
quantum mechanical treatment. The quantum mechanical method is exact
and rigorous but is difcult to use in practice due to computational limitations. As computer technologies have advanced, quantum mechanical
studies have become more numerous. An often-used approach is to
combine the MM and QM formalisms, using one or the other of the two
methods for different aspects of the system under study.

4.13 Importance of Water Molecules in

Cellular Function
Water molecules are essential components of protein, DNA, and RNA
function. Water plays a central role in protein structure and function. In the
absence of water, a protein would not be able to fold into its native state,
nor would it be able to catalyze reactions. Water is an essential component
of many protein-protein, protein-DNA and protein-RNA interfaces, and
without water proteins would not be able to recognize their substrates.
Water surrounds a protein, lls in pockets and grooves on the surface, and
occupies voids in the interior.
When a protein is placed into a water environment it alters the network
of hydrogen bonds. The water molecules in the vicinity of the protein
surface reorient themselves so that positive and negative regions of change
are in opposition. The rotations of the water molecules disrupt the hydrogen bonds between adjacent water molecules and thus alter the network.
The water molecules in the immediate vicinity of the protein surface that
have reoriented themselves are referred to as the rst hydration layer. The
reorientation effect propagates outward from the protein and the next layer
of disrupted hydrogen bonded water molecules is designated as the second
hydration layer.
The same phenomena occur for DNA and RNA. Water molecules in the
vicinity of the DNA and RNA form hydrogen bonds to the molecules.These
bonds are not passive entities but instead contribute to the conformational
stabilization and function of the macromolecules. Hydrogen bonding net-

References and Further Reading

85

works between water and DNA is essential for DNA stability. DNA denatures as it dehydrates. Hydrogen bonds between water and ssRNA are even
more numerous than in the case of dsDNA because of the single strand
character of the RNA leaves bases unpaired and there are additional ribose
oxygen atoms available for bonding.

4.14 Essential Nature of Protein Dynamics

Macromolecules such as proteins are dynamic entities: their internal
motions are essential. They are in continual thermal motion and through
these motions are continually exploring different conformations. When
especially stable states are encountered, the dwell time, the period of time
that the protein remains in such states, increases; when highly unstable
states are populated the dwell time decreases. This continual exploration of
available conformations is central to binding and catalysis.
At the heart of the role of water in the activities of all macromolecules
is its ability to promote rapid conformational changes. Water is a common
element in the active site of enzymes, and is a key mediator of the catalytic
activity of many, if not most, enzymes. When dehydrated these enzymes lose
their catalytic abilities. Yet another place where water seems to be crucial
is in regulation. Proteins such as hemoglobin that use allosteric mechanisms
(this term will be dened and its properties explored in the next chapter)
for regulation operate in a hydrated fashion, and loss of these water molecules impairs the regulatory functioning of the protein. Water, the hydrogen bonds that are continually being made and broken, and the underlying
thermal agitation collectively serve as a lubricant that promotes conformation changes essential to the performance of protein, DNA, and RNA
functions.

General Reference
Brandon C, and Tooze J [1999]. Introduction to Protein Structure, 2nd edition. New
York: Garland Science Publishing.

References and Further Reading

Physical and Electrostatic Properties of Amino Acids and Proteins
Bolen DW, and Baskakov IV [2001]. The osmophobic effect: Natural selection of a
thermodynamic force in protein folding. J. Mol. Biol., 310: 955963.
Dill KA [1990]. Dominant forces in protein folding. Biochem., 29: 71337155.
Myers JK, and Pace CN [1996]. Hydrogen bonding stabilizes globular proteins.
Biophys. J., 71: 20332039.
Pace CN, et al. [1996]. Forces contributing to the conformational stability of
proteins. FASEB J., 10: 7583.

86

4. Macromolecular Forces

Sheinerman FB, and Honig B [2002]. On the role of electrostatic interactions in the
design of protein-protein interfaces. J. Mol. Biol., 318: 161177.
Tsai J, et al. [1999]. The packing density in proteins: Standard radii and volumes.
J. Mol. Biol., 290: 253266.

Complementarity and Interfaces

Glaser F, et al. [2001]. Residue frequencies and pairing preferences at proteinprotein interfaces. Proteins, 43: 89102.
Lo Conte L, Chothia C, and Janin J [1999]. The atomic structure of protein-protein
recognition sites. J. Mol. Biol., 285: 21772198.
Jones S, and Thornton JM [1996]. Principles of protein-protein interactions. Proc.
Natl. Acad. Sci. USA, 93: 1320.
Jones S, et al. [1999]. Protein-DNA interactions: A structural analysis. J. Mol. Biol.,
287: 877896.
Nadassy K, Wodak SJ, and Janin J [1999]. Structural features of protein-nucleic acid
recognition sites. Biochem., 38: 19992017.
Norel R, et al. [1999]. Examination of shape complementarity in docking of unbound
proteins. Proteins, 36: 307317.
Sheinerman FB, Norel R, and Honig B [2000]. Electrostatic aspects of proteinprotein interactions. Curr. Opin. Struct. Biol., 10: 153159.

Hot Spots
Bogan AA, and Thorn KS [1998]. Anatomy of hot spots in protein interfaces. J. Mol.
Biol., 280: 19.
Hu ZJ, et al. [2000]. Conservation of polar residues as hot spots at protein interfaces. Proteins, 39: 331342.

Theoretical Methods: Computer Modeling and Simulation

Cornell WD, et al. [1995]. A second generation force eld for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc., 117: 5179
5197.
Elcock AH, Sept D, and McCammon JA [2001]. Computer simulation of proteinprotein interactions. J. Phys. Chem. B, 105: 15041518.
Honig B, and Nicholls A [1995]. Classical electrostatics in biology and chemistry.
Science, 268: 11441149.
Kollman PA, et al. [2000]. Calculating structures and free energies of complex molecules: Combining molecular mechanics and continuum models. Acc. Chem. Res.,
33: 889897.

Problems
4.1 Atomic motions. Atoms in a protein are constantly in thermal motion.
Assuming an average energy kT of 0.6 kcal/mol for each atom, how fast
is a hydrogen atom moving? How fast is a carbon atom moving? How
long will it take for each of these atoms to move 1 , roughly one bond
length?

Problems

87

4.2 Numerical integration. Numerical techniques, known as nite difference

methods, are used to convert the equations of motion into a form suitable for numerical integration on a computer. The basic idea is to take
the position and momentum of each particle at a given time and
compute how each changes over a small interval of time, the time step
Dt, by calculating the accelerations from the forces, and these from the
potentials of the form given in the chapter. In other words, for each particle i in the system
ai =

1
1 d
Fi =
Ui .
mi
mi dri

A number of computer programs such as CHARMM, AMBER and

GROMOS, are in use that enable a user to carry out MM simulations.
A number of time-stepping algorithms are employed in determining the
future positions from the past positions and forces. These algorithms
are based on expansions such as
2

r (t + Dt ) = r (t ) + v(t )Dt + (1 2)a(t )(Dt ) .

One of the most widely used stepping forms, known as the Verlet algorithm, is
2

r (t + Dt ) = 2r (t ) - r (t - Dt ) + a(t )(Dt ) .
Note that the velocities do not appear in this expression. The positions
at time t + Dt are computed from the positions at the present (t + Dt)
and previous (t) times and from the accelerations at the previous (t)
time. Some algorithms use the velocities explicitly in the computations.
One of these is the velocity Verlet algorithm. Its form is
v(t + Dt ) = v(t ) + (1 2)[a(t ) + a(t + Dt )]Dt .
By making use of the appropriate expansions, and combining terms,
derive both of these Verlet algorithms. What might be an appropriate
time step size? (Hint: Think about the results from Problem 4.1.)

5
Protein Folding and Binding

The world contains a myriad of biological systems. All exhibit a considerable degree of order.They are organized in a hierarchical manner, and order
is present at all levels of the hierarchy. From hydrogen, carbon, nitrogen,
and oxygen, simple atomic groups are formed such as methyl (CH3) and
hydroxyl (OH). These groups are then used to form the basic building
blocks of cellssugars, fatty acids, nucleotides, and amino acids. Simple
sugars (monosaccharides) are organized into short chains (oligosaccharides) or longer ones (polysaccharides). Fatty acids form complexes such as
triglycerides and phospholipids. Nucleotides are used to make RNA and
DNA, and the amino acids give rise to polypeptides, or proteins.
Biological order does not arise out of some mysterious vital force, but
rather is a consequence of the laws of thermodynamics and the character
of the forces in our universe, their strength and their dependence on distance. At rst glance the emergence of highly organized biological entities
seems at odds with the second law of thermodynamics. This law establishes
a thermodynamic arrow of timethe total disorder in the universe
increases as the universe ages, until a terminal stage of disorder is reached
in which the universe suffers a heat or entropy death. Yet, this rst impression is wrong: Order comes about not in spite of the laws of physics but
rather because of them.
Biological systems are open, continually exchanging matter and energy
with their surroundings.They generate order by taking in energy and releasing heat to their surroundings. They absorb radiant energy from sunlight
and from geothermal sources, and they take in foodstuffs that store energy
in high energy chemical bonds. According to the second law of thermodynamics the amount of entropy, or disorder, in a cell and its surroundings
must increase during any process. Thus, the production of order within a
cell is accompanied by the creation of a greater amount of disorder outside
a cell. This is accomplished through the release of heat from the cell at the
same time that the order is produced. Biological entities are not only highly
ordered, but actively generate these states in order to survive and
propagate.
89

90

5. Protein Folding and Binding

One of the central order-creating processes in a cell is the folding of a

protein into its biologically active three-dimensional form. During folding,
nascent proteins change their shape from a rather stretched out conguration to a highly compact form. They develop their secondary structure
alpha helices and beta sheetsand higher order structures with
well-dened signaling roles such as binding motifs and functional domains.
The folding process is the main focus of this chapter. Starting with a brief
review of the thermodynamic conditions for order to emerge, the spontaneous folding of proteins will be examined. Large and complex proteins,
especially those involved in signaling, often require the assistance of a class
of molecules called molecular chaperones to fold and to maintain their
correct form in the cell. Chaperone-assisted folding will be explored next.
That topic will be followed by the third and nal topic in this chapter, the
relationship between the thermodynamic properties of the low-lying stable
states of the folded proteins and their binding and signaling activities.

5.1 The First Law of Thermodynamics:

Energy Is Conserved
The rst law of thermodynamics is expressed in terms of three factors: the
internal energy of a system, the work done by that system, and the heat
absorbed by a system from its surroundings. The internal energy of a system
is the sum of the kinetic and potential energies of its constituents. Work is
done whenever a force is applied to an object to produce a displacement.
Typical examples of systems doing mechanical work are pistons, levers, and
pulleys. The most commonly encountered form of work in chemical systems
is pressure-volume work. In these systems, pressure, or force per unit area,
is usually held constant and there is a change in the volume occupied by
the system doing the work. If two systems are in thermal contact with one
another, energy will ow from the hotter (higher temperature) system to
the colder (lower temperature) system. Heat is the designation given to
the ow, or transfer, of (thermal) energy from one system to another due
to a temperature gradient.
In a chemical reaction that takes place in a cell, or in any other system,
the internal energy E is lowered by the amount of energy used to do useful
work and increased by the amount of heat absorbed in the process. In more
detail, as a system evolves from state a to state b, its internal energy will
change by an amount dE = Eb - Ea. If the amount of work done by the
system on its surroundings is written as W, and the amount of heat absorbed
by the system from its surroundings is designated as Q, then the rst law of
thermodynamics states that
dE = Q - W .

(5.1)

Energy will be gained by a system whenever energy ows into the system
due to temperature gradients and whenever the surroundings do work on

5.2 Heat Flows from a Hotter to a Cooler Body

91

the system. Energy will be lost from a system whenever the system does
work on its surroundings and whenever energy is lost to the surroundings
due to thermal gradients. If we consider pressure-volume work then we may
write this as
dE = Q - PdV ,

(5.2)

where P is the pressure and dV = Vb - Va is the change in volume produced

by the application of the (constant) pressure.

5.2 Heat Flows from a Hotter to a Cooler Body

Heat is associated with random molecular motion. If a hotter body is in
contact with a cooler one in a way that allows matter and energy to ow
from one to the other, temperature differences will be reduced and eventually vanish. The reason for this is a statistical one. There are many more
ways that the contributions of energies of the randomly moving molecules
can add up to a particular internal energy when the temperatures have
equilibrated than when there are large temperature differences between
parts of the whole. If, through random motions or perturbations of the individual molecules, one portion of the system gains an appreciable amount
of thermal energy at the expense of the rest it will not retain it for long.
Instead, the system will relax back to a thermally equilibrated distribution.
The thermally equilibrated distribution thus has the property that it is the
stable distribution. It is also the maximally disordered distribution, in contrast to highly ordered situations where each piece of a system is at some
specic value of the temperature.
These everyday observations are depicted in Figure 5.1. Part (a) of the
gure illustrates mass equilibration and part (b) the similar process of
thermal equilibration. In the case of mass equilibration a system starts out
with most of its mass concentrated in one of two interconnected compartments. The particles are free to diffuse, and over time the masses become
far more evenly distributed between the two compartments. The statistical
character of the process is easy to see. Situations (states) where all or mostly
all of the mass is concentrated in one compartment are rare. In contrast,
there are many ways of distributing half the mass in one compartment and
half in the other, and so under the inuence of random movements of mass
these partitions will occur most, all the time. Situations where all the particles are in one compartment will rarely occur, and when they do the system
will not remain so for long (these states are not stable ones).
The same reasoning applies to thermal equilibration. The rare velocity
distribution, where all the fast particles are in one compartment and the
slow ones in the other, is replaced over time by the usual one where both
compartments containing similar mixes of fast and slow movers. Again,
there are many ways of achieving this kind of distribution and few for the

92

5. Protein Folding and Binding

Figure 5.1. Mass and thermal equilibration: (a) Mass equilibrationIn the left
panel, most of the mass is concentrated in the left compartment. The particles are
free to diffuse to and fro, and through the opening into the adjoining compartment.
Over time, the system will mass equilibrate. In the right panel, roughly half of the
particles are in each compartment. (b) Thermal equilibrationArrows denote
velocity; particles with longer arrows are moving with a greater velocity than those
with shorter arrows. In the left panel, all of the fast particles are in the left compartment, and all the slow particles are in the right compartment. As a result, the
temperature in the left compartment is higher than that in the right compartment.
Again, there is an opening, and the particles can freely move about. Over time, the
system equilibrates so that the temperature in each chamber becomes the same. In
the right panel, each compartment contains a similar mix of slow and fast moving
particles.

other. In the case of thermal equilibration, the temperatures in the two compartments initially differ. The compartment with the fast movers is at a
higher temperature than that containing the slow movers, but over time the
temperatures in the two compartments become the same.

5.3 Direction of Heat Flow: Second Law

of Thermodynamics
The second law of thermodynamics formalizes the observation that heat
ows from a hotter system to a cooler one, and not vice versa. The quantity called entropy gives a measure of the number of ways the molecular
constituents can arrange themselves (i.e., it counts the number of microstates) to achieve a particular value of the macroscopic internal energy. The
entropy increases as the particles approach the distribution that can be
achieved in the largest number of ways, and entropy is maximal when the
system equilibrates over time. The maximization process is interpreted as a
ow of heat.

5.4 Order-Creating Processes Occur Spontaneously

93

The second law of thermodynamics has three parts:

Entropy, like the internal energy, is a property of a system.
In an isolated system, all processes involving transitions from one internal state to another are accompanied by increases in entropy.
In a system that is not isolated from its surroundings, any process
occurring will increase the entropy S in the system by an amount dS
proportional to the quantity of heat absorbed, and the constant of
proportionality is the inverse of the temperature:
dS = Q T .

(5.3)

If a living cell is to increase its internal order it must be in contact with its
surroundings to allow for the exchange of energy. If it is isolated from its
surroundings the amount of disorder within the cell can only increase since
the second part of the second law asserts that
dScell > 0.

(5.4)

When a cell is in contact with its surroundings to allow for the ow of

energy, there are two contributions to the total change in entropy:
dStotal = dScell + dSsurr > 0.

(5.5)

It is now possible for dScell to become negative so that the amount of order
in the cell can increase. This can occur if the increase in disorder in the surroundings is greater than the production of order within the cell.

5.4 Order-Creating Processes Occur Spontaneously as

Gibbs Free Energy Decreases
The rst law of thermodynamics says that if there is no change in volume,
then the change in internal energy of a system is equal to the amount of
heat absorbed from the surroundings. That is, Q = dE. Conversely, if the
pressure is held constant but the volume does change, then the heat
absorbed can be written as Q = (Eb + PVb) - (Ea + PVa). The enthalpy H
of the system is dened as H = E + PV. Thus, at constant pressure and temperature, the heat absorbed is equal to the change in enthalpy, or Q = dH.
The change in entropy of the surroundings is proportional to the amount
of heat released from the system. The inverse of the temperature T is the
constant of proportionality. That is,
dSsurr = -Q T .

(5.6)

In this expression a minus sign has been introduced since Q represents the
amount of heat absorbed from the surroundings. Since at constant temperature and pressure Q = dH, the entropy of the surroundings is simply
dSsurr = -dH/T, and

94

5. Protein Folding and Binding

TdStotal = -dH + TdScell .

(5.7)

It is convenient to introduce another quantity, the Gibbs free energy of the

system, G. This quantity represents the enthalpy minus the amount of
energy tied up internally in random motion and thus not free to do useful
work. It is dened as G = H - TScell so that
dG = dH - TdScell .

(5.8)

Since the right hand side of this last equation is simply the negative of the
right hand side of the previous equation, combining the two expressions
yields the relation
TdStotal = -dG.

(5.9)

This is a remarkable result. It states that the net change in entropy can be
determined from an examination of the change in the Gibbs free energy of
the system alone. Thus, there is no need to evaluate the change in entropy
in the surroundings in order to determine whether a process will occur
spontaneously or not. For a process to occur spontaneously there must be
a net increase in the entropy, or equivalently, the Gibbs free energy of the
system must decrease:
dG < 0.

(5.10)

By considering the Gibbs free energy one can determine whether a process
will occur spontaneously or not. There are two parts to consideran energetic (enthalpic) piece and an entropic one. Crystalline solids are more
ordered than liquids, and liquids, in turn, are more ordered than the gaseous
phase of a given substance. A crystalline solid such as ice can spontaneously
melt to become liquid decreasing the system order. In this process the
energy of the system increases, but this increase is more than offset by the
accompanying increase in entropy or disorder. Conversely, a process that
increases the order within a system can occur spontaneously if the decrease
in entropy is compensated for by a decrease in internal energy. The most
favorable reactions are those where energetic and entropic changes are
aligned, and spontaneous processes do not occur at all when entropic and
energy changes both increase the Gibbs free energy.

5.5 Spontaneous Folding of New Proteins

Newly synthesized proteins spontaneously fold into their physiologically
active three-dimensional shapes. Protein folding is the process whereby
nascent proteins, newly synthesized linear polypeptide chains, spontaneously fold into functional three-dimensional forms. During this process
they develop their secondary structures such as alpha helices and beta

5.5 Spontaneous Folding of New Proteins

95

sheets. The set of similar states into which protein folds is collectively
referred to as the native state. Conversely, the collection of states that a
newly synthesized protein, or an unfolded protein, populates is called the
denatured state. In a series of pioneering experiments in the 1950s and 1960s
Ansnsen showed that protein folding is a reversible process. By varying
conditions in the aqueous environment, proteins were made to go back and
forth between folded and unfolded congurations. Two main conclusions
can be drawn from his experiments. First, all the information needed for
folding is contained in the primary sequence. Second, the native and denatured states are thermodynamically stable statesthey are states of minimum Gibbs free energy.
The most important environmental or physical parameter inuencing
protein stability is temperature. (Two others are pH and salt concentration.)
The native state is a minimum in the Gibbs free energy at physiological
temperatures (and conditions). However, if the temperatures are elevated
above a critical temperature, the denatured state of a protein will lie at a
lower Gibbs free energy than the native state. The reason for this can be
discerned in an examination of the behavior of the two terms in Eq. (5.8)
for the Gibbs free energy. As the temperature is raised, the entropic contribution gains in importance relative to the enthalpic term. The entropic
term is a measure of the number of possible congurations for the main
and side chains. The entropy favors the denatured state because there are
many more ways for the side chains to arrange themselves when unfolded
than when tightly folded into a globular form. The enthalpic contribution,
on the other hand, strongly favors the native state. At low temperatures the
entropic contribution is still appreciable, but the enthalpic term predominates in this regime.
The property of stability is an important one. To be useful a state must
be stable long enough for the biological entity, whether it be a protein, a
DNA molecule, or some larger structure, to carry out its biological function
Such states must be stable in a thermodynamic sense. These states, once
formed, do not change appreciably in time. The effects of small perturbations and of thermal uctuations are rapidly damped out and the behavior
of the system is not appreciably altered. These are equilibrium states in the
language of thermodynamics.
While there are a multitude of states corresponding to the denatured
state, there are usually only a few similar states corresponding to the native
state and its conformation is essentially unique. This aspect is noted in Table
5.1. In order for the native state to be stable there must be an appreciable
gap in energy between the native state and nearby nonnative ones. When
the differences are appreciable, it is difcult for small perturbations and
thermal uctuations to induce transitions to the nearby higher energy
states. Whenever the energy gaps are small, the proteins will be only marginally stable.

96

5. Protein Folding and Binding

Table 5.1. Terms and concepts used to describe protein folding.

Terms and concepts
Denatured state
Energy landscape

Fast folding
Folding funnel

Kinetic trap

Native state

Meaning and signicance

Name given to a large number of high energy congurations of a
newly synthesized or an unfolded protein
A graphical representation of the number of states available to a
protein at each value of the potential energy as a function of a few
signicant degrees of freedom
Submillisecond folding of simple proteins, whose energy landscapes
have few barriers and traps
The overall shape of the potential energy landscape. With many high
energy states and few low energy ones, the surface narrows as the
potential energy (or enthalpy) is reduced
Any set of states forming a local minimum in the energy landscape
that is enclosed by energy barriers large compared to the thermal
energy
Name given to the small number of low energy congurations of a
biologically active protein

5.6 The Folding Process: An Energy Landscape Picture

The process whereby a nascent protein folds into its physiologically viable
3D form can be envisioned in terms of an energy landscape. Each point in
the landscape would represent a possible conformation of the protein.
Similar conformations would be found near one another and dissimilar ones
further apart. The vertical axis in this kind of description would represent
the sum of all contributions to the free energy of the protein except for the
conguration entropy. That is, it represents the enthalpy or potential energy
of the protein. The horizontal axis of the landscape gives the values of the
various degrees of freedom, coordinates such as the dihedral angle measures. Since there are too many coordinates to depict individually, one or
two coordinates, or combinations thereof, are selected that capture the
essential behavior of the protein as it folds. Two representative energy landscapes constructed in this manner are presented in Figure 5.2.
As can be seen in Figure 5.2, the energy landscapes are funnel shaped.
They are broad at the top and narrow at the bottom. The reason for this is
a general one. There are many energetically equivalent states at the top, but
far fewer ones at the bottom. Since each point on the landscape represents
a state, the width of the landscape is proportional to the number of states,
that is, it is directly related to the entropy. This quantity is a rapidly increasing function of the (internal) energy.
The folding process can be depicted as a trajectory connecting many
points on the landscape, denoting the sequence of small conformational
changes that the protein undergoes as it folds. As shown in the gure, a
folding trajectory starts out at a denatured state located at the top of the

5.6 The Folding Process: An Energy Landscape Picture

97

Figure 5.2. Energy landscapes: Each point in the cutaway views of the energy landscapes represents a possible conguration of the protein. In these 3D depictions, the
x, y-axes represent generalized coordinates indicative of the states of the protein.
The vertical axis denotes the potential energy, or equivalently, the enthalpy. The
overall shapebroad at the top and narrow at the bottomgives rise to the description of these surfaces as folding funnels. The funnel on the left is smooth and the
protein trajectories are straight, running down the funnel from the unfolded state
to the native state. The funnel on the right is slightly more complex. A ridge is
present which has to be surmounted before the protein can slide down to the native
state. The trajectories are longer and convolute slightly as they pass over the ridge
and then down the funnel. Even more complex funnels are possible, especially for
multidomain signaling proteins, in which there are mountains and valleys that have
to be traversed during folding.

landscape at a high potential energy and ends at the native state located at
the bottom of the landscape at a low potential energy.
The amount of time required for a protein to fold into its native state is
an important aspect of the process. This is referred to as a kinetic requirement. Not only must a protein fold into its native state, but also it must do
so in a physiologically reasonable time interval. The speed depends critically on the topography of the potential energy surface. If the surface is
studded with deep minima, and the folding trajectories pass close to them,
the rate of folding will be slow. In these situations, the protein will fall into
the minima and must escape before proceeding with its evolution towards
its native state. The deep minima are called kinetic traps because of their
slowing effects on the kinetics, or rates, of folding. Large and complex proteins, especially those involved in signaling pathways, tend to have rugged
landscapes containing minima surrounded by high barriers. On the other
hand, small single domain proteins often have landscapes that are fairly
smooth and these proteins fold rapidly. The difference between smooth and
rough funnels is highlighted in Figure 5.3.

98

5. Protein Folding and Binding

Figure 5.3. Smooth and rough folding funnels: (a) Smooth funnel in which there
are few barriers and all of these are smaller in height than the thermal energy.
(b) Rough funnel possessing several barriers that are difcult to surmount and serve
as kinetic traps because their heights are much larger than the thermal energy.

One of the features present in the rough funnel depicted in Figure 5.3 is
that of a low-lying metastable state. Recall from the discussion at the end
of Section 5.5 that one of the conditions for stability of the native state is
that there be an appreciable energy gap between the native state and those
lying above it. This condition is violated in the rough landscape depicted in
Figure 5.3. The protein will spend an appreciable time in the nearby excited
state. Not only is that state not separated by a large energy gap, but also,
once in that state, the protein must surmount a kinetic trap to get back out
to the native state.

5.7 Misfolded Proteins Can Cause Disease

Not all polypeptide sequences are able to fold in any reasonable amount
of time into a functionally meaningful native state. Rather, almost all randomly selected sequences will fail to do so. The replacement of even a single
residue by another can sometimes convert a protein from a form that is
folding-competent into one that is not. Failures of this kind often lead to
disease. The cystic brosis transmembrane conductance regulator, or CFTR
protein, serves as a prominent example of this sensitivity. Mutations to the
gene encoding the CFTR protein lead to cystic brosis. The CFTR protein
functions as a chloride channel, but when mutated it fails to fold properly

5.8 Protein Problems and Alzheimers Disease

99

and cannot insert in the membrane. The consequence is that the proper
movement of chloride ions is impaired. Among the most prominent symptoms of the disease are the production of salty secretions by the sweat
glands and thick mucus secretions by the lungs.
Another example is how point mutations can lead to impaired folding
and disease is rhodopsin. This protein is found in rods in the retina, where
it functions as the phototransducer. When this protein is mutated in regions
away from the C-terminus it fails to fold properly and doesnt transduce
light. In retinitis pigmentosa, the name for the resulting disorder, the rods
die off; sight worsens progressively, and eventually the subject becomes
blind.
One of the most striking examples of how even a single mutation can
have disastrous consequences on protein folding is that of the blood oxygen
carrier, hemoglobin. In this protein, a substitution of valine for glutamic acid
in the beta chains leads to improper assembly of the four subunits. The
result is sickle cell anemia. The hemoglobin molecules are misshapen and
not only dont transport oxygen adequately, but tend to clump together
causing further impairments. The clumping of misfolded proteins is not
limited to hemoglobin. It is observed in a variety of neurological disorders,
as well.

5.8 Protein Problems and Alzheimers Disease

Alzheimers disease is a neurological disorder that occurs with increasing
frequency late in life. It is the leading cause of senile dementia. This neurodegenerative disorder, rst described by Alois Alzheimer in 1906, can be
identied by the presence of two kinds of lesionsamyliod plaques and
neurobrillary tangles. Amyloid plaques are deposits of the amyloid b (Ab)
protein. These proteins form laments in the extracellular spaces, which are
usually surrounded by abnormal cells and cell structures. The second kind
of lesion occurs within cells. Neurobrillary tangles are composed of a
protein called tau that normally associates with microtubules. When this
protein is hyperphosphorylated it dissociates from the microtubules and
aggregates into insoluble laments, the tangles.
The Ab protein that forms the amyloid plaques is generated from a larger
amyloid b protein precursor (APP) by means of a series of posttranslational
cleavages. APP is a single-pass protein that resides in intracellular membranes. While membrane-bound it undergoes three proteolytic cleavage
operations, rst by an a-secretase, the second by a b-secretase and the third
by a g-secretase. The end product of these nishing operations is the Ab
protein. Mutations in ADD and in a family of proteins called presenilins
lead to familial Alzheimers disease. (The presenilins are 8-pass intramembrane proteins found in complexes that carry out the g-secretase stage of
proteolytic processing.)

100

5. Protein Folding and Binding

5.9 Amyloid Buildup in Neurological Disorders

Amyloid buildups arising from misfolded proteins characterize many neurological disorders. As mentioned above, the amyloid deposits seen in
Alzheimers patients are composed of Ab proteins. The underlying reason
for their aggregation into insoluble clumps is that the proteins are misfolded. The formation of insoluble protein clumps in certain classes of cells
in the brain is not restricted to Alzheimers disease, but rather is encountered in a host of neurological disorders (Table 5.2). One of the most commonly encountered examples is Parkinsons disease. In this neurological
disorder, aggregates of misled alpha synuclein proteins form clumps called
Lewy bodies in dopamergic cells regulating motor function, leading to its
impairment. Another entire set of examples is provided by the spongiform
encephalopathies (SEs), where buildups of prion proteins occur. Mad cow
disease in cattle and CreutzfeldtJakob disease in humans are two forms of
SE. Finally, in Lou Gehrigs disease (amyotrophic lateral sclerosis, or ALS),
CuZn superoxide dismutase (SOD1)-enriched inclusions develop in motor
neurons possibly associated with the buildup of free radicals in the affected
cells.
A clue as to what might be happening in all of these disorders is provided by the observation that the intracellular clumps of proteins that form
in the neurons contain other proteins besides the misfolded ones. Proteins
belonging to the ubiquitin-proteasome system are often found in these
deposits. In Huntingtons disease, the ubiquitin-proteasome system seems
to have broken down, and the normal housekeeping function of removal of
misfolded proteins does not occur. There is growing evidence for a breakdown in at least some forms of Parkinsons disease, as well. It may be that
the ubiquitin-proteasome system, responsible for removal of misfolded proteins, is being overwhelmed, leading over time to cell death as more and
more misfolded proteins accumulate in the long-lived neurons.
Even small clumps of misfolded proteins can be toxic. In Alzheimers
disease, there is either an excessive production of the Ab protein, or the
ratio of the amyloid-prone 42 amino acid residue forms over the less toxic

Table 5.2. Amyloid-producing neurological disorders.

Neurological disorder
Alzheimers disease
CreutzfeldtJakob disease
Huntingtons disease
Lou Gehrigs disease
Parkinsons disease

Amyloid-forming protein
Amyloid b protein
Prion
Huntingtin
CuZn superoxide dismutase
Alpha synuclein

5.10 Molecular Chaperones Assist in Protein Folding

101

40 amino acid residue forms is too high, or there is an alteration of the Ab

proteins biophysical properties promoting clumping. The proteins are partially folded resulting in the exposure of hydrophobic patches of residues
that promote aggregation. The danger inherent in even small oligomeric
assemblies of such proteins can be seen in the hippocampus where small
soluble oligomers consisting of two or three Ab proteins can impair normal
physiological functions of the cells. Small oligomers of misfolded forms of
even nominally harmless proteins can interact in an inappropriate fashion
with other cellular proteins. In sum, small soluble aggregates of misfolded
proteins can be highly toxic to the cells.

5.10 Molecular Chaperones Assist in Protein Folding in

the Crowded Cell
The cell is a crowded place and as a result many polypeptides, especially
large ones, require the assistance of helper molecules, the chaperones, to
reach their native state. Because of crowding, proteins, especially those that
are not completely folded and have exposed hydrophobic surfaces, tend to
aggregate. One of the main functions of the molecular chaperones is to
shuttle large polypeptides to their correct locations in the cell. In the
process they prevent hydrophobic contact-driven aggregates from forming
and assist in the formation of appropriate associations. Because of the
crowding the energy landscape can sometimes be altered to the point where
the proteins cannot reach their native state. Crowding also alters the kinetics, especially those involved in the assembly of multiprotein complexes.The
chaperones perform several useful housekeeping functions. They assist in
folding and in the rescue of proteins that have partially unfolded due to cellular stresses. In eukaryotes, which have a greater proportion of large
polypeptides than prokaryotes, chaperones are among the most abundant
proteins in the cell.
Folding in cells can be simple or complex depending on the nature of the
protein and its cellular destination. Small, rapidly folding proteins can reach
their native, necessary, functional three-dimensional conformation unaided.
As noted by Annsen in his 1973 Nobel Prize lecture, all information
required for folding is contained in the primary sequence. Because of cellular crowding, large and more complex polypeptides may require a protection from the immediate environment; that is, they may need a folding
cage to provide a shielded folding environment. Large and complex
polypeptides, especially those involved in signaling, destined for membrane
insertion and/or attachments, may require the actions of chaperones to
prevent aggregation. Some nascent proteins may be shuttled to their destinations in a form other than their native state and fold into the native state
only upon arrival and insertion into functional multisubunit complexes.

102

5. Protein Folding and Binding

5.11 Role of Chaperonins in Protein Folding

The energy landscapes for protein folding can be fairly smooth or they can
be rough. The landscapes for the fast folding proteins correspond to the
former situation. Large proteins may be able to reach their native state
without any assistance, but may do so on a time scale that is too slow physiologically. These proteins tend to have rough energy landscapes and the
proteins may easily be trapped in local minima. Molecular cheperones
known as chaperonins do two things to alleviate these difculties. First, they
sequester the nascent protein away from other cellular proteins, forming
folding cages. Second, they directly participate in the folding process,
supplying energy through ATP hydrolysis to the substrate protein. The
additional energy supplied to the protein enables that protein to surmount
energy barriers and escape from kinetic traps. The protein is able to fold
far more rapidly into its native state since it can avoid getting stuck in
nonoptimal and nonfunctional states for long periods of time.
The subunits that form the folding cage repeatedly bind and release the
substrate protein. They disrupt the misfolded structures that are kinetically
trapped, and release the protein in a less folded conguration through the
use of mechanical stretching forces. At the end of each round of binding
and release, the protein is free to continue to fold and search for its low
energy native state. The energy-dependent process of binding and release
is called annealing because of its close resemblance to the metallurgical
process of that name.
Chaperonins (Table 5.3) are large, 800- to 1000-kDa barrel-shaped structures that serve as folding cages. The group I GroEL chaperonin contains
Table 5.3. Molecular chaperones provide protected environments for protein
folding, greatly accelerate the folding rate, and stabilize nascent chains.
Family
Group I Chaperonins
Hsp60

Distribution
GroEL in bacteria; Hsp60 in
mitochondria and chloroplasts

Hsp10

GroES in bacteria; Hsp10 in

mitochondria and chloroplasts

Group II Chaperonins

TriC/CCT chaperonins in
archaea and eukaryotic cytosol

Hsp70 Chaperones
Hsp70

Hsp40
Hsp90 Chaperones

DnaK in bacteria; Hsp70 in

eukaryotes
DnaJ in bacteria; Hsp40 in
eukaryotes
HtpG in bacteria; Hsp90 in
eukaryotes

Function
Protected environment for
folding, refolding, recovery
from stress
Co-chaperonin for Hsp60;
assists in folding substrates
bound to Hsp60
Protected environment for
folding, refolding, recovery
from stress
Regulates heat shock response,
stabilizes nascent chains, and
prevents aggregation
Co-chaperone for Hsp70;
regulates activity of Hsp70
Maturation of signal
transduction pathways

5.12 Hsp 90 Chaperones Help Maintain Signal Transduction Pathways

103

Figure 5.4. A GroEL-GroES Annsen cage for protein folding: Fourteen subunits
are arranged into two rinds of seven units each. Each chain contains equatorial,
intermediate, and apical domains. Polypeptide chains are inserted into the cage,
undergo a round of mechanical manipulations (i.e., the chains are annealed to
remove unfavorable bondings), and are ejected. Several rounds of these operations
may take place.

14 identical subunits; the eukaryotic Hsp 60 molecule has 16 identical

subunits. As shown in Figure 5.4 the subunits are arranged into a pair of
rings; in the case of GroEL each ring contains 7 identical subunits. Cochaperonins help in this activity. Group I co-chaperonins form a lid over
the barrel and assist in the folding.

5.12 Hsp 90 Chaperones Help Maintain Signal

Transduction Pathways
The second main group of molecular chaperones consists of the Hsp70
and Hsp90 families. The Hsp70 family of chaperones performs a variety of
tasks in the prokaryotes. DnaK is the outstanding bacterial Hsp70 family
member. It regulates the heat shock stress response and maintains proteins
in their physiologically viable congurations. Eukaryotic polypeptide chains
are some 3040% larger on the average than their prokaryotic counterparts.
Eukarotic Hsp70 chaperones bind newly synthesized proteins. They assist
in transporting these proteins across organelle membranes and into the
endoplasmic reticulum (ER) and mitochondria.They also assist in the insertion of nascent membrane proteins into membranes.
Members of the Hsp90 family are involved in maintaining the functional
integrity of proteins involved in intracellular signaling. These chaperones
often work in association with Hsp70 family members and several other
small ancillary proteins to prevent aggregation and mediate refolding. The
Hsp90 chaperones form complexes with signal molecules, then help in their
translocation to the correct subcellular compartment and into association
with other elements of the signal pathway where they operate.

104

5. Protein Folding and Binding

Members of the Hsp90 family of molecular chaperones are among the

most abundant proteins in the cell. They account for 12% of the total cellular protein even under non-stressful conditions. Hsp90 acts later in the
folding process than the other chaperones. In normal cells, Hsp90 family
members bind to a selective set of proteins operating in the signaling pathways that regulate cell differentiation and embryonic development. The
proteins targeted by Hsp90 typically have low energy conformations that
are only marginally stable. These signaling proteins have difculty remaining in their physiologically competent states and require the assistance of
Hsp90 to stabilize them. For example, in the absence of Hsp90, steroid
hormone receptors are unable to bind their ligands and DNA. Likewise,
bereft of Hsp90, tyrosine kinases such as c-Src lose their ability to function
as kinases and to be acted upon by their regulators.

5.13 Proteins: Dynamic, Flexible, and Ready to Change

Proteins are not static but instead are dynamic structures that exhibit considerable exibility, and readily adopt new shapes. Proteins are dynamic
entities. If one examines the energy landscape in the vicinity of the proteins
native state one nds that there is an ensemble of low-lying states and the
proteins is continually undergoing transitions from one state to another.
This population of states and the barriers separating them play an important role in substrate recognition and catalysis. An example of such as
ensemble of low-lying conformational states is presented in Figure 5.5. In
this gure, none of the barriers are particularly high compared to the
thermal energy kT. In this situation, the proteins will easily undergo transitions from one state to another, but will spend more time in the lowest
energy state than in any of the others. The protein whose energy landscape
is depicted in Figure 5.5 is quite exible. It is able to move back and forth
among a set of different shapes. The energy landscape of a protein that is
completely rigid would be far sparser if it lacked low-lying states that were
easy to get to.

Figure 5.5. Low-lying ensemble of native and nearby state: These states determine
the binding properties of the proteins. Different ligands acting as environmental
factors select one or more of these preexisting states when they bind the protein.

References and Further Reading

105

The energy landscapes are inuenced by environmental factors such as

temperature, pH, ionic concentration, and binding events. Catalysts make
use of protein motions to increase the rate of reactions. Environment
factors, most notably charged groups such as acids, bases, metal ions, and
dipoles belonging to the catalyst generate a shift in the energy landscape.
Two kinds of shifts are possible, kinetic and thermodynamic. In a kinetic
shift, the energy barrier separating two conformational states is lowered,
making possible transitions from a higher energy state to a lower energy
state. The initial kinetic barrier is high compared to the thermal (kinetic)
energy factor kT, and the system may be trapped in the higher, or
metastable, state prior to the kinetic shift.
In a thermodynamic shift (the situation illustrated in Figure 5.5) the
barrier between two states remains the same but the relative energies are
modied so that a formerly higher energy state is now a lower energy state.
The barriers in this scenario low so there are no kinetic barriers, and transitions among the many states can be frequent. In either case, a catalyst generates a shift in the population of conformations towards those that favor
the reaction being catalyzed. This latter utilization of protein exibility is
often referred to in the literature as stabilizing the transition state in the case
of catalysis, and as the induced t model of surface complementarity in the
case of binding. The key point is that protein exibility underlies the ability
of a protein to bind to another moleculeand to entire groups of proteins
of differing shape and sizewith the appropriate degree of specicity. In
all of these situations the ligand may be thought of as selecting out one or
more states from the ensemble of preexisting low-lying states.

References and Further Reading

Annsen Nobel Prize Lecture
Annsen CB [1973]. Principles that guide the folding of protein chains. Science, 181:
223230.

Motions of Proteins
Cavanagh J, and Akke M [2000]. May the driving force be with youWhatever it
is. Nature Struct. Biol., 7: 1113.
Feher VA, and Cavanagh J [1999]. Millisecond-timescale motions contribute to
the function of the bacterial response regulator protein Spo0F. Nature, 400:
289293.
Forman-Kay JD [1999]. The dynamics in the thermodynamics of binding. Nature
Struct. Biol., 6: 10861087.
Frauenfelder H, Sligar SG, and Wolynes PG [1991]. The energy landscapes and
motions of proteins. Science, 254: 15981603.
Kay LE, et al. [1996]. Correlation between dynamics and high afnity binding in an
SH2 domain interaction. Biochem., 35: 361368.

106

5. Protein Folding and Binding

Kern D, et al. [1999]. Structure of a transiently phosphorylated switch in bacterial

signal transduction. Nature, 402: 894898.
Lee AL, Kinnear SA, and Wand AJ [2000]. Redistribution and loss of side chain
entropy upon formation of a calmodulin-peptide complex. Nature Struct. Biol., 7:
7277.
Stock A [1999]. Relating dynamics to function. Nature, 400: 221222.
Zidek L, Novotny MV, and Stone MJ [1999]. Increased protein backbone conformational entropy upon hydrophobic ligand binding. Nature Struct. Biol., 6: 1118
1121.

Protein Folding: The Energy Landscape Picture

Bryngelson JD, et al. [1995]. Funnels, pathways, and the energy landscape of protein
folding: A synthesis. Proteins: Structure, Function and Genetics, 21: 167195.
Chan HS, and Dill KA [1998]. Protein folding in the landscape perspective: Chevron
plots and non-Arrhenius kinetics. Proteins: Structure, Function and Genetics, 30:
233.
Dill KA, and Chan HS [1997]. From Levinthal to pathways to funnels, Nature Structure Biology, 4: 1019.
Leopold PE, Montal M, and Onuchic JN [1992]. Protein folding funnels: A kinetic
approach to the sequence-structure relationship. Proc. Natl. Acad. Sci. USA, 89:
87218725.
Onuchic JN, et al. [1995]. Toward an outline of the topography of a realistic proteinfolding funnel. Proc. Natl. Acad. Sci. USA, 92: 36263630.
Sali A, Shakhnovich E, and Karplus M [1994]. How does a protein fold? Nature,
369: 248251.

Molecular Chaperones and Protein Folding in the Cell

Hartl FU, and Hayer-Hartl M [2002]. Molecular chaperones in the cytosol: From
nascent chain to folded proteins. Science, 295: 18521858.
Pratt WB [1998]. The Hsp90-based chaperone system: Involvement in signal transduction from a variety of hormone and growth factor receptors. Proc. Soc. Exp.
Biol. Med., 217: 420434.
Rutherford SL, and Lindquist S [1998]. Hsp90 as a capacitor for morphological evolution. Nature, 396: 226342.
Sauer FG, et al. [2000]. Chaperone-assisted pilus assembly and bacterial attachment.
Curr. Opin. Struct. Biol., 10: 548556.

Binding Mechanisms
DeLano WL, et al. [2000]. Convergent solutions to binding at a protein-protein
interface. Science, 287: 12791283.
Freire E [1999]. The propagation of binding interactions to remote sites in proteins:
Analysis of the binding of the monoclonal antibody D1.3 to lysozyme. Proc. Natl.
Acad. Sci. USA, 96: 1011810122.
Hilser VJ, et al. [1998]. The structural distribution of cooperative interactions in
proteins: Analysis of the native state ensemble. Proc. Natl. Acad. Sci. USA, 95:
99039908.

Problems

107

Kumar S, et al. [2000]. Folding and binding cassettes: Dynamic landscapes and population shifts. Protein Sci., 9: 1019.
Ma B, et al. [2002]. Multiple diverse ligands binding at a single protein site: A matter
of pre-existing populations. Protein Sci., 11: 184197.
Teague S [2003]. Implications of protein exibility for drug design. Nature Rev. Drug
Dis., 2: 527541.

Problems
5.1 Entropy, density of states, and probabilities. Consider a system of N
atoms, each of which is placed in one of M bins. The atoms are labeled
by the bins into which they are placed, but are otherwise indistinguishable from one another. That is, n1 atoms are placed in bin 1;
n2 atoms are placed in bin 2, and so on up to nM atoms in bin M. The
quantities
pi = ni N
represent the probabilities of nding an atom in a particular bin. The
number of ways a specic conguration can be realized (where conguration means nding n1 atoms in bin 1, n2 atoms in bin 2, and so
on) is, from elementary probability theory, given by the multinomial
coefcient
W ({n1 , n2 , . . . , nM }) =

N!
.
n1 n2 nM

The relationship between number of states k, actually coeffs, and

entropy S was rst established by Ludwig Boltzmann and Max Planck.
Their results are usually presented in the form:
S=

k
ln W ({ni }),
N

and sometimes in the form

M

S = -k pi ln pi .
i =1

Show that the two forms are equivalent; that is,

M
k
ln W ({ni }) = -k pi ln pi .
N
i =1

5.2 Frustration and rugged energy landscapes. Rugged energy landscapes are formed whenever there is a lack of good low energy
conformations. Situations of this type arise when the system of atoms

108

5. Protein Folding and Binding

Figure for Problem 5.2. Nonfrustrated and frustrated squares: Atoms (vertices) are
connected by bonds (sides) with bond (coupling) strengths as indicated. (a) Nonfrustrated square in which all couplings are positive. (b) Frustrated square in which
there is one negative coupling and three positive couplings.

and bonds connecting them becomes frustrated. The term frustration

is intended to convey the inability of the atomic system to fold itself
in such as way that all bond orientations lead to good low energy
states. Instead, for any spatial orientation of bonds, some interactions
will be positive and others will be negative, and in place of a few deep
low energy states there will be a large number of less deep low energy
states.
The notion of frustration can be illustrated using a mini system consisting of four spin 1/2 atoms arranged in a pair of squares as shown
above. Each vertex in a square contains an atom whose spin value is
either +1 or -1. The energy of the systems is the sum of four interactions of the form Jsisj, where si and sj are the spin values of atoms at
neighboring vertices connected by bonds, and J is the bond strength. In
other words, the energy E is given by the expression
E = - J ij si s j ,
and the sum is over the four pairs of neighboring vertices. There are 16
possible states of this system: 2 2 2 2. The nonfrustrated square
on the left possesses two good (deep) low-energy statesone where all
spins are +1 and one where all spins are -1, each with energy -4 J. All
other states have higher energies. (a) Tabulate the distribution of states
and then compare this distribution to that for the frustrated square
shown in the right hand part of the gure. (b) What happens if the top
and bottom couplings are negative while the two side couplings remain
positive?
5.3 Transition rates and their dependence on barrier height. The transition
rate for passage over a barrier from state A to state B is given by the
Arrhenius formula:
v = v 0 e - E0

kT

Problems

109

Figure for Problem 5.3. Transition over a barrier from

one state to another.

In this expression, v is the transition rate, which is equal to the product of

a factor v0 that gives the number of attempts being made on the barrier
(the attempt frequency) and the Boltzmann factor (i.e., the exponential
term) describing the probability for success for any given attempt. Assuming no change in the attempt frequency, how much less likely is a transition
for the case where barrier height is a factor of 3 greater than the thermal
energy kT as compared to a situation where the barrier height is half that
of the thermal energy?

6
Stress and Pheromone Responses
in Yeast

Unicellular organisms such as the budding yeast Saccharomyces cerevisiae

have to cope with continually changing environments and are subject to a
variety of environmental and physiological stresses. These include unfavorable temperatures that produce heat shock, osmotic stresses that disrupt
the water balance, carbon source deprivation that leads to starvation, and
oxidative stresses. Yeast cells respond to these and other unfavorable conditions by altering their patterns of gene expression and by adjusting their
rates of protein synthesis. They may respond in a rather general way to a
stressful situation, or they may respond in a far more specic manner to a
particular stress condition.
The signaling routes that ultimately produce the alterations in gene
expression and protein synthesis begin at the plasma membrane with signal
reception. Signal receptors embedded in the plasma membrane sense
changes in the local environment and receive chemical messages sent by
other cells. The end points of the signaling routes are control points in the
xed infrastructure. It is at these control points that the signals are converted into cellular response. Some signaling routes, especially those found
in bacteria, are fairly short with at most a few elements lying between
sensing and contact elements. Other signal routes are far longer. In these
situations, the routes from cell membrane to the contact points in the cell
interior often involve several intermediate signaling elements and control
points where several signals converge. Whether short or long, a set of signaling elements is activated in a sequential fashion, from cell surface molecules to intracellular signal elements to one or more nal contact elements.
The ensemble of signaling elements activated in this way is referred to as
forming a signaling pathway.
The different kinds of signaling elements that make up the typical
eukaryotic signaling pathway will be introduced in the rst part of this
chapter. Examples will then be given in the second part of the chapter of
how these elements come together to form stress and pheromone signaling
pathways in yeast. The short signaling routes found in bacteria will be
explored in Chapter 7, and an important set of signaling intermediaries,
111

112

6. Stress and Pheromone Responses in Yeast

commonly referred to as second messengers, will be explored in the chapter

after that, Chapter 8.

6.1 How Signaling Begins

Signaling begins at the plasma membrane with receptor-ligand binding and
signal transduction. Transmembrane receptors function as sensors of environmental stresses and as receivers of chemical messages. They transmit,
and, in the process, convert the signals from an external, outside-the-cell
form to an internal, inside-the-cell one that can be understood and further
processed. This process is called signal transduction. Several kinds of transmembrane receptors are presented in Figure 6.1. In each example, part of
the receptor lies in the extracellular spaces enabling it to bind to ligands.
There are also one or more transmembrane segments, portions of the
polypeptide chain that either pass once through the plasma membrane or
wind back and forth several times. Lastly, there is a cytoplasmic segment
that permits the receptor to make contact with signaling molecules inside
the cell, thereby allowing the receptor to transduce a signal.
The notion of signal transduction is a central one. The ligand itself is not
passed into the cell. Rather, a different molecule becomes activated and
conveys the message inside the cell. This conversion process can happen
several times inside the cell with one protein contacting a second one, which
then contacts and activates a third molecule, and so on until the nal signaling element reaches a control point where the signal is converted into a
cellular response. In each of these transfers, one signal molecule replaces
another in a sequential manner to carry the signal. Thus, each step involves
transduction, the conversion of a message from one form to another.

Figure 6.1. Signal receptors: Each receptor has an extracellular region, a transmembrane segment, and an intracellular (cytosol) portion. (a) Single-chain, singlepass receptor; (b) two-chain, single-pass receptor; (c) dimeric arrangement of
two-chain, single-pass receptors; (d) trimeric arrangement of single-chain, singlepass receptors; and (e) single-chain, seven-pass receptor. The N-terminal is usually
located on the extracellular side and the C-terminal on the cytoplasmic side in the
single-chain and seven-pass receptors. In the double-chain receptors, the uppermost
side is N-terminal and the lower side is C-terminal.

6.2 Signaling Complexes Form in Response to Receptor-Ligand Binding

113

Receptors can be grouped into families according to their topology,

structure, and the kinds of ligands they bind. The rst two examples
presented in Figure 6.1 are single-pass receptors. In Figure 6.1(b), two chains
are used rather than a single chain as in Figure 6.1(a). One chain lies entirely
in the extracellular space and is responsible for ligand binding. The other
chain is covalently linked to the rst (through disulde bonds). It contains
transmembrane and cytoplasmic segments and transduces the signal into the
cell.
Receptors form associations with other receptors. One way for a signal
to be conveyed into the cell is through the formation and stabilization of
receptor complexes. The complex may consist of a pair of receptors, as in
Figure 6.1(c), simultaneously bound to a single ligand (1 : 2 stoichiometry)
or to a pair of ligands (2 : 2 stoichiometry). Alternatively a receptor trimer
may form as in Figure 6.1(d) bound to, for example, three ligands. Higherorder complexes may form and these complexes may even involve a mix of
different receptors. The last example, presented in Figure 6.1(e), is for a
seven-pass, single chain receptor. This is the topology of the largest family
of receptors found in the bodythe G protein-coupled receptors (GPCRs).
They sense and transduce sensations such as light and odors, and cell-tocell hormonal and neuromodulatory signals. They are also the targets of
about half of all the drugs commercially produced.

6.2 Signaling Complexes Form in Response to

Receptor-Ligand Binding
Receptor-ligand binding stimulates the assembly of signaling complexes at
and just below the plasma membrane. Ligand binding provokes changes in
the environment around the cytoplasmic portion of the receptor(s) leading
to formation of these complexes. These changes may take one or more
forms. In some receptor families, ligand binding stabilizes the formation of
receptor dimers. The receptors possess an intrinsic kinase activity, which is
turned on when the two chains are brought into close proximity to one
another. The ensuing phosphorylation opens up docking sites for cytoplasmic signaling proteins that, in turn, seed the formation of the signaling
complex. In families such as the GPCRs, ligand binding triggers a changes
in conformation that are sufcient to activate nearby cytoplasmic Gproteins leading to activation of signaling intermediatessecond messengersand the formation of signaling complexes.
Proteins functioning as anchor, scaffold, and adapter proteins help organize these signaling complexes. As depicted in Figure 6.2, anchor proteins
provide platforms for proteins to attach in close proximity to receptors and
other upstream signaling elements. Anchor proteins attach to the plasma
membrane and to membranes of organelles. They allow two or more sig-

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6. Stress and Pheromone Responses in Yeast

Figure 6.2. Assembly of signaling complexes: (a) Signal complex organized by

exposure of a docking site by a receptor aided by the utilization of an adapter
protein; (b) signal complex organized by a cytoskeleton-associated scaffold protein;
and (c) signal complex organized by a membrane-associated anchor protein.

naling proteins to position themselves in close proximity to one another

and to their membrane-associated substrates. Scaffold proteins operate in
a similar fashion to anchor proteins. They too provide platforms for attachment of multiple proteins.They attach to components of the actin cytoskeleton, or alternatively, to microtubules. These attachment sites are usually
located near the plasma membrane and play an important role in relaying
signals from the plasma membrane to downstream control sites. The third
group of nonenzymatic intermediaries is the adapter proteins. These proteins contain protein-protein interaction domains and serve as intermediaries that allow two proteins that would otherwise not be able to communicate to do so.
In order to form a complex, proteins diffuse over to and collect at the
docking sites. The proteins are said to be recruited to the collection point.
The process of recruitment is mostly a passive one driven by diffusion
of proteins localized in the vicinity of docking sites opened up by
receptor-ligand binding. The diffusion is largely planar; that is, it is a twodimensional rather than a three-dimensional process because all signaling
elements are located at and just below the plasma membrane. The adapters,
anchors, and scaffolds localize the necessary signaling elements close to one
another at and just below the plasma membrane. Because the proteins are
localized nearby and because the process is a two-dimensional one, it is
fairly rapid.

6.3 Role of Protein Kinases, Phosphatases, and GTPases

115

6.3 Role of Protein Kinases, Phosphatases, and GTPases

Protein kinases, phosphatases, and GTPases convey signals from the cell
surface to downstream cytoplasmic effectors. The organization of signaling
complexes by anchors, scaffolds, and adapters is illustrated in Figure 6.2. In
each example, proteins designated in a generic sense as protein a, or
protein b, and so on are recruited to the complex. The proteins so indicated are mostly enzymes. Two different kinds of enzymes predominate
protein kinases and protein phosphatases, and these are the primary
intracellular signal transducers. Protein kinases are large proteins that catalyze the transfer of phosphoryl groups to target proteins using ATP as a
donor. Protein phosphatases do the opposite: They catalyze the removal of
phosphoryl groups using ADT as an acceptor. These two operations are
illustrated in Figure 6.3. The substrates that accept and lose phosphoryl
groups are often kinases themselves, so that one kinase activates another,
which then activates a third kinase, and so on.
Protein kinases and phosphatases are not the only categories of enzymes
that participate in signaling. Enzymes called GTPases also contribute to signaling, as do a variety of proteolytic enzymes, or proteases. GTPases are
enzymes that are activated when they bind guanosine triphosphate (GTP)
and are deactivated when they bind guanosine diphosphate (GDP). Two
sets of ancillary enzymes catalyse GDP/GTP binding and release from the
GTPases. As illustrated in Figure 6.4, a guanine nucleotide exchange factor
(GEF) catalyzes the dissociation of GDP from the GTPase. GTP is an abundant cytosolic protein and in response to the actions of the GEF it binds
the GTPase, thereby activating it. The GTPase-activating protein (GAP)
accelerates the enzymatic actions of the GTPase. It also speeds up the
hydrolysis of the bound GTP molecule, leaving a bound GDP molecule in
its place, resulting in the inactivation of the GTPase.

Figure 6.3. Enzymatic actions of protein kinases and protein phosphatases: (a) A
protein kinase catalyzes the transfer of a phosphoryl group to its substrate using
ATP as a donor. (b) A protein phosphatase catalyzes the removal of a phosphoryl
group from its substrate using ADP as an acceptor.

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6. Stress and Pheromone Responses in Yeast

Figure 6.4. Catalytic activities of GTPase-associated factors: (a) A GEF catalyzes

the removal of GDP from a GTPase, which then binds to a GTP molecule. (b) A
GAP catalyzes the hydrolysis of the GTP molecule bound to the GTPase, leaving
a bound GDP molecule.

6.4 Role of Proteolytic Enzymes

Proteolytic enzymes transform, activate, and deactivate signaling elements.
Proteolytic processing is yet another kind of posttranslational modication
that has an important role in signaling. Several different kinds of proteolytic
enzymes, or proteases, are active in the cell. Some of the them catalyze the
cleavage of membrane-associated proteins either on the extracellular side
or the intracellular side of the plasma membrane. The cleavage of a tether
that anchors a signaling protein on the outside of the cell converts that
protein into a diffusible ligand, thereby greatly extending its range of inuence. The intracellular cleavage of the protein either anchored to or embedded in the plasma membrane converts that protein into a messenger that
can relay messages into the cell interior, allowing it to function not only as
an upstream signaling element but also as a downstream one. These operations are illustrated in Figure 6.5(a) and (b).
The enzymes involved in transducing signals are localized where they are
needed in inactive forms. When an initiating event takes place, such as the
arrival of an extracellular ligand that binds a receptor located in the plasma
membrane, the proteins that the signaling pathway comprises are activated.
A series of events take place with elements upstream activating substrates
downstream until the last signaling elements contact the xed infrastructure at a control point.
The signaling elements must be carefully controlled to prevent unwanted
signaling. The signals that convert the signaling molecules from an inactive
to an active form are of several kinds, and these often work in concert to
turn on signaling when and only when appropriate activating signals are
present. One way of accomplishing this is to immobilize a signaling protein
through binding it to a scaffolding protein. If the scaffold protein is proteolytically processed, the signaling protein will be freed up and can convey

6.5 End Points Are Contact Points to Fixed Infrastructure

117

Figure 6.5. Signaling activities of proteolytic enzymes: (a) A protease cleaves the
tether, detaching the ligand from the outer leaet of the plasma membrane allowing it to function as a diffusible ligand. (b) A protease cleaves the polypeptide chain
just below the plasma membrane, allowing the cytoplasmic portion of the protein
to function as a downstream signaling element. (c) A protease degrades a scaffold
protein, freeing up the signaling protein to convey a signal to its downstream substrate. (d) A protease degrades a signaling protein shortening its lifetime and terminating signaling.

a message as shown in Figure 6.5(c). Proteolytic processing can do the opposite and turn off signaling. If a signaling protein rather than a scaffold is
tagged for proteolytic processing as indicated in Figure 6.5(d), the signal
proteins lifetime will be reduced and its signaling activity will be damped
down.

6.5 End Points Are Contact Points to

Fixed Infrastructure
The end points of signaling pathways are control points that make contact
with the xed infrastructure. The furthest downstream signaling elements
in the signaling pathways make contact with one or more components of
the xed infrastructure. The components being contacted may be a receptor or ion channel embedded in a membrane, or they may belong to the
cytoskeleton. They may belong to an intracellular transport organelle, or to
the mitochondria, or to the transcription apparatus in the nucleus, or to the
translation machinery situated in the endoplasmic reticulum.

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6. Stress and Pheromone Responses in Yeast

The most commonly occurring end points are those that contact and regulate the transcription machinery. Proteins that convey regulatory instructions to the transcription machinery are called transcription factors.These are
the last or furthest downstream signaling elements in the various stress and
pheromone pathways. These proteins bind to specic DNA sequences
located in noncoding regions of genes and as a consequence inuence
transcription either positively or negatively. In the former case by makig it
easier to carry out transcription and in the latter case by making it more
difcult to do so.The noncoding gene regulatory regions contain binding sites
for elements of the xed infrastructure to bind and also sites for regulatory
proteins to attach. The control regions are known as promoters and the
sequence-specic control points where transcription factors bind and come
together to regulate transcription are known as responsive elements (REs).

6.6 Transcription Factors Combine to Alter Genes

In the budding yeast, several types of stress response can be evoked. Those
elements of a stress response that are common to all stresses are handled
by the yeast general stress response, while additional elements needed to
treat certain kinds of stresses are handled by a set of specic stress
responses. Organisms such as the yeast respond to stresses by remodeling
their pattern of gene expression, upregulating some genes and downregulating others. Experiments performed using DNA microarrays show that
changes in the patterns of gene expression are global, often involving substantial fractions of the entire genome. The control points are organized in
a way that makes possible these stress responses. Genes that require coexpression because their products must all be present at the same time and
work together possess a common set of responsive elements to which the
transcription factors may bind. That is, multiple copies of each of these
responsive elements are distributed among promoters throughout the
genome. Certain combinations of responsive elements are encountered in
genes that are cotranscribed in response to specic stress and pheremone
signals; other combinations are often encountered in genes coexpressed in
response to a different set of stress signals and some are encountered
in almost all stress responses. By this means a small number of transcription factors can coordinate and control a major remodeling of the cellular
infrastructure.
Transcription factors activated by stresses act in a combinatorial fashion
to trigger genome wide alterations in gene expression. In eukaryotes, these
noncoding DNA sequences almost always contain binding sites for more
than one transcription factor. The transcription factors jointly determine
whether and how strongly to initiate transcription. The process whereby
several transcription factors regulate transcription is known as combinatorial control. This term signies one of the key aspects of the process. Tran-

6.7 Protein Kinases Are Key Signal Transducers

119

scription factors can come together in a variety of ways, acting in different

combinations of twos, threes and so on. A small number of transcription
factors can therefore generate many different patterns of gene expression
in response to stresses and other environmental changes.
Combinatorial control is a synergistic process. In combinatorial control
the effect produced by two positively acting transcription factors is far
greater than the sum of the effects of each individual in the pair. This can
happen in several ways. One way of producing an effect of this sort is for
one of the partners to relieve an autoinhibitory interaction present in its
partner that prevents the partner from acting. Alternatively, one of the partners may act on the DNA substrate to expose or better prepare a binding
site for the other partner. These interactions are all cooperative with the
rst interaction making it easier for the second interaction to occur.
In the remainder of this chapter, the operation of signaling pathways
activated by yeast cells in response to stresses will be examined. The yeast
stress pathways illustrate how the various kinds of signaling proteins come
together to form a pathway. Before examining these pathways the different categories of signaling elements will be looked at in more detail.

6.7 Protein Kinases Are Key Signal Transducers

Protein kinases are central regulators of cellular physiology. These proteins
catalyze the transfer of a gamma-phosphoryl group from an ATP molecule
(usually bound to Mg2+ ion) to a hydroxyl group on the side chain of specic residues in target proteins. In other words they are phosphotransferases that use ATP (and sometimes GTP) as a donor and specic residues
in target proteins as acceptors. Their ability to function as enzymes is due
to the presence of a catalytic domain of some 200 to 300 amino acids. The
kinase domain, along with one or more regulatory domains, comprise the
kinase. Some protein kinases encode their catalytic domains in separate
subunits; in others, the catalytic and regulatory domains are all part of a
single chain molecule.
The protein kinases can be divided into two large groups. In the rst
group, are the protein kinases that target hydroxyl groups on serine/threonine residues. These are called serine/threonine (ser/thr) kinases. In the
second group are the protein kinases that target hydroxyl groups on tyrosine residues, and these are called tyrosine (tyr) kinases.The proteins in each
major grouping can be further placed into a number of families that are
highly conserved across all eukaryotes. Listed in Table 6.1 are ve prominent groups of serine/threonine kinases, organized according to similarities
in kinase domain structure, substrate specicity, and method of regulation/activation. These kinases are encountered in eukaryotes ranging from
yeast to man. Tyrosine kinases are more restricted in their distribution. They
are largely absent from unicellular organisms and appear to have emerged

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6. Stress and Pheromone Responses in Yeast

Table 6.1. Serine/threonine kinases of eukaryotes.

Group
AGC

CaMK

CMGC

Family
Protein kinase A (PKA)/Protein
kinase G (PKG)
Protein kinase C (PKC)
Protein kinase B (PKB), Akt, RAC
G protein-coupled receptor kinase
(GRK)
Ribosomal S6 kinase (S6K)
Phosphoinositide-dependent protein
kinase-1 (PDK1)
CaM kinase (CaMK)
AMP-dependent protein kinase
(AMPK)
Cyclin-dependent kinase (CDK)
Mitogen-activated protein kinase
(MAPK)
Glycogen synthase kinase-3 (GSK-3)
Casein kinase-2 (CK2)

MEK

PIKK

MAP/ERK kinase (MEK)

MAP/ERK kinase kinase (MEKK)
MAP/ERK kinase kinase kinase
(MEKKK)
ATM/ATR; DNA-PKcs
TOR

Comments
Cyclic nucleotide-regulated; basic
amino acid-directed
Lipid-, calcium-regulated
Lipid-regulated
Lipid-, calcium-regulated
Lipid-regulated
Lipid-regulated
Ca2+/calmodulin-regulated; Basic
amino acid-directed
Activated when AMP:ATP is
elevated; basic amino acid-directed
Proline-directed
Substrate of MEKs; proline-directed
Principal substrate of PKB; dual
specicity; proline-directed
Uses both ATP, GTP; Acidic amino
acid-directed; dual specicity
Dual specicity; highly specic
Highly specic
Highly specic
Glutamine-directed
Proline-directed

in response to the need for increased cell-to-cell signaling and control in

multicellular organisms. Tyrosine kinases will be examined in detail in later
chapters.
Serine/threonine kinases have many features in common. The most
important of these similarities from the signaling perspective is their activation through phosphorylation. The kinases become catalytically active
only when crucial residues in a region of their catalytic domain known as
the activation loop becomes phosphorylated. In many, if not most instances
one kinase activates a second kinase, and these kinases, activated sequentially in an upstream to downstream direction, form the core of the signaling pathway. Many of the entries in Table 6.1 can be arranged into pathways
according to their actions on one another. That is, these kinases catalyze the
transfer of phosphoryl groups to other kinases. Many of these are called by
the same name as the downstream kinase, with the addition of another
kinase in the name. An illustration of a kinase signaling cascade whose components are named in this manner is presented in Figure 6.6. Some common
examples of this naming convention are ERK kinase kinases (ERKKs),
AMP kinase kinases (AMPKKs), and CaM kinase kinases (CAMKKs).
Other kinases that act on kinases have their own distinct names. These

6.8 Kinases Often Require Second Messenger Costimulation

121

Figure 6.6. A kinase signaling cascade: A kinase kinase kinase phosphorylates a

kinase kinase, which in turn phosphorylates a kinase, which then phosphorylates its
substrate. In some cascades, the kinases are localized in close proximity to one
another by scaffolding proteins; in others, the upstream element must diffuse to
where the downstream element is tethered.

kinases typically can act on more than one kind of target kinase; that is,
they have broader substrate specicity than the rst group of rather narrowly acting kinase kinases, and consequently have their own name. An
example of this kind of broader acting kinase kinase is phosphoinositide
dependent kinase (PDK). This signaling molecule acts upstream of many,
if not most members of the Protein kinase A, G, and C (AGC) group.

6.8 Kinases Often Require Second

Messenger Costimulation
Phosphorylation by itself may not be sufcient to activate a kinase. Many
of the serine/threonine kinases require the presence of a second messenger
molecule. Second messengers are signaling intermediates connecting events
taking place at the plasma membrane with the intracellular signaling that
eventually converts the signal into a cellular response. Second messengers
are small molecules.There are three main kindscAMP, lipids, and calcium.
These intermediates are sent into the cytoplasm in response to receptor
binding. Members of the AGC group and the CaM kinase family members
are regulated in this way. Binding of a second messenger to the kinase, and
also to the kinase kinase, precedes activation of the signaling pathways by
phosphorylation.
The rst of the second messengers mentioned above, cyclic adenosine
monophosphate (cAMP), is generated from ATP by the enzyme adenylate
cyclase embedded in the plasma membrane. Catalytic and regulatory sites
are located within the cytoplasmic loops and termini of this doubleclustered, multipass molecule (depicted in Figure 8.9). The production of
cAMP is triggered by signals relayed from the cytosolic surface of plasma
membrane-bound receptors, and other intracellular signaling elements
located in its near vicinity. Cyclic AMP binds to protein kinase A and this
kinase is the sole cellular mediator of cAMP stimulated signaling.

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6. Stress and Pheromone Responses in Yeast

Lipid second messengers regulate the activity of a number of kinase

families belonging to the AGC group. The kinases of the AGC group are
activated in a multistep manner. First, there is an initial phosphotransfer
process that induces the migration of the protein kinases to the plasma
membrane where subsequent phosphotransfers serve to activate the catalytic properties of the kinase. Prominent agents of the second messenger
system are lipid kinases and phospholipase C that generate the second messengers, such as diacylglycerol (DAG) that activates protein kinase C, and
other lipid second messengers that trigger the release of Ca2+ from intracellular stores. The lipid second messenger generating system and AGC
kinase activation will be examined in detail in Chapter 8.
The third major kind of second messenger is intracellular calcium. As
noted in Table 6.1 calcium is a regulator of the CaM kinases. It activates the
kinase by rst binding to calmodulin; then the calcium-calmodulin complex
binds and activates the protein kinase and its upstream kinase kinases.
Calcium is not only stored and released from intracellular stores located in
the endoplasmic reticulum, but also enters the cells through ion channels
found in excitable cells such as neurons. Calcium signaling and CaM
kinases, along with many of the AGC kinases, are key agents of neural signaling and for these reasons the CaM and associated signaling pathways
are called learning pathways.

6.9 Flanking Residues Direct Phosphorylation

of Target Residues
A third common theme in kinase actions pertains to the establishment of
substrate specicity. This issue was briey touched upon in the naming of
kinases depending on whether they had a narrow or broad specicity. The
key notion is that specicity is determined not only by the presence or
absence of serine and threonine residues in a potential target protein, but
also by the identity of the residues that ank the potential acceptor of the
phosphoryl group. The anking residues, that is, the amino acid residues
immediately preceding or following the target site, help determine whether
a particular target residue can be phosphorylated or not.
The most general observation that can be made is that kinases belonging to the AGC and CaMK groups tend to be basic amino acid-directed
while those belonging to the CDK, MAPK, GSK3, CK2, and cyclin dependent kinase-like kinase (CMGC) group are mostly proline directed. In more
detail, kinases belonging to the AGC group are more likely to catalyze the
transfer of phosphoryl groups to S/T residues lying near basic amino acids
such as Lys and Arg. The GRKs are an exception to this preference, as they
prefer anking acidic residues. A similar observation holds for the CaMK
group. These kinases too prefer basic amino acid environments. In contrast,
members of the CMGC group are mostly proline-directed, phosphorylat-

6.11 Protein Phosphatases Are Prominent Components

123

ing S/T residues lying within proline-rich regions. The main exceptions to
this rule are the CK2s. These kinases prefer the anking amino acids to be
acidic. Finally, phosphoinositide 3-kinase related kinases (PIKKs) are either
glutamine or proline directed, as listed in Table 6.1.

6.10 Docking Sites and Substrate Specicity

One of the challenges to the reliable operation of cellular control layers is
how to ensure that the kinases phosphorylate the correct substrate at the
right time. The cell is crowded with many proteins, and each of these proteins likely to contain many potential phosphorylation sites. This selection
problem is solved, but only in part, by the presence of specic anking
residues. It turns out that the amino acids lying either just before or just
after the target serines and threonines are not the only amino acid residues
involved in kinase-substrate recognition. Additional residues located well
away from the target phosphorylation sites confer additional substrate
specicity. These sites are referred to as docking motifs or docking domains.
The presence of docking sites on kinase and substrate increases the substrate specicity. These sites are typically located far from the catalytic site
of the kinase and far from the phosphoacceptor site of the substrate. An
example of a kinase family that uses this form of matching is the MAP
kinase family. In this family, the docking sites are located in a C-terminal
domain of the kinase. The docking site is characterized by the presence of
several acidic amino acids, and is matched to a cluster of basic amino acids
in the matching docking sites used by upstream activators, deactivators, and
downstream substrates. Another family of kinases, the glycogen synthase
kinase-3s (GSK-3s), uses a different docking strategy. In this family the
presence or absence of a phosphorylated serine located four residues from
the substrate target serine is a crucial determinant of whether the kinase
can dock at the substrate. A crucial arginine residue serves as the kinase
docking site, and the complementary matching of the arginine residue in
the kinase to the phosphorylated, or primed, site in the substrate stabilizes the kinase in a catalytically active conformation.

6.11 Protein Phosphatases Are Prominent Components

of Signaling Pathways
Reversible phosphorylation, the covalent attachment and removal of phosphoryl groups to and from proteins, is the most widespread method of regulation protein activity in the cell. In addition to a various families of
protein kinases there are several families of protein phosphatases. Protein
kinases catalyze the transfer of phosphoryl groups to target proteins using

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6. Stress and Pheromone Responses in Yeast

ATP as a donor. Protein phosphatases do the opposite. They catalyze the

removal of phosphoryl groups using ADP as an acceptor.
Like the protein kinases, the protein phosphatases fall into two main
groupsthose that catalze the removal of phosphoryl groups from
serine/threonine residues, and those that do the same on tyrosine residues.
Eukaryotic genomes encode a smaller number of protein phosphatases
than protein kinases. The serine/threonine phosphatases can be placed into
two familiesPPP and PPMand the tyrosine phosphatases are placed
into one large familty designated as PTP.The best studied of the serine/threonine phosphatases are the members of the PP1 and PP2 families. These
proteins possess nearly identical catalytic subunits, and it is the regulatory
units that provide substrate specicity. Each phosphatase family member
can associate with any of a number of different regulatory units.

6.12 Scaffold and Anchor Protein Role in Signaling

and Specicity
Scaffold and anchor proteins help organize the signaling pathway and
confer specicity. Spatial localization is a powerful way of ensuring that
protein kinases only phosphorylate the correct substrates. While cytosolic
proteins are, in principle, free to diffuse about in the cytosol, they generally
do not do so. Instead, proteins are restricted to specic locations in the cell,
either in particular organelles or in specic subcellular compartments, that
is, in restricted regions of space in the cytosol. The restriction of signaling
molecules to specic locations in the cell is known as spatial localization or
compartmentalization.
Scaffold and anchor proteins promote spatial localization and substrate
specicity. These proteins do not have any enzymatic activity but rather
serve as platforms that help organize the signaling pathways. The scaffold
and anchor proteins (Figure 6.2) provide binding sites for attachment of the
kinases and their substrates at specic locales in the cell. Scaffolding proteins were rst discovered in the MAP kinase pathways of yeast cells, and
then in the mammalian MAP kinase pathways as well. Since the initial discoveries, several different kinds of scaffold proteins have been identied in
nerve cells, where they play a prominent role in organizing the signaling
machinery.
Anchor proteins are similar to scaffold proteins in that they too help
organize signaling routes, but unlike scaffolds are attached, or anchored, to
the cytoplasmic face of the plasma membrane.They sequester signaling proteins in either active or inactive states, ready for activation or deactivation,
positioned close to their substrates that are usually other plasma membrane-associated signaling proteins. These too are prominent components
of the signaling machinery in nerve cells.

6.14 Pheromone Response Pathway Is Activated by Pheromones

125

6.13 GTPases Regulate Protein Trafcking in the Cell

The transcription machinery resides in the cell nucleus while the translation machinery is located in the cytosol. Among the major consequences of
this compartmentalization is the addition of control points that regulate
nucleocytoplasmic transport. In order for a transcription factor to inuence
transcription it must be located in the nucleus. If it is kept out of the nucleus
then it cannot act. Similarly, a signaling molecule whose target lies in the
cytosol cannot act on that molecule if its location is restricted to the nucleus.
Spatial localization and sequestering is another way of regulating kinase
actions so that they only act on the appropriate targets at the right time. A
hallmark of this sort of regulation is its dynamic character. In response
to regulatory signals, some kinases may migrate from the nucleus into the
cytosol while others translocate in the opposite direction from the cytosol
into the nucleus.
GTPases play a crucial role in regulating protein trafc in a cell. These
are a large family of GTP-binding proteins that function as molecular
switches, operating at key control points in the cell. Members of this family
of control elements are involved in shuttling proteins in and out of the
nucleus. Other family members regulate the movement of cargo throughout the cell, and especially from organelles in the interior to the surface and
back, and still others regulate the organization of the actin cytoskeleton.
GTPases along with a variety of small adapter molecules are often concentrated at and just below the plasma membrane, where they help organize the intracellular signal-transducing pathways leading to the various
control points that serve as interfaces to the xed infrastructure and
mediate the cellular responses to the signals.

6.14 Pheromone Response Pathway Is Activated

by Pheromones
Yeasts respond to mating pheromones and to stresses by altering their patterns of gene expression. Environmental and pheromone signals are sent
from the plasma membrane to the nucleus through a set of parallel pathways named for the last in a series of protein kinases that serve as the
central signal transducers. The kinases that lend their name to the pathways
are mitogen-activated protein (MAP) kinases, members of the CMGC
group of serine/threonine kinases. Six of these pathways have been found
in yeasts and three in mammals.
One of the best-characterized pathways, the pheromone response pathway, is diagrammed in Figure 6.7. As depicted in the gure, this pathway
contains a kinase core that sits between the receptor and associated signal
transduction machinery positioned at the plasma membrane and down-

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6. Stress and Pheromone Responses in Yeast

Figure 6.7. Pheromone response pathway: (a) Pheromones initiate signaling when
they attach to the extracellular ligand-binding portion of the G protein-coupled
receptor (GPCR). Receptor-binding triggers dissociation and activation of the Ga
and Gbg subunits of the G-protein. The Gbg subunit activates the Ste20 kinase and
interacts with the Ste5 scaffold protein resulting in activation of signaling through
a MAP kinase cascade, leading to activation of Ste12 and the subsequent transcription of genes responsive to pheromone signals, and of Far1, a mediator of cell
cycle arrest (in G1). (b) Far1 is an adapter protein and serves as an adapter between
Gbg and the Cdc24 GEF at the plasma membrane.

stream elements that make contact with the transcription machinery to

inuence the program of gene transcription. The transmission of a signal
from the extracellular side of the plasma membrane into the cell interior
and from there to the cell nucleus involves the execution of several activities, or processes, each designed to enhance the specicity, or delity, of the
signaling. These are: transmembrane signal transduction; nucleocytoplasmic

6.14 Pheromone Response Pathway Is Activated by Pheromones

127

shuttling; and recruitment to the cytosolic face, leading to assembly and

activation of the kinase core unit.
Membrane-spanning receptors bind ligands and send signals indicative
of the binding event across the plasma membrane to the cytoplasmic
surface. In the pheromone pathway, members of the G protein-coupled
receptor (GPCR) family perform this task. The GPCR family is a prominent component of the human genome with roughly 600 members identied so far. They serve as sensors for physical and chemical signals such as
light and odors, and for a variety of cell-to-cell, hormonal and neural signals.
These receptors and their methods of action will be explored in detail in
Chapter 12. In brief, when a ligand binds a G protein-coupled receptor, it
produces changes in the electrostatic environment of the cytoplasmic
portions of the receptor. As its name indicates, a GPCR acts through a G
protein to which it is coupled, or tethered. The G proteins are built from
three distinct subunits, called alpha, beta, and gamma, and so are referred
to as being heterotrimeric. The changes in the cytoplasmic portions of the
GPCR cause the dissociation of the G protein alpha subunit from the beta
and gamma subunits, allowing the subunits to move about and activate
other signaling molecules.
The set of actions that follow signal reception and conveyance across the
plasma membrane may be collectively termed recruitment and pathway formation. These activities occur at and just below the plasma membrane. In
the yeast pheromone pathway, the recruitment of two proteins, Ste20 and
Ste5, is promoted primarily by the activated G protein beta subunit. Ste5
continually shuttles between the cytoplasm and nucleus, and in response to
activation of G protein beta subunits, Ste5 starts to accumulate just below
the cell surface. The binding by the G protein beta subunit to Ste5 and
Ste20, the latter a protein concentrated just below the cell surface, is the
crucial step in forming the kinase core. (Ste is an abbreviation for the
term sterile, assigned to proteins whose mutated forms produce nonmating, i.e., sterile phenotypes in the yeast.)
In the pheromone response pathway the kinase core consists of three
kinases, Ste11, Ste7, and Fus3, organized in a linear fashion with one signaling to the next in the chain. The Ste5 protein functions as a molecule
scaffold for the formation of the kinase module. The protein organizes the
kinases into a signaling pathway leading to the activation of the last member
of the module. This assembly step takes place subsequent to binding to the
G protein beta subunit of Ste20, which phosphorylates and activates Ste11,
and to activation of Ste5, which interacts with all members of the MAP
kinase module.
The Fus3 MAP kinase functions as the output unit from the kinase
module. In the absence of pheromone signals, this protein, like the Ste5
scaffold, continually shuttles between nucleus and cytoplasm. (The other
members of the module, Ste7 and Ste11, are cytoplasmic proteins.) In the
presence of pheromone signals, the Fus3 protein assembles into the MAP

128

6. Stress and Pheromone Responses in Yeast

kinase module, along with Ste11 and Ste7, and is activated. It then
translocates back to the nucleus. There it activates the transcription factors
Ste12.
The Fus3 MAP kinase also phosphorylates and thus activates the Far1
adapter protein. This protein has several functions. It is a mediator of cell
cycle arrest (in G1) as indicated in Figure 5.7. Like Ste5, it shuttles between
different subcellular locations. It shuttles the Cdc24 GEF to the plasma
membrane and mediates formation of a complex containing several proteins at the Gbg location where the pheromone signals are strongest. This
action makes the place wherein the cytoskeleton reorients itself in preparation for bud formation.

6.15 Osmotic Stresses Activate Glycerol

Response Pathway
The high osmolarity glycerol (HOG) response pathway, activated by
osmotic stresses, is shown in Figure 6.8. This is another of the yeast stress
pathways. It enables the yeast cells to adapt to changes in external osmolarity by altering their patterns of gene expression. In response to elevated

Figure 6.8. The high osmolarity glycerol (HOG) response pathway: Two receptors,
Sln1 and Sho1, signal through a common MAP kinase cascade and downstream
Hog1 and Msn2/Msn4 transcription factors to inuence the expression of HOGresponsive genes.

6.16 Yeasts Have a General Stress Response

129

external osmolarity, the yeast cells increase glycerol synthesis and decrease
glycerol permeability, thereby increasing their internal osmolarity and
restoring a correct osmotic gradient for water uptake.
The HOG pathway has two distinct osmosensors, Sho1 and Sln1. Sho1
and Sln1 (along with Ypd1 and Ssk1) operate in place of the GPCR and
associated G proteins that sense and transduce pheromone signals. Both
Sho and Sln1 signal through components of the HOG MAP kinase core
module resulting in the activation of the Hog1 MAP kinase. The central
element in the kinase core, Psb2, doubles both as the kinase kinase and as
the scaffold protein for the module. Signals conveyed into the cell through
the Sln1 receptor utilize Ssk2 or Ssk22 as the kinase kinase kinase, while
Sho1 signals are routed via the Ste11 kinase kinase kinase. The last element
in the MAP kinase core, Hog1, like Fus3, shuttles back and forth between
the cytoplasm and nucleus. In response to elevated osmotic stress, the
kinase is phosphorylated by Psb2 and becomes concentrated in the nucleus
along with the Msn2 and Msn4 transcription factors.
The second osmosensor, Sln1, is unusual for a eukaryotic sensor. It is a
histidine kinase, a kind of signal protein that is common in bacteria, but is
not only uncommon in eukaryotes, but also seems to be absent all together
in animals. These systems are often called histidine-aspartate (His-Asp)
phosphorelays because they involve the transfer, or relay, of phosphoryl
groups between histidine and aspartate residues. The mechanistic details of
how His-Asp systems operate in bacterial cells will be a main focus of the
next chapter.

6.16 Yeasts Have a General Stress Response

The term general stress response was coined to describe the expression of
a common set of genes by a number of different stress conditions. Roughly
speaking, the same set of genes was observed to be upregulated in response
to unfavorable temperature shifts, osmotic shock, starvation conditions, and
DNA damage. Among the proteins upregulated in response to these conditions are those such as heat shock proteins that confer general protection
against the stresses. The stimulation of gene transcription of a specic set
of genes is associated with the presence in the promoters of each of these
genes of a stress responsive element, or STRE.
The pathways leading to the transcription initiation sites located in the
nucleus start with a sensing activity taking place in the outer reaches of the
yeast cell. These activities are most often performed by receptors located
in the plasma membrane, but may also be initiated by proteins functioning
as sensors located near or at the cytoplasmic face of the plasma membrane.
As indicated in Figure 6.9 two signaling routes are involved in signaling to
the STREs. One involves glucose sensing by a transmembrane receptor, and
the other involves sensing of internal stresses such as low pH. The crucial

130

6. Stress and Pheromone Responses in Yeast

Figure 6.9. General stress response pathway: Protein kinase A, the core signaling
element, is stimulated by Ras-mediated internal stress signals and by GPCR/G
protein transduced glucose signals, both acting through adenylyl cyclase/cAMP
intermediaries.

element in the internal pathway is the heat shock protein Hsp70 introduced
in the last chapter. Under low pH conditions the number of denatured proteins increases. The Hsp70 proteins must deal with this situation and are
not available to signal to the Ras GEF, Cdc25.
The G protein-coupled receptor (GPCR) functions as a sensor of glucose,
the preferred sugar for yeast cells. In response to the presence of glucose it
stimulates the production of cAMP by adenylate cyclase. These molecules
function as second messengers and have as their cellular role the activation
of protein kinase A (PKA). In the yeast this protein has a pair of regulatory subunits and a pair of catalytic subunits. It is activated when two cAMP
molecules bind to each regulatory subunit. In response, the catalytic subunits dissociate from the regulatory subunits and become activated. The
glucose-activated pathway leading from a GPCR is not the only path to
activation of PKA. Alternatively, Ras may stimulate cAMP production in
response to stress conditions, again leading to activation of PKA.
Activated PKA is a negative regulator of Msn2p and Msn4p, transcription factors (TFs) that bind to the STRE. Under good (logarithmic) growth
conditions these TFs are localized in the cytoplasm, but migrate to the
nucleus when glucose conditions are degraded. The import and export of
Msn2p and Msn4p from the nucleus is regulated by PKA activity and also
independently by signals from the TOR pathway (to be discussed next). The

6.17 Target of Rapamycin (TOR) Adjusts Protein Synthesis

131

overall mechanism is that when growth conditions are poor and the cell is
subjected to stressful conditions the Msn2p and Msn4p proteins accumulate in the nucleus, bind to the STREs, and stimulate transcription of genes
whose protein products protect the cell.

6.17 Target of Rapamycin (TOR) Adjusts

Protein Synthesis
TOR adjusts protein synthesis in accordance with growth conditions. The
process of growth is distinct from that of proliferation, although the latter
often follows from the former. Cell growth refers to the increase in cell size
and mass and is driven by rapid protein synthesis. Cell proliferation is the
result of the progression through the cell cycle resulting in cell division.
Under good growth conditions yeast cells will grow rapidly. The average
lifetime of a ribosome is about 100 minutes, and to maintain an optimal rate
of growth, yeast cells produce 2000 ribosomes per minute. The rRNA genes
account for more than 60% of the total cellular transcription activity. The
yeast genome contains 137 ribosomal protein (RP) encoding genes; there
are some gene duplications and these genes encode 78 different RPs. Their
transcription yields 25% of the total number of mRNA molecules in the
cell.
As a large expenditure of resources is needed to maintain this growth
machinery, when conditions are no longer favorable to growth, cellular
resources must be directed elsewhere. Targets of rapamycin (TOR) proteins
are central controllers of cellular resources. They regulate the balance
between protein synthesis and protein degradation, throttling back the
program of cellular growth in response to reductions in the quality of nitrogen and carbon nutrients. In S. cerevisiae this means shutting down budding,
the growth at a particular location on the cell surface that lends the name
budding yeast to the organism. TOR proteins carry out their resource
management functions by sending out regulatory signals to the translation
apparatus and the transcription machinery.
TOR is a serine/threonine kinase belonging to the PIKK family (Table
6.1). It remains in an active state as long as intracellular amino acid concentrations, most notably, that of leucine, are adequate. Under plentiful conditions it phosphorylates and thus activates a protein called Tap42 (Figure
6.10). This protein is a regulator of phosphatase PP2A activity, functioning
as a subunit of PP2A and PP2A-like phosphatases. The PP2A phosphatase
is composed of three subunits that must come together for the phosphatase
to carry out its dephosphorylation actions on its cognate target proteins.
When Tap42 is activated it binds the catalytic subunit of the phosphatase
PP2A. This binding event prevents the catalytic subunit from associating
with the two other subunits of the phosphatase PP2A and by this means
immobilizes the phosphatase in an inactive form. Whenever the quality of

132

6. Stress and Pheromone Responses in Yeast

Figure 6.10. Regulation of translation initiation through the TOR signaling

pathway: (a) When growth signals are present (good growth conditions) TOR is
phosphorylated by upstream kinases such as PKB, and TOR then phosphorylates
Tap42. In response Tap42 binds the catalytic PP2A subunit, keeping it away from
the other two subunits. The translation inhibitor 4E-BP1 remains in a phosphorylated state and cannot prevent the translation regulator eIF4E from stimulating
translation of mRNAs. (b) In the absence of growth signals, the PP2A phosphatase
is active and dephosphorylates 4E-BP1, which in turn inhibits eIF4E translation
initiation.

carbon and nitrogen nutrients degrades,TOR becomes inactive. It no longer

activates Tap42, which, in turn, no longer prevents activation of PP2A. The
result of turning off TOR and turning on PP2A is that downstream targets
involved in translation initiation and elongation are dephosphorylated and,
by these actions, turns down translation of resident mRNAs.
In yeast, TOR proteins match ribosome biogenesis to nutrient avalilabity.
Not only do the TOR proteins regulate genes that encode metabolic proteins, but also they regulate genes that encode components of the ribosomes
such as the ribosomal proteins mentioned at the beginning of this section.
TOR signaling regulates the transcription of RPs by poly II and also regulates the machinery responsible for transcribing rRNA subunits and tRNAs.
End points of TOR signaling include sites at poly I responsible for transcribing the 35S rRNA subunit, and poly III that transcribes the 5S rRNA
precursor and tRNAs.

6.18 TOR Adjusts Gene Transcription

133

Figure 6.11. Regulation of translation of 5TOP genes through the TOR signaling
pathway: When growth conditions are favorable TOR is active and phosphorylates
the S6K kinase, which in turn phosphorylates the ribosomal factor S6 thereby
stimulating transcription of mRNAs bearing a 5TOP sequence in their upstream
untranslated (regulatory) region (UTR).

When growth conditions are favorable, TOR phosphorylates a

serine/threonine kinase called S6K (Figure 6.11). This kinase, a member of
the AGC family (Table 6.1), is a key regulator of ribosomal biogenesis.
When activated it phosphorylates S6 ribosomal protein resulting in
increased translation of mRNAs that encode components of the translation
apparatus. The genes encoding these ribosomal proteins are referred to as
5TOP genes because they contain a characteristic 5TOP (terminal oligopyrimidine tract) sequence in their mRNA regulatory region.

6.18 TOR Adjusts Gene Transcription

TOR adjusts gene transcription in accordance with nutrient conditions.
Under poor nutrient conditions, transcription factors are activated that
regulate genes encoding proteins involved in metabolism. The specic transcription factors involved in adjustments to poor quality nitrogen supply
are the Gln3p and Gat1p, and for poor carbon conditions the transcription
factors are Msn2p and Msn4p. The other set of TOR end points, besides
beings elements of the translation initiation and elongation regulatory
apparatus, are regulators of these transcription factors. In a manner analogous to the immobilization of the catalytic subunit of PP2A, these transcription factors are kept out of the nucleus when growth conditions are

134

6. Stress and Pheromone Responses in Yeast

Figure 6.12. Regulation of transcription in response to nitrogen conditions through

the TOR signaling pathway: (a) Under good nitrogen nutrient conditions, TOR
catalyzed phosphorylation of Tap42 leads to the immobilization of Sit4, a PP2A-like
phosphatase. The transcription factor Gln3 is sequestered in the cytoplasm by the
Ure2 scaffold and does not stimulate transcription. (b) Under poor nitrogen conditions, TOR does not phosphorylate Tap42. Sit2 is able to dephosphorylate Gln3,
which then decouples from Ure2 and enters the nucleus, where it stimulates transcription of genes in response to the poor nutrient conditions.

good, and allowed to translocate to the nucleus when the conditions no

longer favor growth.
Two anchor proteins, Ure2p and a 14-3-3 family member, are involved
in this regulation. Under good conditions (Figure 6.12) TOR stimulates
Tap42p, which inhibits Gln3p and Gat1p translocation. TOR also directly
stimulates Ure2p, which inhibits the migration of the two transcription
factors into the nucleus. When conditions are poor these multiple inhibitory
inuences are lifted and the transcription factors are able to migrate into
the nucleus and activate gene transcription. Similarly, the Msn2p and Msn4p
proteins are inhibited by 14-3-3 anchor proteins (not shown) in a nutrient
supply-dependent way.

6.19 Signaling Proteins Move by Diffusion

The proteins responsible for signaling in the cell are mobile; they move from
one cellular location to another. For example, the Fus3 protein (Figure 6.7
and Section 6.13) involved in the pheromone response not only translocates
from the plasma membrane and the nucleus, but also shuttles back and forth
between the cytoplasm and nucleus. Some of the pathways discussed in this
chapter end at transcription sites in the nucleus, while other pathways terminate at the translation machinery in the cytoplasm. Mobility is central to

6.19 Signaling Proteins Move by Diffusion

135

the signaling activities of many, if not most, signaling proteins in the cell.
The predominant means of movement for signaling proteins is passive
diffusion.
Particles diffuse from one location to another through random movements known as Brownian motion. Brownian motion is named after the
English botanist Robert Browning who in 1826 studied and puzzled over
the zig-zag movements of dust particles suspended in a uid that he
observed using a light microscope. Albert Einstein provided an explanation
of this phenomenon in 1905. In his analysis, Einstein showed that Brownian motion arises from random collisions of the dust particles with the molecules of the uid.
The cell interior is an aqueous medium lled with ions, biomolecules,
a cytoskeleton, and organelles. The speed at which a particular signaling
molecule can diffuse depends on three factors:
viscosity of the medium,
binding, and
crowding effects.
Diffusive motion is slower than inertial motion. In diffusion, the medium
exerts a drag or frictional force on the molecules that slow them down. The
difference between inertial and diffusive motion is reected in the mean
square displacements x2 produced by each kind of motion, namely,
x 2 =

m
kT 2
t , t <<
,
m
a

(6.1)

x 2 =

m
2kT
t , t >> .
a
a

(6.2)

and

In the rst expression, corresponding to inertial motion, the mean square

displacement grows as the square of the time, while in the second, that of
diffusive motion, the mean square displacement increases only linearly with
time. The overall scale is established by the ratio of the particle mass m to
the friction coefcient a. At small times the distances traveled by the particles are so small and they have not yet collided with any other particles.
At large times the particles have undergone a number of random collisions
with particles in their surroundings, changing direction each time. This type
of random movement, Brownian motion, underlies the diffusion process as
rst noted by Einstein.
It is customary to discuss diffusion in terms of a diffusion coefcient
rather than a friction coefcient. The diffusion coefcient D represents the
ratio of thermal energy to the friction coefcient:
D=

kT
.
a

(6.3)

136

6. Stress and Pheromone Responses in Yeast

This expression formalizes the notion that as the viscosity (friction) goes
up the mobility represented by the quantity D goes down. The cellular
medium is viscous but not excessively so. Small and moderately sized biomolecules diffuse about four times slower in the cytosol as they would in a
pure water medium.
As might be expected the larger the molecule the slower it will diffuse.
This aspect of diffusive motion is encapsulated in the StokesEinstein
relationship:
a = 6 phr0 ,

(6.4)

where h is the viscosity and r0 is the hydrodynamic (particle) radius.

Biomolecules can be slowed down by binding effects and by crowding,
beyond the factor of 4 reduction in rate mentioned above and the hydrodynamic dependence on the radius. Organelles and the cytoskeleton can
impede rectilinear motion, as can other biomoelcules through steric hindrance effects. Large macromolecules, especially those with masses greater
than about 200 kDa, might be slowed down considerably. Binding effects
can come into play not only to slow down movement but also to halt it
entirely. For example, calcium ions are rapidly bound by calmodulin, which
acts as a buffering agent to prevent large-scale diffusion of the ions. Binding
reactions taking place before the biomolecules reach their targets can also
hinder if not stop the biomolecules entirely. And of course, signaling proteins become immobilized when they reach their cellular targets and bind
them.

References and Further Reading

Global Patterns of Gene Transcription and Combinatorial Control
Chu S, et al. [1998]. The transcriptional program of sporulation in budding yeast.
Science, 282: 699705.
Causton HC, et al. [2001]. Remodeling of yeast genome expression in response to
environmental changes. Mol. Cell Biol., 12: 323337.
Gasch AP, et al. [2000]. Genomic expression programs in the response of yeast cells
to environmental changes. Mol. Cell Biol., 11: 42414257.
Holstege FCP, et al. [1999]. Dissecting the regulatory circuitry of a eukaryotic
genome. Cell, 95: 717728.
Pilpel Y, Sudarsanam P, and Church GM [2001]. Identifying regulatory networks by
combinatorial analysis of promoter elements. Nature Genet., 29: 153159.
Spellman PT, et al. [1998]. Comprehensive identication of cell cycle-regulated
genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol.
Cell Biol., 9: 32733297.

Protein Phosphatases
Cohen PTW [2002]. Protein phosphatase 1Targeted in many directions. J. Cell Sci.,
115: 241256.

References and Further Reading

137

Janssens V, and Goris J [2001]. Protein phosphatase 2A: A highly regulated family
of serine/threonine phosphatases implicated in cell growth and signaling.
Biochem. J., 353: 417439.

Mitogen-Activated Protein Kinases

Banuett F [1998]. Signalling in the yeasts: An informational cascade with links to
lamentous fungi. Microbiol. Mol. Biol. Rev., 62: 249274.
Estruch F [2000]. Stress-controlled transcription factors, stress-induced genes and
stress tolerance in budding yeast. FEMS Microbiol. Rev., 24: 469486.
Grner W, et al. [1998]. Nuclear localization of the C2H2 zinc nger protein
Msn2p is regulated by stress and protein kinase A activity. Genes Dev., 12:
586597.
Gustin MC, et al. [1998]. MAP kinase pathways in the yeast Saccharomyces
cerevisiae. Microbiol. Mol. Biol. Rev., 62: 12641300.
Herskowitz I [1995]. MAP kinase pathways in yeast: For mating and more. Cell, 80:
187197.
Widmann C, et al. [1999]. Mitogen-activated protein kinase: Conservation of a threekinase module from yeast to human. Physiol. Rev., 79: 143180.

Scaffold Proteins and MAP Kinase Cascades

Garrington TP, and Johnson GL [1999]. Organization and regulation of mitogenactivated protein kinase signaling pathways. Curr. Opin. Cell Biol., 11: 211
218.
Madhani HD, and Fink GR [1998]. The riddle of MAP kinase signaling specicity.
Trends Genet., 14: 151155.
Schaeffer HJ, and Weber MJ [1999]. Mitogen-activated protein kinases: Specic
messages from ubiquitous messengers. Mol. Cell. Biol., 19: 24352444.
Whitmarsh J, and Davis RJ [1998]. Structural organization of MAP-kinase signaling modules by scaffold proteins in yeast and mammals. Trends Biochem. Sci., 23:
481 485.

Shuttling
Elion EA [2001]. The Ste5p scaffold. J. Cell Sci., 114: 39673978.
Mahanty SK, et al. [1999]. Nuclear shuttling of yeast scaffold Ste5 is required for
its recruitment to the plasma membrane and activation of the mating MAPK
cascade. Cell, 98: 501512.
Van Drogen F, et al. [2001]. MAP kinase dynamics in response to pheromones in
budding yeast. Nature Cell Biol., 3: 10511059.

TOR Central Controller

Gingras AC, Raught B, and Sonenberg N [2001]. Regulation of translation initiation
by FRAP/mTOR. Genes Dev., 15: 807826.
Schmelzle T, and Hall MN [2000]. TOR, a central controller of cell growth. Cell, 103:
253262.
Shamji AF, Kuruvilla FG, and Schreiber SL [2000]. Partitioning the transcriptional program among the effectors of the TOR proteins. Curr. Biol., 10:
15741581.

138

6. Stress and Pheromone Responses in Yeast

Diffusion
Luby-Phelps K [2000]. Cytoarchitecture and physical properties of cytoplasm:
Volume, viscosity, diffusion, intracellular surface area. Int. Rev. Cytol., 192: 189221.
Verkman AS [2002]. Solute and macromolecular diffusion in cellular aqueous
compartments. Trends Biochem. Sci., 27: 2733.

FRAP
Lippincott-Schwartz J, Altan-Bonnet N, and Patterson GH [2003]. Photobleaching
and photoactivation: Following protein dynamics in living cells. Nature Cell Biol.,
5: S7S14.

Problems
6.1 Diffusion rates of various biomolecules can be measured inside the cell
using GFPs in a technique called uorescence recovery following photobleaching (FRAP). In this approach, a region inside the cell is subjected to intense laser light, which photobleaches any uorescence in
that region. GFPs located outside the exposed region then diffuse back
into the bleached region, and this movement is studied using low intensity laser light.
What is the hydration radius of a GFP molecule in water at a thermal
energy kT = 0.6 kcal/mol? Use the StokesEinstein relationship, Eq.
(6.4) together with Eq. (6.3) assuming viscosity h = 0.01 P = 0.01 g/cm
s, and a GFP diffusion coefcient D = 87 mm2/s. How long will it take
for the GFP proteins to diffuse 10 microns? Hint: Note from Eqs. (6.2)
and (6.3) that
x 2 = 2 Dt .
(This result holds in one dimension. In two dimensions, a factor of 4
appears in place of the factor of 2, and in three dimensions a factor of
6 appears.)

7
Two-Component Signaling Systems

Bacteria such as Escherichia coli are able to sense their external environments and, in response to changes in these conditions, alter their physiology. Lifetimes of bacterial proteins are generally short, and bacteria alter
their metabolism and reproductive strategies by turning on some genes and
turning off others. The corresponding signal pathways start at the plasma
membrane, where sensing takes place, and terminate at DNA control
points, where transcription is initiated. The pathways in bacteria are generally short and typically involve two core elements, a membrane-bound
sensor and signal transmitter, and a receiver that establishes contact with
the transcription apparatus.
Several representative examples of bacterial two-component signaling
pathways are listed in Table 7.1. Two kinds of signaling pathways are presented. The rst kind of pathway is represented in the table by the chemotaxis pathway. This pathway controls a piece of xed infrastructure in the
cell directly. In the case of chemotaxis, it controls the operation of the
agellar motor and terminates at a motor control point. The second kind
of pathway, represented by all other entries in the table, regulates gene
expression and by that means controls the xed infrastructure too. These
pathways terminate at DNA promoters, and the second components, the
response regulators, serve as transcription factors. Like the yeasts discussed
in the last chapter, bacteria cannot control their environments and so must
continually sense and respond to changes in their surroundings by altering
their patterns of gene expression.
Bacteria are not the only organisms that use two-component systems.
Plants use these systems to respond to hormones and to light. In the rst
part of this chapter, the bacterial chemotactic system will be examined in
detail. Binding and catalysis will be discussed and the notion of a linear
pathway, introduced in the last chapter, will be broadened to include
feedback. These explorations will be followed by a discussion of twocomponent signaling in plants.

139

140

7. Two-Component Signaling Systems

Table 7.1. Representative two-component signaling systems and their cellular

roles.
Sensor

Response regulator

Tar/CheA
EnvZ
FixL

CheY, CheB
OmpR
FixJ

KinA, KinB
NarX, NarQ
NtrB

Spo0A, Spo0F
NarL, NarP
NtrC

PhoR

PhoB

Function
Regulates chemotaxis
Osmoregulation; controls porin gene expression
Regulates nitrogen xation genes in symbiotic
bacteria
Regulates stress-induced sporulation gene expression
Regulates nitrate/nitrite metabolism gene expression
Nitrogen assimilation; controls glutamine synthetase
gene expression
Phosphate uptake gene expression

7.1 Prokaryotic Signaling Pathways

Two-component signaling and phosphorelays form the core of prokaryotic
signaling pathways. The signaling pathways used by the bacteria to relay
signals to the transcription machinery and to morphological structures such
as agellar motors during chemotaxis are built in a fashion similar to those
found in eukaryotes. Like the eukaryotic pathways, protein kinases and
phosphotransfer processes lie at the heart of the signaling routes. In place
of the serine, threonine, and tyrosine residues used by eukaryotes as attachment sites for phosphoryl groups, two other amino acids, histidine and
aspartate, are used. The basic signaling unit has a histidine kinase sensor
and an aspartate-based response regulator that mediates the cellular
response to the signal event. Like all signaling systems the design is a
modular one; as a result many different combinations of the two components and their modular domains can be formed. The more elaborate versions of the basic two-component system are called phosphorelays.
The arrangements of elements of a basic two-component signaling system
and a typical phosphorelay are presented in Figure 7.1. The rst signaling
element in the two-component system is the sensor/histidine kinase. It has
a receptor (input) unit and a transmitter. The receptor spans the plasma
membrane, and in response to ligand binding, transduces a signal across the
membrane from the extracellular side to the cytoplasmic side. Some receptors respond to osmolarity conditions. Others sense nutrient conditions or
respond to harmful chemicals such as heavy metals or respond to signals
sent by other bacteria. The transmitter unit may be part of the same
polypeptide chain as the receptor, or it may be encoded as a separate
protein. When signaled to do so by the receptor, the transmitter autophosphorylates itself on a conserved histidine residue. The phosphoryl group is
then transferred to the response regulator, the second element in the twocomponent signaling system. The response regulator, like the sensor/histidine kinase, has two functional units, and these usually belong to a single

7.2 Catalytic Action by Histidine Kinases

141

Figure 7.1. Phosphotransfer systems: (a) Two-component system; and (b) phosphorelay. The dark circles in the units symbolize histidine and aspartate residues
serving as binding sites for phosphoryl groups. Phosphotransfer steps are denoted
by squared-off arrows accompanied by circled Ps.

polypeptide chain. The rst is the receiver. It possesses a conserved aspartate residue that binds, or receives, the transferred phosphoryl group. The
second part of the response regulator chain is the output unit. Once fully
activated, the response regulator diffuses to, and establishes contact with,
the transcription machinery.
The second part of Figure 7.1 illustrates the operation of a typical phosphorelay. There are two additions to the system depicted in the rst part of
the gure. The sensor unit is more complex; it now becomes a hybrid sensor,
containing an extra module, an aspartate-bearing receiver functionally
identical to the receiver unit in a response regulator. Sandwiched between
the sensor unit and the response regulator is a small histidine phosphotransfer (HPt) protein. It is functionally identical to the catalytic portion of
the transmitter unit and contains the conserved histidine. As a result of
these two additions, the two-step His-Asp phosphotransfer becomes a fourstep His-Asp-His-Asp phosphorelay.

7.2 Catalytic Action by Histidine Kinases

Histidine kinases catalyze the transfer of a phosphoryl group rst to its own
histidine residue and then to a conserved aspartate residue in a response
regulator. Histidine kinases (HKs) must carry out a number of binding
operations. These operations are sequestered in specic domains resulting

142

7. Two-Component Signaling Systems

Figure 7.2. Domain organization of the CheA histidine kinase: A number of conserved residues characterize the histidine kinases. The conserved residues and their
surrounding 5 to 12 amino acid residues are known as homology boxes. The conserved residues are used, in one-letter amino acid code form, to name the boxes.
Four of these, the N, G1, F, and G2 homology boxes are involved in forming the
cleft that holds the ATP molecule in position. The fth, the H box, contains the conserved histidine residue involved in phosphotransfer.

Table 7.2. Domain organization of the CheA histidine kinase.

Domain
P1
P2
P3
P4
P5

Function
H domain; contains the conserved histidine (H), the site of autophosphorylation
Regulatory domain; binds CheY and CheB
Dimerization domain; binds CheA
Kinase domain; contains the asparagine (N), phenylalanine (F), and glycine-rich
(G1 and G2) boxes; binds ATP
Interaction domain; binds receptors and CheW

in a highly modular protein organization. The domain organization of

several histidine kinases has been determined using X-ray crystallography
and NMR. Among the proteins studied are CheA, used in chemotaxis, and
EnvZ, used in osmosensing. The CheA and EnvZ proteins are representative members of two classes of histidine kinases. In one class (CheA) the
histidine residue that functions as the site for phosphotransfer is located
in the C-terminal region; in the other class (EnvZ) it is found in the Nterminal region.
There are ve CheA domains (Figure 7.2 and Table 7.2).These are named
in order from the N-terminal to the C-terminal as P1 to P5. The P1 domain
contains the conserved histidine (H box) that is the site for the phosphotransfer. The P2 domain is a regulatory domain and binds the CheY and
CheB response regulators. The P3 domain is a dimerization domain. This
domain is present because the autophosphorylation process requires two
HK proteins. One HK molecule catalyzes the phosphorylation at the appropriate histidine residue in the second HK molecule (Figure 7.3). The P4
domain contains the homology boxes that facilitate nucleotide (ATP)
binding and catalysis of the phosphotransfer from ATP to the histidine
residue with the exception of the H box.The last domain, P5, mediates interactions with the receptor and the CheW scaffold (Figure 7.2).

7.3 The Catalytic Activity of HK Occurs at the Active Site

143

Figure 7.3. Structure of the CheA dimer: Shown are the P3 dimerization domain,
P4 kinase domain and P5 regulatory domains arranged in a dimer. The gure was
prepared using the Protein Explorer with atomic coordinates as determined by Xray crystallography and deposited in the PDB le under accession code 1b3q.

As noted above, the CheA domain organization is not the only one found
in histidine kinases. A second, rather common organization is for the H box
to be located in the C-terminal region rather than the N terminal one. In
this mode of organization, the H box containing the conserved histidine
residue is found in close proximity to the dimerization domain and the N,
G1, F, and G2 homology boxes to form a catalytic core.

7.3 The Catalytic Activity of HK Occurs at

the Active Site
The part of a histidine kinase responsible for catalyzing the transfer of
phosphoryl groups is referred to as the active site. The active site of the
histidine kinase is centered about the residue that receives the phosphoryl
group. This residue, along with a small number of other highly conserved
residues that surround the phosphoacceptor and assist in binding, denes
the active site. In CheA, the active site includes residues comprising the
boxes of the P4 kinase domain. These form a cleft that holds the ATP molecule in the correct orientation for transfer of the g-phosphoryl group from
the ATP donor to the His48 acceptor located in the P1 domain.
There are several different kinds of catalysis. The type of catalysis
carried out by the histidine kinases, called covalent catalysis, requires the

144

7. Two-Component Signaling Systems

presence of a nucleophile to break bonds. A nucleophile is an atom or

molecule that can donate an electron pair. A nucleophilic (nucleus-loving)
process is one in which an electron pair is donated to another atom or
molecule. There are a number of common nucleophiles. Deprotonated
water molecules can serve as a nucleophiles. Alternatively, solvent-exposed
oxygen atoms in hydroxyl groups in amino acid residue side chains can
function as nucleophiles. The nucleophile in histidine kinase action is the
imidazole ring structure on the histidine side chain. In this instance, the
imidazole ring is said to attack the terminal phosphoryl group of the ATP
molecule.
A key feature in the catalytic actions of the kinases is the presence of
Mg2+ ions that facilitates the breaking of the bond holding terminal phosphoryl groups in place in ATPs. The Mg2+ion binds the ATP molecule prior
to catalysis. During catalysis, the Mg2+-bound ATP and the substrate come
together in a small region of space, usually the groove, pocket, or cleft
formed by the kinase that was mentioned in the last paragraph. The combined actions of physical positioning and electrostatic shaping greatly
accelerate the phosphotransfer reaction. These actions are said to stabilize
the transition state, lowering the barrier for transfer of the phosphoryl
group from donor to acceptor. (See Problem 7.1 for a further discussion of
catalysis.)

7.4 The GHKL Superfamily

Histidine kinases along with a large number of ATPases form the GHKL
superfamily that is quite distinct from the eukatyotic kinase STTE superfamily. The phosphoaccepting histidine, His48, is surrounded by glutamate,
and lysine residues. The histidine, glutamate, and lysine residues form a
hydrogen-bonding network. This arrangement and that of the four-helix
bundle (Figure 7.4) also characterize the histidine-containing phosphotransfer (HPt) domains that are key components of phosphotransfer
systems. The amino acid residues that make up the HPt domains fold in an
up-down, up-down manner into four-helix bundles. The second helix in the
bundle contains a highly conserved histidine residue that is exposed to the
solvent and serves as the acceptor site for transfer of the phosphoryl group.
The phosphoryl group covalently attached to the histidine residue is subsequently transferred to an aspartate residue located in a response regulator protein.
The ensemble of alpha helices and beta sheets that forms the threedimensional structure of the kinase core differs markedly from that of
the serine/threonine and tyrosine kinases (STTKs). The histidine kinases
closely resemble a number of ATPases. ATPases are proteins that couple
metabolic energy stored in the high-energy bonds of phosphoryl groups to
operate as pumps and motor proteins, all of which require that work be
done. These proteins, including the histidine kinases, form the GHKL super-

7.5 Activation of Response Regulators by Phosphorylation

145

Figure 7.4. The CheA P1 domain: The structure

revealed by X-ray crystallography is that of a fourhelix bundle (A through D), plus a anking helix
(E). A box shows the location of the conserved
His48 residue plus conserved anking residues on
helix B. The gure was prepared using Protein
Explorer with atomic coordinates from PDB le
under accession code 1i5n.

Table 7.3. Domain structures of the response regulators.

Family

Domain structures

CheY
OmpR
FixJ
NtrC

Receiver domain
Receiver domain, winged helix-turn-helix output domain
Receiver domain, four-helix bundle output domain
Receiver domain, ATPase domain, DNA-binding domain

family. They each have a characteristic fold consisting of ve helices and

three sheets, and possess the conserved residues that dene the N, G1, F,
and G2 boxes. Crystal structures for ATPases such as DNA gyrase B, Hsp90,
and MutL closely resemble those for CheA and EnvZ.

7.5 Activation of Response Regulators

by Phosphorylation
Response regulators can be placed into families according to their domain
structure. Most transcription factors contain at least two domains. There
is an N-terminal domain that functions as a receiver, and there is a Cterminal domain that operates as an output unit (Table 7.3). Receivers function as protein kinases that catalyze the transfer of the phosphoryl groups
from the conserved histidines on histidine kinases to one of their own conserved aspartate residues. Output domains typically contain DNA-binding
and regulatory sequences that enable the response regulators to function
as transcription factors. Some response regulators, such as the chemotactic
CheY and CheB proteins (to be discussed later in this chapter) contain only
the receiver domain and do not bind DNA. However, most proteins diffuse
to DNA control points once the phosphoryl group is transferred to the
receiver.
Proteins are continually in motion and undergoing shape (conformational) changes. There are atomic, group, and residue movements, backbone

146

7. Two-Component Signaling Systems

motions, side-chain motions, shifts in secondary structure elements, and

domains movements. These motions enable a protein to continually sample
a population of states, and this ability is important for binding and release,
catalysis, and signaling. Rapid motions occur on picosecod-nanosecond
time scale, involving only a few atoms and small energy changes. Slower
motions occurring on longer microsecond to millimicrosecond time scales,
involving appreciable numbers of amino acid residues and far larger energy
changes.
NMR spectroscopy has been used to explore protein motions and conformational changes. Using these techniques, it has been observed that the
active site of an enzyme undergoes conformation changes taking place on
a time scale of microseconds to milliseconds.The regions that undergo these
motions correlate with the region involved in protein-protein interactions.
In examining what happens to the NtrC protein, it was found that NtrC
dimers form in response to phosphorylation and then oligomers form that
activate gene transcription. The picture that emerges from studies of NtrC
and several other response regulators is one in which response regulators
and activated in an allosteric manner by phosphorylation.
There are two populations of states. One population of states corresponds
to the inactive protein, the other to the active form. Phosphorylation shifts
the equilibrium between the two populations of states. Both populations
preexist and are continually sampled, but phosphorylation shifts the balance
in favor of the active form. Shifts in equilibria between two preexisting
populations of conformational states, produced either by ligand binding or
by covalent attachment of phosphoryl groups, are known as allosteric
modications. In an allosteric modication, binding at one location in the
molecule alters how other portions of the molecule respond to their binding
partners. These alterations may be thought of as a consequence of the
conformational changes accompanying the shifts in equilibrium.

7.6 Response Regulators Are Switches Thrown at

Transcriptional Control Points
The commonality with pumps and other ATPases and GTPases seen with
histidine kinases extends to response regulators. One of the key observations of how the pumps work is that they utilize a highly conserved aspartate residue in the active site along with several other residues. Like the
pump ATPases, an aspartate functioning as the nucleophile, working along
with two other aspartate residues, a lysine, a threonine or serine, and a pair
of water molecules, form a dense network of bonds in this region of the
molecule. Phosphorylation of the conserved aspartate is a key intermediate step in the energy-generating hydrolysis process carried out by the
pumps. As they did for the histidine kinases, the common properties of the
residues provide useful insights into how the response regulators function.

7.7 Structure and Domain Organization of Bacterial Receptors

147

Response regulators are switches. The energy stored in the high-energy

acyl phosphate bond is released when the switch is thrown. The response
regulators involved in transcription activation form complexes that jointly
drive a series of conformational changes leading to transcription activation.
The throwing of the switch is achieved through hydrolysis of the bond, that
is, the response regulators catalyze their own dephosphorylation. This
occurs when response regulators and their target proteins come together
so that the energy released through hydrolysis is used to drive conformational changes in the complexes that activate transcription.

7.7 Structure and Domain Organization of

Bacterial Receptors
Bacterial receptors have a stereotypic structure and domain organization.
Most bacterial sensor units are constructed from a single polypeptide chain
that passes through the plasma membrane twice. The N-terminal lies in the
cytoplasm at the end of a short segment. The chain threads out and then
back through the plasma membrane as shown in Figure 7.5. The extra-

Figure 7.5. Tar receptor: (a) Tar monomer with labels denoting the various
regionsThe presence of several regulatory (methylation) sites in the cytoplasmic
domain is denoted. (b) Tar dimerThe basic structure is that of a homodimer that
appears as a 35-nm long helical bundle oriented perpendicular to, and passing
through, the plasma membrane. The outer, ligand-binding, portion has the form of
two four-helix bundles, one per dimer subunit. Two helices from each bundle span
the plasma membrane. The cytoplasmic portion is arranged into a four-helix bundle,
formed by two helical hairpins, one from each subunit.

148

7. Two-Component Signaling Systems

cytoplasmic looping portion serves as the ligand-binding region. The

portion that is C-terminal to the transmembrane segments contains a linker
followed by a long signaling domain. While most receptors conform to this
plan, there are some receptors, most notably those involved in cell-to-cell
signaling, such as the quorum-sensing (AgrC) receptor and the competence
(ComD) receptor, that pass through the plasma membrane six times. The
two- and six-pass receptors are bacterial counterparts to the one- and
seven-pass receptors found in eukaryotes.
The EnvZ sensor unit is a representative example of the single chain,
two-pass membrane topology. This form differs from the bacterial chemotaxis receptor Tar, which signals to a separate histidine kinase CheA
protein. Tar is a two-pass receptor of the form described above, but some
of the signaling pieces such as the histidine kinase domain are sequestered
on a separate protein. NtrB represents a third kind of sensor unit; it is a
soluble protein and does not attach to the plasma membrane at all.

7.8 Bacterial Receptors Form Signaling Clusters

Bacterial receptors form dimers, trimers, and higher order oligomers. The
two subunits of the Tar dimer are linked through multiple bonds, and
form a tightly packed and relatively inexible set of parallel helices. The
multiple bonds limit the possible relative movements of the signal helix
in response to ligand binding, especially since the energy effects of ligand
binding are small. The movements used to transmit a signal over a distance
of 35 nm from the outside to the histidine kinase on the inside are modest.
In response to ligand binding, the signal helix undergoes a 0.1 to 0.2 nm
sliding, or piston-like, displacement towards the cytoplasm. The mechanism
is an allosteric one in which ligand binding shifts the equilibrium towards
a population of displaced conformations.
The cytoplasmic signaling module of the Tar receptor forms associations
with two kinds of proteins. One of these is CheW that functions as a molecular scaffold. The other is CheA, the histidine kinase. CheA proteins are
linked to the Tar signal helix through the CheW scaffold. Both CheW and
CheA contain modules termed SH3 domains. These modules serve as interfaces for the protein-protein interactions leading to the assembly of not just
a pair of receptors, scaffolds, and histidine kinases, but rather for the formation of an extended network of such units. The Tar receptors are not the
only ones in these signaling clusters. Tar receptors are intermingled with
another major receptor, the Tsr serine receptor, and with three less prominent chemotactic sensors. The signals sent through the receptors converge
upon and are integrated by the CheA histidine kinase.
The net result of the interactions between receptors, scaffolds, and
protein kinases is the formation at one pole of the bacterium of a signaling
mesh. In this mesh, the extracellular portions of the receptors bind to

7.9 Bacteria with High Sensitivity and Mobility

149

ligands, while the cytoplasmic portions of the receptors composed of sets

of helical coils form a set of signaling whiskers. These whiskers bind to
the scaffolds and through them to the protein kinases. The overall system
functions much like a primitive nose, exhibiting considerable sensitivity to
external stimuli over a broad range of concentrations.
The signaling complex operates through ligand-induced expansions and
contractions. In the absence of ligand binding, the receptors bind CheA and
thus stimulate the kinase (autophosphorylation) activity leading to structural changes within the array. Ligand binding inhibits these interactions.
When ligands bind the receptors, the pistonlike movements of the signal
helices cause the elements of the array to move apart, or disperse. This
expansion is sufcient to shut down the autophosphorylation of histidine
residues in CheA proteins and the attendant downstream signaling. The
formation of large signaling complexes consisting of multiple transmembrane receptors is not restricted to bacteria. Lymphocytes belonging to the
vertebrate immune system and neurons utilize extensive signaling complexes and meshes on their surfaces, too.

7.9 Bacteria with High Sensitivity and Mobility

Bacteria such as Escherichia coli and Salmonella typhimurium are highly
mobile, free-swimming organisms. Bacteria such as Escherichia coli and Salmonella typhimurium are able to sense nutrients and noxious substances in
their local environments, and, in response, swim towards the nutrients and
away from dangerous chemicals. The essential components of the system
that makes possible these chemotactic responses lie at the poles of the cell.
A agellar motor complex resides at one pole, and a sensor complex lies at
the other. About 50 genes are involved in chemotaxis. Roughly 10 genes are
needed to encode the sensor and signaling complex, and about 40 genes are
needed to encode assembly and structural proteins of the agellar motor.
These two systems are coupled to one another. Signals are sent from the
sensor system to control point at the agellar motor switch.
The agellar motor converts an electrochemical gradient into a mechanical torque that drives the bacterium forward at speeds up to 25 m/s. The
propulsive force is provided by 6 to 10 agella organized into a bundle. Each
agellum can either rotate clockwise (CW) or counterclockwise (CCW). A
agellum has an intrinsic handedness. When all agella undergo CCW rotation, the bundle movement is concerted and the cell moves uniformly. If
one or more agella rotate in a CW fashion, the bundle ies apart, the
movement is disorganized, and the cell tumbles. Because of its small size,
Brownian effects limit the effective straight-line distance that can be
traversed without readjustments to the trajectory. The movement of a swimming bacterium consists of a series of smooth runs punctuated by periods
of tumbling in which a new direction for running is chosen randomly. The

150

7. Two-Component Signaling Systems

seemingly random movements of the bacterium are biased towards attractants or away from repellents through modications in the tumbling frequency arising from the sensory input signals.

7.10 Feedback Loop in the Chemotactic Pathway

The chemotactic pathway contains a feedback loop that promotes robust
behavior. The Tar, CheW, and CheA proteins constitute the sensor/transmitter unit of the two-component bacterial chemotaxis system. The CheY
protein functions as the response regulator. The overall arrangement of the
various units in the chemotactic system is depicted in Figure 7.6, and their
functions are summarized in Table 7.4. CheY is a diffusible messenger molecule. Upon activation through phosphorylation it diffuses to the motor
complex where it binds to motor switch protein FliM to promote CW
motion and tumbling. CheY is phosphorylated by activated CheA, and
dephosphorylated by the protein phosphatase CheZ. In the absence of

Figure 7.6. Tar chemotactic signaling pathway: The receptor (Tar) and histidine
kinase (Che A) are encoded on separate chains. CheW is a scaffold that mediates
the interactions between sensor and histidine kinase. CheY is the aspartate-bearing
response regulator that mediates the cellular response (contact with agellar
motor).

Table 7.4. Tar chemotactic pathway.

Component
Tar
CheW
CheA
CheY
CheZ
CheR
CheB

Function
Receptor
Scaffold
Histidine kinase transmitter
Aspartyl response regulator
Regulatory (phosphatase)
Regulatory (methyltransferase)
Regulatory (methylesterase)

7.10 Feedback Loop in the Chemotactic Pathway

151

CheZ, CheY dephosphorylation would take about 10 s, a period of time that

is too long for rapid adaptation to changes in nutrient concentration. When
CheZ is present it binds to CheY and reduces the time for dephosphorylation from 10 s to 1 s.
The remaining two proteins, CheB and CheR regulate the activity of the
Tar receptor. Tar, like many receptors, is a conduit for two-way communication between outside and inside. Ligand binding on the outside alters the
binding properties and catalytic actions of the cytoplasmic part of the
protein. Binding events taking place in the cytoplasmic portion of the molecule inuence the receptors outside ligand binding properties. The cytoplasmic part of the Tar signal helix contains binding sites for attachment of
methyl groups. As a consequence, Tar and receptors like it are known as
methyl-accepting chemotactic proteins (MCPs). CheR catalyzes the transfer
of methyl groups to glutamate residues on the Tar signal helix using Sadenosyl methionine as the methyl donor. This activity is continual, and the
addition of these methyl groups on the helix progressively reduces the
binding afnity of the Tar receptors for their ligand. CheB does the opposite.
It removes methyl groups from Tar and restores a high binding afnity.
CheB is activated by phosphorylated CheA, thereby forming a feedback
loopTar signals to CheA, which signals to CheB, which signals back to
Tar. As the ligand concentration builds up, and more and more Tar receptors are bound, CheA is less and less phosphorylated, CheB remains inactive, and only a few methyl groups are removed from Tar. Thus, at high
ligand concentrations, the sensitivity of Tar to its ligand is reduced. In the
absence of ligand binding, that is, at low ligand concentrations, CheA is
phosphorylated and can activate CheB. CheB removes methyl groups from
Tar, and returns Tar to a condition of high binding afnity and sensitivity
to its ligand. By this means, the methylation level tracks the concentration.
At low concentrations, Tar has few attached methyl groups, and it has a high
afnity for its ligand. At high concentrations, many methyl groups are
added, and Tar has a low binding afnity.
Tar can maintain its sensitivity to concentration gradients over some ve
orders of magnitude through this feedback mechanism. This process is
called exact adaptation; the steady state tumbling frequency in a homogeneous ligand environment (no signal) is insensitive to the ligand concentration over a broad range of attractant or repellent concentrations. The
chemotactic process is independent of specic values of the concentrations
over a broad range. As a result, the bacterium can sense small spatial
changes in concentration, i.e., it can detect small concentration gradients
over many orders of magnitude in mean concentration. This ability is
referred to as robustness. It is a consequence of the network connectivity,
particularly that of the crucial feedback loop. This feedback loop compensates for the effect of ligand binding, thereby maintaining a robust performance regardless of the ligand concentration.
A bacterium is too small to be able to detect concentration gradients by
comparing concentrations at different points along its body. Instead of using

152

7. Two-Component Signaling Systems

a spatial strategy, bacteria adopt a temporal one: They determine concentration gradients by comparing concentrations at slightly different times.
This is equivalent to determining a spatial concentration gradient, since
there is translational motion of the bacterium during the measurement time
interval. The key to the temporal or time derivative method is to compare
two different quantities, each measuring in some way concentration. One
quantity is the concentration at an instant in time, represented as a percent
of receptor occupancy. The second quantity is the receptor concentration a
few seconds earlier, represented by the level of receptor methylation. There
is a lag between receptor binding and the adjustment in methylation
CheA must be phosphorylated, and then CheB must be phosphorylated
before there is an adjustment in receptor methylation. The lag between
receptor binding and methylation of a few seconds serves as a memory that
enables determination of temporal gradients.

7.11 How Plants Sense and Respond to Hormones

Plants use two-component systems and phosphorelays for sensing and
responding to hormones. At certain times in its life a plant will undergo
senescence, the state in which somatic tissues no longer required are broken
down and their components reused in younger tissues. At other times a
plant will undergo organ abscission, in which organs such as seeds and
fruit are detached from the main body of the plant in a developmentally
regulated manner. Plants, like animals, undergo wound-healing and mount
defenses against pathogens.These developmental and defense processes are
regulated by plant hormones such as ethylene (C2H4) and cytokinins.
Arabidopsis is a small weed, and it was the rst plant species to have its
genome sequenced. The Arabidopsis genome encodes 12 histidine kinases,
22 response regulators, and 5 HPt proteins. Histidine kinases used for
sensing and responding to ethylene, cytokinins, and for osmosensing are
listed in Table 7.5. All of these with the exception of ERS1 are hybrids. They
contain a receiver domain and signal through HPt proteins.
The Arabidopsis response regulators (RRs) can be partitioned into two
groups.A-type ARRs contain the required Asp phosphorylation site, as well

Table 7.5. Hormone and osmosensing in plants.

Histidine kinase
ETR1
ERS1
CRE1/AHK4/WOL
AHK2
AHK3
AtHK1

Sensing function
ethylene
ethylene
cytokinins
cytokinins
cytokinins
osmosensor

7.11 How Plants Sense and Respond to Hormones

153

as several other required residues, but lack the DNA-binding domain needed
for operation as transcription factors. A-type ARRs are found in increased
numbers shortly after a cell is exposed by cytokinins. B-type ARRs contain a
DNA-binding domain in their C-terminal regions; nuclear localization
signals (NLSs) needed for entry into the nucleus; and modules associated
with Asp response regulation and signaling. Thus, the type-B ARRs contain
all the components needed for operation as transcription factors.
Cytokinin ligands are recognized by the sensor histidine kinases CRE1,
AKH2 and AKH3. Receptor-binding activates a phosphorelay from CRE1
to an HPt protein called AHP, which translocates to the nucleus and transfers its phosphoryl group to a type B response regulator. The type B RRs
activate transcription of genes encoding Type A response regulators. The
type A RRs operating in conjunction with AHPs and histidine kinases
modulate the activities in other signaling pathways (Figure 7.7).
The ethylene-signaling pathway is erected in a way that combines histidine kinase action with MAP signaling cascades. Ethylene receptors are
histidine kinases, but these proteins do not appear to transfer phosphoryl
groups to aspartyl response regulators, but rather signal through one or
more MAP-like serine/threonine kinases. The signaling mechanisms appearing in the Arabidopsis ethylene pathway bear a striking resemblance to
those of the yeast high osmolarity (HOG) pathway discussed in the last
chapter. In the ETR1 pathway, ethylene is a negative regulator of its recep-

Figure 7.7. Cytokinin signal transduction in Arabidopsis thaliana: Binding of the

cytokinin ligand to the Cre1 receptor/histidine kinase activates a His-Asp phosphorelay. Signals are relayed through AHP1/2 types of HPt intermediates, and a
B-type Arabidopsis response regulator (ARR), resulting in the expression of
cytokinin-responsive genes including A-type of ARRs. These together with B-type
ARRs and AHPs regulate downstream cellular targets.

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7. Two-Component Signaling Systems

Table 7.6. Light-sensitive proteins and their distribution.

Sensor protein

Chromophore

lmax (nm)

Distribution

Rhodopsins
Phytochromes
Cryptochromes

Retinal
Tetrapyrrole
Flavins

500
665, 730
400500

Animals
Bacteria, protists, plants
Plants, insects, mammals

tor. If the ligand is not present, the receptor is in an on state and sends
signals through the MAP3 kinase CTR1. The activated CTR1 proteins negatively regulate transcription of ethylene response genes. When ethylene is
present in the environment, the receptor state is off, and it can no longer
activate CTR1. Turning off CTR1 relieves the repression by CTR1 of transcription of ethylene response genes.

7.12 Role of Growth Plasticity in Plants

Plants retain a great deal of plasticity in their growth and developmental
programs and tie these programs to environmental cues such as lighting. In
plants, light sensing is used to guide developmental, morphological, and
physiological responses, whereas in animals light sensing guides behavioral
responses. Plants are responsive not only to light intensity, but also to its
orientation and duration, and its spectral properties. Light sensing enables
plants to project growth into well-illuminated spaces and away from regions
shaded by other plants. Light sensing allows plants to synchronize, or
entrain, their growth and developmental rhythms with daily and seasonal
cycles. It enables plants to arrive at decisions on when to germinate and
when to ower, for example.
Light sensing by plants is an activity distinct from photosynthesis. Sensing of light in plants is achieved using two kinds of photoreceptors
phytochromes and cryptochromes (Table 7.6). These molecules, like the
rhodopsin molecules found in the rods of the animal eye, contain a light
absorbing group, or chromophore, that alters its conformation upon absorbing light of the appropriate wavelength. The conformational changes are
sent on to other signaling elements to complete the transduction of the light
signal into intracellular signal events.

7.13 Role of Phytochromes in Plant Cell Growth

Phytochromes are red/far-red photoreceptors that initiate developmental
and proximity responses to light. Phytochromes exist in two forms. One
form operates as a red photoreceptor with peak absorption at 665 nm, and
the other as a far-red photoreceptor with peak absorption at 730 nm. Light
absorption triggers the conversion of one form to the other. The red light

7.13 Role of Phytochromes in Plant Cell Growth

155

absorbing form is designated as Pr and the far red light form as Pfr. When
the red form (Pr) absorbs light it is converted into the far-red form Pfr, and
similarly, when the far-red form (Pfr) absorbs light it is converted to the red
form Pr. The ratio of these two forms is a measure of the amount of red
light incident on the plant compared to the amount of far red light. Direct
sunlight provides more red than far red light, but the light characteristics
change when surrounding foliage shades the plant. Then the light shifts
towards the far red. Thus, during exposure to direct sunlight Pfr will predominate in the leaves and when shaded Pr will be more abundant.
The signaling pathway leading to alterations in gene expression has been
explored in Arabidopsis. The pathway is direct and short. Using green uorescent protein it has been found that the Pfr form, but not the Pr form,
translocates to the nucleus. Upon arrival, the Pfr form interacts with a transcription factor PIF3. The interaction of Pfr with PIF3 triggered by the red
light inuences a large number of cellular signaling and metabolic pathways. As observed using microarrays, some pathways are turned on while
others are turned off (Figure 7.8a).
Phytochromes are not limited to plants. They are found in photosynthetic
bacteria and in protists (algae). Cyanobacterial phytochromes are histidine

Figure 7.8. Phytochrome and cytochrome signal transduction: (a) Phytochrome signaling, in which the active form of the light receptor Pfr acts in conjunction with the
transcription factor PIF3 to regulate the expression of light-regulated genes.
(b) Cytochrome signaling, in which expression of (blue) light-sensitive genes is
turned off under dark conditions.The blue light receptor in conjunction with the regulator COP1 translocates to the nucleus where COP1 stimulates the proteolysis of
the transcription factor HY5. Under blue light conditions the Cry1/COP1 complex
remains in the cytosol and COP1 cannot inhibit HY5-mediated transcription.

156

7. Two-Component Signaling Systems

kinases that relay signals to aspartyl response regulators. The plant

phytochromes appear to have replaced a histidine kinase activity with a
serine/threonine kinase one. The transformed kinases still exhibit many of
the characteristics of histidine kinases, supporting the notion of a bacterial
origin. These signaling proteins are thus regarded as divergent histidine
kinases that along with some of the ethylene receptors lack some of the
amino acid residues necessary for histidine kinase activity.

7.14 Cryptochromes Help Regulate Circadian

Rhythms
Cryptochromes are blue/ultraviolet-A photoreceptors that help regulate
circadian rhythms. They have a peak absorption in the range 400 to 500 nm.
They are found not only in plants, but also in insects and mammals. The
cryptochrome signaling pathways regulate physiological processes such as
the setting of period and phase of an organisms internal circadian clock so
that internal body rhythms match those present in the external environment (daily and seasonal). This process is called entrainment.
Like the phytochrome signaling pathway, the cryptochrome signaling
pathway is direct and short. The control point at the end of the signaling
pathway is one that regulates transcription, but it is not a transcription
factor at the promoter site as in the case of the phytochromes. Instead blue
light photoreceptors remain bound to a transcription regulator called
COP1. The COP1 protein regulates the activity of transcription factor HY5
that stimulates transcription of a large number of genes. When it is activated, COP1 tags HY5 for degradation by proteolytic proteins in the
nucleus, and thus COP1 inhibits the ability of the HY5 protein to stimulate
transcription.
The C-terminal domain of Cry1, Ctt1 binds to COP1 under both dark
and light conditions. In the dark COP1 is localized predominantely in the
nucleus and in light it becomes mostly cytosolic. The blue-light transcription factor HY5 is localized in the nucleus under both kinds of illumination. When blue light strikes the Cry1 photocenter, conformational changes
occur in COP1 altering its cellular location and turning off its inhibition of
HY5. In blue light conditions, HY5 escapes being tagged for destruction,
and is then able to stimulate transcription (Figure 7.8(b)).
In this form of regulation, the presence or absence of blue light modulates the effective lifetime of the HY5 protein. This form of regulation is a
fairly common one. It is fast because no lengthy protein synthesis steps are
required. Rapid modulatory actions in a signaling pathway can take one of
several forms. They can involve immobilizing a signal protein at a particular location, or they may involve proteolytic degradation of a signaling
element as in the case of cryptochrome signaling. One form of regulation
by localization already encountered in this and the previous chapter is the

References and Further Reading

157

sequestering of transcription factors in the cytosol and away from the

nucleus until the appropriate signal is received.

References and Further Reading

Histidine Kinases
Bilwes AM, et al. [1999]. Structure of CheA, a signal-transducing histidine kinase.
Cell, 96: 131141.
Mourey L, et al. [2001]. Crystal structure of the CheA histidine phosphotransfer
domain that mediates response regulator phosphorylation in bacterial chemotaxis. J. Biol. Chem., 276: 3107431082.

Response Regulators
Baikalov I, et al. [1996]. Structure of the Escherichia coli response regulator NarL.
Biochem., 35: 1105311061.
Birch C, et al. [1999]. Conformational changes induced by phosphorylation of the
FixJ receiver domain. Structure, 7: 15051515.
Feher VA, and Cavanagh J [1999]. Millisecond-timescale motions contribute
to the function of the bacterial response regulator protein Spo0F. Nature, 400:
289293.
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Chemotaxis Receptors
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Feedback, Methylation, and Robust Behavior

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7. Two-Component Signaling Systems

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Receptor Clusters and Formation of a Primitive Nose

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Plant His-Asp Signaling

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Problems
7.1 A signicant number of the proteins discussed in the last two chapterskinases, phosphatases, GTPases, and proteasesare enzymes.
Catalysts increase the rate of the reactions by many orders of magnitude, but are not themselves altered during catalysis. In their absence
the biochemical reactions to be catalyzed are too slow because of unfavorable mixes of positive and negative charges that have to be brought
into close proximity at the transition state long enough for the reaction
to occur. As was discussed in the chapter, the histidine kinases not only
grip and position the ATP in a favorable orientation for transfer of the
gamma phosphoryl group, they also modify the electrostatic environment. Negative charges of the phosphoryl group are countered by the
positive charges of the divalent magnesium cation; a nucleophile is
present to break bonds, and the cleft at the active site in laced with
charged residues. These activities help stabilize the transition state, and
in the process lower the activation barrier for the transition to the nal
state. This lowering is depicted schematically in the gure shown below.

Problems

159

Figure for Problem 7.1. Uncatalyzed and catalyzed transition states.

The equation shown to the right of the gure denes the three rates
involved in the catalytic process. Kinetic equations governing the catalysis
can be constructed by applying the law of mass action. The resulting expressions in terms of enzyme and substrate molar concentrations are
d[E ]
= -k1 [E ][S] + k-1 [ES] + k2 [ES],
dt
d[S]
= -k1 [E ][S] + k-1 [ES],
dt
d[ES]
= k1 [E ][S] - k-1 [ES] - k2 [ES],
dt
where the ks are the reaction rates.
The amount of enzyme is usually far less than the amount of substrate
and is completely bound up by the substrate. As a result one can make the
steady state assumption that
d[ES]
= 0.
dt
Using the above formulas, derive the MichaelisMenton equation
V0 =

Vmax [S]
,
K M + [S]

where Vmax is equal to k2 ([ E] + [ES]), the Michaelis constant KM = (k1 +

k-1)/k2, and the steady state velocity V0 is k2[ES].
Plot V0 versus [S]. What happens at high [S]? It is customary to determine the constants appearing in the MichaelisMenton equation by making
measurements of V0 values at different substrate concentrations [S] and
arranging the data in the form 1/V0 versus 1/[S]. What does a plot of 1/V0
versus 1/[S] look like? What is the slope and what is the intercept?

8
Organization of Signal Complexes
by Lipids, Calcium, and Cyclic AMP

Cytoplasmic signaling proteins are recruited to the cell plasma membrane

in response to signals sent from other cells. The recruited signal molecules
are organized into modules and complexes, and the rst steps in converting the extracellular signals into cellular responses are taken. Several kinds
of small signaling molecules, most notably, lipids, calcium, and cyclic adenosine monophosphate (cyclic AMP, or cAMP), act as signaling intermediaries. They tie together events taking place subsequent to ligand binding by
helping to recruit and organize the proteins that function as the primary
intracellular signal transducers. Because of their role as signaling intermediaries, the small molecules are commonly termed second messengers, the
rst messengers being the extracellular signal molecules, and the third messengers being the large protein kinases and phosphatases that are recruited
to the plasma membrane.
Localization plays an important role in the organization of signaling pathways, especially those that lead from the cell surface and end at control points
at the nucleus, cytoskeleton, and elsewhere. First, second, third and higher
order messengers do not diffuse about the cell but rather act in restricted
portions of space. The plasma membrane contributes to the localization.
It does far more than simply separate outside from inside and provide a
uid medium for the insertion, uniform lateral movement, and tethering of proteins; it assumes an active role in the signaling processes taking
place.
In the rst part of this chapter, the composition and organization of the
plasma membrane will be examined. This exploration will be followed by a
discussion of how lipid, calcium, and cAMP second messengers are generated. Second messengers stimulate the activities of serine /threonine kinases
belonging to the AGC family. Signaling by these kinases will be explored
in the third part of the chapter.

161

162

8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

8.1 Composition of Biological Membranes

Biological membranes are composed of phospholipids, glycolipids and
cholesterol. Membrane lipids are linked together through the cooperative
effects of multiple weak noncovalent interactions such as van der Waals forces
and hydrogen bonds,and as a result there is considerable uidity of movement
within the membranethe constituents are free to diffuse laterally and
rotationally. The overall structure is that of a uid of lipids and membraneassociated proteins undergoing Brownian motion. The motions of the molecules are not completely unrestricted, but instead are limited to specic
regions of the membrane, giving rise to a mosaic of membrane compartments.
Three classes of lipids are found in biological membranesphospholipids,
glycolipids, and cholesterol. Phospholipids are the primary constituents. The
four most common phospholipids are listed in Table 8.1. Phosphoglycerides
contain a glycerol backbone that is linked to a phosphoryl group bonded
to a phosphorylated alcohol group. A different backbone component,
sphingosine, is used in the sphingolipids. Of the four commonly occurring
phospholipids, all except phosphatidylserine have uncharged head groups.
Phosphatidylserine has a negatively charged head group and is found
exclusively in the cytoplasmic leaet. Phosphatidylinositol is of special
importance in metazoans. It is reversibly phosphorylated at one or more OH
sites on the inositol ring by lipid kinases to generate lipid signal molecules
that coordinate a number of cellular processes including cytoskeleton
control and motility, insulin signaling (glucose and lipid metabolism), and
growth factor-promoted cell survival.
The glycolipids, like the phospholipids, have a backbone connected to
fatty acyl chains. They differ from the phospholipids in that they contain
one or more sugar groups in place of the phosphoryl-alcohol bearing headgroup of the phospholipids (Figure 8.1). Glycolipids are found in the exoplasmic leaet of the plasma membrane, and are believed to promote
cell-to-cell recognition. The sugar residues that form the hydrophilic head
extend out from the cell surface.

Table 8.1. Lipid constituents of the plasma membrane: ExoplasmicOuter;

CytoplasmicInner.
Type
Phospholipids

Glycolipids
Cholesterol

Lipid
Phosphatidylcholine (PC)
Phosphatidylethanolamine
Phosphatidylserine
Sphingomyelin
Phosphatidylinositol

Comments
Exoplasmic leaet
Cytoplasmic leaet
Cytoplasmic leaet, negatively charged head
group
Exoplasmic leaet, sphingosine backbone
Major role in signaling
Exoplasmic leaet, cell-to-cell recognition
Inuences uidity and membrane organization

8.2 Microdomains and Caveolae in Membranes

163

Figure 8.1. Schematic representations of phospho- and glycolipids and cholesterol:

(a) A phospholipid such as PC consisting of a pair of acyl chains in the tail region,
a backbone, which in this case is glycerol, and a head region consisting of a phosphoryl group (circle) plus an alcohol group (rectangle). (b) A phosphatidylinositol
molecule consisting of a tail region, a glycerol backbone, and a phosphoryl group
coupled to a hexagonal inositol ring in the head region. (c) A glycolipid in which
the head group consists of one or more sugar groups (square). (d) A cholesterol
molecule is composed of a fatty acyl tail connected to a rigid steroid ring assembly
with an OH group at the terminus that serves as its polar head.

8.2 Microdomains and Caveolae in Membranes

Biological membranes contain microdomains and caveolae specialized for
signal transduction. Lipids found in biological membranes vary in chain
length and degree of saturation. Chains vary in length, having an even
number of carbons typically between 14 and 24, with 16, 18, and 20 most
common in phospholipids and glycolipids. Chains with one or more double
bonds are unsaturated. These bonds are rigid and introduce kinks in the
chain. In a fully saturated acyl chain the carbon-carbon atoms are covalently linked by single bonds. Each carbon atom in such a chain can establish a maximum possible number of bonds with hydrogen atoms, hence the
term saturated. Such chains are free to rotate about their carbon-carbon
bonds, and can be packed tightly. In contrast, the kinks present in an array
of unsaturated lipids cause irregularities or voids to appear in the array;
these molecules cannot be packed as tightly.
The degree of saturation of the acyl chains and the cholesterol content
inuence the melting point and uidity of the lipids in the membrane.
This point can be illustrated by some everyday examples. Fats, oils, and
waxes are examples of lipids. Butter, a saturated lipid, is a solid gel at
room temperature, while corn oil, an unsaturated lipid, is a liquid at the
same temperature. Cholesterol plays an important role in determining the
uidity of the membrane compartments. It is smaller than the phospho- and
glycolipids and is distributed between both leaets. As the concentration
of cholesterol increases, the lipid membrane becomes less disordered,
gel-like and more like an ordered liquid in which the lipids are more tightly
packed together, especially when saturated sphingolipids are present
(Figure 8.2).

164

8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

Figure 8.2. Lipids and the lipid bilayer: (a) Membrane bilayers contain a mixture
of amphipathic lipids. Each lipid molecule has a hydrophilic (polar) head region and
a two-pronged hydrophobic tail region oriented as shown. Tails are fatty acyl chains,
hydrocarbon chains with a carboxylic acid (COOH) group at one end and (usually)
a methane group at the other terminus. In cells, the polar head of each lipid molecule is surrounded by water molecules and thus is hydrated. The density of water
molecules drops off rapidly in the hydrophobic interior. (b) Small cholesterol molecules are situated in between the larger lipid molecules. Lipids differ from one
another in the number, length, and degree of saturation of the acyl chains, and in
the composition of their head groups. The overall packing density is greater in (b)
than in (a) due to the presence of cholesterol and of lipids with straighter saturated
acyl chains. Signaling proteins (not shown) carrying a GPI anchor attach to the exoplasmic leaet while signaling proteins bearing acetyl and other kinds of anchors
attach to the cytoplasmic leaet. These proteins congregate in cholesterol and
sphingolipid-enriched membrane compartments.

The plasma membranes of eukaryotes are not uniform, but rather contain
several kinds of lipid domains, each varying somewhat in its lipid composition. Compartments enriched in cholesterol and/or sphingolipids contain
high concentrations of signaling molecules: GPI-anchored proteins in their
exoplasmic leaet and a variety of anchored proteins in their cytoplasmic
leaet. Two kinds of compartmentscaveolae and lipid raftsenriched in
cholesterol and glycosphingolipids, are specialized for signaling.
Caveolae (little caves) are detergent-insoluble membrane domains
enriched in glycosphingolipids, cholesterol, and lipid-anchored proteins.
Caveolae are tiny ask-shaped invaginations in the outer leaet of the
plasma membrane. They play an important role in signaling as well as in
transport. Caveolae may be at, vesicular, or even tubular in shape, and may
be either open or closed off from the cell surface. They are detergent insoluble and are enriched in coatlike materials, caveolins, which bind to cholesterol. Cholesterol- and sphingolipid-enriched microdomains can oat
within the more diffuse lipid bilayer.
The second kind of cholesterol- and sphingolipid-enriched compartment
is a lipid raft. It does not have a cave-like shape and does not contain caveolins, but instead is rather at in shape. The uid and detergent-insoluble
properties of both the rafts and the caveolae arise from the tight packing
of the acyl chains of the sphingolipids and from the high cholesterol
content. The cholesterol molecules not only rigidify the compartment but

8.4 Generation of Lipid Second Messengers from PIP2

165

Table 8.2. Phosphoinositide nomenclature.

Phosphoinositide
D3

D4
D5

Members
Phosphatidylinositol-3 phosphate
Phosphatidylinositol-3,4 biphosphate
Phosphatidylinositol-3,5 biphosphate
Phosphatidylinositol-3,4,5 triphosphate
Phosphatidylinositol-4 phosphate
Phosphatidylinositol-4,5 biphosphate
Phosphatidylinositol-5 phosphate

Designation
PtdIns(3)P
PtdIns(3,4)P2
PtdIns(3,5)P2
PtdIns(3,4,5)P3 or PIP3
PtdIns(4)P
PtdIns(4,5)P2 or PIP2
PtdIns(5)P

also facilitate the formation of signaling complexes and the initiation of signaling by them.

8.3 Lipid Kinases Phosphorylate Plasma

Membrane Phosphoglycerides
The plasma membrane phosphoglyceride known as phosphatidylinositol
plays an important role in signaling, cytoskeleton regulation, and membrane
trafcking. The inositol ring of the phosphatidylinositol molecule contains
a phosphoryl group at position 1 that is tied to the glycerol backbone. All
other OH groups of the inositol ring can be phosplorylated except those at
positions 2 and 6. Just as protein kinases catalyze the transfer of phosphoryl groups to selected amino acid residues, lipid kinases catalyze the transfer of phosphoryl groups to specic sites on lipids. Several lipid kinases
catalyze the phosphorylation of phosphatidylinositol.
The lipid kinase phosphoinositide-3-OH kinase (PI3K) catalyzes the
transfer of a phosphoryl group from an ATP molecule to the OH group at
position 3 of the inositol ring of the lipid. Other lipid kinases, PI4K and
PI5K, catalyze the transfer of phosphoryl groups to the other available sites,
positions 4 and 5, on the ring. An entire ensemble of phosphoinositides can
be produced through the addition and subtraction of phosploryl groups
from positions 3, 4, and 5 of the inositol rings. These phosphorylated lipid
products, their placement into D3, D4, and D5 phosphoinositide groups, and
their common abbreviations are listed in Table 8.2.

8.4 Generation of Lipid Second Messengers from PIP2

Two lipid second messengers are generated from PIP2 by phospholipase
C. Phosphatidylinositol 4,5 biphosphate (PIP2) serves as the source of
two lipid second messengers: diacylglycerol (DAG) and inositol 1,4,5triphosphate (designated Ins(1,4,5)P3 or IP3). The plasma membrane functions as a cellular repository for the PIP2 and other phosphoinositides.

166

8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

Figure 8.3. Structure of phosphatidylinositol 4,5 biphosphate (PIP2): Cleavage of

this molecule by PLC generates the lipid second messengers diacylglycerol (DAG)
and inositol 1,4,5-triphosphate (IP3). The insert shows in block form the organization of the molecule into tail, backbone, linker phosphoryl group, and inositol head
groups.

Phospholipase C (PLC) cleaves the membrane-situated PIP2 into DAG

which contains the acyl chains plus the glycerol backboneand IP3which
contains the rest of the head group (Figure 8.3).
A large number of plasma membrane receptors use PLC as an intermediary to signal and activate downstream kinases. Prominent among these
are G protein-coupled receptors (GPCRs) and growth factor receptors.
There are three PLC subtypes, designed as PLCb, PLCg, and PLCd. These
enzymes have a modular organization that supports their (a) localization
at the plasma membrane, (b) activation by upstream receptor signals, and
(c) catalytic activities (Figure 8.4). The Pleckstrin homology domain and the
C-terminus SH2 domain mediate binding to the plasma membrane PtdIns.
The upstream, signaling elements such as G protein subunits bind to and
activate PLC, and the SH3 and N-terminal SH2 domain mediates interactions with upstream growth factor receptors. Once formed by PLC acting
on PIP2, IP3 diffuses to intracellular stores located in the endoplasmic reticulum (ER) and triggers the release of Ca2+. The calcium ions together with
DAG activate protein kinase C, the main downstream target of PLC signaling (Figure 8.5).

8.5 Regulation of Cellular Processes by PI3K

167

Figure 8.4. Organization of phospholipase C: The domain structure of the three

classes of PLC isozymes is shown. Each type of PLC has a Pleckstrin homology
(PH) domain in its N-terminus. PH domains bind to plasma membrane PtdIns
proteins. The PH domains are not all the same; they vary their binding afnities
among the three classes. The X and Y domains form the catalytic domain of the
enzyme.

Figure 8.5. Signaling through PLC: Activation of PLC by ligand-GPCR binding

stimulates dissociation of the G protein subunits, which then activate PLC. The PLC
proteins tether to the plasma membrane by binding PIP3 lipids, and cleave PIP2 into
DAG and IP3. The latter translocates to the intracellular stores (IS) triggering
release of calcium ions, which along with DAG bind to and stimulate protein kinase
C activity.

8.5 Regulation of Cellular Processes by PI3K

Phosphoinositide-3-OH kinase (PI3K) helps regulate a variety of cellular
processes. PI3Ks mediate cellular responses to GPCRs, growth factors and
insulin, activation by cell adhesion molecules called integrins, and leukocyte
(white blood cell) function. An important characteristic of the proteins that
interact with lipid second messengers is the presence of one or more lipid-

168

8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

Figure 8.6. PI3 kinase domain structure: (a) Classes of catalytic subunits. (b) Examples of regulatory subunits that associate with Class IA subunits of PI3 kinases.
Abbreviations: Adapter-binding (AB) domain; Ras-binding (RB) domain; Src
homology domain-3 (SH3); Src homology domain-2 (SH2); BCR-homology GTPase
activating (BH) domain.

binding domains in their regulatory regions. Several kinds of lipid-binding

modulesPH domains, C2 domains, and FYVE domainsmediate the
binding of lipid second messengers to their downstream targets.
There are three classes of mammalian PI3Ks (Figure 8.6). Class I PI3Ks
are heterodimers composed of a 100-kDa catalytic subunit and an 85-kDa
or 55-kDa joint regulatory/adapter subunit. There are two kinds of Class I
PI3Ks, determined by the presence or absence of an adapter-binding (AB)
domain in its N-terminal region (Figure 8.4a) and by kinds of receptor
binding events that activate them. The adapter for the Class IA PI3Ks binds
to growth factor receptors, while Class IB PI3Ks are activated primarily by
G protein coupled receptors operating through the associated Gbg subunits.
Class II PI3Ks contain a C2 domain in their C-terminal region. Class III
PI3Ks may be constitutively active in the cell and help regulate membrane
trafcking and vesicle formation, two housekeeping activities carried out
all the time. As shown in Figure 8.7, PI3K functions as a key intermediary
to activate protein kinase B.

8.6 PIPs Regulate Lipid Signaling

There is a corresponding set of lipid phosphatases that catalyzes the
removal of phosphoryl groups from inositol rings. Perhaps the most prominent of these is PTEN (phosphatase and tensin homolog deleted on chromosome 10). PTEN acts in opposition to PI3K and catalyzes the removal

8.7 Role of Lipid-Binding Domains

169

Figure 8.7. Signaling through PI3K: Ligand growth factor receptor binding
stimulates the dissociation of the regulatory and catalytic subunits of PI3K from
each other. The catalytic subunit phosphorylates the PIP2 proteins at the 3 position,
thereby making PIP3, which then diffuses to and binds PDK1 and protein kinase B.

of phosphoryl groups from position 3 on inositol rings. It acts on PIP3 to

return it to a PIP2 form, thereby reversing the catalytic effects of PI3K.
The importance of dephosphorylating actions is made apparent by the
high frequency of either mutated or missing forms of PTEN in at least one
kind of brain cancer (glioblastoma), in prostate cancer, and in endometrial
(uterine) cancer. The major downstream target of the PIP3 lipids is protein
kinase B (Figure 8.7). This kinase supplies what may best be termed a survival signal in response to growth factor-binding to receptors such as the
insulin receptor. In the absence of growth signals such as insulin, plateletderived growth factor, and neural growth factor, the levels of activated
protein kinase B remain low. They increase in response to the just
mentioned growth signals. When PTEN does not throttle back the survival
signaling to a baseline level by dephosphorylating the lipid second
messengers, the cells undergo uncontrolled growth and proliferation. The
actions taken by PTEN tend to suppress the cancer-promoting actions of
overly active protein kinase B. For this reason PTEN is referred to as a
tumor suppressor.

8.7 Role of Lipid-Binding Domains

Lipid-binding domains facilitate the interactions between proteins and
lipids. As has been discussed in the last few sections, a number of different
lipid-binding domains mediate the interactions between proteins and lipid
bilayers. Four of these domainsthe PH, C1, C2, and FYVE domains
are especially prominent. They are found in hundreds of proteins and
mediate protein recruitment to lipid membranes. These domains and their
properties are summarized in Table 8.3. As indicated in the table, the PH
domain also mediates protein-protein interactions. Two modules that
appear in Table 8.3the SH2 and PTB domainsare primarily known as

170

8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

Table 8.3. Lipid-binding domains found on proteins.

Designation

Domain name

C1
C2

Protein kinase C homology-1

Protein kinase C homology-2

FYVE
PH

Fab1p, YOTB, Vac1p, Eea1

Pleckstrin homology

PTB

Phosphotyrosine-binding

SH2

Src homology-2

Description
~50 amino acid residues; binds DAG
~130 amino acid residues; binds acidic
lipids
~70 amino acid residues; binds PI3P
~120 amino acid residues, binds PIP3
headgroup, PIP2 and its headgroup; binds
proteins
~100 amino acid residues; binds
phosphorylated tyrosine residues; binds
phospholipids in a weak nonspecic manner
~100 amino acid residues; binds
phosphorylated tyrosine residues; binds
phospholipids

phosphotyrosine binding domains. These domains possess phospholipidbinding properties, and for that reason have been included in Table 8.3.

8.8 Role of Intracellular Calcium Level Elevations

Transient elevations in intracellular calcium levels serve as a second messenger. As is characteristic of a second messenger, local increases in intracellular calcium concentration are triggered by the binding of signal
proteins to receptors embedded in the plasma membrane. In the absence
of triggering signals, intracellular calcium levels are maintained at a low
level, no more than 0.1 mM in some cell types. Unlike cAMP and cellular
lipids, calcium is not synthesizesd by cells. Instead, there are two reservoirs
of calciumthe extracellular spaces outside the cell and the calcium stores
located within the cell.The calcium concentration in the extracellular spaces
is on the order of 2 mM, some 20,000 times greater than the resting levels
within the cell. Extracellular calcium enters a cell through ion channels
located in the plasma membrane. Calcium is sequestered within the cell in
intracellular stores (IS), regions enriched in calcium buffers located in the
lumen of the endoplasmic reticulum, the matrix of mitochondria, and in
the Golgi. In response to the appropriate signals, calcium is released into
the cytosol from the stores.
Signals that trigger the entry of extracellular calcium through ion channels and the release of intracellular calcium from stores are sent through
two kinds of receptors embedded in the plasma membrane. The rst kind
of receptor is the voltage-gated ion channel. These are opened and closed,
or gated, through changes in membrane voltage. These channels are found

8.9 Role of Calmodulin in Signaling

171

in cells whose membranes are excitable. Whenever the membrane is depolarized the ion channels open allowing calcium ions from the extracellular
spaces to diffuse through and enter the cell. The second kind of membrane
signal molecule is the ligand-gated receptor such as the G protein-coupled
receptor. When a ligand binds the GPCR receptors, phospholipase C is activated. As discussed earlier in this chapter, PLC hydrolyzes PIP2 to IP3 and
DAG. IP3 diffuses over to, and binds to, IP3 receptors located in the ER.
This event serves as a release signal, resulting in the movement of Ca2+ out
of the stores and into the cytosol.
The duration of a calcium signal is short. Intracellular calcium levels are
restored to their base values fairly rapidly. Buffering agents bind calcium
ions before they can diffuse appreciably from their entry point. Free
calcium path lengths, the distance traveled by calcium ions before being
bound, average less than 0.5 m, which is far smaller than the linear dimensions, 10 to 30 m, of typical eukaryotic cells. In addition to being buffered,
ATP-driven calcium pumps located in the plasma membrane rapidly
remove calcium ions from the cell, and other ATP-driven pumps transport
calcium back into the intracellular stores. The take-up of calcium by buffers
along with its rapid pumping out of the cytosol and into the stores produces
a sharp localization of the signaling both in space and time.

8.9 Role of Calmodulin in Signaling

Calmodulin is a calcium sensor involved in activating many signaling pathways. Calmodulin is an abundant protein, consisting of 0.1% of all the
protein present at any given time in the cell. It functions as a calcium sensor,
and to carry out this role it is distributed throughout the cytosol and
nucleus. Calcium is an extremely important regulator of cells in the brain,
and the cytosolic concentrations of calmodulin in neurons may reach 2%.
Calmodulin is a small protein, consisting of only 148 amino acid residues.
It is organized into two lobes connected by a exible helix giving it a fairly
elongated dumbbell shape. As shown in Figure 8.8, calmodulin has four
calcium-binding sites; two are in the N-terminal lobe and two are in the Cterminal lobe. Calcium binding produces a shift in the population of equilibrium states from a fairly closed to a more open elongated structure. The
shift in population exposes a number of hydrophobic patches that serve as
attachment sites to downstream signaling partners.
Calmodulin serves as a key intermediary in a number of signaling pathways. When bound to calcium it promotes the activity of PI3K, nucleotide
phosphodiesterases and adenylyl cyclases (both to be discussed shortly),
protein kinases such as multifunctional CaM-dependent protein kinase II
(CaMKII), protein phosphatases such as CaM-dependent protein phosphatase 2B(PP2B), and a number of cytoskeleton regulators.

172

8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

Figure 8.8. Solution NMR structure of calmodulin, free and bound to calcium:
(a) Calcium-free calmodulin consisting of an (upper) N-terminal domain and a
(lower) C-terminal domain. (b) Ca2+4-bound calmodulin. The four calcium ions are
depicted as dark gray spheres. The two prominent hydrophobic patches that are
exposed in this more open conformation are bound by W7 molecules shown in a
space-lled model. The gure was prepared using Protein Explorer with atomic
coordinates deposited in the PDB under accession numbers 1dmo (a) and 1mux (b).

8.10 Adenylyl Cyclases and Phosphodiesterases Produce

and Regulate cAMP Second Messengers
Adenylyl cyclase is an integral membrane enzyme that catalyzes the conversion of intracellular ATP into cyclic adenosine monophosphate (cyclic
AMP or cAMP). The organization of the adenylyl cyclase molecule is
depicted in Figure 8.9. As can be seen there are two transmembrane (TM)
regions (M1 and M2) and two large cytoplasmic regions (C1 and C2). Each
transmembrane region consists of six highly hydrophobic membranespanning helices connected by short loops. One of the cytoplasmic regions
lies topologically in-between the TM regions. The other cytoplasmic region
is a situated C-terminal to the second membrane-spanning region. The
overall structure of the adenylyl cyclase molecule resembles a dimer, each
unit consisting of a TM region followed by a cytoplasmic region. However,
monomer-like structures are not functional. The cytoplasmic regions
together form the catalytic core of the molecule. The relative orientation of
C1 relative to C2 is important, and both C1 and C2 are required for binding
and catalysis.
The magnitude and duration of cyclic nucleotide second messenger signaling is regulated by another class of enzymes, namely, nucleotide phosphodiesterases (PDEs). As shown in Figure 8.10, cAMP has a phosphate
group attached to both the 3 carbon and 5 carbon of the ribose. PDEs are
enzymes that catalyze the hydrolytic cleavage of 3 phosphodiester bonds

8.11 Second Messengers Activate Certain Serine/Threonine Kinases

173

Figure 8.9. Organization of adenylyl

cyclase: The cylinders denote transmembrane segments. These are organized into a
repeated set of six segments (M1 and M2).
The cytoplasmic C1 and C2 catalytic
domains consist of a compact region (C1a
and C1b) and a broad loop (C1b and C2b).

Figure 8.10. Adenosine triphosphate and cyclic adenosine monophosphate:

(a) ATP molecule consisting of an adenine joined to a ribose to which are attached
three phosphoryl groups, named in the manner shown. (b) cAMP showing the cyclic
structure.

in cAMP resulting in its degradation to inert 5AMP. They also carry out
the same operation in cGMP to yield inert 5GMP. The PDEs terminate
second messenger signaling. They modulate these signals with regard to
their amplitude and duration, and through rapid degradation restrict the
spread of cAMP to other compartments in the cell.

8.11 Second Messengers Activate Certain

Serine/Threonine Kinases
Second messengers acting in the vicinity of the plasma membrane help
organize the signaling pathways. They exert their inuences by activating
and regulating a large number of serine/threonine kinases, among which are

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8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

Table 8.4. Members of the AGC family of serine/threonine kinases: Different geneencoded isozymes and alternatively spliced isoforms are listed in column 3. Second
messengers required for the activation of the kinases are listed in column 4.
AGC kinase family
Protein kinase A
Protein kinase B

Protein kinase C
Classical
Novel
Atypical

Structure
2 regulatory subunits;
2 catalytic subunits
Single chain; PH,
catalytic, regulatory
domains
Single chain; catalytic,
regulatory domains
Single chain; catalytic,
regulatory domains
Single chain; catalytic,
regulatory domains

Forms

Regulation

RIa, RIb, RIIa, RIIb

Ca, Cb, Cg
a, b, g1, g2

cAMP

PKC-a, PKC-b1,
PKC-b2, PKC-g
PKC-d, PKC-e,
PKC-h, PKC-q
PKC-z, PKC-i,
PKC-l

Ca2+, DAG,
phosphatidylserine
DAG,
phosphatidylserine

PI3K lipid products

those belonging to the AGC family. Three subfamilies of AGC kinases are
included in Table 8.4 along with the second messengers involved in their
activation. The kinases sequester their catalytic activities within a catalytic
domain (or subunit) and similarly combine their regulatory activities into
one or more regulatory domains (or subunits). Some of the kinases possess
a separate lipid-binding PH (Pleckstrin homology) domain, while others
incorporate lipid-binding structures such as C1 and C2 domains into their
regulatory regions.
The kinases all have a common structure and similar modes of activation. Second messengers and upstream kinases activate them. Binding of
the second messengers to PH domains and regulatory motifs induces the
movements of the kinases to the plasma membrane near the sites of second
messenger release. This step is followed by phosphorylation by an upstream
kinase. In the case of protein kinase B and protein kinase C the upstream
kinase kinase has been identied. It is called phosphoinositide-dependent
kinase-1 (PDK1). This enzyme may also be the one responsible for activating protein kinase A. In all cases, the role of the upstream kinase is
to catalyze the transfer of a phosphoryl group to a crucial residue situated
in the activation loop of the AGC kinase. When this occurs the amino
acid residues involved in catalysis are unblocked and can carry out their
functions.

8.12 Lipids and Upstream Kinases Activate PKB

Protein kinase B is the primary target of signals relayed from membranebound signal receptors via lipid second messengers. It is activated in two
stages. In the rst stage the PI3K product PIP3 binds to PKB through its

8.12 Lipids and Upstream Kinases Activate PKB

175

PH domain. In response the kinase migrates to the plasma membrane

where it is phosphorylated by 3-phosphoinositide-dependent kinase-1
(PDK-1) and then again at a second location either by another kinase or
by itself. Once it is recruited to the plasma membrane and phosphorylated,
the PKB enzyme is fully activated. As discussed earlier PIP3-mediated
signaling is terminated by a protein phosphatase PTEN that converts PIP3
to PIP2.
Protein kinase B is centrally involved in insulin signaling. One of its
immediate downstream targets is glycogen synthase kinase 3 (GSK3). In
the absence of insulin signaling, GSK3 is active, and when it phosphorylates
glycogen synthase it inhibits its enzymatic stimulation of glycogen synthesis. Signaling is fairly rapid. Within a few minutes of insulin binding, PKB
is activated and phosphorylates GSK3, thereby inactivating it so that it
cannot phosphorylate glycogen synthase. The latter becomes dephosphorylated and consequently is better able to stimulate glycogen production.
Another effect of insulin binding leading to GSK3 inactivation is that of an
increase in protein translation. This, too, is slowed down by GSK3, but
insulin signaling frees the protein translation initiation regulator eIF2B
from its inhibition by GSK3 (Figure 8.11).

Figure 8.11. Insulin signaling through protein kinase B: (a) In the absence of PKB
activity GSK3 inhibits the stimulation of glycogen synthesis by glycogen synthase
(GS). (b) When PKB is activated it binds to and prevents GSK3 from inhibiting GS.
(c) In the absence of PKB activity GSK3 inhibits the initiation of protein synthesis
by eIF2B. (d) When PKB is activated it binds to and prevents GSK3 from inhibiting eIF2B.

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8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

8.13 PKB Supplies a Signal Necessary for Cell Survival

Protein kinase B is activated by a number of signals, not just insulin. It is
activated through PI3K when growth factors bind to members of the receptor tyrosine family to which the insulin receptor belongs. The effect on the
cell of protein kinase B signaling is to enhance cell survival. It mainly does
so by inhibiting proteins that promote cell suicide, or apoptosis. Two examples of this kind of action are presented in Figure 8.12. The rst example is
inhibition of Bad signaling. Bad is a member of a group of apoptosis regulators known as the Bcl-2 family that will be explored in Chapter 15. Some
Bcl-2 family members promote apoptosis while others inhibit it. These proteins are regulated by phosphorylation. The Bad protein, in particular, promotes apoptosis, but this activity is turned off by phosphorylation. When
PKB is fully activated, it diffuses over to and phosphorylates Bad, thereby
preventing its proapoptosis actions.
The second example presented in Figure 8.12 is inhibition of Forkheadmediated transcription. The Forkhead protein is a transcription factor.
Protein kinase B catalyzes the phosphorylation of substrates at sites characterized by the consensus sequence RXRXXS/T. Among the PKB

Figure 8.12. Survival signaling through protein kinase B: (a) In the absence of PKB
signaling Bad translocates from the cytoplasm to the mitochondria where it promotes apoptotic responses. (b) When PKB is activated it phosphorylates Bad, which
then binds to a 14-3-3 protein and becomes immobilized in the cytoplasm. (c) In the
absence of PKB-signaling, the Forkhead (FH) transcription factor translocates from
the cytoplasm to the nucleus where it promotes the expression of proapoptotic
genes. (d) When PKB is activated it phosphorylates FH, which then binds to a 143-3 protein and becomes immobilized in the cytoplasm.

8.14 Phospholipids and Ca2+ Activate Protein Kinase C

177

substrates possessing this consensus sequence are the transcription factors

belonging to the Forkhead (FH) family. As was the case for Bad, phosphorylating these proteins inactivates them. In the absence of PKB signaling,
the FH proteins translocate from the cytoplasm to the nucleus where they
stimulate transcription of apoptosis-promoting genes. When protein kinase
B is actived it can phosphorylate FH resulting in its binding to, and immobilization by, cytoplasmic 14-3-3 proteins. Binding to the 14-3-3 proteins
prevents FH from carrying out its anticell-cycle progression actions, and
thus again promotes cell survival. In both of these examples, the cellular
response to lipid-mediated protein kinase B signaling is survivalthe suppression of the cell suicide program, and continued progression through the
cell cycle.

8.14 Phospholipids and Ca2+ Activate Protein Kinase C

There are several subfamilies of protein kinase C (PKC), each characterized by a slightly different domain structure. As depicted in Figure 8.13, the
different isozymes of PKC belong to either the classical, novel, or atypical
subfamilies. The pseudosubstrate (PS) is a portion of the chain that interacts with and blocks the activity of the catalytic domain, but activation
relieves the block. There are three requirements for full activation of
protein kinase C. The rst is phosphorylation. Protein kinase C, like protein kinase B, is activated by phosphorylation. PKC is phosphorylated by
upstream kinases such as PDK1. The second requirement is presence of
subfamily-specic second messengers acting as coactivators. The various
isozymes of protein kinase C are listed in Table 8.4 along with the associated coactivators.

Figure 8.13. Structure of protein kinase C and protein kinase B: (a) Protein kinase
C familiesConventional (c), novel (n), and atypical (a) protein kinase C proteins.
(b) Protein kinase B and PDK1 proteins. Abreviations: pseudosubstrate (PS); Pleckstrin homology (PH); octicopeptide repeat (OPR).

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8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

The best-characterized PKC proteins are the classical (conventional)

isozymes. As indicated in Table 8.4, these enzymes are activated by calcium
along with DAG. Their C1 domain binds DAG while their C2 domain binds
Ca2+. Two other parts of the protein, C3 and C4, form the ATP and
substrate-binding lobes of the catalytic core. The steps leading to second
messenger signaling have been discussed. Binding of a ligand to a receptor
activates PLC, which then splits PIP2 to create DAG and IP3. The latter
stimulates the release of Ca2+ from intracellular stores and together the
Ca2+ and DAG activate protein kinase C. Binding of DAG to the C1 domain
and Ca2+ to the C2 domain facilitates the recruitment of the protein to
the plasma membrane, where it binds to the negatively charged phosphatidylserine molecules concentrated in the cytoplasmic leaet. In this
process the C1 domain is mostly responsible for the recognition of phosphatidylserine while the C2 domain confers a more general specicity for
lipid membranes.

8.15 Anchoring Proteins Help Localize PKA and PKC

Near Substrates
Anchoring proteins were introduced in Chapter 6. These proteins do not
function as enzymes but instead help localize the kinases near their substrates. They provide sites for the tethering of protein kinase A and protein
kinase C, and protein phosphatases PP1 and PP2B, to the cytoplasmic face
of the plasma membrane and similarly to organelle membranes. There are
several prominent families of kinase-anchoring proteins, among which are
the A-kinase anchoring proteins (AKAPs) and receptors for activated C
kinase (RACKs).
The AKAPs localize protein kinase A close to its substrates. The AKAPs
contain an amphipathic helix that binds to specic regulatory subunits of
protein kinase A, and the AKAPs have a targeting sequence that directs
the anchor protein to a specic subcellular location. Sites of AKAP attachment include cell membranes, cytoskeleton, nuclear matrix, and endoplasmic reticulum. The AKAPs serve as control points where the primary
protein kinase-signaling elements, and their regulators and modulators, are
brought together near or at their xed infrastructure targets. The AKAPs
serve as platforms for assembly and integration of several signaling proteins, thereby serving as points of control of their substrates.
The RACKs bind protein kinase C proteins once they have been partially activated by their upstream kinases and coactivators. Different
isozymes of the PKCs localize to different subcellular locations. In response
to production of cofactors such as Ca2+ and DAG, different PKC isozymes
translocate from the plasma membrane to distinct subcellular locations,
aided by the RACKs. Some PKCs are localized by their RACKs to the
plasma membrane, where they phosphorylate L-type calcium channels,

8.16 PKC Regulates Response of Cardiac Cells to Oxygen Deprivation

179

ligand-gated ion channels (discussed in Chapter 18), and other membranebound signaling proteins. Other PKCs are localized to the nucleus or to the
mitochondria or to other cellular compartments where they phosphorylate
their substrates.

8.16 PKC Regulates Response of Cardiac Cells to

Oxygen Deprivation
Cells in any tissue of the body will not survive prolonged periods of oxygen
deprivation. In oxygen deprived heart muscle (myocardium) cells shift from
oxidative phosphorylation to anaerobic glycolysis. The amount of available
ATP drops, and cellular pH rises. Contractile forces are reduced, and energy
dependent ion pumps are impaired. Myocardial cells (and others, too)
respond to brief periods of oxygen deprivation by adjusting their cellular
processes, and as a result are be able to tolerate longer periods of poor
oxygen conditions. This kind of response is known as ischemic preconditioning (IP). The key signaling events responsible for IP are sketched in
Figure 8.14. As can be seen in the gure, signaling starts with release of
adenosine by stressed cells resulting in their binding to adenosine receptors, members of the G-protein coupled receptor (GPCR) family. The heterotrimeric G proteins activated by these receptors stimulate the hydrolysis
of PtdIns(4,5)P2 to DAG and IP3; the DAG acting as a cofactor for e and d
members of the nPKC subfamily, which are anchored nearby the receptors
along the plasma membrane, stimulates their activation.
Once activated the PKC e and d proteins change their cellular location,
translocating from the plasma membrane to the mitochondria. They
become anchored there, and associate with and activate members of the Src
family of protein tyrosine kinases such as Lck leading to activation of a
MAP kinase cascade.The downstream PKC e-signaling targets are the mitochondrial ATP-dependent potassium channel proteins, which are phosphorylated by the last MAP kinase in the cascade. The PKC e and d have
opposing effects. While PKC e promotes preconditioning responses, PKC d
stimulates proapoptotic responses. The cellular response to oxygen deprivation will depend at least in part on the balance between these two signals.
Anchoring proteins such as the RACKs help localize the PKC isozymes
in their proper locations. This aspect is illustrated in Figure 8.14 where
several RACKs are depicted localizing the PKCs to the plasma membrane,
mitochondria, and nucleus. At the plasma membrane the RACKs position
the kinase near its substratecardiac L-type calcium channels. In the
nucleus, the RACKs serve a similar role, placing the kinase near transcription factors that it regulates by means of phosphorylation. The RACKs not
only help localize the proteins near their substrates, but also help organize
signaling complexes. This aspect is represented in the gure by the binding
of Src/Lck along with PKC e to the mitochondrial RACK.

180

8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

Figure 8.14. Protein kinase C signaling in cardiac cells: Signaling is initiated by

binding of adenosine to a G protein-coupled receptor (GPCR) resulting in dissociation of the G protein tethered nearby into its Ga and Gbg subunits. The Ga subunit
activates phospholipase C, leading to formation of IP3 and DAG second messengers. IP3 triggers the release of calcium from intracellular stores (IS). Calcium and
DAG activate cPKCs and DAG activates nPKCs.The RACKs position these protein
kinases near their substrates along the plasma membrane and in the mitochondria
and nucleus. (To keep the gure from getting too cluttered, phosphorylation of the
PKCs by upstream kinases is not shown.)

8.17 cAMP Activates PKA, Which Regulates Ion

Channel Activities
Cyclic AMP is a second messenger that acts through cAMP-dependent
protein kinase A (PKA) to form the cAMP signaling pathway. This pathway
is used to relay a variety of hormonal and neuromodulatory signals to
the transcription machinery in the nucleus, to ion channels embedded in the
plasma membrane, and to other targets such as the actin cytoskeleton. The
cAMP signaling pathway consists of a receptor such as a GPCR, an intermediary such as a G protein, adenylyl cyclases and phosphodiesterases that
form and regulate cAMP production, and protein kinase A. In more detail,
ligand binding to a GPCR activates a G protein (the intermediary) tethered to the cytoplasmic region of the plasma membrane close enough to
the receptor to be activated by its cytoplasmic region. Once activated, the
G protein translocates to and stimulates the production of cAMP by adenylyl cyclases. The cAMP molecule, in turn, binds to and activates protein
kinase A.

8.17 cAMP Activates PKA, Which Regulates Ion Channel Activities

181

Protein kinase A contains a pair of catalytic (C) subuints and a pair

of regulatory (R) subunits. In its inactive form, the regulatory units bind to
the catalytic site and inhibit its activity. Binding of cAMP to the regulatory
subunits results in a conformational change that permits the dissociation
of the regulatory units form the complex. Once freed of its regulatory
subunits, the catalytic subunits are able to catalyze the transfer of
phosphoryl groups to their targets using ATP as the phosphoryl group
donor. Protein kinase A is able to phosphorylate a number of different
proteins. The choice of target is dictated by the choice of regulatory units
and by its subcellular location. The specic location in the cell is determined
by an AKAP.
The AKAP complexes incorporate not only protein kinases but also
protein phosphatases that after a time turn off what the kinases turn on. In
Figure 8.15, PKA together with protein phosphatases PP1 or PP2A regulate the opening of an ion channel embedded in the plasma membrane. An
AKAP called Yotiao helps regulate the opening and closing of ion channels called NMDA receptors in neurons in the brain. The manner of this is
depicted in the gure.
AKAP79 is a well-studied member of the A-kinase anchoring protein
family. This protein binds two AGC kinases, protein kinase A and protein
kinase C along with a prominent serine/threonine protein phosphatase
called calcineurin (CaN), or as it is sometimes known, protein phosphatase
2B (PP2B). These enzymes are capable of binding many different substrates

Figure 8.15. PKA signaling through AKAPs regulating ion channel openings:
Binding of a neuromodulator to a G protein-coupled receptor initiates signaling in
the neuron. The activated Ga subunit stimulates production of cAMP by adenylyl
cyclase. The cAMP molecules bind to the regulatory subunits of protein kinase A.
In response, the regulatory subunits dissociate from the catalytic subunits, which
then phosphorylate the ion channel, overcoming its inhibition (closed state) by the
protein phosphatases. After a time, the protein kinases are no longer able to maintain the channel in its phosphorylated state and the ion channel closes again (not
shown).

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8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

with high afnity. The desirable property of high afnity is combined with
another important property, substrate specicity, through use of an AKAP.
The AKAP79 situates the three signaling proteins near their targets: plasma
membrane receptors and ion channels. The enzymatic functions of these
proteins are disabled by their association with the anchoring protein.
Ca2+/CaM supplies the on signal for these enzymes. When this complex
binds protein kinase C and calcineurin, the enzymes detach, and diffuse to
and phosphorylate/dephosphorylate their nearby targets. Similarly, cAMP
binding will enable protein kinase A to detach from the AKAP79 and phosphorylate its targets.
In the brain, protein kinase A participates in the learning pathway
along with several other protein kinases. The main endpoint of the learning pathway is the nucleus where kinases functioning as transcription
factors regulate gene expression. Signaling interactions of a short term
nature involving the regulation of ion channels, and long term natureproducing changes in gene expression, are central to brain function. They
will be examined in Chapter 21.

8.18 PKs Facilitate the Transfer of Phosphoryl Groups

from ATPs to Substrates
The core unit of protein kinase A is representative of the core units of
all serine, threonine, and tyrosine kinases. As shown in Figure 8.16, it
consists of a small lobe, a linker, and a large lobe. A cleft formed by
elements of the small and large lobes operates as the catalytic site. The
large lobe binds the substrate peptide or protein, while the small lobe
supplies the main site for attachment of the ATP molecule. The ATP
molecule sits at the base of the cleft and provides structural support, helping
to x and maintain the orientations of the large and small lobes with
respect to one another. The cleft is bordered by the glycine-rich loop
and the C helix from the small lobe and by a beta sheet from the large
lobe. As shown in Figure 8.16, the phosphoryl groups attached to the
adenosine are oriented towards the cleft with the gamma phosphoryl group
at the end. The loops and beta sheet position this group for transfer to the
substrate.
A common feature in protein kinases is the presence of metal ions. As
was the case for the histidine kinases discussed in the last chapter, these
positively charged ions help position and stabilize the cells assembly. The
Mg ion-positioning loop highlighted in the gure assists in positioning the
magnesium ions.
There are two phosphorylation sites on the core unitThr197 and
Ser338. In the active state conformation, the cleft is opened up and the core
unit is catalytically competent. Phosphorylation at Thr197 and Ser338 helps
stabilize the open conformation.

References and Further Reading

183

Figure 8.16. Crystal structure of the conserved core of the catalytic subunit of
protein kinase A: The core unit consists of a small lobe, a linker and a large lobe.
The small lobe contains a ve-stranded antiparallel beta sheet and a conserved helix
(the C helix). The large lobe consists mainly of helices plus a grouping of four beta
strands at the bottom of the active site cleft. The phosphoryl groups covalently
attached at Thr197 and Ser338 are depicted as space-lled model. The ATP molecule is also shown with a space-lled representation. The gure was prepared using
Protein Explorer with atomic coordinates deposited in the Protein Data Bank
(PDB) under accession code 1ATP.

References and Further Reading

Plasma Membrane Organization
Brown DA, and London E [1998]. Structure and origin of ordered lipid domains in
biological membranes. J. Mem. Biol., 164: 103114.
Sheets ED, Holowka D, and Baird B [1999]. Critical role for cholesterol in Lynmediated tyrosine phosphorylation of FceRI and their association with detergentresistant membranes. J. Cell Biol., 145: 877887.
Simons K, and Ikonen E [2000]. How cells handle cholesterol. Science, 290: 1721
1726.
Simons K, and Toomre D [2000]. Lipid rafts and signal transduction. Nature Rev.
Mol. Cell Biol., 1: 3141.
Smart EJ, et al. [1999]. Caveolins, liquid-ordered domains, and signal transduction.
Mol. Cell Biol., 19: 72897304.
Vereb G, et al. [2003]. Dynamic, yet structured: The cell membrane three decades
after the SingerNicolson model. Proc. Natl. Acad. Sci. USA 100: 8055380558.

Lipid Signaling, Phosphoinositide-3-OH Kinase, and PIP2 Signaling

Honda A, et al. [1999]. Phosphatidylinositol 4-biphosphate kinase a is a downstream effector of the small G protein ARF6 in membrane rufe formation. Cell,
99: 521532.

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8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

Raucher D, et al. [2000]. Phosphatidylinositol 4,5-biphosphate functions as a second

messenger that regulates cytoskeleton-plasma membrane adhesion. Cell, 100:
221228.
Toker A, and Cantley LC [1997]. Signaling through the lipid products of
phosphoinositide-3-OH-kinase. Nature, 387: 673676.
Vanhaesebroeck B, and Watereld MD [1999]. Signaling by distinct classes of
phosphoinositide 3-kinases. Exp. Cell Res., 253: 239254.

Lipids and Lipid-Binding Domains

Czech MP [2000]. PIP2 and PIP3: Complex roles at the cell surface. Cell, 100: 603
606.
Hurley JH, and Meyer T [2001]. Subcellular targeting by membrane lipids. Curr.
Opin. Cell Biol., 13: 146152.
Lemmon MA, and Ferguson KM [2000]. Signal-dependent membrane targeting by
Pleckstrin homology domains. Biochem. J., 350: 118.

Calcium/Calmodulin
Berridge MJ, Lipp P, and Bootman MD [2000]. The versatility and universality of
calcium signaling. Nature Revs. Mol. Cell Biol., 1: 1121.
Carafoli E [2002]. Calcium signaling: A tale for all seasons. Proc. Natl. Acad. Sci.
USA, 99: 11151122.
Chin D, and Means AR [2000]. Calmodulin: A prototypic calcium sensor. Trends
Cell Biol., 10: 322328.

Adenylyl Cyclases
Cooper DMF, Mons N, and Karpen JW [1995]. Adenylyl cyclases and the interaction between calcium and cAMP signaling. Nature, 374: 421424.
Hurley JH [1999]. Structure, mechanism, and regulation of mammalian adenylyl
cyclase. J. Biol. Chem., 274: 75997602.
Taussig R, and Gilman AG [1995]. Mammalian membrane-bound adenylyl cyclases,
J. Biol. Chem., 270: 14.

Cyclic AMP
Houslay MD, and Milligan G [1997]. Tailoring cAMP-signaling responses through
isoform multiplicity. Trends Biochem. Sci., 22: 217224.
Rich TC, et al. [2001]. A uniform extracellular stimulus triggers distinct cAMP
signals in different compartments of a simple cell. Proc. Natl. Acad. Sci. USA, 98:
1304913054.
Schwartz JH [2001]. The many dimensions of cAMP signaling. Proc. Natl. Acad. Sci.
USA, 98: 1348213484.

Cyclic Nucleotide Phosphodiesterases

Beavo JA [1995]. Cyclic nucleotide phosphodiesterases: Functional implications of
multiple isoforms. Physiol. Rev., 75: 725748.
Francis SH, Turko IV, and Corbin JD [2001]. Cyclic nucleotide phosphodiesterases:
Relating structure and function. Prog. Nucl. Acid Res. Mol. Biol., 65: 152.

Problems

185

PTEN
Yamada KM, and Araki M [2001]. Tumor suppressor PTEN: Modulator of cell signaling, growth, migration and apoptosis. J. Cell. Sci., 114: 23752382.

Anchoring Proteins
Colledge M, and Scott JD [1999]. AKAPs: From structure to function. Trends Cell
Biol., 9: 216221.
Feliciello A, Gottesman ME, and Avvedimento EV [2001]. The biological function
of A-kinase anchoring proteins. J. Mol. Biol., 308: 99114.
Mochly-Rosen D, and Gordon AS [1998]. Anchoring proteins for protein kinase C:
A means for isozyme selectivity. FASEB J., 12: 3542.

Protein Kinases B and C Signaling

Dudek H, et al. [1997]. Regulation of neuronal survival by the serine-threonine
protein kinase Akt. Science, 275: 661665.
Newton AC, and Johnson JE [1998]. Protein kinase C: A paradigm for regulation of
protein function by two membrane-targeting modules. Biochim. Biophys. Acta,
1376: 155172.
Ron D, and Kazanietz MG [1999]. New insights into the regulation of protein kinase
C and novel phorbel ester receptors. FASEB J., 13: 16581676.
Shepherd PR, Withers DJ, and Siddle K [1998]. Phosphoinositide 3-kinase: The key
switch mechanism in insulin signaling. Biochem. J., 333: 471490.
Vanhaesebroeck B, and Alessi DR [2000]. The PI3K-PDK1 connection: More than
just a road to PKB. Biochem. J., 346: 561576.

Protein Kinases
Huse M, and Kuriyan J [2002]. The conformational plasticity of protein kinases. Cell,
109: 275282.
Taylor SS, et al. [1999]. Catalytic subunit of cyclic AMP-dependent protein
kinase: Structure and dynamics of the active site cleft. Pharmacol. Ther., 82:
133141.

Problems
8.1 As discussed in the chapter, biological membranes can be described as
uids in which lipids and proteins freely diffuse within the plane of the
membrane. Singer and Nicholson [Singer SJ and Nicholson GL [1972].
The uid mosaic model of the structure of cell membranes. Science, 175:
720731] presented the basic features of this picture in a 1972 paper
that appeared in Science. The membranes are not homogeneous structures but rather are organized into domains, each characterized by a
somewhat different mix of lipids and proteins. A typical value for the
diffusion coefcient for lateral diffusion in the plane of the lipid bilayer
in the uid phase is D = 3 10-8 cm2/s. When the cholesterol content is

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8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

increased to high values the diffusion coefcient decreases to D = 2

10-9 cm2/s. How far will the lipids diffuse in 1 s in the uid phase?
8.2 Proteins diffuse more slowly than lipids. Some proteins, especially those
involved in signaling, are not free to diffuse at all. These proteins
are immobilized through interactions with each other and with the
cytoskeleton elements. Protein diffusion coefcients are consequently
quite variable, but in those instances where the proteins can diffuse
freely within a domain, diffusion coefcients ranging from D = 4
10-9 cm2/s to D = 2 10-10 cm2/s are observed. Given these numbers,
how does the viscosity of the lipid bilayer compare to the viscosity of
water?

9
Signaling by Cells of the
Immune System

The human body has three super signaling and control systemsthe
immune system, the endocrine system, and the nervous system. Each system
has a myriad of cells extending throughout the body and specialized for
signaling. Cells of the immune system communicate using cytokines; cells
of the endocrine system send out hormones and growth factors, and the
cells of the nervous system utilize neurotransmitters and neuromodulators.
The immune system, the subject of this chapter, consists of several organs,
colonies of leukocytes, and large numbers of extracellular messengers. The
immune systems job is to identify and destroy pathogens, entities that enter
the body, establish themselves in a specic locale, or niche, multiply, cause
damage, and exit. Leukocytes, the white blood cells, are highly motile, shortlived cells that move through the cardiovascular and lymphatic systems into
damaged tissues. The leukocytes kill bacterial, protozoan, fungal, and multicellular pathogens; they destroy cells infected with viruses and bacteria, and
eliminate tumor cells.
Leukocytes are highly mobile cells that can migrate from blood into
tissues and back again into blood. They respond to infections by attacking and destroying the causative agents, or pathogens. They carry out inammatory responses, innate immune responses, and adaptive immune
responses. The inammatory response to an infection involves the triggering of physiological responses such as fever and pain, redness and swelling,
and a buildup of white blood cells, the leukocytes, at the infection site. A
local environment is formed that promotes migration of leukocytes to the
infection site, the destruction of the invasive agents, and the repair of
damaged tissues. It takes several days for the adaptive immune response to
develop, and during that the inammatory response contains the infection.
The innate immune response is a phylogenetically ancient form of
defense by multicellular organisms against pathogens. It involves recognition by host cell surface receptors of molecules situated on the outer surface
of pathogens. Such molecules are characteristic of the pathogen. Because
the receptors are encoded by the host genome and thus innate, the recognition response is termed an innate immune response. Lipopolysaccharides
187

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9. Signaling by Cells of the Immune System

(LPS) are an example of molecules that are recognized by receptors

expressed on cells of host multicellular organisms. They are prominent
components of the outer membrane of gram-negative bacteria such as
Escherichia coli.
The adaptive immune response is unique to vertebrates. It involves the
production of antibodies and enables the host to respond to pathogens that
have eluded the innate immune response, and to pathogens have not been
encountered before. There are two basic situations: Pathogens may reside
outside of the host cells, or they may hide within cells of the body. Antibodies are receptors that recognize and bind antigens, foreign substances
uniquely derived from the pathogens. Antigens (antibody generators) may
be molecules or structures located on the surface of pathogens, toxins
secreted by a pathogen, foreign RNA, or any other molecule identied by
the host as not belonging to the host (not self). In situations where the
pathogens hide within the cell the solution used by the immune system is
to present peptide fragments of the pathogen, i.e., antigens, on the surface
of the host cells where they be seen and can trigger immune responses.Antigens that are encountered extracellularly are taken up by leukocytes specialized for their ingestion, and these are presented on the cell surface of
the leukocytes.
This chapter will begin with a review of the different kinds of leukocytes
found in the body and the signaling proteinsthe cytokinesused by them
to communicate with one another. The signaling pathways used by each of
the ve classes of cytokines will then be examined. The chapter will conclude with an examination of signaling through the T cell receptor.

9.1 Leukocytes Mediate Immune Responses

An immune response involves detection, the marshalling and movement of
resources (cells) between tissue and blood, creation of a protected environment, production of leukocytes needed for ghting the infection, and
destruction of the invaders and diseased cells. Colonies of leukocytes are
formed from hematopoietic (blood-forming) stem cells in several developmental stages in a number of locations in the bodyin bone marrow, in
lymphoid tissue, in the circulating blood, and in body tissues. There are two
main categories of leukocytes: Cells that migrate in and out of the lymphatic
system and mature and differentiate there are categorized as lymphocytes.
These cells are the key players in the adaptive immune response. Members
of the second group of cells differentiate and mature in bone marrow. These
myeloid cells are key mediators of inammation and innate immune responses and are categorized as in Table 9.1 as myeloid-derived phagocytes/
granulocytes.
Dendritic cells and mast cells are distributed throughout the body, serving
as sentinels whose purpose is to detect the presence of pathogens. As

9.1 Leukocytes Mediate Immune Responses

189

Table 9.1. Leukocytes: Cells of the immune system.

Leukocyte
Lymphocytes
Dendritic cells
B cells
T cells
NK cells and cytotoxic T cells
Myeloid-derived phagocytes/granulocytes
Basophils
Eosinophils
Mast cells

Macrophages
Neutrophils

Function
Antigen-presenting cells (APCs); sentinels
distributed throughout the body
Produce antibodies, mediate adaptive immune
responses; antigen-presenting cells
Mediate adaptive immune responses; help B
cells respond to antigens
Kill virally infected and cancerous cells

Circulate in the bloodstream; mediate

inammatory responses and recruit leukocytes
Reside in submucosal tissues; kill multicellular
parasites
Sentinels broadly distributed throughout the
body; initiate inammatory and allergic
responses and recruit luekocytes
Antigen-presenting cells; engulf and digest
bacteria, fungi, dead/dying cells
Engulf and digest viruses, bacteria, protozoa,
fungi, viral infected cells, and tumor cells

already noted, antigen presentation is the way cells respond to pathogens

such as viruses that hide within host cells, thereby eluding direct detection
by the sentinels. The immune system deals with these pathogens by shipping and displaying antigens on the external surfaces of antigen-presenting
cells (APCs), where T cells can recognize them through ligand-receptor
binding. In their role as sentinels, dendritic cells are able to take foreign
materials from the extracellular spaces and display them on their surfaces
leading to activation of T cells. They along with B cells and macrophages
are called professional APCs. All nucleated cells in the body are capable
of displaying peptides derived internally in the cytosol from invading
pathogens. This kind of activity is constitutive so that in the absence of
pathogens only self-molecules are displayed on the surface. These surface
display and recognition processes are the essence of self- versus non-self
recognition. If not dealt with in the treatment of patients, they lead to the
rejection of tissue grafts and organ transplants.
Many of the leukocytes listed in Table 9.1 contain vesicles lled with histamines, hormones, and other inammatory agents; with cytokines for signaling; and with enzymes that mediate the destruction of pathogens. They
release the contents of their vesicles (granules) in response to the appropriate signals, and as a result they are called granulocytes, while leukocytes
that are able to engulf pathogens are referred to in Table 9.1 as phagocytes.

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9. Signaling by Cells of the Immune System

9.2 Leukocytes Signal One Another Using Cytokines

Leukocytes continually send and receive messages from one another, acting
in many ways like a highly mobile nervous system whose job it is to identify and destroy pathogens. Cytokines are small, secreted molecules, usually
less than 30 kDa in mass. They are synthesized and secreted by leukocytes,
most commonly macrophages and T cells. They convey messages to other
leukocytes and to nonhematopoietic cells such as neurons. The cytokine
signals instruct leukocytes to grow, differentiate, mature, migrate, and die.
The signals stimulate antiviral and antitumor activities, and stimulate and
regulate the three kinds of immune responses.
Cytokines, like many proteins belonging to the control layer, are
pleiotropic, or multifunctional, in their actions. The specic physiological
response elicited by a given cytokine is dependent upon cellular context.
Different responses are produced in different cellular environments, that is,
in cells expressing different mixes of proteins. Furthermore, the specic
effect of a cytokine on a leukocyte not only depends upon the type of leukocyte but also upon the leukocytes physiological state. For example, it will
depend upon the cells state of maturation.
Cytokines often act in a redundant manner in which several different
cytokines produce the same physiological response in a cell. Several different cytokines are usually produced at the same time and they act synergistically. The receipt of messages conveyed by cytokines often triggers
another burst of cytokine signals by the recipient thereby creating a cascade
of signaling events. Cytokines convey signals in a targeted fashion. They
usually operate over short distances and have short half-lives. However,
they can convey signals over long distances too. For example, they can send
messages to bone marrow to instruct cells to make more leukocytes. The
overall result of the cytokine signaling is to rapidly activate and recruit cells
of the immune system in response to the onset of an infection.
Interleukins are cytokines secreted mostly by T cells. After T cells, monocytes (and macrophages) are the most prolic interleukin conversationalists. The messages are received by other leukocytes, but especially by B and
T cells, and instruct the recipients to growth and proliferate and to differentiate. A representative sampling of the interleukins is presented in Table
9.2, while a more extensive but compressed listing of cytokines is presented
Table 9.3. As can be seen from an examination of the entries in Table 9.2,
T cells send out multiple cytokines. These signaling proteins work synergistically with one another in cell-dependent ways to induce a variety of
changes in the recipients.
Although colonies of different leukocytes are present at all times, their
numbers are increased when an infection occurs, and then their numbers
are reduced once the infection has been treated. Many of these cells are
maintained in an immature state awaiting signals that instruct them to differentiate into specic, more mature forms. This aspect is reected in the

9.2 Leukocytes Signal One Another Using Cytokines

191

Table 9.2. A sampling of leukocyte-to-leukocyte signaling proteins (interleukins).

Interleukin

Sending cell

Receiving cell

Instructions

IL-1
IL-2

Monocytes and DCs

Th1 cells

Th cells
Activated B, T cells

IL-3
IL-4

Th cells
Th2 cells

Stem cells
Activated B, T cells

IL-5

Th2 cells

Activated B cells

IL-6

Monocytes and other

cells
Stromal cells
T cells
B, T cells, monocytes
Bone marrow stromal
cells
Monocytes and other
APCs

Activated B cells
Stem cells
T cells
Monocytes, Th cells
B cells

Proliferation
Growth and proliferation; Ig
production
Growth and differentiation
Proliferation and differentiation;
promotes Th2 differentiation;
Ig production
Maturation, proliferation and
differentiation; Ig production
Proliferation and differentiation;
Ig production
Growth factor for pre-T, B cells
Growth
Inhibits cytokine production
Growth and proliferation

Th1 cells

Promotes Th1 differentiation

IL-7
IL-9
IL-10
IL-11
IL-12

Table 9.3. Signaling molecules of the immune system.

Family and representative members
Toll/IL-1: IL-1, TLR1-10, Toll

Tumor necrosis factor (TNF): FasL, TNFa,

TRAIL, Apo3L
Hematopoietic (Class I cytokine receptors):
Prolactin, EPO, GH, GM-CSF, TPO, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11,
IL-12
Interferon/IL-10 (Class II cytokine receptors):
IFNa, IFNb, IFNg, IL-10
Chemokine: IL-8, Rantes, MIP-1

Main role(s)
Mediates the innate immune response to
bacterial pathogens; mediates
inammatory responses and stimulates
lymphocytes
Mediates adaptive immune responses of
growth, proliferation, and death
(apoptosis)
Key mediators of the adaptive immune
response; regulates and coordinates
leukocyte activities; functions as
leukocyte-specic growth hormones,
promoting growth and differentiation
Mediates antiviral and antitumor responses,
and promotes the adaptive immune
response
Leukocyte chemoattractants

table by the frequent presence of differentiation in the list of instructions

conveyed by the interleukins. Yet another kind of instruction conveyed by
interleukins is to express a specic set of cell surface molecules, for example,
those belonging to the immunoglobulin (Ig) family of antigen-binding
receptors.

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9. Signaling by Cells of the Immune System

9.3 APC and Nave T Cell Signals Guide Differentiation

into Helper T Cells
Interactions between dendritic cells and nave T cells leading to differentiation into helper T cells is illustrative of interleukin signaling. Immature
dendritic cells serve as sentinels in the body. Their morphology is specialized for capturing antigens from their surrounding. When they encounter
an antigen denditic cells migrate to the secondary lymphoid organs. They
differentiate into antigen-presenting dendritic cells, and interact with nave
T cells thereby stimulating their differentiation into either T1 helper cells
or T2 helper cells (Figure 9.1). T1 and T2 helper cells perform different
immune functions. T1 helper cells secrete cytokines that stimulate actions
by macrophages in inammatory responses. T2 helper cells secrete a mix of
cytokines that triggers the differentiation of B cells into antibody-releasing
plasma cells.

Figure 9.1. Helper T cell development: The rst steps in the specication involve
interactions between the immature dendritic cells and the pathogen they have
encountered. These interactions determine the precise nature of the receptor complexes expressed on the surface of the antigen-presenting dendritic cells and the
type of cytokines secreted when they interact with the nave T cells in the secondary lymphoid tissue. Differentiation is triggered by interactions between the dendritic cell (DC) and a nave T cell (Thp). The Thp expresses IL-2, which, acting in
an autocrine manner on itself, leads to its differentiation into either a Th1 cell or a
Th2 cell. IL-12 production stimulates differentiation into Th1 cells and inhibits formation of Th2 cells. IL-4 production stimulates differentiation into Th2 cells and
inhibits formation of Th1 cells.

9.4 Five Families of Cytokines and Cytokine Receptors

193

9.4 Five Families of Cytokines and Cytokine Receptors

There are ve families of cytokines and cytokine receptors. The ve families, representative members, and their most prominent activities are listed
in Table 9.3. The rst family in the table is the Toll/IL-1 family, named for
the interleukin-1 (IL-1) cytokine, the familys founding member. IL-1 is primarily sent from macrophages to lymphocytes stimulating lymphocyte
activation. When received by granulocytes, IL-1 promotes inammatory
responses. The Toll receptors are key mediators of the innate immune
response. Their ligands are not cytokines but instead are molecular markers
found on the surfaces of pathogens.
The next family of cytokines is the tumor necrosis factor (TNF) family.
The tumor necrosis factor superfamily consists of a group of ligands and
receptors that coordinate and regulate growth, proliferation, and survival
of leukocytes. They coordinate the development of lymphoid organs and
temporary inammatory structures, an essential part of the adaptive
immune response. One group of these proteins conveys death signals that
instruct cells to suicide, or die by apoptosis (discussed in Chapter 15). A
key function of these signals is homeostasis, the maintenance of stable
(baseline) populations and the preservation of a balance between different
subpopulations of cells ready to respond to future infections.
Hematopoietin receptors are sometimes referred to as Class I cytokine
receptors and the interferon/IL-10 receptors as Class II cytokine receptors.
The hematopoietins are a family of more than 20 different signal molecules
that are especially prominent in lymphocyte signaling. As already mentioned, interleukins, secreted by macrophages, T cells, and bone marrow
stromal cells stimulate growth, proliferation, and differentiation of several
cell types. In response to these signals, stem cells differentiate into progenitor B and T cells; B cells differentiate into antibody-secreting plasma cells
and B cells and macrophages express core components of their antigenspecic responses.
Interferons mediate antiviral responses by alerting other leukocytes that
a virus attack is underway and by upregulating genes whose products have
antiviral activities. In addition, interferons act as antitumor agents by downregulating genes important for cell proliferation, and they regulate the
expression of many genes essential for the adaptive immune response.
Lastly, leukocytes are guided to the site of an infection by chemokines, a
family of small, 7- to 15-kDa protein chemoattractants. These molecules are
secreted by several different kinds of cells at an infection site in order to
recruit leukocytes from blood to sites of infection in tissue. Neutrophils,
monocytes, broblasts, endothelial cells, and epithelial cells secrete
chemokines. The chemokines form chemical concentration gradients that
guide the migration of monocytes, neutrophils, eosinophils, and lymphocytes, and they arrest their movement at the appropriate location.

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9. Signaling by Cells of the Immune System

9.5 Role of NF-kB/Rel in Adaptive Immune Responses

The NF-kB/Rel signaling module plays an important role in the inammatory, innate, and adaptive immune responses. The signaling pathway activated in response to Toll receptor binding has several features in common
with the pathway activated in response to tumor necrosis factor receptor
binding. Both utilize signaling through the NF-kB signaling module; both
convey signals through MAP kinase modules, and both use adapters belonging to the TRAF family to link upstream receptor binding to downstream
intracellular signaling.
NF-kB proteins are transcription factors that regulate genes for cytokines,
chemokines, adhesion molecules, antimicrobial peptides, and other factors
important for immune responses. In the absence of activating signals these
proteins form an inactive cytosolic module with their IkB inhibitors, and a set
of IKK regulatory kinases.The IKK proteins consist of two catalytic subunits,
IKKa and IKKb, and a regulatory subunit, IKKg, also called NF-kB essential
modulator (NEMO). Both of the IKK catalytic subunits are competent to
phosphorylate IkB proteins.The IKKs are themselves activated by upstream
kinases,and are the key point of convergence of a variety of regulatory events
triggered by extracellular signals and intracellular stresses.
There are ve members of the NF-kB family of proteins: NF-kB1
(p50/p105), NF-kB2 (p52/p100), c-Rel, RelB, and RelA (p65). In the
absence of activating signals, these proteins are sequestered in inactive
forms in the cytosol by inhibitory IkB factors. When the repressive effects
of the IkBs are lifted, the NF-kBs form homo- and heterodimers, the most
common combination being p65/p50, and translocate to the nucleus where
they function as transcription factors (Figure 9.2).
The NF-kB1 and NF-kB2 proteins are formed and sequestered in the
cytosol as p105 and p100 precursors. They are proteolytically processed to
make the functional p50 and p52 forms. The C-terminus portion that is

Figure 9.2. Binding of a NF-kB heterodimer to DNA: Shown is a p65/p50

NF-kB heterodimer (ribbon model)
bound to an IFNb enhancer (ball-andstick representation) viewed looking
down along the DNA axis. The gure
was prepared using Protein Explorer
with atomic coordinates deposited in the
PDB under accession numbers 1le5.

9.5 Role of NF-kB/Rel in Adaptive Immune Responses

195

Figure 9.3. The NF-kB node: Extracellular stimuli and intracellular stresses activate upstream protein kinases, which phosphorylate the IKKs. In response, the IKKs
phosphorylate the IkB proteins, triggering either ubiquitination/degradation and/or
nucleocytoplasmic shuttling, depending on the upstream signals and subunits
present. The NF-kB proteins form dimers that translocate to the nucleus where they
stimulate transcription of NF-kB responsive genes.

removed from p105 and p100 contains a series of ankyrin repeats that
are also present in the IkB proteins and mediates their sequestration.
Ankyrin repeats are 33 amino acid residues, protein-protein interaction
modules. They are found in many signaling proteins, usually as four or more
tandem-arranged repeated copies. The chain of steps leading to mobilization of the NF-kB proteins is depicted schematically in Figure 9.3.
In the absence of activating signals, the IkB proteins bind to sites in
the Rel homology domain (RHD) of the NF-kBs, thereby masking their
nuclear localization sequences (NLSs) and forcing their retention in the
cytosol. The IkBa proteins contain nuclear export sequences (NESs) and,
when they are bound to nuclear NF-kB dimers, they promote their export
to the cytosol. The inhibition on NF-kB by the IkB proteins is relived by
phosphorylation, which induces the ubiquitin-mediated degradation of the
IkBs by the 26S proteasome (Figure 9.3). IkB proteins contain N-terminal
serines that are the substrates of IkB kinases (IKKs).
The NF-kB module turns on and then turns off. One of the genes upregulated by the NF-kB proteins encodes IkBa. Signaling through the TNF
receptor leads to increased migration of NF-kB proteins to the nucleus
where they stimulate transcription of the gene for IkBa, which once synthesized binds to NF-kB, thereby shutting down its response to TNF.
Because of the time delays inherent in the signaling pathway the NF-kB
protein activity levels move up and down over time. The other IkB subunits
do not depend on the NF-kB proteins for their transcription. The subunits
activity remains fairly constant over time, enabling them to smooth out the
oscillations in NF-kB activity brought on by the negative feedback.

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9. Signaling by Cells of the Immune System

9.6 Role of MAP Kinase Modules in

Immune Responses
MAP kinase modules convey cytokine and stress signals in the inammatory, innate, and adaptive immune responses. Mitogen-activated protein
(MAP) kinase modules convey cytokine, stress, and growth signals that are
sent to them from the plasma membrane to the nucleus where they inuence transcription of target genes. There are three mammalian MAP kinase
pathways. One of these, the extracellular signal-regulated kinase (ERK)
pathway, primarily carries growth signals. The other two, the c-Jun NH2terminal kinase (JNK) and p38 MAP kinase pathways, relay inammatory
cytokine and stress signals.
As was the case for the yeast MAP kinases discussed in Chapter 6, there
are three kinases in a MAP kinase module. The rst of these is a serine/
threonine kinase. It phosphorylates the second kinase in the module, which
is a dual specicity kinase. The middle kinase phosphorylates the third
kinase in the module at threonine and tyrosine residues, hence the name
dual specicity. The target residues are arranged as Thr-X-Tyr, where X is
either Glu, Pro, or Lys for the ERK, JNK, and p38 pathways, respectively. Once they are phosphorylated, the third and last kinases in the
module typically stimulate the transcriptional activity of members of
several families of transcription factors. The three families, their upstream
activating signals, and their downstream targets are depicted schematically
in Figure 9.4.
The Ras and Rho GTPases function as molecular switches that relay
growth, cytokine, and stress signals from receptors and their adapters and
other intermediaries to the MAP kinase modules. Ras is a crucial regulator of growth signals. When its gene is mutated at certain residues it can get
stuck in the on position and will continually send inappropriate growth
messages to the ERK MAP kinase module. Ras will be discussed in more
detail along with the other GTPases and their upstream growth factor
receptors in Chapter 11 and again in Chapter 14, on cancer.

9.7 Role of TRAF and DD Adapters

TRAF and DD adapters transduce signals from TNF receptors into the
cell. Scaffolds, anchors and adapters were introduced in Chapter 6. These
proteins are not enzymes, but rather they organize the signaling nodes
and control points where the enzymes can interact with one another and
with the xed infrastructure. Scaffolds that help organize MAP kinase
cascades were examined in Chapter 6; anchors that tether serine/threonine
kinases to membranes were discussed in the last chapter, and now an

9.7 Role of TRAF and DD Adapters

197

Figure 9.4. Mammalian MAP kinase modules:The upstream activators fall into two
groups. (a) The ERK MAP kinase module is activated by growth and mitogenic
signals relayed to it by the Ras GTPase. (b) The JNK and p38 MAP kinase modules
are activated by proinammatory cytokines and stress signals relayed to them by
the Rho family of GTPases.

important class of adaptersthe TRAF proteinswill be introduced.

The tumor necrosis factor (TNF) receptor-associated factors, or TRAFs,
help organize signaling nodes in pathways that mediate innate and
adaptive immune responses and stress responses. These proteins contain a
TRAF domain in their C-terminus. The TRAF domain consists of two
portions: a TRAF-C domain that interfaces to the receptors and a coiledcoil domain that mediates interactions with other TRAFs. These are shown
in Figure 9.5.
The TRAF adapters act as key intermediaries between the receptors and
downstream signaling elements. The N-terminal domains of the TRAFs
contain motifs called zinc and RING ngers. These motifs consist of
arrangements of four cysteine and/or histidine residues that bind zinc ions,
facilitating the formation of a compact domain that, along with the Cterminus coiled-coil domain, mediates downstream signaling. There are six
mammalian TRAFs, named TRAF1 through TRAF6. The TRAF2 and
TRAF6 proteins are crucially involved in TNF and Toll signaling, where
they function as adapters that link the receptors to downstream NF-kBs,
AP-1 signaling, and MAP kinase cascades.

198

9. Signaling by Cells of the Immune System

Figure 9.5. Structure of the TRAF domain of the TRAF2 adapter determined by
X-ray crystallography: (a) Top viewShown is a trimeric arrangement of TRAF
domains that transduce signals from a 3 : 3 arrangement of TNF receptors and
ligands. (b) Side viewThe overall shape of the signaling unit resembles a mushroom with the C-TRAF domains forming the cap and the coiled-coil domains
making up the stalk. The gure was prepared using Protein Explorer with atomic
coordinates deposited in the PDB under accession number 1ca9.

9.8 Toll/IL-1R Pathway Mediates Innate

Immune Responses
A group of plasma membrane-bound receptors called the Toll/IL-1R family
plays a key role in leukocyte responses to bacterial lipopolysaccharide
(LPS). The Toll/IL-1R signaling pathway activated in response to ligand
binding by receptors in this family is an ancient one. It has been identied
in plants, in insects (Drosophila), where it is known as the Toll/Dorsal
pathway, and in vertebrates where it is referred to as the IL-1R/NF-kB
pathway.
LPS is a prominent component of the outer membrane of gram-negative
bacteria such as E. coli. The sensing of the presence of LPS by leukocytes,
most notably, macrophages, activates the Toll/IL-1R signaling pathway. The
focus of this pathway is the nuclear factor-kB (NF-kB) and a MAP kinase
cascade leading to activation of members of the AP-1 family of transcription factors such as c-Jun (Figure 9.6). Ligand binding results in the rapid
activation and subsequent translocation of NF-kB to the nucleus, where it
and c-Jun upregulate the expression of genes for IL-1, IL-6, interferons,
TNF, the cell adhesion molecules ICAM-1 and E-selectin, and the
chemokine IL-8 (not shown). Signaling through this pathway not only starts
an infection-containing innate immune response, but also launches the
adaptive immune response.

9.9 TNF Family Mediates Homeostasis, Death, and Survival

199

Figure 9.6. Signaling through the IL-1/Toll pathway: Shown are the IL-1R and its
associated IL-1RAcP receptor along with several adapter proteins, namely, MyD88,
Tollip, and ECSIT, which assist in recruiting kinases and other signaling elements
to the plasma membrane. The rst step in their activation is the recruitment of the
adapter proteins to the plasma membrane. The serine/threonine kinase IRAK (IL1R-associated kinase) and the TRAF6 adapter are then assembled at the site. These
proteins activate a number of upstream kinases collectively designated in the gure
as Tak1/Tab1/NIK, which then activate the NF-kB pathway and the JNK/p38 MAP
kinase pathways.

9.9 TNF Family Mediates Homeostasis, Death,

and Survival
Cell suicide, or apoptosis, instructions are an important part of the immune
response. Immature T lymphocytes that do not respond properly to selfantigens are eliminated in the thymus through apoptosis. Other thymocytes
that do not have the correct arrangement of their T cell receptors (TCRs)
are disposed of in this way, too. At the end of an immune response, superuous, mature, activated T cells are removed, and the immune system is
returned to its basal level ready to respond to the next infection. The maintenance of basal levels of the different kinds of leukocytes when there are
no infections are present is referred to as cellular homeostasis. It relies on
apoptosis to remove the superuous leukocytes.
Apoptosis signaling mechanisms are not only used for elimination of
incorrectly functioning and superuous cells. It provides a mechanism for
ensuring that cells do not survive outside of the tissue to which they belong.
It is used by cytotoxic T cells to kill virus-infected cells and cancerous cells.

200

9. Signaling by Cells of the Immune System

Figure 9.7. Upstream signaling in the TNF pathways: Trimeric arrangements of

ligands, receptors, and adapters convey messages to signal enzymes. (a) Apoptosis
signals are conveyed by death domain-bearing receptors and adapters among which
are Fas-associated death domain (FADD) proteins, TNF-R-associated death
domain (TRADD) proteins and receptor-interacting proteins (RIPs). (b) Inammatory cytokine- (e.g., IL-6 and IFNg) promoting signals.

Some tissues such as the eye, testis, and parts of the central nervous system
cannot tolerate inammation. These are privileged sites that insulate themselves from immune responses. If a lymphocyte comes into contact with a
cell in any of these sites it will rapidly undergo apoptosis. Tumor cells trying
to evade the immune response use this mechanism, as well.
The TNF receptors can be divided into two groups according to the kind
of adapter proteins they use. Members of one group of TNF receptors have
TRAF-binding motifs in their cytoplasmic domain, which mediate binding
to TRAFs, while members of the second group have death domain (DD)
motifs and bind to DD-bearing adapters. Receptors belonging to the second
group are called death receptors because they convey cell suicide (apoptosis) instructions to the recipient. Upstream signaling events in these two
TNF pathways are depicted in Figure 9.7. Most TNF ligands are single-pass
transmembrane proteins with their C-terminus outside the cell and their Nterminus in the cytoplasm. They contact TNF receptors expressed on the
surface of an opposing cell. The arrangement of receptors and ligands is
3 : 3. Three receptors arranged symmetrically contact three ligands. This
signaling arrangement is facilitated by a trimeric arrangement of adapters
of the form illustrated in Figure 9.5.

9.10 Role of Hematopoietin and Related Receptors

Hematopoietin receptors bind most of the interleukins, while a closely
related set of receptors binds the interferons. Members of the hematopoietin family of cytokines are leukocyte hormones and growth factors.
Although all members of this family of ligands and receptors have similar

9.10 Role of Hematopoietin and Related Receptors

201

Table 9.4. Hematopoietic and interferon receptors, Jaks and STATs:

Abbreviationserythropoietin (EPO); prolactin (PRL); thrombopoietin (TPO);
growth hormone (GH); granulocyte macrophage colony stimulating factor
(GM-CSF).
Receptor
Class I-Hematopoietin cytokines:
Homodimeric receptors:
EPO, PRL, TPO
GH
Receptors that share gc:
IL-2, IL-7, IL-9, IL-15
IL-4
Receptors that share bc:
GM-CSF, IL-3, IL-5
Receptors that share gp130:
IL-6, IL-11

Jak

STAT

Jak2
Jak2

STAT5a, STAT5b
STAT5a, STAT5b, STAT3

Jak1, Jak3
Jak1, Jak3

STAT5a, STAT5b, STAT3

STAT6

Jak2

STAT5a, STAT5b

Jak1, Jak2, Tyk2

STAT3

IL-12:
IL-12Rb1, IL-12Rb2

Jak2, Tyk2

STAT4

Class II-Interferon/IL-10:
IFNa, IFNb
IFNg
IL-10

Jak1, Tyk2
Jak1, Jak2
Jak1, Tyk2

STAT1, STAT2
STAT1
STAT1, STAT3, STAT5

structures, there are some differences in structure and subunit composition

that allow grouping the family members into subfamilies. This breakdown
is presented in Table 9.4. Hematopoietic receptors are assembled from two
or three subunits, each a single-pass glycoprotein. Each receptor contains a
ligand-binding subunit that is unique for that receptor type, plus one or
more subunits that are common, or shared, by several other receptor types.
A number of cytokines have a common beta chain (bc); others share a
gamma chain (gc); still others have a common gp130 subunit. The IL-12
group of cytokines listed in the table departs somewhat from the overall
pattern. This group consists of cytokines that bind to homodimeric chains.
Interferon and IL-10 receptors (Class II) are constructed from multiple
unique subunits and in this way differ from the other grouping in the table,
which are collectively called Class I receptors.
Cells regulate the binding activity of their plasma membrane receptors
by varying the subunit composition. Cells can switch between low, medium,
and high afnity-binding by differentially expressing different receptor
subunits. Several examples of this form of control are supplied by the
cytokine receptors. IL-2 is composed of IL-2Ra, IL-2b, and IL-2g subunits.
The IL-2g chain is expressed widely while the other two subunits are
expressed in a restricted fashion. By varying the subunit composition the
receptor afnity for its cognate ligand can change from high afnity to low
afnity with one or more intermediate afnity complexes.A similar depend-

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9. Signaling by Cells of the Immune System

ence of binding afnity upon receptor subunit composition occurs in the

IL-12 and IL-6 receptor systems.
Cytokine ligands are typied by their structure, a four-helix bundle fold
consisting of two pairs of antiparallel a-helices linked together by loops.
Some are short chain cytokines; IL-2, 3, 4 are constructed from short
a-helices, 8 to 10 residues in length. Long chain cytokines such as GH, EPO,
G-CSF, and the gp130 cytokines are built from chains that are longer, 10 to
20 residues in length. A third group of cytokine ligands, notably IL-5 and
IFN-g, are formed as a doubled four-a-helix fold, and contain eight a-helices.

9.11 Role of Human Growth Hormone Cytokine

The human growth hormone (hGH) cytokine and its receptor serve as a
model system for cytokine receptor-ligand recognition. The hematopoietin
chains bind their ligands in characteristic ways. In Figure 9.8, one molecule
of the growth hormone ligand simultaneously binds two growth hormone
receptor chains. This 1 : 2 binding is fairly typical of the entire group. An
examination of the human growth hormone provides some insights into
how this happens. Each hGH molecule contains two receptor-binding sites.
One interface is located in helix 4 and the other is formed from helices 1
and 3. Thus, a single molecule of hGH forms a homodimeric complex with
a pair of hGH receptor molecules.
Not all residues in the hGH ligand-hGH receptor interface contribute
equally to the binding energy. Instead, a few residues located in the vicinity

Figure 9.8. Structure of the human growth hormone (hGH) ligand bound to two
hGH receptors: Shown are two extracellular domains of the hGH receptors bound
to a single hGH ligand. The hGH ligand binds in the cleft between the two extracellular domains of the receptors. The gure was prepared using Protein Explorer
with atomic coordinates deposited in the PDB under accession number 1hwg.

9.12 Signal-Transducing Jaks and STATs

203

of the center of the interface contribute most of the binding energy.

These residues form a functional epitope, or hot spot, that mediates
ligand-receptor binding. Ligand engagement occurs when residues belonging to ligand and receptor portions of the hot spot come into close proximity.
During the binding process an initial contact between the ligand and receptor surfaces is established. The contact is followed by a random diffusion
stage where one surface moves (rolls) relative to the other until the motion
is stabilized. This happens when the two portions of the functional epitope
make contact. The maintenance of close surface contact through rolling
diffusion greatly accelerates the association rate over that which would
result from a single collision followed by a separation and then another
elastic collision, and so on, until the correctly oriented surfaces are engaged.
The 1 : 2 association of the hGH ligand with its receptors is more efcient
than a 2 : 2 association. The 1 : 2 complex is formed in a stepwise fashion.
In the rst step, a ligand molecule nds and establishes contact with one
receptor molecule. The 1 : 1 complex then makes contact with the second
(unbound) receptor molecule to create a stable 1 : 2 signaling unit. The
advantage to this mechanism is that the second step involves diffusion in
two dimensionsthe second receptor molecule moves along membrane
surface to contact the bound pairrather than three.This may be compared
to the case where the second step is another single ligand-single receptor
binding event followed by association of the pairs. In this latter 2 : 2 scenario
a second three-dimensional diffusion process would be required.
Many of the cytokines bind their receptors in a manner similar to that of
the human growth hormone. Like hGH, the ligands for the bc family of
receptors have two binding sites. Members of the bc family form high afnity complexes stepwise in the following manner. The ligand rst binds the
Ra subunit. The bc chain then binds to the bound pair to form a 1 : 1 : 1
complex. Two complexes then come together to form a dimerized complex
that activates the intracellular components of the signaling machinery.
Hematopoietin receptors such as IL-2R that belong to the gc family are
composed of three distinct subunits. A single ligand molecule possesses
three binding sites and so is able to bind the three subunits when forming
the high afnity 1 : 1 : 1 : 1 complex. A similar process is used by the gp130
cytokines to form stable complexes that are signaling-competent.

9.12 Signal-Transducing Jaks and STATs

Jaks and STATs transduce signals into the cell from the plasma membrane.
The reason for receptor dimerization can be understood by noting that
there is considerable exibility in the membrane-spanning polypeptide
chains. Under these conditions the binding of a ligand to the extracellular
portion of a chain will not produce a large and long-lasting (stable) shift in
conformation of the cytoplasmic portion of the chain needed to serve as a

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9. Signaling by Cells of the Immune System

Figure 9.9. Domain organization of Jaks and STATs: (a) Janus tyrosine kinases, or
Jak proteins. (b) Signal transducer and activator of transcription (STAT) proteins.

signal. The solution to this problem of how to transduce a signal into the
cell is solved by dimerization. The combined effect of ligand binding and
dimerization alters the intracellular electrostatic environment sufciently
to stabilize a different mix of conformational states that will activate the
Jaks, triggering phosphorylation and the subsequent recruitment of the
STATs and other signaling elements to the receptors. The Jaks (tyrosine
kinases) and STATs (transcription factors) are then able to transduce the
hematopoietin signals into the cell responses.
The domain organization of the Jak and STAT proteins is presented in
Figure 9.9. As shown in part (a) of the gure, the Janus kinases possess
kinase and pseudokinase domains that face one another, hence the name
Janus kinase (Jak), named after the Roman god of gates and doorways.They
also possess a receptor-binding region that mediates their binding to recognition motifs in the cytoplasmic terminal of the cytokine receptors. Once
recruited to the receptors, autophosphorylation occurs, resulting in their
activation. The activated kinases then phosphorylate the receptors, thereby
exposing docking sites for the STATs. As depicted in part (b) of the gure,
the STATs possess a dimerization domain, an SH2 domain, a tyrosine phosphorylation site, and a DNA-binding domain. The signal transducer and
activator of transcription proteins are transcription factors, and they are
recruited to the activated signaling complex formed by the phosphorylated
cytoplasmic receptor terminals and Jaks. They bind the phosphorylated
tyrosine residues in the receptors through their SH2 domains, and they are
phosphorylated on their tyrosine residues by the Jaks. Once these recruitment and activation steps take place, the STATs are able to form heterodimer and homodimers, and translocate to the nucleus where they
stimulate transcription of their target genes.

9.13 Interferon System

205

9.13 Interferon System: First Line of Host Defense in

Mammals Against Virus Attacks
Interferons are secreted into the bloodstream where they circulate to all
cells in the body to alert them that a virus attack is underway. They trigger
an organism-wide, or global, response to the virus attack in cells that have
not been attacked along with those that have been invaded. Invasion of a
virus and the resulting viral replication stage produce dsRNA (doublestranded RNA) molecules. These are present in the cell only when a virus
has invaded and started replication. The presence of dsRNA sets off a series
of regulatory events. A resident cellular sensor of dsRNA called protein
kinase R (PKR) is activated by the dsRNAs. The PKR proteins activate a
NF-kB, and IFN regulatory factor (IRF), which binds within the IFN stimulated responsive element (ISRE) to promote transcription of the antiviral
proteins including the interferons. In addition, PKR signals other control
proteins to halt all protein synthesis (Figure 9.10). This action blocks the
virus ability to replicate and make proteins that it needs. The overall process operates though a negative feedback loop. The presence of dsRNA is
required for action by PKR, and when this quantity is reduced the block
on protein synthesis is relieved.
Interferon signal transduction is initiated when a ligand binds to an
interferon receptor dimer (Figure 9.11). As is typical of the hematopoietins this event stimulates the recruitment to the plasma membrane and activation of members of the Janus family of protein tyrosine kinases.
Phosphorylation stimulates the further recruitment of the STAT proteins, which are phosphorylated by the Jaks. In the case of IFNa/bmediated signaling, Jak1 and Tyk2 phosphorylate STAT1 and STAT2,
which then form heterodimers that further associate with an IRF protein.

Figure 9.10. Antiviral activities of PKR: Protein kinase R, a sensor of dsRNAs,

stimulates production of interferons by activating NF-kB and IRF (not shown) transcription factors, and halts protein synthesis by activating eIF2a, a negative regulator of protein synthesis.

206

9. Signaling by Cells of the Immune System

Figure 9.11. Interferon signaling: Displayed in the gure are the main components
of the IFNa/b and IFNg pathways from the plasma membrane to the nucleus. Distinct a and b chains of each receptor jointly bind a ligand. Janus family kinases are
recruited and are activated. They create docking sites for the STATs by phosphorylating the receptors. The STATs are recruited and are phosphorylated by the Jaks.
STATs form dimers (trimers) and translocate to the nucleus where they bind to
interferon-responsive DNA transcriptional sites, the ISREs and interferm-gamma
activated sites (GASs).

The trimers translocate to the nucleus where they stimulate transcription of IFNa/b-responsive genes IFNg utilizes a different set of signal
transducers. Jak1 and Jak2 are recruited to the plasma membrane and
phosphorylate STAT1s, which form homodimers that translocate to the
nucleus where they stimulate transcription of IFNg-responsive genes.
Cytokines such as the interferons mount a multilayered response. Besides
arrest of protein synthesis and cell multiplication, they stimulate the
production of enzymes that kill (degrade) the viruses, recruit cells that
attack virus-containing cells, and organize other elements of the adaptive
immune response.

9.14 Chemokines Provide Navigational Cues

for Leukocytes
Chemokines are chemoattractants that guide the movement of leukocytes.
Some chemokines are inammatory chemokines while others are lymphoid
chemokines. Inammatory chemokines attract neutrophils, macrophages,
and other leukocytes central to the innate immune response. Inammatory
chemokines are secreted by several different kinds of cells including leukocytes and endothelial cells that line the blood vessels. Lymphoid chemokines
help regulate leukocyte trafc and cell compartmentalization within lym-

9.15 B and T Cell Receptors Recognize Antigens

207

phoid tissues. Thus, they act to maintain homeostasis among the various
populations of lymphocytes. Stromal cells, endothelial cells, and other cells
residing in the lymphoid organs produce these signaling molecules.
Chemokines have a positive charge and bind to heparin and heparin
sulfate, negatively charged polysaccharides found within the extracellular
matrix and on the surface of endothelial cells. The heparin and heparin
sulfate molecule act as receptors for the chemokines. The chemokines
become immobilized when bound to the polysaccharide-covered substrates
and form stable gradients of chemokine concentration. The migratory
leukocytes navigate up the gradients to the target sites.
Chemokines are bound at the cell surface by G protein-coupled receptors (GPCRs), seven-pass transmembrane receptors coupled to G proteins.
Their method of transducing signals into the cell was examined briey in
the last chapter and will be discussed in detail in Chapter 12. There are four
families of chemokine receptors. These families are distinguished from one
another by their structure and by their chromosomal locations. The distinguishing structural feature is the presence of four conserved cysteine (C)
residues in specic positions. The CXC and CC families are fairly large with
several members each, while the two small families, designated as CX3C
and C, have a single known member each. The CXC family has an amino
acid located between the rst and second cysteines; in the CC family the
two cysteines are directly adjacent to one another. There is considerable
redundancy; within each ligand-receptor family, the chemokines can bind
to more than one type of receptor, and the receptors bind to more than one
type of chemokine.
Lymphoid chemokines form chemical highways within lymphoid organs
that guide the migration of lymphocytes from site to site bringing together
lymphocytes that must interact with one another in the adaptive immune
response. The B-cell attracting chemokine (BCA-1), and secondary lymphoid chemokine (SLC) are representative lymphoid chemokines. They
bind the CXCR5 and CCR7 receptors, respectively. Gradients of BCA-1
chemokines guide the entry of T cells and dendritic cells into lymphoid
organs. Similarly, gradients of SLC are crucial for compartmental homing
of B cells and T cells, helping to establish distinct territories within the secondary lymphoid organs.

9.15 B and T Cell Receptors Recognize Antigens

The rst stage of the lymphocyte immune response is the production by B
cells of antibodies, receptors that bind to specic antigens. There are two
classes of antigen-recognizing receptors, one set expressed on B cells and
referred to as B cell receptors (BCRs), and the other set expressed on T
cells and referred to as T cell receptors (TCRs). Major histocompatability
complexes (MHCs) also recognize antigens, and these may be regarded as

208

9. Signaling by Cells of the Immune System

a third class of antigen-recognizing receptors. All are members of a large

family of glycoproteins known as immunoglobulins (Igs) that share a similar
structure and fold.
Each B cell expresses on its surface a large number of a single kind of
Ig molecule. When a B cell encounters an antigen that it recognizes (i.e.,
that it binds), it matures and differentiates into a plasma cell that produces
many more antibodies of that specic type. These antibodies are no longer
membrane-bound receptors but instead are secreted and become free to
diffuse and bind to their specic antigen whenever that antigen is exposed
on the surface of the bacterium or other pathogen. This binding event not
only tags that object for destruction by other leukocytes, but also inactivates viruses and bacterial toxins by impeding their ability to attach to their
cellular targets.
At least two distinct signals are needed to elicit cellular responsiveness.
Antigen binding to the BCR is the rst signaling step. The B cell ingests the
antigen-antibody complex, processes the antigen, and presents it bound to
an MHC Class II molecule on its cell surface. Activated helper T (Th) cells
possessing receptors that recognize that specic antigen convey the second
signal. Signals relayed into the Th cell trigger the production and release of
cytokines by the Th cell. The cytokines are bound to cytokine receptors on
the B cell. This second signal triggers B cell differentiation and proliferation and the release of antibodies from the resulting plasma cells.
T cells target pathogens such as viruses and small bacteria that hide inside
other cells. Antigen receptors on TCRs recognize short peptide sequences,
typically 8 to 15 residues long, belonging to an antigen that has been
processed and bound to MHC molecules expressed on the surface of MHCpresenting cells. The TCR recognition process differs from BCR antigen
recognition since the latter targets entire molecules, either denatured or
native forms of proteins or cell-bound carbohydrates. Both the BCR and
TCR molecules have distinct antigen-binding and signaling units. Unlike
the BCR, TCRs are not later made in quantity and secreted but instead
remain membrane-bound. The signaling tail of the T cell receptor associates with a set of chains collectively termed CD3. The CD3 chains, and a
pair of disulde-linked z chains, are immunoglobulin family members that
assist the TCR molecule in transducing signals into the cell.

9.16 MHCs Present Antigens on the Cell Surface

Almost all cells in the human body express MHC proteins on their surface.
Between 104 and 106 Class I MHCs are present on the surface of a typical
nucleated cell, and similar numbers of Class II MHC proteins are expressed
on the surfaces of MHC Class II-expressing cells. Each MHC allele can bind
some 1000 to 2000 different self-peptides represented at greater than 10
copies per cell and up to several hundred in some cases. Pathogen proteins
are encountered at far smaller numbers. 102 to 104 copies accumulate on

9.17 Antigen-Recognizing Receptors Form Signaling Complexes

209

the cell surface. Pathogen peptides trigger responses even though they are
only a small fraction of the total peptide fragment populationfrom 0.01
to 0.1%. Thus, the MHC must exhibit high sensitivity and selectivity of
foreign peptides while maintaining low responsiveness to self-peptides.
Different individuals express different mixes of these molecules. These
proteins lie at the core of self versus not-self recognition. The job of an
MHC is to present antigens to T cells and to NK cells. Class I MHCs interact with CD8 receptors on cytotoxic T cells and with KIR receptors on NK
cells. Class II MHCs interact with CD4 receptors on helper T cells. Class I
members bind peptides derived from molecules encountered in the cytosol
while Class II members bind peptides from molecules broken down in the
endosomes.
What the MHCs of both classes have to do is present the broadest range
of peptides possible while simultaneously satisfying the requirement that
these peptides be presented in a way that maximally stimulates TCR
responses. The minimal size of a peptide fragment needed to discriminate
between self and foreign is about 8 or 9 amino acid residues. One way of
satisfying the broadness requirement is to have several binding sites along
the MCH. However, this would violate the TCR stimulation requirement.
To maximally induce a TCR response, there should be a single peptidebinding site with each peptide of a given sequence binding to a given MHC
allele in exactly the same way. The solution is to have a single binding
pocket and a way to generate a spectrum of different MHCs utilizing a combination of conserved and nonconserved (variable region) residues in the
binding pocket.

9.17 Antigen-Recognizing Receptors Form Signaling

Complexes with Coreceptors
The mixes of proteins expressed on the surfaces of leukocytes are unique
markers of the population or subpopulation to which the leukocytes belong.
The different ensembles of proteins reect not only the kind of leukocyte
but also characterize the different stages of leukocyte development and
activation/inactivation. The proteins that can be so used are given a cluster
of differentiation, or CD, designation because of their association with specic lineages or stages of differentiation. CD proteins function as receptors,
coreceptors, and as cell adhesion molecules.
Transmembrane-signaling proteins that form signaling complexes with
the BCRs and TCRs are well represented in the CD listing. The two main
classes of T cells are distinguished from one another by the presence of
either CD4 or CD8 molecules on their surfaces. CD4 cells such as helper T
cells signal other leukocytes to converge on the site of an infection. The
CD4 molecula acts in concert with T cell receptors to bind peptides derived
from antigens bound to MHC-II molecules. (The TCR binds the peptide,
while the CD4 molecule binds MHC-II). The T8 group of T cells, such as

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9. Signaling by Cells of the Immune System

Figure 9.12. T cell receptor (TCR) signal transduction pathways leading to expression of genes activated by AP-1, NF-kB, and NFAT transcription factors working in
concert: In response to ligand binding at the TCR, CD45 (a receptor tyrosine phosphatase) and CD4 activate Fyn and Lck. Fyn and Lck phosphorylate the g, d, and e
chains of the CD3 complex and the z dimer. After phosphorylation, ZAP-70 containing two SH2 domains is recruited and binds to the phosphatyrosines and is then
activated through phosphorylation by Lck. ZAP-70 then interacts with PLC-g, which
subsequently cleaves PIP2 to produce IP3 leading to the aforementioned increased
concentration of intracellular calcium. DAG activates PKC, which serves as the
upstream kinase for activation of NF-kB, while ZAP-70 acts through a RasGEF,
Ras, and a MAP kinase cascade leading to activation of AP-1. Intermediate steps
involved in activation of NF-kB by PKC have been omitted for simplicity.

cytotoxic T cells, express CD8 proteins on their surfaces. The CD8 proteins
act in concert with the T cell receptors to bind peptides derived from antigens bound to MHC-I molecules.
Several transmembrane proteins must associate with one another for efcient signaling into the cell. The upstream signaling starts with ligand
binding and association of the TCRs with their CD4 or CD8 coreceptors
and with CD3 chains (Figure 9.12). CD3 consists of several separate chains,
denoted as d, g, and e. In addition, there is a fourth kind of chain, called z.
These chains form homo- and heterodimers that associate with the a/b
chains of the TCR. The CD3 and z chains possess immunoreceptor tyrosine-based activation motifs, or ITAMs, in their cytoplasmic regions. These
enable the CD3 chains to recruit and bind Fyn and Lck protein tyrosine
kinases (protein tyrosine kinases will be discussed in detail in Chapter 11),
triggering a series of steps leading to activation of AP-1, NF-kB, and NFATresponsive genes.
Signaling through the TCR and its associated proteins culminates in the
transcription of genes-encoding receptors, signal transducers, cell adhesion

9.18 Costimulatory Signals Between APCs and T Cells

211

molecules, and cytokines. One of the most prominent of the cytokines

upregulated through TCR signaling is IL-2. Compared to the straightforward signal transduction pathways utilized by cytokines, signaling through
the TCR is far more complex. The steps leading to IL-2 production, along
with many other gene products, involve activation of the lipid second messenger pathways discussed in the last chapter. Intracellular calcium levels
are increased through stimulation of phospholipase C activity leading to a
greater IP3 production, which increases the concentration of intracellular
calcium. The calcium ions along with calmodulin activate the protein phosphatase, calcineurin, which, in turn, dephosphorylates and thus activates the
nuclear factor of activated T cells (NFAT) transcription factors. The NFATs
work in cooperation with members of the AP-1 family of transcription
factors and the NF-kB transcription factor. These transcription factors stimulate the transcription of genes leading to the differentiation and proliferation of the T cells.

9.18 Costimulatory Signals Between APCs and T Cells

The task of discriminating between self and non-self is a difcult one.
Several failsafe mechanisms operate to ensure that inappropriate actions
are not taken by B cells and T cells. One of the mechanisms is the requirement that there be two independent activating signals before T and B cells
are fully activated. The rst signals are conveyed by the BCRs and TCRs
in association with their CD4 and CD8 coreceptors. The second set of
signals is sent into the cell via costimulatory pathways. The most prominent
of the costimulatory pathways are the CD28/B7 system and the
CD40/CD40L signaling pathway (Table 9.5).
The rst entry in Table 9.5 is the CD40 receptor and its ligand. They
are members of the TNF superfamily. Costimulatory signals supplied via
CD40 are an important part of the help supplied by helper T cells. The
CD40 ligand is found on CD4+ and CD8+ T cells, while the CD40 receptor
is found on APCs such as B cells and dendritic cells. Signaling through
Table 9.5. T lymphocyte costimulators: AbbreviationsInducible costimulator (ICOS); programmed
death-1 (PD-1).
Receptor

Ligand

Comments

CD40

CD40L

TNF superfamily

CD28/B7
CD28
CTLA-4
ICOS
PD-1

B7-1, B7-2
B7-1, B7-2
ICOSL
B7-L1, B7-L2

Ig superfamily
Positive regulator
Negative regulator
Positive regulator
Negative regulator

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9. Signaling by Cells of the Immune System

CD40/CD40L activates resting B cells and primes the dendritic cells to adequately stimulate the T cells.
The remaining receptors and ligands all belong to the CD28/B7 branch
of the immunoglobulin (Ig) superfamily. The rst of these, CD28, is constitutively expressed on the surface of most T cells. Signaling through CD28
helps determine how the T cells will differentiate. In the absence of the costimulatory signals the T cells undergo anergy, becoming nonresponsive, or
tolerant, of antigens presented on the surface of the antigen-presenting cells
(APCs). In this state the T cells are inactive. They do not proliferate nor do
they send out cytokines signals. The co-stimulator signals not only prevent
anergy but also help determine whether the activated T cell will become a
helper T cell or an effector T cell.
CD28 and CTLA-4 both bind to a pair of ligands, B7-1 and B7-2. The
CTLA-4 receptor is transiently expressed. It has a higher afnity for the B7
ligands than does CD28, and when it is expressed it shuts down the signaling.
A second pair of positive and negative regulators, ICOS and PD-1, also
appears in the table.These glycoproteins are not constitutively expressed but
are inducible through CD28 signaling and act later during immune responses.
The positive regulator, ICOS, helps promote B-T cell interactions while
PD-1, like CTLA-4, halts cytokine production and the cell cycle progression.

9.19 Role of Lymphocyte-Signaling Molecules

Lymphocyte-signaling molecules form immunological synapses. In order to
carry out the kind of signaling required for an immune response several
kinds of molecules must come together at and just below the proximal cell
surfaces of the T cell and the APC. Signaling into the leukocyte cell interior is triggered at the plasma membrane by surface contact (cell adhesion),
by antigen-signaling through the T cell receptors and coreceptors, and by
costimulatory signals. Cell adhesion molecules important for this process
include integrins such as LFA-1, and cell adhesion molecules belonging to
the Ig superfamily, such as the ICAMs. Cell adhesion molecules play a
crucial role in leukocyte motility and will be examined in detail in the next
chapter.
The transmission of messages across the plasma membrane and into the
cell interior is best thought of as a process. It is carried out in several distinct steps. It takes a certain amount of time and covers a region of space.
Depending on the character of the message, the transmission can be fairly
straightforward or can require several intermediate steps in order to elicit
the appropriate cellular response. In either case signal transduction is not
carried out by a single gene product but rather by multiple gene products
working in close physical proximity and contact with one another. In the
immune response the receptors themselves are composed of a number of
polypeptide chains. Some of the chains are involved in recognition, others

9.20 Kinetic Proofreading and Serial Triggering of TCRs

213

with transducing a signal from the extracellular face to the cytoplasmic face.
The receptors work together with coreceptors and with cell adhesion molecules to relay information into the cell.
In the immune systems receptors cluster and rearrange themselves.
Signals received at the cell surface help shape the clusters; the character of
the cluster in turn shapes the signal transduced into the cell interior. The
lipid bilayer is not passive but instead plays an active role in these adaptive
rearrangements. These procedures are utilized by leukocytes, by nerve cells,
and, as was discussed in Chapter 7, in the bacterial chemotactic system.
The proteins required for signaling between T cells and APCs are organized into signaling structures called immunological synapses (ISs). The term
synapse denotes a junction between cells where information is transmitted. Synapses are highly enriched in signaling molecules that not only transmit signals but also adaptively control the properties of the relayed
messages such as their strength. The term synapse originates in studies of
the transmission of signals between nerve cells. The literal meaning of the
term, coined by Sir Charles S. Sherrington in 1897, is to clasp together.
Synapses between nerve cells will be explored in Chapter 21.
Immunological synapses bring together a host of proteins required to
support sustained signaling. Among the components of the synapse are
receptors, cytoplasmic kinases, and adapters and anchors that link events at
the cell surface to the cytoskeleton. The TCRs and MHCs lie in the middle
of immunological synapse. As discussed earlier, the TCRs associate with
cytoplasmic CD3 complexes, protein tyrosine kinases such as Lck and Fyn,
and costimulatory molecules such as CD28 and CD40. Other proteins
belonging to the immunological synapse include ICAM-1 immunoglobulins
and LFA-1 intergrins, which bind one another, and members of the CD2
grouping of Ig adhesive molecules, which include CD48 (rat) and CD58
(human). This second group of mainly adhesive proteins surrounds the
central portion of the immunological synapse.

9.20 Kinetic Proofreading and Serial Triggering of TCRs

The TCR utilizes kinetic proofreading and serial triggering to discriminate
between self and non-self antigens. The TCR is remarkable. It can tell self
from non-self antigens even when the latter are buried in a sea of self antigens. At rst glance, the TCR assemblage looks unwieldy, if not outright
baroque. A whole series of diffusion, phosphorylation, and binding events
are required to elicit a proper cellular response to antigen binding. Involved
are several cytoplasmic proteins, namely, Lck, Fyn, and ZAP-70, a number
of adapters, a cluster of transmembrane chains, some of which, most notably
the z-z chains associated with CD3, are subjected to multiple phosphorylations, and nally PLC and the RasGTPase. It turns out that these two
aspects of TCR signaling are tied together.

214

9. Signaling by Cells of the Immune System

Completion of the series of phosphorylation steps, including those of the

z-z chains, depends on the nature of the peptide gripped by the MHC molecule and bound to the TCR. If the ligand is not a non-self antigen, the activation series will not run to completion before the ligand dissociates from
the receptor. The dissociation will abrogate the sequence of diffusion and
phosphorylation events before a strong (optimal) signal can be generated.
By this means differences in lifetime of the receptor-ligand binding are converted to differences in signaling because of the presence of many intermediate time- and energy-consuming steps.
This process is called kinetic proofreading. This term was rst introduced
in the 1970s to explain how high levels of delity could be achieved in operations such as transcription and translation. In these processes, the error
rates are exceedingly low, on the order of 104 to 109. These low error rates
are hard to understand using thermodynamic arguments alone because the
energy differences between correct and incorrect steps are miniscule.
This situation is comparable to that of the TCR, which can tell self
from non-self antigens when the latter are present in as few as 1 part in
10,000.
Kinetic proofreading occurs along with another chain of steps called serial
triggering. The term triggering in this case refers to the generation of
a signal by the TCR at the end of the series of internal steps. In serial
triggering, a single peptide-bound to an MHC molecule (pMHC) is able to
bind to several TCRs, one after the other in a serial fashion. Once the series
of diffusions and phosphorylation is completed the TCR is internalized and
the pMHC is free to engage another TCR. By this means the effect of a small
number of non-self peptides is amplied. Serial engagement favors short
lifetimes so that many receptors can be engaged, while kinetic proofreading
favors longer ones to increase the delity.The result is a balance between the
two processes and a dissociation rate optimized for TCR signaling.
The formation of an immunological synapse permits optimal signaling
through the TCR. As noted above, in an immunological synapse, a ring of
adhesive molecules called the peripheral supramolecular activation cluster
(pSMAC) surrounds a central ring of TCRs and associated signaling proteins, collectively referred to as the central supramolecular activation cluster
(cSMAC). This clustering together of TCRs into a cSMAC accomplishes
two things. It promotes serial triggering, and it helps to rapidly turn off signaling through receptor internalization once the signaling has reached its
appropriate level.

General References
Benjamini E, Coico R, and Sunshine G [2000]. Immunology (4th edition) New York:
John Wiley and Sons.
Janeway CA, Jr., Travers P, Walport M, and Capra JD [1999]. Immuno Biology (4th
edition) London: Current Biology Publications.

References and Further Reading

215

References and Further Reading

Lymphocytes
Abbas AK, Murphy KM, and Sher A [1996]. Functional diversity of helper T lymphocytes. Nature, 383: 787793.
Fearon DT, and Locksley RM [1996]. The instructive role of innate immunity in the
acquired immune response. Science, 272: 5054.
Glimcher LH, and Murphy KM [2000]. Lineage commitment in the immune system:
The T helper lymphocyte grows up. Genes Dev., 14: 16931711.
Goldrath AW, and Bevan MJ [1999]. Selecting and maintaining a diverse T-cell
repertoire. Nature, 402: 255262.
Pulendran B, Palucka K, and Banchereau J [2001]. Sensing pathogens and tuning
immune responses. Science, 293: 253256.
Rissoan MC, et al. [1999]. Reciprocal control of T helper cell and dendritic cell differentiation. Science, 283: 11831186.

NF-kB Signaling Node

Hoffmann A, et al. [2002]. The IkB-NF-kB signaling module: Temporal control and
selective gene activation. Science, 298: 12411245.
Li QT, and Verma IM [2002]. NF-kB regulation in the immune system. Nature Rev.
Immunol., 2: 725734.
Senftleben U, et al. [2001]. Activation by IKKa of a second, evolutionary conserved,
NF-kB signaling pathway. Science, 293: 14951499.
Silverman N, and Maniatis T [2001]. NF-kB signaling pathways in mammalian and
insect innate immunity. Genes Dev., 15: 23212342.

MAP Kinase Modules

Cowan KJ, and Storey KB [2003]. Mitogen-activated protein kinases: New signaling
pathways functioning in cellular response to environmental stress. J. Exp. Biol.,
206: 11071115.
English J, et al. [1999]. New insights into the control of MAP kinase pathways. Exp.
Cell Res., 253: 255270.

TRAFs
Arch RH, Gedrich RW, and Thompson CB [1998]. Tumor necrosis factor receptorassociated factors (TRAFs)A family of adapter proteins that regulate life and
death. Genes Dev., 12: 28212830.
Chung JY, et al. [2002]. All TRAFs are not created equal: Common and distinct
molecular mechanisms of TRAF-mediated signal transduction. J. Cell Sci., 115:
679688.

Toll and Toll-Like Receptors

Aderem A, and Ulevitch RJ [2000]. Toll-like receptors in the induction of the innate
immune response. Nature, 406: 782787.
Hemmi H, et al. [2000]. A Toll-like receptor recognizes bacterial DNA. Nature, 408:
740745.

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9. Signaling by Cells of the Immune System

Medzhitov R, and Janeway C, Jr. [1997]. Innate immunity: The virtues of a nonclonal
system of recognition Cell, 91: 295298.
Medzhitov R, Preston-Hulburt P, and Janeway CA, Jr. [1998]. A human homologue
of the Drosophila Toll protein signals activation of adaptive immunity. Nature,
388: 394397.

TNFs
Bodmer JL, Schneider P, and Tschopp J [2002]. The molecular architecture of the
TNF superfamily. Trends Biochem. Sci., 27: 1926.
Locksley RM, Killeen N, and Lenardo MJ [2001]. The TNF and TNF receptor superfamilies: Integrating mammalian biology. Cell, 104: 487501.

Jaks, STATS, and Hematopoietins

Guthridge MA, et al. [1998]. Mechanism of activation of GM-CSF, IL-3, and IL-5
family of receptors. Stem Cells, 16: 301313.
Heinrich PC, et al. [2003]. Principles of interleukin (IL)-6-type cytokine signaling
and its regulation. Biochem. J., 374: 120.
Levy DE, and Darnell JE, Jr. [2002]. STATs: Transcripional control and biological
impact. Nature Rev. Mol. Cell Biol., 3: 651662.
Ortmann RA, et al. [2000]. Janus kinases and signal transducers and activators of
transcription: Their roles in cytokine signaling, development and immunoregulation. Arthritis Res., 2: 1632.

The Interferon System

Goodbourn S, Didcock L, and Randall RE [2000]. Interferons: Cell signalling,
immune modulation, antiviral responses and viral countermeasures. J. Gen. Virol.,
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Chemokines
Baggiolini M [1998]. Chemokines and leukocyte trafc. Nature, 392: 565568.
Cyster JG [1999]. Chemokines and cell migration in secondary lymphoid organs.
Science, 286: 20982102.
Rollins BJ [1997]. Chemokines. Blood, 90: 909928.

T Cell Costimulation
Lanzavecchia A [1998]. License to kill. Nature, 393: 413414.
Schwartz RH [2001]. It takes more than two to tango. Nature, 409: 3132.
Sharpe AH, and Freeman GJ [2002]. The B7-CD28 superfamily. Nature Rev.
Immunol., 2: 116126.
Viola A, et al. [1999]. T lymphocyte costimulation mediated by reorganization of
membrane microdomains. Science, 283: 680682.

Immunological Synapses
Coombs D, et al. [2002]. Activated TCRs remain marked for internalization after
dissociation from pMHC. Nature Immunol., 3: 926931.

Problems

217

Dustin ML, and Cooper JA [2000]. The immunological synapse and the actin
cytoskeleton: Molecular hardware for T cell signaling. Nature Immunol., 1: 2329.
Grakoui A, et al. [1999]. The immunological synapse: A molecular machine controlling T cell activation. Science, 285: 221227.
Krummel MF, et al. [2000]. Differential clustering of CD4 and CD3z during T cell
recognition. Science, 289: 13491352.
Lee KH, et al. [2003]. The immunological synapse balances T cell receptor signaling and degradation. Science, 302: 12181222.
Monks CRF, et al. [1998]. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature, 395: 8286.

Problems
9.1 There are two basic approaches used in theoretical studies of how signaling pathways operate: macroscopic and microscopic. The starting
point for the macroscopic approaches is the law of mass action, and for
the microscopic approach it is the principle of detailed balance. In both
classes of approaches one constructs a set of equations that captures
the salient features of the signaling process in terms of rates and other
quantities that can be related to experiment. One then follows the evolution of the system on a computer to answer questions posed by the
experimental data. In many situations, modeling a system and then simulating its behavior on a computer is the only way to see just what the
biomolecules are doing over time.
The law of mass action is a statement that the rate of a chemical reaction is proportional to the concentrations of the reactants. To illustrate
how this rule works, consider a reaction of the form

The rate of change of the product [C] is

d[C]
= k1 [A][B] - k-1 [C][D].
dt

(9.1)

In the above, the rate of change of a reactant is expressed as the difference between its rate accumulation and its rate of loss through
degradation and additional/reverse reactions. Expressions of this form
can be simplied if some of the concentrations do not change appreciably over time, if some intermediates are formed rather transiently
compared to the other steps, and if the reaction only proceeds in one
direction or the other. For example, if the concentration of D does not
change over time the second term in Eq. (9.1) becomes a linear one in
which [D] is absorbed into the constant. An intermediate step, where a

218

9. Signaling by Cells of the Immune System

complex between A and B is formed, has been omitted in writing the

above under the transient formation assumption.
One can use the law of mass action to construct models of signaling
pathways. Consider a set of kinases arranged in a pathway so that one
kinase activates a second kinase and so. Let XiP denote the ith activated
kinase in the pathway. Let Xi+1 be its substrate kinase, and let Y denote
a protein phosphatase that acts on Xi+1P to return it to an unphosphorylated state. The following pair of expressions can describe the joint
actions of XiP and Y on Xi+1:

The rate equation governing the buildup of the phosphorylated (i + 1)th

kinase is

d X i +1

dt

] = k [X ][X
p

i +1

] - ji+1 [X i+1 ],
p

i +1

(9.2)

where [Y] has been assumed to be constant and was absorbed into the
phosphatase rate constant j. Other equations can be constructed that
describe interactions between ligands and receptors, between receptors
and kinases/adapters, and between adapters and kinases. The result is
a model of each of the steps in the signaling pathway.
(a) Derive the above expression, Eq. (9.2), for the rate of change in
[Xi+1P]. This equation can be solved to determine the steady-state (ss)
concentration of Xi+1P.
(b) Show that the result, taking the concentration of the unphosporylated plus phosphorylated forms of Xi+1 to be a constant T, is
X T
.
] = [ ]
( j k ) + [X ]
p

[X

p
i +1

ss

i +1

i +1

(9.3)

This steady-state formula, Eq. (9.3), relates the stimulus [XiP] to the
response [Xi+1P]. What is the behavior of the resulting stimulusresponse curve at low [XiP], and at high [XiP]? How does this compare
to MichaelisMenten kinetics discussed in the problems for Chapter 7?
9.2 The reaction kinetics models based on the law of mass action make up
a macroscopic description of the biochemical processes taking place.
Alternatively one could employ a microscopic description that deals
with the movement and interactions at the molecular and atomic levels
rather than dealing with macroscopic quantities such as the concentra-

Problems

219

tions. The starting place for the microscopic approaches is the principle
of detailed balance, which states that the rate at which a system makes
a transition into a particular microstate from a preceding state is equal
to the rate that the system will transition back out of that particular
microstate to its predecessor. In mathematical terms the principle is
pi Pij = p j Pji ,

(9.4)

where pi is the probability that the ith microstate is occupied (i.e., the
occupation probability), and Pij is the transition probability from
microstate i to microstate j. The above expression is a statement that
the laws of physics are the same going forward or backward. In particular, the expression states that the probability that a state is occupied
times the rate at which there is a transition out of that state to another
is equal to the probability that the entered state is occupied times the
rate at which that state is exited back to its predecessor. The occupation probabilities can be dened in terms of the Boltzmann factor b:
pi =

1 -bEi
e ,
Z

(9.5)

where b = 1/kBT, Ei is an energy that characterizes the microstate i, and

Z is a normalization factor. With this denition one can introduce a
sampling algorithm that allows a researcher using a computer to simulate the evolution of a biochemical system over time in order to study
its properties and see how it works.The sampling algorithm for the transitions is
e -bDEij, E j > Ei ,
pij =
1, E j Ei .

(9.6)

In the simulations, one starts out with the system in some initial state,
then randomly picks a new microstate and calculates the energy difference DEij = Ej - Ei. If the energy difference is negative then the transition leads to a state of lower energy and the move is allowed. If the
energy difference is positive, then the transition increases the energy of
the system. One then selects a random number between 0 and 1. If that
random number is less than the calculated Pij, the transition is allowed.
Otherwise the move is rejected and another proposed transition is
selected. Note that in this approach moves that increase the energy
become possible allowing the system to transition over barriers and out
of pockets in the energy landscape. This would not be possible in situations where only energy decreasing steps were allowed.
This algorithm, known as the Metropolis algorithm, was introduced
in a landmark paper in 1953. The general method of randomly sampling
a set of moves of a microsystem according to a probability distribution
is known as the Monte Carlo method and is extensively used in several

220

9. Signaling by Cells of the Immune System

different forms to study how complex physical and chemical systems

evolve over time.
Show that the Metropolis algorithm, Eq. (9.6), satises the detailed
balance condition, Eq. (9.4), using Eq. (9.5). What are the results of this
sampling procedure when the energy of state j just barely exceeds that
of state i? What happens when the energy differences between the two
states are large? The quantity Z appearing in the denition of the occupation probabilities is known as the partition function in statistical
physics. Write an expression for it in terms of the Boltzmann factor b.
Theoretical (Modeling) Studies
Asthagiri AR, and Lauffenburger DA [2001]. A computational study of feedback
effects on signal dynamics in a mitogen-activated protein kinase (MAPK)
pathway model. Biotechnol. Prog., 17: 227239.
Heinrich R, Neel BG, and Rapoport TA [2002]. Mathematical models of protein
kinase signal transduction. Mol. Cell, 9: 957970.
Tyson JJ, Chen KC, and Novak B [2003]. Sniffers, buzzers, toggles and blinkers:
Dynamics of regulatory and signaling pathways in the cell. Curr. Opin. Cell Biol.,
15: 221231.

10
Cell Adhesion and Motility

Movement and adhesive contacts between cells and opposing surfaces are
necessary for embryonic development and normal adult functioning of cells
in the body. During development, cells do more than grow, multiply, and
differentiate. They also segregate, migrate, and aggregate in forming tissues
and organs. In adult life, vascular endothelial cells and broblasts migrate
during wound healing, leukocytes migrate in response to infections, and
cancerous cells migrate during metastasis.
Cells establish and maintain contacts with other cells and with the extracellular matrix. These contacts stabilize the aggregates of cells and enable
them to work together to carry out their functions. Adhesive contacts
underlie axon outgrowth during development of the nervous system. Paralleling the steps taken by migrating cells, growth cones, motile sensory
structures enriched in adhesion molecules, guide the formation of connections in the nervous system.
Several different families of cell adhesion moleculesintegrins, cadherins, immunoglobulin superfamily cell adhesion molecules (IgCAMs),
and selectinsmediate adhesive contacts between surfaces in the body.
These molecules, acting as receptors and counterreceptors/ligands on
opposing surfaces, are responsible for establishing and maintaining physical contact and communication between the extracellular matrix, the cell
surface, and the cytoskeleton. These families of adhesion molecules, along
with several different kinds of diffusible molecules and their receptors, also
mediate axon outgrowth in the nervous system. Leukocyte adhesion and
mobility will be examined in the rst part of this chapter and axon outgrowth in the second.

10.1 Cell Adhesion Receptors: Long Highly

Modular Glycoproteins
The extracellular regions of cell adhesion receptors are mosaics of domains,
strung together in a linear fashion. The domains may be composed of a
number of short identical subdomains, referred to as repeats, or they may
221

222

10. Cell Adhesion and Motility

be erected from a mixture of two or more different domains, or they may

be built from both repeats and single domains. Some of the domains in the
extracellular regions are specic to a particular family and dene membership in that family. Examples are cadherin domains in cadherins, and lectin
domains in selectins. Other domains, most notably the immunoglobulin(Ig) like domains and bronectin type III repeats, occur in many different
proteins. A representative sampling of extracellular regions of adhesionpromoting proteins is presented in Figure 10.1.
The extracellular regions of these proteins are attached to several
different kinds of membane-associated segments. The most commonly
encountered form of attachment is to a single-pass transmembrane segment
that is followed by a short cytoplasmic tail. All of the proteins appearing is
Figure 10.1 attach in this manner. There are three families of selectins
vascular endothelial (E), leukocyte (L), and platelet (P). All of these attach
by means of transmembrane segments. So do 5/2 IgCAMs such as NCAM,
6/5 IgCAMs such as L1, and 4/6 IgCAMS such as deleted in colorectal
cancer (DCC), but IgCAMS such as Contactin attach by means of a GPI
anchor. Most cadherins attach through a single-pass TM segment, but cadherins such as Flamingo attach by means of a seven-pass transmebrane
chain and may signal through G proteins.
During an inammatory/immune response, leukocytes emigrate out of
the bloodstream and converge upon the injured tissue. The emigration
process occurs in several stages. Leukocytes rst establish a loose contact
with the endothelial cells lining the vessel walls and begin rolling. Next, a
hard adhesive contact is established, and nally the leukocytes crawl out of
the blood vessel and migrate into the injured tissue. Adhesion receptors

Figure 10.1. Representative extracellular regions of proteins involved in cell adhesion: (a) Classical cadherins containing 5 cadherin domains N-terminal to the transmembrane segment shown in black. (b) Neural cell adhesion molecules (NCAMs)
containing 5 Ig-like domains and 2 bronectin type III (FNIII) repeats. (c) Eselectins containing an amino terminal lectin domain, an EGF domain, 6 short consensus repeats (SCRs), and a membrane proximal cleavage region.

10.2 Integrins as Bidirectional Signaling Receptors

223

work synergistically with receptors for cytokines and soluble growth factor.
They signal through many of the same pathways as growth factors and
together promote cell survival, proliferation, and differentiation. The
cytoplasmic domains of cell adhesion receptors make contact with the
cytoskeleton, and the receptors help regulate cell shape and polarization,
and cytoskeleton organization and motility. During metastasis cancer cells
utilize a similar ensemble of mechanical and adhesion receptor signaling
processes. In metastasis, cells detach from the surrounding tissue, migrate
to remote sites elsewhere in the body using the lymphatic and circulatory
systems, reattach, and then establish a colony. Signaling between the cell
surface and the nucleus coordinates the expression of specic cell adhesion
molecules at different stages in metastasis.
Cell adhesion receptors are both large and exible and because of these
properties present challenges in their study using high resolution NMR and
X-ray crystallography methods. The solution to this technical challenge is
to take advantage of their inherent modularity and characterize portions
fragments and domainsof these molecules. The resulting NMR and X-ray
crystallography data together with electron microscopy results provide a
core body of structural information on these essential proteins.

10.2 Integrins as Bidirectional Signaling Receptors

Integrins are membrane-spanning glycoproteins composed of noncovalently attached a and b subunits. In vertebrates, 18 distinct alpha subunits
and 8 different beta subunits have been identied so far. Not all of the 18
8 = 144 combinations of alpha and beta subunits can be formed. Instead,
a far smaller number, namely, 24, ab heterodimers can occur. Each integrin
subunit contains a large extracellular domain, a single transmembrane
segment, and a short cytoplasmic domain. There is one exception to this
rule: The b4 subunit has a large cytoplasmic domain. The a subunits are
larger than the b subunits. The a subunits vary in size from 120 to 170 kDa
(up to 1114 amino acid residues) while b subunits range in size from 90 to
100 kDa (up to 678 residues).
Integrins are multidomain proteins. The extracellular parts of the alpha
and beta chains each contain ve or more domains and some of these
domains may consist of multiple subdomains. The domain organization of
a representative integrin alpha chain and of a typical beta chain are
depicted in Figure 10.2. In part (c) the two chains of the integrin molecule
are bent in a V-shape that supports low afnity ligand binding. Not all integrin molecules contain inserted (I) or I-like domains. These ligand-binding
domains are included in the model integrin depicted in the gure. The most
important feature of this gure is the bent shape. The integrin molecule
undergoes massive rearrangements in response to allosteric signals, and the
far more open shapes that result mediate intermediate and high afnity

224

10. Cell Adhesion and Motility

(a)

(b)

(c)

Figure 10.2. Domain organization and assembly of the extracellular portions of

an integrin containing inserted (I) and I-like ligand-binding domains: (a) Domain
organization of the alpha subunit. (b) Domain organization of the beta subunit.
(c) Assembly of the alpha and beta chains in a V-shaped conformation showing the
exposed ligand binding I and I-like domains at the end of the molecule. Abbreviations: b-tail (bT); hybrid (H); integrin-epidermal growth factor (I-EGF, or E);
plexin/semaphorin/integrin (PSI).

ligand binding. X-ray crystallography data revealing this bent shape are
presented in Figure 10.2.
Integrins and cadherins differ from most transmembrane signal transducers that transmit signals in one direction, from outside the cell inward.
Integrin and cadherin receptors transmit signals in both directionsfrom
outside the cell inward (outside-in) and from inside the cell outward
(inside-out). In outside-in signaling, binding of the integrins to the ECM
triggers changes in the pattern of gene expression. Inside-outside signals
produce changes in the integrin conformation resulting in changes in adhesiveness. Integrins tie the ECM to the cellular cytoskeleton and anchor cells
in a xed position, while cadherins are a primary element of cell-to-cell
junctions.

10.3 Role of Leukocyte-Specic Integrin

The leukocyte-specic integrin LFA-1 mediates the migration of T cells.The
migration of T cells occurs in several stages. In the rst stage, lamellipodia,
broad at structures, form and extend out from the leading edge of the cell.
This stage is followed by a step in which new adhesive contacts are established and stabilized. The cell body then contracts, and the following edge,
or tail, detaches. T cells use the aLb2 integrin, also called the leukocyte

10.4 Most Integrins Bind to Proteins Belonging to the ECM

225

Figure 10.3. Integrin crystal structure: (a) V-shaped, or bent, closed conformation
of the aVb3 integrin. The alpha chain is shown in light gray and the beta chain in
black. (b) Complex formed between two aL inserted (I) domains (black) and a pair
of ICAM-1 ligands (light gray). The gure was prepared using Protein Explorer with
atomic coordinates deposited in the PDB under accession numbers 1jv2 (a) and
1mq8 (b).

function-associated antigen-1 (LFA-1) integrin, to contact ICAM-1 proteins

on opposing endothelial cells (Figure 10.3). LFA-1 is also a main component of the pSMAC rings of immunological synapses.
Contacts between LFA-1 integrins and ICAM-1s trigger the formation
of signaling complexes and signaling through Rho GTPases to downstream
kinases, most notably myosin light chain kinase (MLCK) and Rho kinase
(ROCK). These kinases are restricted to specic locations in the cell.
MLCK is concentrated near the leading edge of the cell, and ROCK is localized to the tailing edge. These serine/threonine kinases act on myosin light
chains (MLCs) which when phosphorylated promote actin-myosin-ber
contractions. When coordinated over time, signaling through the adhesion
receptors produces leading edge attachments, cell body contractions, tailing
edge detachments, and T cell movement.

10.4 Most Integrins Bind to Proteins Belonging to

the ECM
The extracellular matrix (ECM) is intercellular material, mostly glycoproteins and proteoglycans, that surrounds cells and forms the connective
tissue in the body. Examples of ECM connective tissue include teeth and
bone, cartilage and tendons, and skin. The ECM encompasses the various
basement membranes that provide structural support for tissues and organs.
A typical basement membrane has a basal lamina formed by glycoproteins
secreted by the attached cells and a reticular lamina formed by protein
bers from the underlying connective tissue. Collagens, glycine-rich glyco-

226

10. Cell Adhesion and Motility

proteins, are the primary ECM elements. Collagens are long ropelike linear
molecules composed of three chains arranged in a triple helix. Typical
lengths of collagens are 300 to 400 nm. Two families of adapter molecules
are present in the basal laminalaminins and bronectins. These molecules
connect ECM collagens to adhesive cellular proteins.
The extracellular matrix has considerable inuence over the metazoan
cell. Both mechanical and chemical signals are sent from the ECM to the cell
surface, and from there they are transduced into the cell interior. Integrins
have as their ligands the aforementioned ECM adapter molecules laminin
and bronectin. Binding to these proteins triggers the formation of adhesive
contacts at specic locations along the plasma membrane. Binding to
the matrix proteins stimulates integrin clustering, signaling to proteins
embedded in the plasma membrane,and to proteins located in the cytoplasm.
The cytoplasmic tails of integrin beta chains contain motifs of the form
NPxY (arginine-proline-X-tyrosine) that are recognized by phosphotyrosine-binding (PTB) domains of cytoplasmic adapter molecules, nonreceptor tyrosine kinases such as Src, focal adhesion kinase (FAK), and the actin
cytoskeleton-binding protein talin. The binding of the beta chains to talin
and other proteins establishes mechanical linkages called focal adhesions
between the ECM, the cell surface, and the cytoskeleton that facilitates clustering of integrins and the onset of signaling. Once the focal adhesions are
formed positive feedback signals to the integrin receptors stimulate their
further clustering and additional signaling from them to the focal adhesions.
The result of this two-way signaling is the tying together, or integration, of
the ECM with the cytoskeleton of the cell. The integrative properties of
these receptors led to their being given the name integrins.

10.5 Cadherins Are Present on Most Cells of the Body

Cadherins (calcium dependent adherins) are a large family of transmembrane proteins. In mammalian species there are at least 80 family members.
A large number of these proteins is expressed preferentially in the nervous
system. Cadherins contain an extracellular domain, a transmembrane
segment, and a cytoplasmic tail. The dening characteristic of this family is
the presence of a cadherin motif, or EC domain, in the extracellular domain.
These motifs are tandemly repeated anywhere from 2 times to 30 or more
times.The most N-terminal of these domains is the adhesive site, while other
portions of the extracellular domain serve as spacers and supply multiple
binding sites for calcium ions. Like integrins, cadherins form clusters when
they bind their ligands, in this case, counterreceptors on opposing surfaces.
The juxtamembrane portion of the cytoplasmic domainthe part of the
cytoplasmic domain nearest the plasma membraneinteracts and contributes to the clustering and adhesive strengthening taking place upon
ligand binding.

10.5 Cadherins Are Present on Most Cells of the Body

227

Figure 10.4. Ectodomain of a classical cadherin: Shown

are the ve domains, labeled EC1 through EC5, from
the N-terminus towards the C-terminus, which is nearest
plasma membrane. Small single and paired spheres represent calcium ions, which are necessary for proper functioning of the receptor. Clusters of spheres denote the
locations of N-linked and O-linked sugars. The gure
was prepared using Protein Explorer with atomic coordinates deposited in the PDB under accession number
1l3w.

Classical cadherins such as E-cadherin (epithelial cadherin) and Ncadherin (neural cadherin), contain about 750 amino acid residues. Their
extracellular domain consists of ve repeats of a 100-amino acid EC
domain, which extends out 250
from the cell surface (Figure 10.4). These
cadherins attach to the actin cytoskeleton by means of a linker protein
called catenin. The cytoplasmic domain of these cadherins is approximately
150 amino acid residues in size, and contains a catenin-binding site. There
are two catenin subunits. The b subunit binds to the catenin domain of the
cadherin and to the a subunit of the catenin. The a subunit, in turn, connects to the actin cytoskeleton. This cadherin-catenin complex is referred
to as a zonula adherens junction in epithelial cells and as an adherens junction in neurons. In epithelia, these junctions link cells together laterally to
permit them to form stable sheets, and they separate the apical (top) region
from the basolateral (bottom) region of each of the cells. In neurons, the
adherens junctions connect the pre- and postsynaptic cells, thereby providing mechanical stability and a means of conveying signals in both directs
across the synapse.
Cadherins operate in a homophilic manner, and have a role in maintaining tissue boundaries and stabilizing synapses. Cells that segregate into
distinct tissues express different cadherins, so that a cadherin receptor on
the surface of one cell binds to a cadherin receptor of the same type on the
surface of an opposing cell. Calcium binding is necessary for clustering, or
oligomerization, of cadherin receptors as well as for ligand binding. An
example of how this requirement might serve a useful function is provided
by N-cadherin binding in synaptic junctions. Intense synaptic activity generates long-lasting changes in synaptic transmission. Remodeling of the
synapse underlies these changes. These changes might occur through the
following sequence of steps: Intense activity depletes the supply of calcium
in the vicinity of the synapse. This lowering in the local calcium concentration weakens the links between cadherin molecules allowing remodeling

228

10. Cell Adhesion and Motility

activities to proceed. This step is followed by reestablishement of rm

contact when the calcium concentration returns to its basal levels.

10.6 IgCAMs Mediate CellCell and

CellECM Adhesion
Cell adhesion molecules (CAMs) of the immunogloulin superfamily (Ig-SF)
mediate cellcell and cellECM adhesion. Cell surface receptors belonging
to this superfamily, the IgCAMs, are characterized by the presence of one
or more immunoglobulin-like domains in their extracellular region. These
adhesion molecules mediate cell-to-cell contact by binding to cell surface
counterreceptors, and help establish and maintain contact with the extracellular matrix by binding to ECM constituents. Unlike cadherins, calcium
binding is not required either for ligand binding or for clustering to occur.
Some IgCAMs bind in a homophilic manner while others bind in a heterophilic way.
One of the main kinds of IgCAM is the neural IgCAM, or NCAM,
expressed on neurons (Table 10.1). These receptors contain one or more
repeats of the Ig fold plus a number of bronectin type III folds, in their
extracellular domain as depicted in Figure 10.1. The NCAMs are grouped
into families according to the organization of the extracellular domains.
NCAM contains ve immunoglobulin-fold repeats plus a pair of bronectin
type III (FnIII) repeats proximal to the plasma membrane. It is thus classied as a 5/2 IgCAM. Another important and well-studied neural IgCAM
is L1. This cell adhesion molecule is a 6/5 IgCAM family member. A third

Table 10.1. Members of the IgCAM group of cell adhesion receptors:

AbbreviationsICAM (intercellular cell adhesion molecule); LFA (lymphocyte
function-associated antigen), NCAM (neural cell adhesion molecule), PECAM
(platelet endothelial call adhesion molecule), VCAM (vascular cell adhesion
molecule), CD (cluster of differentiation).
Ig-SF CAM

CD designation

Ligand

Distribution/role
Lymphocytes, endothelial
cells when inammation is
present
Recirculating leukocytes
Leukocytes
Lymphocytes
Broad distribution
Neural tissue
Platelets, leukocytes
Lymphocytes, endothelial
cells when inammation is
present

ICAM-1

CD54

LFA-1, Mac-1, brinogen

ICAM-2
ICAM-3
LFA-2
LFA-3
NCAM
PECAM-1
VCAM-1

CD102
CD50
CD2
CD58
CD56
CD31
CD106

LFA-1, Mac-1
LFA-1
LFA-3
LFA-2
NCAM, collagen, heparin
PECAM-1
a4b1 and a4b7 integrins

10.7 Selectins Are CAMs Involved in Leukocyte Motility

229

group of IgCAMs are the DCC family members. Named for the protein
DCC (deleted in colorectal cancer) these are 4/6 IgCAMs. Molecules closely
related to DCC have a prominent role in the development of the nervous
system. They are found on growth cones where they bind a family of diffusible chemoattractants called netrins that guide growing axons during
embryonic development, as will be discussed later in this chapter.
The two most prominent families of IgCAMs are the NCAMs and ICAMs
(intercellular cell adhesion molecules). The ICAMs bind to integrins and
thus mediate heterophilic binding. NCAMs bind to identical receptors on
opposing cells and thus mediate homophilic binding. Besides their role in the
immune/inammatory system, these proteins play an important role during
embryonic development and in the nervous system both during development and in adult life. NCAMs are also expressed in the heart, gonads, and
skeletal muscle. Because of their role in signaling and maintaining tissue
integrity they have a prominent role in several forms of cancer.

10.7 Selectins Are CAMs Involved in Leukocyte Motility

Leukocytes circulate in blood vessels and the lymphatic system, and when
an infection is detected they converge to infection sites. Selectins mediate
the movement of leukoctyes within blood vessels to sites of infection,
and out of the blood vessel into the surrounding inamed tissue. They are
expressed on the surface of leukoctes, the endothelium lining the blood
vessels, and platelets that form adhesive plugs, or clots, at sites of wounds.
There are three kinds of selectins: L-selectins are expressed on leukocytes,
E-selectins are expressed on the endothelial cells, and P-selectins are
expressed on platelets on the endothelium. These adhesion molecules
mediate the capture of the circulating leukocytes by the walls of the blood
vessels and make possible their subsequent rolling.
Selectins are mosaic proteins with a common structural organization.
They each have an NH2 terminal lectin domain followed by an epidermal
growth factor (EGF) domain followed by a number of consensus repeats
(CRs) of a complement-like binding sequence, a single transmembrane
segment, and a short cytoplasmic tail. The lectin domain is a carbohydratebinding domain that enables the selectin to bind to carbohydrate structures
on its ligand. The CRs are thought to function as spacers that extend the
molecule a distance that supports optimal rolling. Deletion of portions of
this segment impairs rolling. The three selectins vary in the number of CRs.
Human P-selectin has nine CRs, while E-selectin has six and L-selectin just
two.
The lectin domain situated near the NH2 terminus is a carbohydratebinding domain. Selectin ligands such as GlyCAM-1 and CD34 are
members of the mucin family. These are long rodlike glycoproteins with
multiple serine/threonine residues and are heavily glycosylated (O-linked).

230

10. Cell Adhesion and Motility

Table 10.2. Selectins, cells, and structures that express them, their functions, and
ligands: Ligand abbreviations are as follows: PSGL-1 (P-selectin glycoprotein
ligand-1); MAdCAM-1 (mucosal addressin cellular adhesion molecule-1);
GlyCAM-1 (glycosylation-dependent cell adhesion molecule-1).
Selectin

Expression

L-selectin

Leukocytes

P-selectin
E-selectin

Platelets, endothelium
Endothelium

Functions
Leukocyte trafcking,
rolling adhesion
Rolling adhesion
Rolling adhesion induced
by inammation

Ligands
PSGL-1, GlyCAM-1,
MAdCAM-1, CD34
PSGL-1
PSGL-1

Another ligand, MAdCAM-1, is an IgCAM and directs selectins to mucosal

tissues. The primary P-selectin ligand is PSGL-1. Both ligands and selectins
support rapid bond formation and dissociation.
Selectins are the rst cell adhesion receptors to be activated in an inammatory response. In the absence of an injury E-selectins and P-selectins are
not expressed on the cell surface while L-selectins are constitutively active
in order to promote continual leukocyte homing. This activity is guided
by the appearance of its ligand in the vicinity of an inammation. The
expression of E-selectin on the surface of endothelial cells is triggered by
cytokines such as TNF-a, IL-1, and lipopolysaccharide (LPS). P-selectin
is stored in granules inside platelets (a-granules) and endothelial cells
(WeibelPalade bodies). P-selectin can be shipped to the outer surface in
minutes in response to histamines and other triggering signals. Cytokines
induce synthesis of P-selectin mRNAs.
Platelets are cytoplasmic fragments of bone marrow cells called
megakaryocytes. Platelets are released from the bone marrow and circulate
in the bloodstream. Their role in the inammatory response is to form clots
or plugs that block blood ow at sites of injury. Adhesion and aggregation
are central to platelet function, and they express integrins, selectins, and
IgCAM receptors. Microvilli are adhesion molecule-rich extensions of the
cell surface. They are formed at the outside facing, or apical, surface in a
variety of different cell types in order to increase the effective surface area.
The primary platelet selectin ligand is the P-selectin glycoprotein-1 (PSG1).
It is constitutively expressed on the tips of microvilli of leukocytes such as
neutrophils and monocytes.

10.8 Leukocytes Roll, Adhere, and Crawl to Reach the

Site of an Infection
Leukocytes are highly mobile cells that migrate from blood in and out of
lymphatic organs and into tissues, and then back out again into circulation.
They respond to infections by attacking and destroying the causative agents.

10.9 Bonds Form and Break During Leukocyte Rolling

231

The leukocytes do not passively oat along the blood vessels, but instead
remain in contact with the cells lining the walls of the blood vessels. They
move along the walls by rolling in a controlled way. This mode of locomotion enables them to stop at the right location and then exit by crawling out
between the cells. Adhesion is critically important, enabling the leukocytes
to rst roll, and then crawl, and nally to kill the pathogens.
In an inammatory response, the physiology of the blood vessels is
altered to better enable the leukocytes to reach the site of infection. Postcapillary venules are the main locus of vascular inammatory activity during
an inammatory response. These structures, some 30 to 40 microns in diameter, consist of a layer of endothelial cells on the inside and a supporting
basement membrane on the outside. Blood ow in small vessels such as the
postcapillary venules takes place under low ow rates, reasonably high
viscosities, and small vessel diameter. Under these conditions the velocity
of the blood ow is greatest in the center of the vessel and decreases to
zero as the vessel walls are approached. During inammation the blood
vessels dilate and the overall ow rate is slowed. Red blood cells aggregate
into large assemblies called rouleaux that collect into the center of the
vessels displacing white blood cells. In response the leukocytes move from
the center of the blood vessel to the vicinity of the vessel walls. When this
happens cell surface receptors expressed on the plasma membranes of the
endothelial cells and leukocytes can engage one another to promote rolling
and then hard adhesion.

10.9 Bonds Form and Break During Leukocyte Rolling

Shear stresses are forces generated when adjacent layers of a uid slip over
each other. Because of the shear stresses that are present, leukocytes will
either tumble or roll in the direction of the blood ow. Tumbling motion is
a rotary movement done without any contact with the cell wall. Rolling,
in contrast, is a rotary movement carried out while in contact with the
endothelial cells of the vessel wall. The observed motions of leukocytes are
jerky. Periods of rapid motion are interrupted by periods in which the
motions are far slower. The periods of slow motion correspond to tethering of the leukocytes to the membranes of the endothelium and the periods
of rapid motion to contact-free ow.
Bonds form and break during leukocyte rolling (Figure 10.5). Bond lifetimes must be long enough to permit formation of multiple bonds. If the
off (bond dissociation) rates are too high multiple bonds cannot form. One
bond will dissociate before another can be formed. At the other extreme,
too tight a bonding will either immobilize the cell or will lead to situations
where large forces can pull a receptor molecule out of the membrane. The
association and dissociation rates should be rapid, but not too rapid. In
leukocyte rolling, the bonds that tether leukocytes to the endothelial wall

232

10. Cell Adhesion and Motility

Figure 10.5. Bond under shear forces during leukocyte rolling: Depicted are the compression, stretching, and nally dissociation of bonds between a cell
adhesion molecule and its ligand during rolling of a
leukocyte along the inner wall of a blood vessel.

are subjected to stretching, or tensile, forces as a result of the shear stresses.

Forces have an important effect on the bond lifetimes. They shorten the
lifetimes of the bonds. The enhancements can be appreciablethe off-rate
rises exponentially with increasing force. The enhanced dissociation rates
arising from the tensile forces shift the bond lifetimes into the range needed
to ensure proper rolling.
The off-rate amplication is a consequence of the lowering of the energy
barriers by the applied forces. The dependence of bond lifetimes on the
presence and strength of applied forces is not specic to rolling or leukocytes. Instead, these considerations apply equally well to other bonds
between receptors and ligands. The acceleration in dissociation rates due to
the presence of applied forces provides a general mechanism for transducing mechanical stresses into signaling responses. Membrane, cytoskeletal,
and signaling elements coming together at control points will sense and
respond to mechanical stresses by dissociating far more rapidly than would
be the case in the absence of stresses.

10.10 Bond Dissociation of Rolling Leukocyte as Seen

in Microscopy
Rugged energy landscapes describe bond dissociation during leukocyte
rolling. The stretching properties of bonds between cell adhesion molecules
and their ligands have been explored using atomic force microscopy (AFM)
and optical tweezers. In a typical AFM experiment, the adhesion molecules
of interest are attached to a sharp tip, one having a diameter of no more
than a few microns. The tip is mounted on a exible cantilever. When the
adhesion molecules are brought into close proximity to their ligands,
mounted on a separate surface, the cantilever will bend and undergo a displacement. These displacements are measured by sweeping a laser beam

10.11 Slip & Catch Bonds Between Selectins & Carbohydrate Ligands

233

Figure 10.6. Energy landscape for dissociation of bonds between adhesive molecules and their ligands: Abbreviationstransition state 1 (TS1); transition state 2
(TS2); intermediate state (IS).

across the cantilever and measuring the angle and orientation of the
reected light. In an optical tweezers experiment, one uses a laser beam to
trap dielectric particles. External forces applied to the trapped particles can
be determined by monitoring the angular deection of the laser light. The
adhesive molecules are mounted on a pedestal attached to a surface and
the ligands are attached to optically trapped beads.
Experiments of several different adhesion molecule/ligand combinations
have been studied, and the results placed within an energy-landscape perspective. A representative landscape deduced from these experiments is
presented in Figure 10.6. This landscape has two prominent features. First,
it is a rugged landscape. The pockets are deep, several times the thermal
energy, resulting in the presence of at least one metastable intermediate
state. The situation shown in the gure is that of two transition states.
Second, two different outer barriers heights are presentone of these corresponds to low afnity binding and the other to high afnity binding. The
high inner barrier is responsible for high strengths seen at short time scales
and the major portion of the activation energy. The lower barriers located
further out extend bond lifetimes when forces are absent.

10.11 Slip and Catch Bonds Between Selectins and

Their Carbohydrate Ligands
The thrust of the previous section was that externally applied forces reduce
the lifetimes of receptor-ligand bonds by lowering the energies of the
transition barriers. The force-driven reductions in lifetime allow rolling

234

10. Cell Adhesion and Motility

leukocytes to detach from surface tethers at the right time while maintaining good adhesion to that place on the surface at earlier times. The corresponding bonds are called slip bonds because they allow the rolling cells to
slip away. These are not the only kinds of bonds that form. An observation
often made not only of rolling leukocytes, but also migrating bacteria, is
that shear stresses promote good surface contact. The physical mechanism
underlying this phenomenon is captured by the notion of a catch bonda
bond that is strengthened by the external forces rather than weakened. The
selectins that mediate leukocyte rolling exhibit both kinds of bonds. The
catch bonds are formed rst. They enable the leukocytes to be caught by
the surface. Once the leukocytes are caught and begin rolling, a transition
takes place from a catch to a slip bond regime so that the latter can mediate
proper detachment from the surface.
Selectins are not only expressed by migrating leukocytes. They are
expressed early during embryonic development and enable embryos to
attach to the lining of the uterus. Failure to properly lodge in the uterus is
a prominent cause of fertilization and implantation failures. In the attachment process, carbohydrate ligands upregulated and expressed on the
surface of the uterus bind the L-selectin molecules expressed on the surface
of the embryo. The adhesion takes place under shear stresses created by
mucin secretions and, once established, sets off a chain of signaling events.

10.12 Development in Central Nervous System

Development of the central nervous system involves many of the same
operations as used in body development. The development of the central
nervous system, with its laminar structure and organization into dozens of
anatomically and functionally distinct areas, is one of the most remarkable
and dramatic processes in nature. It takes place in several stages. It begins
with the production of immature neurons, or neurites, and glia, cells that
eventually supply neurons with growth factors, nutrients, protection (astrocytes); and electrical insulation (Schwann cells). The neurites multiply, grow,
differentiate, and migrate to various sites where they aggregate into distinct
cortical layers and regions. During this period support structures such as
the ECM are erected from molecules secreted by the developing cells. Cell
growth, maturation, and circuit formation follows arrival of the neurites at
their cortical destinations. The growing cells within each of the layers and
areas develop their morphologically and electrophysiologically distinct
axonal and dendritic structures, and they establish initial circuit-forming
synaptic contacts. The initial contacts are then rened, and unwanted connections and cells are removed. At the end of this process more than 1010
neurons will have formed some 1015 precise synaptic connections.
Once the neurites reach their cortical destination and their migration
ceases, they send out processes that become axons and dendrites that form
the synapses and neural circuits. The growth cone (Figure 10.7) is a motile

10.13 Diffusible, Anchored, and Membrane-Bound Glycoproteins

235

Figure 10.7. Structure of a growth cone: Microtubules are represented by thick

lines and the actin cytoskeleton by thin lines.

sensory structure located at the tip of an advancing axonal or dendritic

process. It contains dynamic extensions of its leading margin called lopodia and lamellipodia that can extend and retract, change position, and differentially adhere to surfaces. Paralleling the actions taken during the
earlier cell migrations, the growth cone explores, interacts with, interprets,
and responds to signaling molecules in its local microenvironment. It can
navigate over distances of up to several centimeters corresponding to more
than 1000 cell body diameters. This key structure contains a dense actin
cytoskeleton and is enriched in adhesive and signaling proteins.

10.13 Diffusible, Anchored, and Membrane-Bound

Glycoproteins in Neurite Outgrowth
The molecules that guide growth cones to their cortical targets include
members of the same families as those involved in leukocyte movement.
Integrins, cadherins, and IgCAMs are expressed in growth cones and all
contribute to growth cone navigation. In addition to these cell adhesion
molecules, several families of diffusible, anchored and membrane-bound
signaling glycoproteins direct growth cone navigation by supplying attractive and repulsive signals. These additional molecules involved in growth
cone guidance are evolutionarily ancient.They have been found and studied
in the nematode worm C. elegans, in the fruit y D. melanogaster, in the
chick, and in mammals including humans. They are listed in Table 10.3.
The diffusible signaling molecules function as molecular markers.
Growth cones maintain contact with surfaces while they navigate. Cells
serving as signposts for the growth cones secrete diffusible markers. The
markers may delineate forbidden regions where the growth cones are not

236

10. Cell Adhesion and Motility

Table 10.3. Diffusible navigation markers and their receptors: The receptors are
grouped according to the ligandsnetrins, slits, semaphorins, and ephrins.
Ligand-receptor systems

Description

Netrins
DCC family
UNC-5 family

Short or long range attraction; growth cone guidance

Short or long range repulsion; growth cone guidance

Slits
Roundabout

Long range repulsion; growth cone guidance, axonal branching

Semaphorins
Plexins
Neuropilins
Scatter factor receptors

Axonal repulsion
Neuronal sorting; axonal repulsion
Short range interactions; invasive growth; attraction

Ephrins
Eph receptors

Contact-dependent repulsion; cell sorting, growth cone guidance

to enter, or mark intermediate targets toward which the growth cones are
to turn, or provide a marker for the nal destination. The way directional
information is supplied is through concentration gradients. The basic idea,
known as the chemoafnity hypothesis, is that chemical markers are laid
down on the surfaces over which the axons navigate. These markers form
concentration gradients that are read by the growth cones to obtain the
correct heading.

10.14 Growth Cone Navigation Mechanisms

A number of mechanisms operate in concert in growth cone navigation.
Long range guidance by the secretion of diffusible molecules from signpost
and target cells is supplemented by local (short range) positional cues and
by contact-mediated cues. As indicated in Table 10.3 some of the guidance
molecules attract growth cones while others repulse them. The designation
long range refers to the large distance between the cells that secrete diffusible molecules such as netrins, slits, and semaphorins and the growth
cones that sense these molecules when they are deposited in their local
microenvironment. In many instances intermediate target cells secrete
these molecules, and navigation is in short steps of several hundred microns.
The intermediate target cells are referred to as guidepost or signpost cells,
terms already mentioned. The immobilization of the diffusible molecules
within the extracellular matrix or attached to accessible surfaces such as
those of glial cells allows for detections of small variations in concentration
by the growth cones. These molecules together with the cell adhesion molecules create highways for the growth cones to travel upon.

10.15 Molecular Marking

237

The term short range usually refers to diffusion that spreads markers no
more than a few cell diameters from the source. The distance over which a
marker will diffuse depends on the spatial distribution of receptors. If receptors for the marker are highly expressed by nearby cells the distance traveled will be reduced over that which results from a sparse distribution of
receptors. The term contact-dependent is used to designate binding by
membrane-associated receptors on the surface of the growth cones to
membrane-associated ligands or counterreceptors expressed on the surface
of adjacent cells, and to components of the extracellular matrix such as
laminin. The distinction between a ligand and a counterreceptor is based on
whether the signaling is one-way or two-way. If it is two-way then both signaling partners are receptors. If one of the signaling partners supplies a
signal but does not transduce signals either directly or indirectly across its
own plasma membrane, then that partner is a ligand.
The modern form of the chemoafnity hypothesis has been greatly
expanded to incorporate short and long range mechanismsattraction,
repulsion, and bifunctionalityand pathnder axons, guidepost cells, and
selective fasciculation. Here is how the last process is described: Axons in
organisms such as Drosophila can grow on tracks laid down by earlier
pathnder axons. These later axons form axon bundles, or fascicles. The
axons select a particular axon bundle, bypassing inappropriate bundles in
the process, guided by molecular markers expressed on the surfaces. The
overall mechanism is thus called selective fasciculation. As the axons
approach their cellular targets they separate, or defasciculate, allowing the
axons to individually form connections.

10.15 Molecular Marking by Concentration Gradients

of Netrins and Slits
Concentration gradients of diffusible netrins and slits, and their receptors,
serve as molecular markers. The idea advanced in the 1950s and 1960s that
concentration gradients of diffusible chemical markers guide growth cones
to their targets gained support with the discovery of netrins in the mid
1990s. Netrins and slits function as diffusible chemoattractants and repulsants. Their receptors are all multidomain mosaic proteins containing
several immunoglobulin-like domains along with several bronectin
domains in their extracellular region along with a transmembrane segment
(Figure 10.8). Netrins and slits are bifunctional. They can act either as
chemoattractants or as chemorepellants. The specic response to a netrin
or slit molecule is dictated by the receptor to which it binds, and in particular it depends upon the properties of the cytoplasmic domain of the receptor. When binding to a DCC receptor, netrins provide a positive signal, but
when bound to an UNC-5 receptor they impart a repulsive cue. While the
extracellular domain of a netrin receptor determines the binding specicity,

238

10. Cell Adhesion and Motility

Figure 10.8. Netrins, slits, and their receptors: (a) Netrins consist of a laminin-like
domain, 3 EGF domains, and a netrin C-terminus domain. (b) There are two netrin
receptors. The rst (left) is the DCC receptor contains 4 immunoglobulin (Ig)
domains followed by 6 FNIII domains. The second receptor (right) is the UNC-5
receptor. It has 2 Ig domains plus 2 thrombospondin domains. (c) Slits consist of 4
leucine-rich repeats followed by 6 EGF domains, plus an agrin-laminin-perlecan-slit
domain, plus 3 more EGF repeats and a cys knot. (d) The slit receptor Roundabout
(Robo) contains 5 Ig domains followed by 3 FNIII repeats.

the cytoplasmic domain determines the cellular response elicited by the

guidance cue. This aspect has been demonstrated in experiments where the
cytoplasmic portion of a slit receptor was combined with the extracellular
portion of a netrin receptor and vice versa: The functionalityattraction or
repulsiontransferred with the cytoplasmic domain.
Both kinds of actionsattraction and repulsionare required. An
example of why this is so is provided by studies of commissural axons. These
are axons that cross the midline of the developing central nervous system
of bilaterally symmetric animals in order to contact neurons on the other
side. Midline glial cells concurrently express netrins and slits. The netrins
function as attractants and the slits as repellants. While navigating towards
the midline the growth cones express the netrin DCC receptors more
strongly than the slit Robo receptors, and the net effect is attractive. To
avoid endlessly wandering back and forth across the midline due to netrinmediated attraction, Robo expression is rapidly increased once the midline
is crossed. Responsiveness to netrins is reduced, the net effect becomes
repulsive, and the growth cone is sent on its way.

10.17 Their Eph Receptors Mediate Contact-Dependent Repulsion

239

10.16 How Semaphorins, Scatter Factors, and Their

Receptors Control Invasive Growth
Semaphorins and their plexin and neuropilin receptors provide guidance
cues and work together with scatter factors and their receptors to control
invasive growth. The semaphorins are a large family of secreted, GPIanchored and transmembrane guidance proteins. Based on structural considerations they have been grouped into seven or eight classes. Classes 1
and 2 consist of invertebrate semaphorins, while the others classes contain
vertebrate semaphorins. The dening characteristic of all semaphorins is
the presence of a 500-amino acid residue long Sema domain in the extracellular region. Two families of receptors for semaphorinsplexins and
neuropilinshave been found. The cytoplasmic domains of receptors
belonging to these families are short and thus have limited signaling capabilities. The receptors trigger intracellular responses either through association with coreceptors, or by directly coupling to intracellular signaling
proteins such as GTPases.
Scatter factor is also known as hepatocyte growth factor (HGF). It, like
the ephrins to be discussed in the next section, is a polypeptide growth
factor that binds to receptor tyrosine kinases, the main subject of the
next chapter. Scatter factors are not only involved in neural growth cone
guidance and growth, but also are central participants in invasive growth
and branching morphogenesis. The term branching morphogenesis
encompasses the growth, invasion, and proliferation of cells forming
branched tubular structures that carry uids in the vasculature, lungs,
kidneys, and mammary glands. It is a type of invasive growth that encompasses organ development and regeneration, wound healing, axon
guidance, and metastasis. The scatter factor receptor Met contains a Sema
domain in its extracellular region, and next to that a short Met-related
sequence (MRS), as do the semaphorins and plexins. Semophorin receptors
and ligands interact with each other and with Met and other growth factor
receptors and their ligands to transduce guidance and growth signals. For
example, the semaphorin 4D receptor plexin B1 interacts with Met to
control invasive growth.

10.17 Ephrins and Their Eph Receptors Mediate

Contact-Dependent Repulsion
Eph receptors make up a large subfamily of tyrosine kinases that are
involved in growth cone navigation, in directing migratory neurons during
development, and in vascular cell assembly. These receptors and their
ligands, the ephrins, are membrane-bound, and direct embryonic development by contact inhibition (repulsion). Eph receptors and ephrins are

240

10. Cell Adhesion and Motility

Figure 10.9. EphB receptor-ephrinB ligand
tetrameter: The gure was prepared using
Protein Explorer with atomic coordinates
deposited in the PDB under accession
number 1kgy.

widely expressed by cells in the vertebrate embryo. There are at least 14

different Eph receptors and 8 different ephrin ligands. The ability of ephrins
and their receptors to guide axons has been studied in the developing chick.
Graded distributions of ephrins help guide axons from the chick retina to
their visual cortex (tectum). They also assist in regulating axonal branchings and arborization in this retinotectal neural pathway.
Eph receptors fall into two groupsEphA and EphB. The extracellular portion of an EphB receptor contains an immunoglobulin-like
domain in the N-terminal region followed by a cysteine-rich sequence
followed by two bronectin domains proximal to the plasma membrane.
There is a single transmembrane chain and a cytoplasmic tyrosine
phosphorylation domain. EphA receptors lack the transmembrane and
cytoplasmic portions and instead are attached to the outer leaet of the
plasma membrane by a GPI anchor. The ephrins-fall into two groups,
as well. The ephrin-A group contains GPI-anchored ephrins and the
ephrin-B group consists of ephrins possessing a transmembrane segment
and a cytoplasmic tail. In general, EphA receptors bind ephrinAs and EphB
receptors bind ephrinBs.
A key step in activating the catalytic activity of the receptor is oligomerization. Like the scatter factor receptors just discussed, as well as other
receptor tyrosine kinases, ligand binding brings together two or more receptor molecules (Figure 10.9). The formation of a dimer or a higher order
oligomer brings the cytoplasmic domains into close physical proximity to
one another and this triggers their autophosphorylation leading to recruitment of signaling molecules and the formation of a chain of signaling
events ending at one or more control points. The chains of signaling events
launched by binding of growth factors to receptor tyrosine kinases are a
main focus of the next chapter.

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Brose K, et al. [1999]. Slit proteins bind Robo receptors and have an evolutionalily
conserved role in repulsive axon guidance. Cell, 96: 795806.
Goodhill GJ [2003]. A theoretical model of axon guidance by the Robo code. Neural
Comput., 15: 549564.

Problems

243

Hong KS, et al. [1999]. A ligand-gated association between cytoplasmic domains of

UNC5 and DCC family receptors converts netrin-induced cone attraction to
repulsion. Cell, 97: 927941.
Kidd T, Bland KS, and Goodman CS [1999]. Slit is the midline repellent for the Robo
receptor in Drosophila. Cell, 96: 785794.
Ming GL, et al. [2002]. Adaptation in the chemotactic guidance of nerve growth
cones. Nature, 417: 411418.

Semaphorins, Scatter Factors, Their Receptors, and Invasive Growth

Giordano S, et al. [2002]. The semaphorin 4D receptor controls invasive growth by
coupling with Met. Nature Cell Biol., 4: 720724.
Goshima Y [2002]. Semaphorins as signals for cell repulsion and invasion. J. Clin.
Investig., 109: 993998.
Maina F, and Klein R [1999]. Hepatocyte growth factor, a versatile signal for developing neurons. Nature Neurosci., 2: 213217.
Tamagnone L, and Comoglio PM [2000]. Signaling by semaphorin receptors: Cell
guidance and beyond. Trends Cell Biol., 10: 377383.

Eph Receptors and Ephrins

Holder N, and Klein R [1999]. Eph receptors and ephrins: Effectors of morphogenesis. Development, 126: 20332044.
Kullander K, and Klein R [2002]. Mechanisms and functions of Eph and ephrin signaling. Nature Rev. Mol. Cell Biol., 3: 475486.
Mellitzer G, Xu QL, and Wilkinson DG [1999]. Eph receptors and ephrins restrict
cell intermingling and communication. Nature, 400: 7781.
Orioli D, and Klein R [1997]. The Eph receptor family: Axonal guidance by contact
repulsion. Trends Genet., 13: 354359.
Wilkinson DG [2001]. Multiple roles of Eph receptors and ephrins in neural development. Nature Rev. Neurosci., 2: 155164.

Problems
10.1 Bond dissociation strengths are not constant quantities but instead are
sensitive to the presence or absence of external forces. This dependence is captured by the expression
0
koff (F ) = koff
exp [-E (F ) kBT ].

(10.1)

In this equation, the usual dependence of the rate on a constant

barrier height E has been replaced by a force-dependent barrier E(F).
The quantity F represents the applied force on the bond formed
during rolling. The applied force amplies the off-(dissociation) rate
for the receptor-ligand complex by reducing the height of the transition barrier. This force-driven reduction in barrier heights by an
external force is depicted in the gure presented below. The amplication in dissociation rate due to the presence of an applied force is
found by inserting the simplied expression for E(F) into the off-rate
equation. This gives

244

10. Cell Adhesion and Motility

0
koff (F ) = koff
exp [Fxav kBT ],

(10.2)

where koff(0) is the unstressed rate; that is,

0
koff (0) = koff
exp [-E kBT ]

(10.3)

is the conventional value for the off-rate in the absence of an applied

force. In the above, the angle-dependent expression has been replaced
by the quantity xav representing a thermally averaged distance between receptor and ligand interfaces over which the bond weakens
but does not yet break The expression for the amplication in the offrate is known as Bells equation. According to Bells equation the offrate rises exponentially with increasing force. In situations where the
barriers are sharp, the principal consequence is the linear lowering of
the barrier heights as a function of distance, with little change in shape
or location of the peaks and valleys. The inuences of an applied force
on the energy landscapes are illustrated in the gure for the cases
where there are two barriers.
Dissociation rates are inuenced not only by applied forces but also
by how rapidly they are applied. A bond that will appear strong if the
forces are rapidly ramped up to a maximum value will appear far
weaker and more easily broken if the same external forces are applied
slowly. One may dene an unbinding force as that force required to
just break a bond. In experiments performed in the laboratory, con-

Figure 1 for Problem 10.1. Lowering of the activation barrier by an applied force:
The applied force F acts along a direction oriented at an angle q with respect to the
reaction coordinate x, reducing the energy E by an amount Fxc os q. Because of
the dependence on the reaction coordinate x the outer barrier is driven down while
the inner barrier is barely affected. If the forces are strong enough the outer barrier
can be reduced below the height of the inner barrier, thereby revealing information
about the latters characteristics.

Problems

245

Figure 2 for Problem 10.1. Receptor-ligand bond dissociation data:The most probable unbinding forces are plotted against the logarithm of the loading rate. (a) The
data fall along a single line. (b) There is a sharp break in the data, producing two
linear regimes.

stant loading forces are applied to the bonds; that is, forces F of the
form F = rt, where r is the loading rate and t is the time. Under these
conditions the most probable unbinding force F* is related to the rate
and to the characteristic length by
F* =

kBT
ln
x

r
kBT
koff (0)
x

(10.4)

When the most probable unbinding forces of receptor-ligand pairs are

plotted against the logarithm of the loading rate the data typically fall
along a straight line. Two commonly encountered situations are
depicted below. In (a), the data are distributed about a single straight
line. What quantity does the slope of this line represent, and how does
it relate to an energy landscape? In (b), there is a sharp break in the
straight line so that there are two slopes. Interpret these results within
an energy landscape picture, i.e., what does the energy landscape look
like? Recall that typical thermal energies are on the order of 0.6
kcal/mol. For a distance x of 0.5 what is the corresponding force,
expressed in Newtons?
References on the Theory of Slip and Catch Bonds
Bell GI [1978]. Models for specic adhesion of cells to cells. Science, 200: 618627.
Dembo M, et al. [1988]. The reaction-limited kinetics of membrane-to-surface adhesion and detachment. Proc. R. Soc. Lond. B, 234: 5583.

11
Signaling in the Endocrine System

The endocrine system consists of a number of glands containing specialized

secreting cells that release signaling molecules into the bloodstream, other
bodily uids,and extracellular spaces.Glands of the endocrine system include
the adrenal gland, the hypothalamus, and the parathyroid, pineal, pituitary,
and thyroid glands. Hormone-secreting cells are found in many locations
in the body. The thymus produces a variety of hormones that regulate
lymphocyte growth, maturation and homeostasis. The pancreas contains
groups of hormone-secreting cells organized into pancreatic islets (the Islets
of Langerhans) that secrete insulin, glucagon, and somatostatin. Hormonesecreting epithelial cells are strategically situated in the gastrointestinal tract,
gonads, kidneys and placenta, where they direct the mitogenic activities of
cells and tissues that wear down and require constant replenishment, and
angiogenetic activities that produce new blood vessels.
Cells of the endocrine system produce several different kinds of hormonespeptide and polypeptide hormones, steroids synthesized from
cholesterol, hormones derived from amino acids, and hormones made from
fatty acids. Some hormones are lipophilic while others are water soluble.
Lipophilic hormones such as steroids, retinoids, and thyroids are small,
relatively long-lived hormones able to pass through the plasma membrane
and enter the cell where they bind nuclear receptors.Water soluble hormones
are fairly short lived and bind to receptors embedded in the plasma membrane of the target cells. Two kinds of transmembrane receptors bind
nonlipophilic hormones: Polypeptide hormones that promote growth and
wound-healing are bound by receptor tyrosine kinases; all others are bound
by G protein-coupled receptors, which will be discussed in the next chapter.
Polypeptide hormones are small compact molecules, ranging in size from
6 to 45 kDa. Epidermal growth factor (EGF) and nerve growth factor
(NGF) were the rst polypeptide growth factors to be discovered. These
and other growth factors bind to receptor tyrosine kinases expressed by
the appropriate recipient cells. Receptor tyrosine kinases are single chain
proteins that pass through the plasma membrane once. They, along with
nonreceptor tyrosine kinases, are exclusively found in metazoans. Several
247

248

11. Signaling in the Endocrine System

different classes of proteins help transduce polypeptide hormone signals

into the cell. These elements include, besides receptor tyrosine kinases
and the aforementioned nonreceptor tyrosine kinases, a variety of highly
modular adapters, and several families of GTPases. Each of these classes of
signal transducers will be examined in this chapter.

11.1 Five Modes of Cell-to-Cell Signaling

In the last chapter, signaling between cells was characterized as being either
contact-mediated or short range or long range. This catalog of signaling
modes can be generalized to encompass cytokine signals in the immune
system, long range hormone signals in the endocrine system, and neurotransmitters and neuromodulators in the nervous system. The expanded
ensemble of signaling modes contains ve entriesendocrine, paracrine,
autocrine, juxtracrine, and synaptic. In endocrine signaling, the sending and
receiving cells can be far apart. As noted in the introductory remarks, the
signals are conveyed from sending glands to distant points in the body by
bodily uids.
Paracrine signaling refers to signaling in which molecules are secreted
directly into the extracellular spaces by an originating cell, and these molecules travel no more than a few cell diameters to reach a target cell. The
signaling molecules are often immobilized by elements of the extracellular
matrix and bind to receptors on the surface of their cellular targets. This
mode of signaling is utilized by hormone-releasing epithelial cells, by
cytokine-releasing endothelial cells and broblasts, by chemotropic
factor-releasing cells that direct growth cones and migrating cells, and by
morphogen-releasing cells in organizing centers that direct cell fate during
development (to be discussed in Chapter 13).
Some hormones that are secreted act back on the cells releasing them.
In this autocrine mode of signaling, positive feedback serves to amplify
the initial weak signals. This mode is encountered in the immune system
(see, for example, signaling by IL-2 shown in Figure 9.1) and also in the
endocrine system as part of growth factor signaling. It is made use of during
development, and if not properly regulated leads to uncontrolled growth
and proliferation in a variety of cancers.
Cell-to-cell (juxtacrine) signaling is frequently conveyed by direct contact
between a receptor on one cell and a cell surface-bound ligand, or counterreceptor, on an adjacent cell. The nondiffusible signaling molecules participating in juxtacrine signaling may pass through the plasma membranes
or may be tethered to the outer leaet of the plasma membranes by GPI
anchors. This mode of signaling includes situations where cell surface receptors bind ligands that are components of the extracellular matrix.
Synapses are junctions formed to promote sustained signaling between
cells. Signaling through chemical synapses is the preeminent form of sig-

11.2 Role of Growth Factors in Angiogenesis

249

naling between nerve cells. This synaptic signaling involves the diffusion of
signaling molecules (neurotransmitters) across a synaptic cleft between the
membranes of a pair of pre- and postsynaptic nerve cells. The membrane
and submembraneous regions of synapses are specialized structures highly
enriched in many different kinds of signaling molecules. Like synaptic signaling between neurons, immune system T cells use synaptic signaling when
contacting antigen-presenting cells.

11.2 Role of Growth Factors in Angiogenesis

Several families of growth factors coordinate and control the formation of
new blood vessels during angiogenesis. Whenever new tissue is made additional blood vessels must be created to supply oxygen and nutrients and
remove waste products. The process of vascular development takes place in
several stages. In the earlier stages, called vasculogenesis, precursor vascular endothelial cells migrate, differentiate, proliferate and assemble into an
initial set of vascular connections of uniform size. In the later stages, termed
angiogenesis, the initial latticework of tubules is rened through further differentiation, sprouting and branching to form a mature vascular system. The
fully developed network contains large arteries that branch into progessively smaller blood vessels terminating in capillaries, and it contains a
return system of progressively larger venous structures.
The endothelial and smooth muscle cells that form the lining and sheathing of blood vessels coordinate their activities by sending and receiving a
variety of chemical signals. Some of the signaling molecules are vascular
endothelial cell-specic; others such as platelet-derived growth factor
(PDGF) and basic broblast growth factor (bFGF) are not. Members of the
vascular endothelial growth factor (VEGF) family are prominent among
the vascular specic growth factors. They are required for vasculogenesis
and also play a role in angiogenesis. A second family of growth factors,
the angiopoietins, binds to endothelial cell line-specic Tie receptors. They
work together with endothelial-specic ephrins and with the VEGFs to
coordinate and control angiogenesis. The VEGFs and angiopoietins operate
through a paracrine mechanism to signal and recruit nearby cells. The
ephrins remain attached to the plasma membrane of their originating cell
and signal bidirectionally through a juxtacrine mechanism.
As shown in Figure 11.1, the VEGF, Tie, and Eph receptors involved in
vascular development and repair are mosaic proteins. Their extracellular
regions are composed of multiple domains arranged in a linear fashion.They
differ from the receptors discussed in the last chapter by the presence of a
tyrosine kinase domain in their cytoplasmic segment. The angiopoietins and
ephrins promote the remodeling and branching, vascular maturation, and the
attachment to surrounding support cells and extracellular matrix. For
example, Ang1 promotes vascular maturation and stabilization while Ang2

250

11. Signaling in the Endocrine System

Figure 11.1. Angiogenesis receptors: The VEGFR-1/2 proteins contain 7 Ig-like

domains followed by a transmembrane segment and a cytoplasmic kinase domain
split by an insert. The VEGFR-3 chain differs slightly from VEGFR-1/2 in that the
set of tandem Ig domains is shortened by one and contains a disulde bridge. Tie
receptors 1 and 2 contain an Ig-like domain followed by 3 EGF-like repeats and
then another Ig domain followed by 3 FNIII repeats, a transmembrane segment,
and the split kinase domain. Eph receptors contain an Ig domain, a cysteine-rich
region, plus 2 FNIII repeats, a transmembrane segment, and a cytoplasmic kinase
domain.

maintains the vascular system in a plastic state. The ephrins were discussed
earlier in connection with growth cone navigation. The vascular-specic
Ephrin-B2 ligand and EphB4 receptor function in a roughly analogous
manner in angiogenesis. They are differentially expressed in endothelial
cells. The Ephrin B2 ligand functions as an arterial marker, and the EphB4
receptor operates as a venous marker.These signaling proteins help delineate
the boundary between blood vessels that become arteries and those that
become veins.

11.3 Role of EGF Family in Wound Healing

Growth factors of the EGF family coordinate and control wound healing
and the replacement of cells in tissues that undergo rapid turnover. Wound
healing is a complex process. Even in the case of a simple skin cut it requires
the participation of agents of the nervous system to generate a pain signal,
and elements of the immune system to generate an inammatory and
immune response. New tissue must be grown to replace the damaged material. The old damaged vasculature must be taken down and new vascula-

11.4 Neurotrophins Control Neuron Growth, Differentiation, & Survival

251

ture installed, and the underlying connective tissue must be remodeled and
restored.
In a skin cut, gloss keratinocytes (skin cells) migrate into the region of
injury, and upon arrival proliferate and mature. Signaling by members of
the EGF family of ligands and their receptors is essential for the proper
activities of keratinocytes during wound healing. Several EGF family
ligands are involved in repairing a skin wound. Among these are TGFa,
HB-EGF, AR, and betacellulin. These ligands are synthesized by keratinocytes, along with their EGF receptors and supply proliferation and
migratory signals, through an autocrine mechanism. In response to these
signals the keratinocytes increase their production of integrins. Binding by
these integrins to the ECM mediates substratum adherence by the keratinocytes and triggers the expression of collagesase-1.This enzyme degrades
the ECM, a step required for remodeling and repair of damaged tissue.
Continued autocrine signals maintain the expression of collagenase-1
during migration and repair.
Besides the skin, wound repair occurs in the gastronintestinal tract, in
muscle tissue following injury, and at sites of chronic inammation, to name
just a few common examples. Primary sources of epithelial growth factors
include the submaxillary salivary gland, the duodenal Brunners gland,
epithelial cells in the mammary gland, and the kidneys. Growth factors are
released into bodily uidssaliva, serum, milk, and urineand travel to
sites of injury and growth. Epithelial cells at many places in the body are
continuously renewed and growth factors are released locally near these
sites as well. In these situations the growth factors operate in autocrine and
paracrine manners rather than through an endocrine mechanism.

11.4 Neurotrophins Control Neuron Growth,

Differentiation, and Survival
Programmed cell death is an important process in the developing nervous
system. It is used to remove cells that are extraneous, cells that were transiently produced to help in development but are no longer needed, and cells
that failed to wire up properly to other cells. Neurotrophins are trophic
factors, chemicals that stimulate growth and development. Neurotrophins
such as NGF regulate the decision to survive or die, promote axonal and
dendritic outgrowth, branching and remodeling, and help control synapse
formation and plasticity. Other examples of trophic factors are ciliary
neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF),
PDGF, and bFGF.
The neurotrophin family of ligands and receptors contains four ligands
and two receptors. The four ligands are NGF, brain derived neurotrophic
factor (BDNF), neurotrophic factor 4 (NT4), and NT3. These ligands bind
to two kinds of neurotrophin receptors, namely, p75NTR receptors and Trk
receptors. As shown in Figure 11.2, the p75NTR receptor contains a cyto-

252

11. Signaling in the Endocrine System

Figure 11.2. Neurotrophin receptors: The p75NTR receptor has four cysteine-rich
repeats (CR1CR4), a transmembrane segment, and a cytoplasic death domain. The
Trk receptors have a cysteine segment (C1), 3 leucine-rich repeats (LRRs), a second
cysteine segment (C2), 2 immunoglobulin-like domains (Ig1, Ig2), an insert transmembrane segment, and a cytoplasmic kinase domain. All of the neurotrophins can
bind p75NTR. NGF binds the TrkA receptor; BDNF and NT4 bind the TrkB receptor, and NT3 binds the TrkC receptor.

plasmic death domain, while the Trk receptors possess a cytoplasmic tyrosine kinase domain. The two receptors operate using different sets of
adapter proteins to convey a variety of messages into the cell as indicated
in Figure 11.3. The Trk receptors utilize Shc and Grb2 adapters, while p75
employs TRAFs and a set of four or more adapters (Figure 11.3b) to convey
death and arrest messages. In general, p75NTR receptors convey apoptotic
messages and Trk receptors convey survival messages, but as illustrated in
Figure 11.3 the full picture is far richer. These and other growth factor
receptors work together along with each other and with integrins and
cadherins to convey messages to most or all control points in the cell. These
include regulatory sites for gene expression (to promote growth and differentiation), sites of focal contact between cells and ECM (to control
movement, adhesion, cell shape, and outgrowth), and mitochondrial control
sites for apoptosis (to regulate death versus survival decisions).

11.5 Role of Receptor Tyrosine Kinases in

Signal Transduction
Receptor tyrosine kinases transduce signals from polypeptide growth
factors into the cell. Except for receptors such as p75, polypeptide growth
factors bind to members of the receptor tyrosine kinases (RTKs), singlepass transmembrane receptors possessing an intrinsic tyrosine kinase
activity. These receptors contain three functional regions: an N-terminal

11.5 Role of Receptor Tyrosine Kinases in Signal Transduction

253

Figure 11.3. Ligand-bound Trk and p75 neurotrophin receptors, and associated
adapters: (a) NGF-bound Trk receptor. Second messengers and associated signaling intermediates involved in activating the PKB and PKC have been omitted to
keep the gure from becoming too cluttered. (b) Neurotrophin-bound p75 receptor. Abbreviations: p75NTR-associated cell death executor (NADE), neurotrophin
receptor-interacting melanoma-associated antigen (MAGE) homolog (NRAGE),
neurotrophin receptor-interacting factor (NRIF), Schwann cell factor-1 (SC1).

extracellular region that contains one or more ligand-binding sites; a transmembrane segment; and a C-terminal cytosplasmic region that contains a
catalytic domain and several phosphorylation sites.
The RTKs that bind polypeptide growth factors are highly modular linear
arrays of domains. Among the domains present in the extracellular regions
of these glycoproteins are Ig domains, bronectin type III domains, cysteine-rich domains and the EGF ligand-binding domains. A juxtamembrane
region is situated just inside the transmembrane helix region and, as mentioned above, there is a tyrosine kinase domain near the C-terminal region.
Docking sites are opened when (auto)phosphorylation of tyrosine residues
in the activation loop of the kinase domain turns on that domains catalytic

254

11. Signaling in the Endocrine System

activity resulting in a subsequent phosphorylation of tyrosine residues.

When this happens, proteins containing phosphotyrosine recognition
modules are able to dock at these sites.
Like other single pass transmembrane receptors, ligand binding in the
extracellular N-terminal region does not perturb the electrostatic environment enough to stabilize a conformation sufciently different in its Cterminal cytoplasmic region to serve as a signal. Instead ligand binding
promotes the formation of stable receptor dimers and oligomers. It is the
bringing together of two or more cytoplasmic domains that initiates
signaling inside the cell. In the case of RTKs, the close proximity of the two
cytoplasmic kinase domains and accompanying phosplorylation sites enable
cross or autophosphorylation of tyrosine residues.
The transmission of a signal into the cell interior by a receptor tyrosine
kinase following ligand binding occurs in two stages. The rst step is the
autophosphorylation, or cross phosphorylation, of tyrosine residues in the
activation loop of the catalytic domain. The bringing together of the cytoplasmic portions of the RTK triggers this step with the result that the catalytic activity of the kinase domain is turned on. The kinase domain then
catalyzes the phosphorylation of one or more tyrosine residues in the cytoplasmic region outside of the catalytic domain. This second step makes
available docking sites for signal proteins. Key to this second function is the
presence of protein-protein recognition modules, compact domains that
recognize short peptide sequences containing phosphorylated tyrosine,
serine, and threonine residues.
Receptor dimers and oligomers can be formed in response to ligand
binding in more than one way. Ligand-mediated dimerization triggers
human growth hormone (hGH) signaling. As discussed in Chapter 9, a
single ligand rst binds to one receptor and then attaches to the second
receptor to form a bridge that holds together a 1 : 2 ligand-receptor
complex. A different strategy is observed in EGF-induced dimerization. In
this case, the extracellular portions of the two receptors form the bridge
resulting in a 2 : 2 complex. In more detail, EGF-EGFR binding triggers
conformational changes that expose a loop on each receptor molecule.
These loops bind to each other to form the bridge. As noted earlier, the
ErbB2 receptor does not require a ligand for its activation. This happens
because the bridging loop on this receptor is constitutively in the open for
bridging conformation. The formation of a bridge by a pair of loops in
contact with a ligand dimer is depicted in Figure 11.3.

11.6 Phosphoprotein Recognition Modules Utilized

Widely in Signaling Pathways
Signaling proteins diffuse from one location to another, are activated and
turned off by binding and posttranslational modications, and are recruited,
assembled, and disassembled into signaling complexes at cellular control

11.6 Phosphoprotein Recognition Modules

255

points. The arrangements of these events into signaling pathways so that a

sequence of signaling steps can occur in the correct order at the right time
in the right place is made possible through the use of modular signaling
domains. Some of these domains supply a sort of glue that enables one
protein to bind another, and for that operation to be followed by yet
another binding operation leading to the recruitment of proteins and formation of signaling complexes. Other domains are specically designed to
recognize the presence of posttranslational modications such as the addition of phosphoryl groups. To see the utility of this kind of modular domain,
consider the receptor tyrosine kinases just discussed. The result of ligand
binding is the presence of one or more phosphorylated tyrosine residues
near the cytoplasmic C-terminus of the receptor. If this event cannot be
recognized and responded to, signaling ends.
Src homology-2 (SH2) and phosphotyrosine binding (PTB) domains
recognize short peptide sequences containing phosphotyrosine residues.
The SH2 domain is the prototypic recognition module. It consists of approximately 100 amino acid residues, and is found on proteins that assemble and
disassemble in the vicinity of the plasma membrane in response to autoand cross-phosphorylation of tyrosine residues in the cytoplasmic portion
of transmembrane receptor molecules. It is the rst discovered and largest
family of modules that recognize phosphorylated tyrosines. SH2 domains
recognize phosphorylated tyrosines and a specic anking sequence of
three to six amino acid residues located immediately C-terminal to pY.
Phosphotyrosine binding (PTB) domains also recognize short peptide
sequences containing phosphotyrosine, but, in contrast to SH2 domains,
amino acid residues N-terminal and not C-terminal to pY belong to the
recognition sequence. PTB domains recognize turn-loop structures and, in
particular, hydrophobic amino acid residues ve to eight residues located
N-terminal to pY.
14-3-3 proteins are small, 28 to 30 kDa in size. There are at least seven
isoforms of the mammalian 14-3-3 proteins; they are abundant and are
expressed in all eukaryotic cells. Unlike most of the other domains discussed in this section, 14-3-3 domains are not embedded in larger proteins
but exist as independent units that form homodimers. These proteins bind
with high afnity to peptide sequences containing phosphoserine residues
followed by a proline two positions towards the C-terminal. As dimers they
are able to bind to signaling molecules such as Raf-1 containing tandem
repeats of phosphoserine motifs. The 14-3-3 proteins localize their binding
partners within the cytoplasmic compartment and keep them sequestered
from the nucleus and membranes. These proteins operate in the growth and
apoptosis pathways and in cell cycle control.
Forkhead-associated (FHA) domains are conserved sequences of 55
to 75 amino acid residues. Whereas SH2 and PTB domains recognize
sequences containing phosphorylated tryosines, FHA domains can recognize peptide sequences containing phosphorylated threonines, phosphorylated serines, and phosphorylated tyrosines with a pronounced afnity for

256

11. Signaling in the Endocrine System

phosphothreonines. They are found in kinases, phosphatases, RNA-binding

proteins, and especially in nuclear proteins such as transcription factors and
proteins involved in detecting and repairing DNA damage.

11.7 Modules that Recognize Proline-Rich Sequences

Utilized Widely in Signaling Pathways
Proline has several properties that favor its utilization in signaling pathways. First it exhibits a rather limited range of conformations since it alone
of the amino acids has a side chain that closes back onto the backbone
amino nitrogen atom. This restriction in conformation extends to the
residue immediately preceding it and the result is a preference for a particular secondary structure called a polyproline (PP II) helix. This helix has
an extended structure with three residues per turn, and the prolines form
a continuous hydrophobic strip about the helix surface and also expose
several hydrogen binding sites. All in all, the result is an amphipathic structure sometimes referred to as a sticky arm. At the same time the binding
is relatively weak and complexes so formed are easy to disassemble. Small
changes in binding sequence or by phosphorylation produce large changes
in the dissociation constant.
SH3,WW, EVH1, and GYF domains all recognize proline-rich sequences,
but each recognizes a slightly different sequence or group of sequences. All
recognize some variant of the sequence PxxP that forms a left handed PP
II helix. The WW domain contains a pair of tryptophans (W) spaced some
20 to 22 amino acids apart and a block of two to four aromatic amino acids
situated in between the two tryptophans, hence the name WW domain.
There are several kinds of WW domains, each specic for a certain kind of
proline-rich sequence. Group IV WW domains recognize sequences containing phosphorylated serine/threonine residues.

11.8 ProteinProtein Interaction Domains Utilized

Widely in Signaling Pathways
Regulator-of-G protein-signaling (RGS) proteins contain a domain approximately 120 amino acids in length that acts as a G recognition module. It
functions as a GAP to accelerate the GTPase activities of Ga subunits of
heterotrimeric G proteins coupled to GPCRs. This module has been found
in proteins containing other signaling modules such as PDZ domains. The
next entry in Table 11.1 following RGS domain is the sterile alpha motif
(SAM) domain. This domain forms homo- and hetero-oligomers with other
domains and is encountered in a variety of signaling proteins. For example, it
is found in the C-terminus of all Eph receptors.

11.8 ProteinProtein Interaction Domains

257

Table 11.1. Protein interaction and phosphoprotein recognition modules:

Phosphoprotein recognition motifs are denoted using p-Tyr, p-Ser, and p-Thr
notation. Proline (P)-based protein interaction motifs are described using single
letter codes.
Name of module
Src homology-2
Phosphotyrosine binding
Src homology-3
WW
Enabled/vasodilator-stimulated
phosphoprotein homology-1
Gly-Tyr-Phe
14-3-3
Forkhead-associated
Regulator-of-G protein signaling
Sterile a motif
Death domain
PSD-95, DLG, ZO-1

Abbreviation

Recognition motif(s)

SH2
PTB
SH3
WW
EVH1

Phos-Tyr
Phos-Tyr, NPxY
PxxP
PPxY; PPLP; P-R; Phos-Ser
FPPPP

GYF
14-3-3
FHA
RGS
SAM
DD
PDZ

PPPPGHR
Phos-Ser
Phos-Thr
Ga
SAM
DD
PDZ, C-terminal motifs

The next-to-last entry in the table is the death domain (DD). Proteins
containing this domain convey death (apoptotis) instructions. The p75NTR
receptor shown in Figure 11.1 contains a cytoplasmic death domain (DD).
This domain enables cytoplasmic proteins bearing similar death domains
to attach via a DD-to-DD linkage. The DD is not the only module of this
sort. Instead there is an entire family of six (or seven)-helix bundle death
domains that includes the DDs, death effector domains (DEDs), caspase
recruitment domains (CARDs), and Pyrin domains (PYDs). Death
domains will be discussed further in the chapter on apoptosis.
The nal entry in the table is for the PDZ domain, named for the rst
three proteins found with this domainthe postsynaptic density protein of
95 kDa (PSD-95) found in postsynaptic terminals of neurons, the Discs
Large (DLG) protein of Drosophila, and the zona occludens 1 (ZO-1)
protein found in epithelial cells. These domains promote protein-protein
interactions through PDZ-PDZ binding and also recognize specic
sequences in the C-terminals of target proteins. Proteins containing PDZ
domains are especially prominent in synaptic terminals where they combine
with other domains to form scaffolding proteins. Members of the Shank
family of proteins serve as examples of this theme. As shown in Figure 11.4,
Shank proteins contain ve domains. Their SH3, PDZ, and proline-rich
regions either directly or indirectly bind to the three main kinds of glutamate receptors found in postsynaptic terminals, providing an anchorage
for their assembly and clustering. This assembly is anchored to the actin
cytoskeleton by intermediates that bind the ankrin repeats in the Nterminus and the proline-rich region in the middle. Finally, the SAM

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11. Signaling in the Endocrine System

Figure 11.4. Domain organization of a Shank protein: From the N-terminus to the
C-terminus the Shank protein has a set of ankrin repeats (Ank), an SH3 domain, a
PDZ domain, proline-rich region, and a SAM domain.
Table 11.2. Families of nonreceptor tyrosine kinases.
Family
Abl
Csk
FAK
Fes
Jak
Src
Syk
Tec

Family members
Abl, Ars
Csk, Ctk
CAKb, FAK
Fps (Fes), Fer
JAK1-3, Tyk2
Blk, Fgr, Fyn, Hck, Lck, Lyn, Src, Yes, Yrk
Syk, ZAP70
Bmx, Btk, Itk/Tsk, Tec, Txk/Rlk

domains near the C-terminus promote the linking of one Shank protein to
another through SAM-SAM binding.

11.9 Non-RTKs Central in Metazoan Signaling

Processes and Appear in Many Pathways
Metazoans make extensive use of tyrosine phosphorylation in their signal
transduction pathways and in addition to the receptor tyrosine kinases
there are several families of nonreceptor tyrosine kinases (NRTKs). These
signaling protein tyrosines range in size from 50 to 150 kDa. They are the
archtypical examples of modular proteins. In addition to possessing catalytic domains, the NRTKs possess in various combinations phospholipid,
phosphotyrosine, and proline-rich sequence recognition domains. Like an
RTK, the signaling (catalytic) activity of an NRTK is triggered by tyrosine
phosphorylation in the activation loop of the kinase domain. This can
happen through autophosphorylation or through phosphorylation by
another kinase.
The NRTKs can be grouped into eight families (Table 11.2) according to
their catalytic domain sequences, domain composition, and posttransla-

11.10 Src Is a Representative NRTK

259

tional modications. The Src family of NRTKs is the largest with nine
members while several other families have as few as two members. Some
NRTKs tether to the cytoplasmic face of the plasma membrane and are
covalently modied to permit attachment. Most are cytoplasmic proteins,
but Abl family members possess a nuclear localization signal (NLS) and are
found both in the cytoplasm and in the nucleus.

11.10 Src Is a Representative NRTK

The Src family is representative of the NRTKs and the discussion will
mostly focus on this family and on focal adhesion kinase (next section). The
organization of the catalytic domain of Src follows the general pattern
discussed above for the RTKs. The catalytic domain is bilobed with an
active site cleft and an activation loop containing a critical tyrosine residue.
Phosphorylation of this tyrosine activates the kinase domain. The overall
structure of Src is as follows. The N-terminal region contains a myristylation
site and sometimes a palmitoylation site. An SH3 domain, an SH2 domain,
the catalytic domain, and nally the COOH terminal region containing a
second critical tyrosine residue follow initial segment. Phosphorylation of
the tyrosine (Tyr527) in the COOH tail by regulatory proteins such as Csk
deactivates the protein kinase.
The SH2 and SH3 domains are located on the backside of the catalytic
domain of Src and do not impede activation and catalysis (Figure 11.5). The
SH2 domain of Src, as well as the SH2 domains of Fyn, Lck, and Fgr, select
peptide sequences of the form pYEEI; but they also bind the sequence
pYAEI of FAK and other similar sequences in other proteins. The SH3

Figure 11.5. The Src protein in open and closed conformations: (a) The Src protein
is in an open conformation in which a crucial tyrosine residue in the kinase domain
is exposed and phosphporylated. (b) The site of tyrosine phosphorylation in the
kinase domain is blocked and the binding surfaces of the SH2 and SH3 domains are
sequestered.

260

11. Signaling in the Endocrine System

Figure 11.6. SH2- and SH3-bearing proteins: (a) Organization of c-Src nonreceptor tyrosine kinase. (b) Organization of the Grb2 adapter protein. Proteins such as
Grb2 that lack domains that carry out enzymatic activities are referred to as
adapters. If they are very large and can bind multiple proteins they are called scaffolds. Sites where the protein can be phosphorylated on tyrosine residues are
labeled by the abbreviation p-Tyr.

domain of Src binds a number of PXXP-like sequences forming left handed

PP II helices. Although these two domains are located on the backside of
the catalytic domain they cooperate with one another to inhibit Src catalytic activity. In the simplest model of how this occurs Src has two states,
an inactive closed conformation and an active open conformation (Figure
11.6). In its closed conformation the SH2 domain binds to pTyr527 in the
COOH tail and the SH3 domain binds to a PP II helix in a portion of the
linker between SH2 domain and the catalytic domain. These binding events
are sufcient to shift the catalytic domain into a conformation where it
cannot bind ATP and release ADP, and cannot bind its protein substrate.
Thus phosphorylation at Tyr527 by Csk turns off the kinase activity of Src
through cooperative autoinhibitory actions of Srcs own SH2 and SH3
domains.The inhibitory interactions are relatively weak, and Src is activated
when phosphotyrosine- and polyproline-containing sequences in other
signal proteins favorably compete for Src SH2 and SH3 binding. Primary
activators of Src include PDGF and other RTKs such as EGF, FGF, and
NGF. These are not the only transmembrane receptors that signal through
Src family members. Other signal pathways linked to these NRTKs include
integrins, G protein-coupled receptors, and immune system receptors.
Proliferation is a highly regulated process. For proliferation to occur the
appropriate adhesive and growth factor signals must be sent and received.
Integrin receptors and receptor tyrosine kinases are localized in the plasma
membrane close to one another and together work in signaling growth. The
adhesive signals conrm that the cell remains in adhesive contact with the
extracellular matrix, and thus growth is permitted. Paxillin, a 68-kDa
protein associated with focal adhesions, contains multiple binding sites and
serves as a platform for gathering adhesive signals relayed through integrin
receptors and growth factor signals sent by receptor tyrosine kinases.
Several NRTKs participate in the relay of messages from the transmem-

11.11 Roles of Focal Adhesion Kinase Family of NRTKs

261

brane receptors to paxillin; prominent among these is the focal adhesion

kinase (FAK).

11.11 Roles of Focal Adhesion Kinase Family

of NRTKs
The FAK family of NRTKs promotes the assembly of signaling complexes
at focal adhesions, and regulates motility and growth factor signaling. Focal
adhesions are points of contact and adhesion between the cell and its supporting membranes. Focal adhesions are control points where growth and
adhesion signals are integrated together and coordinated across multiple
points of ECM-to-cell surface contact to govern the overall growth and
movement of the cell. These control points not only regulate the assembly
and disassembly of the focal adhesions but also convey signals that control
cellular growth, proliferation, differentiation, and survival.
Extracellular matrix proteins, transmembrane proteins, and the actin
cytoskeleton proteins participate in the adhesive contacts. Integrins and
growth factor receptor co-localize at focal adhesions. The integrins bind to
ECM proteins such as laminin and the growth factor receptors bind to
growth factor ligands. In response to ligand binding by these receptors, a
number of nonreceptor tyrosine kinases and adaptor/scaffold proteins
are recruited to the plasma membrane. Among the nonreceptor tyrosine
kinases recruited are Src, its negative regulator Csk, and focal adhesion
kinase. These kinases along with paxillin, a key adapter, link the integrin
and growth factor receptor-signaling to the actin cytoskeleton.
Paxillin is a fairly small protein; as mentioned previously it is only 68 kDa
in mass, but it contains a large number of binding sites. Its structure is shown
in Figure 11.7a. It possesses two tyrosine phosphorylation sites that are targeted by the nonreceptor tyrosine kinases such as Src, Csk and FAK, and
bound by SH2 domain-bearing proteins subsequent to phosphorylation. A
proline-rich region serving as an attachment site for SH3 domains is located
in the same vicinity. These N-terminal sites provide a linkage to upstream
integrins and growth factor receptors, and also downstream to proteins
associated with the actin cytoskeleton through the ve LD repeats. The Cterminal LIM domains anchor the paxillin protein at the plasma membrane.
The domain composition of FAK is presented in Figure 11.7b. It does not
have any SH2 or SH3 domains but instead provides phosphorylation and
anchoring sites for proteins with these domains. In place of the SH2 and SH3
domains FAK has two large domains of about 400 amino acids each, one on
either side of the catalytic domain. FAK possesses six tyrosine phosphorylation sites. Two of these, Tyr397 and 407, lie just N-terminal to the kinase
domain; two other, Tyr576 and 577, lie inside the kinase domain, and the last
two, Tyr861 and 925, lie in the COOH terminal region N-terminal to the
FAT. Autophosphorylation at Tyr397 exposes an SH2 docking site for Src.

262

11. Signaling in the Endocrine System

Figure 11.7. Focal adhesion proteins paxillin and FAK: (a) PaxillinThe four
Lin-11, Isl-1, Mec-3 (Lim) protein-protein interaction domains in the C-terminus
mediate targeting to focal adhesions (FAs). The leucine-rich sequences, or LD
repeats, sequences of the form LDXLLXXL, where X is any amino acid residue,
found in the N-terminus domain bind FAK and proteins such as vincullin associated with the cytoskeleton. The N-terminal domain contains a number of motifs
(tyrosine phosphorylation sites and proline-rich regions) that bind to Sh2- and SH3bearing proteins. (b) Focal adhesion kinase (FAK)This protein has a focal adhesion targeting (FAT) domain in its C-terminus, and several tyrosine phosphorylation
sites and proline-rich regions in its N-terminal domain.

Phosphorylation by Src at Tyr407, 576, and 577 maximally activates the

kinase domain, and phosphorylation at the sixth site,Tyr925, provides a Grb2
docking site. The domain structure of Grb2 was presented in Figure 11.6b. It
is an adapte protein that links FAK to the MAP kinase pathway. There are
also two proline-rich regions in the COOH terminal domain that provide
docking sites for adapte proteins bearing SH3 domains. Thus, like paxillin,
FAK serves as integrator of adhesive and growth signals. The COOHterminal region of FAK contains a focal adhesion targeting (FAT) sequence
of about 160 amino acids that provides binding sites for paxillin and talin.

11.12 GTPases Are Essential Regulators of

Cellular Functions
Since 1982, an ever-increasing number of GTPases have been found in
eukaryotes. Most of these belong to the Ras superfamily. The Ras GTPases
are small, 2040 kDa monomeric proteins that bind guanine nucleotides,

11.13 Signaling by Ras GTPases from Plasma Membrane and Golgi

263

Table 11.3. The Ras superfamily of GTPases.

Family
Ras
Rho
Ran
Rab
Arf

Function
Operates in the pathway that relays growth, proliferation, and differentiation
signals to the nucleus
Relays coordinating signals to the actin cytoskeleton
Shuttles mRNAs and proteins in and out of the nucleus
Regulates the targeting and docking of cargo vesicles to membranes
Regulates the formation of cargo vesicles

either GDP or GTP. More than 100 Ras superfamily GTPases have been
identied to date in eukaryotes. Each of these can be placed into one of
ve familiesRas, Rho, Ran, Rab, or Arf (Table 11.3). There are several
other groups of proteins that operate as GTPases. Chief among these are
the Ga subunits of the heterotrimeric G proteins that associate with G
protein-coupled receptors and the EF-Tu elongation factors that help
regulate protein synthesis.
Members of the Ras superfamily of GTPases carry out a variety of essential regulatory tasks. They regulate transcription, migration and focal adhesion, transport through the nuclear pore complex, assembly of the nuclear
envelope and mitotic spindle, and vesicle budding and trafcking. Ras
family members act as upstream switches in the pathway that conveys
growth and differentiation signals to the nucleus. Rho family members Rho,
Rac and Cdc42 coordinate growth and adhesion signaling. They regulate
the reorganization of the actin cytoskeleton, and coordinate its structure
with gene expression in response to extracellular signals. Ran GTPases regulate the inport and export of molecules from the cytoplasm to the nucleus.
They regulate the assembly of the nuclear envelope and also that of the
mitotic spindle. Rab proteins with over 30 family members are the largest
family of the Ras superfamily of GTPases. They are regulators of vesicle
trafcking, while Arf GTPases control vesicle budding.

11.13 Signaling by Ras GTPases from Plasma

Membrane and Golgi
Ras is a major regulator of cell growth. Ras GTPases convey growth, proliferation, and differentiation signals to the nucleus from the plasma membrane and from the Golgi apparatus. Ras is implicated in a large number
of human cancers, and in this context it will be examined further in Chapter
15. As shown in Figure 11.3 Ras operates as a master switch in the pathway
connecting upstream receptors for growth factors with downstream MAP
kinases that relay these signals to the transcription machinery in the
nucleus. Once Ras becomes GTP-bound it activates the serine/threonine
kinase Raf, which belongs to the Raf/MEK/ERK MAP kinase module.

264

11. Signaling in the Endocrine System

Signaling through this module activates downstream transcription factors

that stimulate growth and differentiation.
These downstream steps are preceded by upstream activation of receptor tyrosine kinases through ligand binding that creates binding sites for the
Grb2 adapter protein. This adapter enables the RasGEF Son-of-sevenless
(Sos) to bind and then accelerate the formation of GTP-bound Ras proteins. In the absence of GTP-bound Ras, the Raf kinase resides in the
cytosol. GTP-bound Ras brings Raf to the plasma membrane. The Raf Nterminal domain binds Ras, thereby enabling the Raf C-terminal domain
to bind and phosphorylate MEK.
Ras signaling is not restricted entirely to the plasma membrane. It can be
activated at the Golgi through a calcium-dependent pathway involving a
second pair of RasGEFs and RasGAPs. In place of the plasma membrane
sequence of steps where the adapter Grb2 links receptor kinases to the
RasGEF Sos, there is a pathway leading through Src. This nonreceptor
tyrosine kinase activates phospholipase Cg (PLCg), which acts on PIP2 to
produce DAG and IP3. The latter stimulates the release of Ca2+ from the
intracellular stores. In response to release of DAG and Ca2+, the RasGEF
RasGRP1 translocates to the Golgi where it activates Ras, while at the same
time the RasGAP calcium-promoted Ras inactivator (CAPRI) relocates
to the plasma membrane where it deactivates any plasma membraneassociated Ras.

11.14 GTPases Cycle Between GTP- and

GDP-Bound States
All GTPases function in a similar manner, passing through an assisted cycle
of GDP/GTP-binding and release. Recall from Chapter 6 and Figure 6.4
that GTPases bind either GTP or GDP. They are converted from GDPbound forms to GTP-bound forms by the catalytic actions of guanine
nucleotide exchange factors (GEFs). GTPase-activating proteins (GAPs)
catalyze their subsequent hydrolysis. In more detail, the GEFs kick out
the GDP molecule. In most situations GTP is far more abundant than GDP
and it readily binds the vacated pocket. In catalyzing the hydrolysis of the
GTP, a molecule of a phosphoryl group is removed leaving a GDP molecule bound to the GTPase.
GTPases such as Ras function as molecular switches. These switches are
turned on when GTP is bound and turned off when GDP is bound. In
the on state the GTPases are continually active and become inactive only
when turned off by a GAP. Ras is inactive when GDP-bound and it remains
so until upstream signals conveyed by RasGEFs trigger the dissociation of
GDP from Ras. Since the cellular concentration of GTP is far greater than
that of GDP, the binding of GTP to Ras follows almost immediately. At this
point Ras is activated and can bind to a downstream target, or effector,

11.14 GTPases Cycle Between GTP- and GDP-Bound States

265

Figure 11.8. GTPase cycles: (a) Switch cycle in which GTP-binding turns on the
GTPase, which can then interact with multiple effector molecules. The GTPase will
stay on until turned off by hydrolysis. (b) Assembly-controller cycle in which GTPbinding initiates interactions with an effector molecule. If the assembly is not
correct, the assembly step is aborted prior to hydrolysis, a process that is necessary
for disengagement from the substrate.

molecule. It remains active (on) until it undergoes hydrolysis. During the

time it remains on it can activate many Raf proteins, as illustrated in
Figure 11.8a.
The GEF for plasma membrane associated Ras is Sos (Son of sevenless).
It works in the following way to speed up the dissociation of GDP from
Ras: The Ras molecule, consisting of 188 amino acids residues, contains two
exible regions on its surface. These surface regions can alternate between
several conformational substates, and they are referred to as Switch 1 and
Switch 2. In its inactive state the Ras molecule binds GDP tightly with a
dissociation rate of 10-5/s. Sos-binding produces a tertiary Sos-Ras GDP
complex that stabilizes Ras in a conformation in which Switch 1 has swung
out to open the binding pocket. A portion of Sos stabilizes Switch 2 in an
alternative conformation, and this change plus an altered electrostatic environment further weakens the binding of the phosphate group of GDP and
its associated magnesium ion to Ras. The changes in the switch regions
increase the dissociation rate by several orders of magnitude and allow
GDP to exit the binding pocket in less than a second.
The Ras GAPs work in the following manner. Arginine and lysine
residues have long side chains and are positively charged under physiological conditions.When bound to the Ras-GTP complex the Ras GAPs extend
an arginine nger into the active site that neutralizes negative charges
in its vicinity. A network of hydrogen bonds form, stabilizing the transition
state and promoting the cleavage of the phosphodiester bond-linking

266

11. Signaling in the Endocrine System

gamma phosphate group to the GDP molecule. The result is an increase in

the rate of hydrolysis from one GTP molecule every 35 minutes to 102 to
103 molecules per minute.

11.15 Role of Rho, Rac, and Cdc42, and

Their Isoforms
Rho, Rac, and Cdc42, and their isoforms, coordinate the reorganization of
the actin cytoskeleton in response to extracellular signals. Members of
the Rho family of GTPasesRho, Rac, and Cdc42regulate cell polarity,
cell morphogenesis and shape, and cell motility. Each of these GTPases
operates in a different pathway. In broblasts, Rho promotes the formation
stress ber bundles, Rac proteins regulate actin polymerization resulting in
the formation of lamellipodia, and Cdc42 proteins control the formation
of lopodia. All three regulate the formation of focal adhesion complexes.
These observations are not limited to broblasts but rather are believed
to take place in all eukaryotic cells. In addition to their role in remodeling
the actin cytoskeleton, Rho family members regulate gene transcription.
Signaling through receptor tyrosine kinases, G protein-coupled receptors
and cytokine receptors activate Rho GTPases. In many instances integrins
and Rho family members work together to regulate the actin cytoskeleton.
An examination of the steps leading to bud formation in yeasts provides
some insight into how cell polarity develops and is regulated by the Rho
family GTPases.The Cdc42 GTPase, its GEF called Cdc24, its GAP, of which
there are several, and a scaffold protein named Bem1 contribute to the start
of budding.As is the case for all GTPases, Cdc42 goes through a cycle of GDP
release catalyzed by its GEF immediately followed by GTP binding. This
state is followed some time later by hydrolysis catalyzed by its GAP in which
GTP is cleaved leaving GDP bound to the CDc42 protein. Unlike Ras, the
Cdc42 protein does not act on multiple substrate proteins in its GTP-bound
state. Rather it goes through repeated cycles of GDP-GTP-GDP binding and
hydrolysis to regulate the polymerization of actin bers. The difference
between the Ras switchlike behavior and Cdc42 assembly controller-like
behavior is depicted in Figure 11.8. In the Ras mode of cycling, the GTPase
is on and can inuence many substrate proteins before being switched off
by hydrolysis. In the Cdc42 mode of operation (Figure 11.8b), hydrolysis is
required. Substrate interactions are again initiated by GTP binding but the
action is not completed until the hydrolysis part of the cycle is executed.
The EF-Tu GTPase uses this method of repeatedly cycling on and off during
the translation elongation process.This second mode allows for some checking and quality control over the process; the assembly step can be aborted
prior to hydrolysis if errors are detected.
Cell polarization (and bud formation) is typically driven by environmental cues such as gradients of chemoattractants or by signals from neighboring cells. These cues tell the cell which way is up and which way is

11.16 Ran Family Coordinates Trafc In and Out of the Nucleus

267

down. In bud formation, the adapter protein Bem1 is recruited to the site
of bud growth where it interacts with Ccd42 and its Cdc24 GEF. The GEF
is recruited and stabilized at the plasma membrane by the Gbg subunit activated by GPCR signaling. Both Cdc42 and Cdc24 bind to the scaffold and
a positive feedback loop is set up in which additional Bem1 proteins are
recruited. A crucial element in the choice of bud site selection is the presence of position landmarks in the cell that identify the location of the poles.
If these landmarks are absent the yeast cell is still able to form buds by a
process in which a bud site is randomly selected.

11.16 Ran Family Coordinates Trafc In and Out

of the Nucleus
While small molecules can rapidly and freely diffuse in and out of the
nucleus, macromolecules of size 40 to 50 kDa or greater cannot. Instead they
are transported through selective and facilitated diffusion through nuclear
pore complexes. Nuclear pore complexes (NPCs) are large units built up
from more than 100 different proteins. The NPCs function as selective gates.
Only the larger macromolecules containing the correct targeting or localization sequence are allowed through. Nuclear pore complexes have no
motors to actively transport cargo. Instead, import and export occur by
means of facilitated diffusion. This process does not consume energy and
is nondirectional. Proteins, mRNAs, tRNA, ribosomal subunts, and other
macromolecules are transported bound to transport molecules called
importins and exportins that recognize the nuclear localization signals
(NLSs) and nuclear export signals (NESs). These transport receptors
together with adapter molecules, Ran GTPases, Ran GEFs, and Ran GAPs
facilitate the diffusion of the macromolecules in and out of the nucleus.
Transport receptors belonging to the importin b family shuttle cargo in
and out of the nucleus through the NPC. These receptors are encountered
in two forms: as importins that shuttle cargo into the nucleus and as
exportins that chaperone cargo the other way. The loading and release of
cargo by the importin b receptors is regulated by the Ran GTPases. Ran, a
25-kDa protein, is an abundant gene product found in all eukaryotic cells.
The key element in their ability to regulate cargo movement is the formation of a Ran concentration gradient across the nuclear envelope. This gradient in GTP-bound Ran is established and maintained by the Ran GEFs
and Ran GAPs. The Ran GAPs are restricted to the cytoplasm and cannot
enter the nucleus, while the Ran GEFs are localized exclusively in the
nucleus. As a result the concentration of GTP-bound Ran is low in the
cytoplasm and high in the nucleus.
The combined actions of importin b receptors and Ran GTPases are illustrated in Figure 11.9. As is the case for all GTPases its main actions occur
during the GTP-bound part of the cycle, when it shuttles exportin + cargo
and unbound importin molecules through the NPC from the nucleus to the

268

11. Signaling in the Endocrine System

Figure 11.9. Ran GTPase-mediated import and export through nuclear pore complexes (NPCs): Importin (dark square) shuttles its cargo (dark circles) through the
NPC into the nucleus. In the nucleus, GTP-bound Ran GTPase triggers the release
of the cargo from the importin and shuttles the importin back out to the cytoplasm.
The GTP-bound Ran GTPase shuttles exportin (light squares) plus its cargo (light
circles) through the NPC from the nucleus to the cytoplasm where other proteins
(not shown) help dissociate the complex. The unbound exportin then diffuses back
to the nucleus.

cytoplasm. It differs from the Ras switching and Cdc42 cytoskeleton assembly in so far as its GEF and GAP actions take place in different compartments. Hydrolysis is not required for movement through the pore and
release of the cargo, but this aspect changes when Ran mediates assembly
of the nuclear envelope. In those assembly operations hydrolysis is
necessary.

11.17 Rab and ARF Families Mediate the Transport

of Cargo
Recall that in eukaryotes secreted soluble molecules and plasma membrane
lipids and proteins are transported from compartment to compartment by
transport vesicles. Newly synthesized proteins and lipids move from the
ER to the Golgi apparatus and from there to the plasma membrane. The
vesicles that transport these molecules are produced by budding from the
membrane of the donor compartment, and upon arrival to the acceptor
compartment fuse with the membrane of the acceptor compartment
thereby transferring the cargo. The process of delivering cargo from the
internal compartments to the plasma membrane is known as exocytosis.
Cargo moves in two directions, outward and inward. Many membrane components are rapidly turned over. Vesicles transport macromolecules from

11.17 Rab and ARF Families Mediate the Transport of Cargo

269

Figure 11.10. Rab GTPase cycle: The

Rab GTPase cycle synchronizes vesicle
transport and fusion between the membranes labeled A and B. In the cycle, a
GDP-bound Ran protein is shuttled by
its GDI to membrane A where GDP is
released and GTP is bound in its place.
While in its active form, a cargo vesicle
pinches off from the membrane and is
conveyed to membrane B where it
docks and then fuses. Hydrolysis occurs
and, assisted by the GDI, the GDPbound Ran protein is released from the
membrane and returned to the cytosol.

the plasma membrane either to the lysosomes, where they are degraded, or
to endosomes, where they are recycled back to the plasma membrane. The
process of transporting plasma membrane molecules to lysosomes for
degradation is known as endocytosis, and the outward and inward movement of plasma membrane molecules is said to take place in the secretory
and endocytic pathways.
The Rab family with more than 30 known members is the largest group
of Ras superfamily GTPases. These proteins coordinate the docking and
fusion of cargo vesicles operating in the secretory and endocytic pathways.
Reciprocal pairs of proteins called v-SNAREs and t-SNAREs mediate the
fusion of cargo vesicles. The binding of the SNAREs is preceded by tethering/protection protein-binding steps that ensure that only the appropriate fusion events takes place. The Rab proteins are localized to the
cytoplasmic face of organelles and vesicles and participate in the preliminary binding operations.
The Rab protein cycle of GDP and GTP binding and release is synchronized with the movement and fusion of cargo vesicles. The combined set of
steps is illustrated in Figure 11.10. In this gure the GEFs and GAPs are
joined by a third set of accessory regulatorsGDP dissociation inhibitors,
or GDIs. These molecules bind and maintain pools of inactive Rab proteins
in the cytosol and serve as recycling chaperones. They rst help to release
the GDPases from the membranes and then shuttle them back to their site
of origin in the cytosol in preparation for their next cycle of use.
ADP-robosylation factors (Arfs) make up the fth family of Ras
GTPases. These 20-kDa proteins help regulate the formation of cargo vesicles through budding from donor membranes. In order for budding to take
place, coat proteins must be assembled over the membrane surface. These
binding agents, coat protein I (COPI), coat protein II (COPII), and clathrin,
mediate the mechanical forces that pull a membrane into a bud, and they

270

11. Signaling in the Endocrine System

help capture membrane receptors and cargo. The Arf proteins recruit the
coat proteins to the donor membrane.

References and Further Reading

Angiogenesis
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development. Genes Dev., 13: 10551066.
Jones N, et al. [2003]. Tie receptors: New modulators of angiogenic and lymphangiogenic responses. Nature Rev. Mol. Cell Biol. 2: 257267.
Neufeld G, et al. [1999]. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J., 13: 922.
Yancopoulos GD, et al. [2000]. Vascular-specic growth factors and blood vessel formation. Nature, 407: 242248.

Neurotrophins
Chao MV [2003]. Neurotrophins and their receptors: A convergence point for many
signaling pathways. Nature Rev. Neurosci., 4: 299309.
Lee R, et al. [2001]. Regulation of cell survival by secreted proneurotrophins.
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Zheng YZ, et al. [2000]. Cell surface Trk receptors mediate NFG-induced survival
while internalized receptors regulate NFG-induced differentiation. J. Neurosci.,
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Ligand-Induced, Receptor-Mediated Dimerization

Schlessinger J [2002]. Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell, 110: 669672.

Phosphoprotein Recognition
Berg D, Holzmann C, and Reiss O [2003]. 14-3-3 proteins in the nervous system.
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Durocher D, and Jackson SP [2002]. The FHA domain. FEBS Lett., 513: 5866.
Forman-Kay JD, and Pawson T [1999]. Diversity in protein recognition by PTB
domains. Curr. Opin. Struct. Biol., 9: 690695.
Pawson T, Gish GD, and Nash P [2001]. SH2 domain, interaction modules and cellular wiring. Trends Cell Biol., 11: 504511.
Tzivion G, and Avruch J [2002]. 14-3-3 proteins: Active cofactors in cellular regulation by serine/threonine phosphorylation. J. Biol. Chem., 277: 30613064.
Yaffe MB [2002]. Phosphotyrosine-binding domains in signal transduction. Nature
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Recognition of Proline-Rich Motifs

Ball LJ, et al. [2002]. EVH1 domains: Structure, function and interactions. FEBS
Lett., 513: 4552.

References and Further Reading

271

Freund C, et al. [1999]. The GYF domain is a novel structural fold that is involved
in lymphoid signaling through praline-rich sequences. Nature Struct. Biol., 6:
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Kay BK, Williamson MP, and Sudol M [2000]. The importance of being proline: The
interaction of proline-rich motifs in signaling proteins with their cognate domains.
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Macias MJ, Wiesner S, and Sudol M [2002]. WW and SH3 domains, two different
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Mayer BJ [2001]. SH3 domains: Complexity in moderation. J. Cell Sci., 114:
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Zarrinpar A, and Lim WA [2000]. Converging on proline: The mechanism of WW
domain peptide recognition. Nature Struct. Biol., 7: 611613.

ProteinProtein Interaction Domains

Aravind L, Dixit MV, and Koonin EV [1999]. The domains of death: evolution of
the apoptosis machinery. Trends Biochem. Sci., 24: 4753.
Harris BZ, and Lim WA [2001]. Mechanism and role of PDZ domains in signaling
complex assembly. J. Cell Sci., 114: 32193231.
Hepler JR [1999]. Emerging roles for RGS proteins in cell signaling. Trends Pharmacol. Sci., 20: 376382.
Hofmann K [1999]. The modular nature of apoptotic signaling proteins. Cell. Mol.
Life Sci., 55: 11131128.
Hung AY, and Sheng M [2002]. PDZ domains: Structural modules for protein
complex assembly. J. Biol. Chem., 277: 56995702.
Schultz J, et al. [1997]. SAM as a protein interaction domain involved in developmental regulation. Protein Sci., 6: 249253.
Zhong H, and Neubig RR [2001]. Regulator of G protein signaling proteins: Novel
multifunctional drug targets. J. Pharmacol. Exp. Ther., 297: 837845.

Src Nonreceptor Tyrosine Kinase

Brown MT, and Cooper JA [1996]. Regulation, substrates and function of Src.
Biochim. Biophys. Acta, 1287: 121149.
Frame MC, et al. [2002]. v-Srcs hold over actin and cell adhesions. Nature Rev. Mol.
Cell Biol., 3: 233245.

Focal Adhesions
Ilic D, Damsky CH, and Yamamoto T [1997]. Focal adhesion kinase: At the crossroads of signal transduction. J. Cell Sci., 110: 401407.
Schlaepfer DD, Hauck CR, and Sieg DJ [1999]. Signaling through focal adhesion
kinase. Prog. Biophys. Mol. Biol., 71: 435478.
Turner CE [2000]. Paxillin and focal adhesion signaling. Nature Cell Biol., 2:
E231E236.
Renshaw MW, Ren XD, and Schwartz MA [1997]. Growth factor activation of MAP
kinase requires cell adhesion. EMBO J., 16: 55925599.

Ras Family of GTPases

Campbell SL, et al. [1998]. Increasing complexity of Ras signaling. Oncogene, 17:
13951413.

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11. Signaling in the Endocrine System

Hancock JF [2003]. Ras proteins: Different signals from different locations. Nature
Rev. Mol. Cell Biol., 4: 373384.
Kolch W [2000]. Meaningful relationships: The regulation of the Ras/Raf/MEK/
ERK pathway by protein interactions. Biochem. J., 351: 289305.

Rho Family of GTPases

Gladfelter AS, et al. [2002]. Septin ring assembly involves cycles of GTP loading and
hydrolysis by Cdc42p. J. Cell Biol., 156: 315326.
Hall A [1998]. Rho GTPases and the actin cytoskeleton. Science, 279: 509514.
Irazoqui JE, Gladfelter AS, and Lew DJ [2003]. Scaffold mediated symmetry breaking by Cdc42p. Nature Cell Biol., 5: 10621070.
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Ran Family of GTPases

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Rab and Arf Families of GTPases

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Recycling, and Signaling in the Endocytic Pathway

Gonzlez-Gaitn M [2003]. Signal dispersal and transduction through the endocytic
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Problems
11.1 (a) Binding interactions. One of the standard questions addressed in
investigations of signal transduction asks what interactions are
possible in a particular molecular setting. A number of proteins

Problems

273

Figure for Problem 11.1. Phosphorylated threonine and tyrosine residues are
identied by a P enclosed in a circle. The symbol PP refers to proline-rich
regions. Threonine and tyrosine phosphorylation sites with the correct anking
sequences for recognition by their kinases are indicated by the symbols p-Thr and
p-Tyr. Note that phosphorylation at the sites located in the center of the catalytic
domains of the NRTKs is required for activation.

and membranes are shown in the accompanying gure. Identify

the possible interactions in a mixture containing multiple copies of
each element (a) through (j).
(b) Build your own. Starting at the plasma membrane construct one
or more signaling pathways using the elements (a) through (j).
11.2 Nerve cells are highly polarized structures, and their terminals are
located far from the cell body where the nucleus is located. Signals
sent from axon terminals to the cell body must travel distances of up
to a meter.These signals are referred to as retrograde signals since they
move in a direction opposite to action potentials, which travel down
the axon to the nerve terminal. How might a neuron generate a rapid
(i.e., faster than one conveyed by passive diffusion) retrograde signal,
observing that receptor tyrosone kinases such as the Trks (and also G
protein-coupled receptors) are internalized following ligand binding?

12
Signaling in the Endocrine and
Nervous Systems Through GPCRs

The grouping formed by the G protein-coupled receptors (GPCRs) is the

largest of its kind in the body. Recent estimates of the number of GPCRs
in humans fall in the range of 800 or so. This number is divided roughly in
half between receptors that bind sensory signals originating from outside
the body (exogenous ligands) and signals produced internally by other cells
(endogenous ligands). GPCRs transduce a remarkably diverse spectrum of
messages into the cell. Among the signals transduced are light (phototransduction), extracellular calcium ions, tastants (gustatory), odorants
(smell), pheromones (mating signals), warnings (pain), immunological
(chemokines), endocrine (hormones), and neural (neuromodulators and
neurotransmitters).
Members of the GPCR superfamily are sometimes referred to as the
7TM receptors because their chains pass back and forth through the plasma
membrane seven times. The GPCRs transmit messages into the cell by activating heterotrimeric G proteins and by sending signals through growth
pathways. In the absence of GPCR ligand binding, heterotrimeric G proteins remain in close association with the GPCRs. Ligand binding leads to
G protein dissociation and signaling through second messengers that target
protein kinases/phosphatases and ion channels. In the rst part of the
chapter, the general characteristics of GPCR signaling will be examined,
and second messengers initially discussed in Chapter 8 will be looked at in
more detail.
The GPCRs are the targets of many therapeutic drugs due to the receptors prominent involvement in the endocrine and nervous systems. It is estimated that some 40 to 60% of all therapeutic drugs target GPCRs. Some
drugs act as agonists and others as antagonists. An agonist induces the same
response in the receptor as that triggered by the natural ligand. An antagonist binds to the receptor but the receptor does not transmit a signal in
response to the binding event. By binding the receptor the antagonist blocks
access of the natural ligand to the receptor and thus prevents transmission
of a signal. Drugs that bind in an antagonistic fashion are known as blockers. Prominent examples are beta blockers, which antagonize the beta275

276

12. Signaling in the Endocrine and Nervous Systems Through GPCRs

adrenergic receptor, and antihistamines, which inhibit the histamine H1

receptor. The second part of the chapter will provide an overview of signaling using hormones, neurotransmitters, and neuromodulators (endogenous ligands) and how light and other exogenous ligands are transduced
into cellular responses.

12.1 GPCRs Classication Criteria

G protein-coupled receptors share little sequence homology with one
another yet all are organized in a remarkably similar fashion. All are single
chain polypeptides with a topology as depicted in Figure 12.1. The transmembrane segments are composed of hydrophobic amino acid residues
arranged into an alpha helix. The helices are connected to one another by
three extracellular loops E1E3 and three cytoplasmic loops C1C3.
Each GPCR has an extracellular N-terminal region and a cytoplasmic Cterminal region. The extracellular and cytoplasmic regions can be quite
large. The extracellular loop E2 and the N-terminus segment are especially
well situated for ligand binding. These structures vary in size from one class
of GPCR to another. The intracellular loops plus the cytoplasmic ends of
the transmembrane helices participate in protein binding and activation.
The cytoplasmic tail and third intracellular loop (C3), in particular, provide
multiple sites for protein docking and interactions.
GPCRs can be grouped into families according to the most prominent
characteristics of the extracellular and intracellular domains, loop properties, and the presence of structurally important disulde bridges. GPCRs
possess a number of highly conserved sequences that inuence the structure and binding properties of the GPCRs. These, too, enter into the classication process. There are three main families of GPCRs and a growing

Figure 12.1. Stereotypic representation of a rhodopsin family (Class A) G proteincoupled receptor: Shown are the seven membrane-spanning alpha helices H1H7,
three extracellular loops E1E3, and three cytoplasmic loops C1C3.

12.1 GPCRs Classication Criteria

277

Table 12.1. Three main classes of G protein-coupled receptors: Receptor subtypes

are listed to the right of the ligand. G protein-coupled receptors are classied into
families according to their structure and the presence or absence in them of a
number of highly conserved sequences.
Class A: Rhodopsin family

Class B: Secretin family

Adrenergic a1, a2, b1, b2

Angiotensin AT1A, AT1B, AT2

Calcitonin CTR
Glucagon GR

Dopamine D1 to D5

Latrotoxin CL1, CL2, CL3

Histamine H1, H2
Melanocortin MC1, . . . , MC5
Melatonin ML1A, ML1B
Muscarinic acetylcholine
m1, . . . , m5
Neurokinen NK1, NK2, NK3
Opioid delta (d), kappa (k),
mu (m)
Prostanoid EP1, . . . , EP4,
DP, FP, IP, TP
Purine A1, A2A, A2B, A3,
P2Y1, . . . , P2Y6
Serotonin 5-HT receptor
subtypes
Somatostatin SST1, . . . , SST5
Vasopressin V1A, V1B, V2

PTH/PTHrP PTH/PTHrPR
Secretin SCTR
VIP/PACAP VIP1, VIP2

Class C: Glutamate family

Calcium CaR
GABA GABABR1 and
GABABR2
Glutamate mGluR1 to
mGluR8

list of smaller groups with a few members each. Some of the more prominent G protein-coupled receptors belonging to the three major families are
listed Table 12.1. Most of these receptors form small subfamilies. Each
receptor has several subtypes and each subtype may be expressed in one
of a number of alternative spliced forms.
Class A contains most of the known GPCRs. It is frequently referred to
as the rhodopsin family, named for the rhodopsin molecule responsible for
transducing photons in rod cells of the retina. Receptors for a diverse spectrum of ligands including most of the amide and peptide hormones and
neuromodulators belong to this family. Class A receptors bind a number of
glycoprotein hormones such as LH, TSH, and MSH using a large extracellular domain. The glycoprotein-binding receptors differ from the GPCRs
for the smaller ligands. The latter receptors rely on extracellular loops or
utilize a binding pocket formed by the H3 to H6 helices.
Class B GPCRs, the secretin family, include calcitonin, a 32-amino acid
peptide hormone released by C cells in the thyroid gland that regulate
calcium concentrations in the blood. Secretin, a hormone released by S
cells in the small intestine, stimulates the pancreas to secrete uids that regulate acidity during digestion. Parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP) regulate bone and mineral

278

12. Signaling in the Endocrine and Nervous Systems Through GPCRs

homeostasis. Many ligands for Class B GPCRs such as glucagon, secretin,

and VIP/PACAP (vasoactive intestinal peptide/pituitary adenylate cyclaseactivating polypeptide) are fairly large. Receptors in this family utilize an
extracellular domain that is far larger than the one depicted in Figure 12.3,
plus nearby extracellular loops, for ligand binding.
Class C receptors include the metabotropic glutamate and GABAB
receptors mentioned earlier. This collection of GPCRs is also known as the
glutamate family. Receptors that signal the presence of extracellular
calcium (CaRs) also belong to this group as do a number of mammalian
pheromone receptors. An extensive extracellular domain and a large intracellular domain characterize this group. The extracellular domain of these
proteins contains two lobes that clamp down on the ligand. This extracellular domain is homologous to bacterial periplasmic binding proteins
(PBPs). These are proteins found in the periplasmic space between the
outer and inner membranes in gram-negative bacteria. It is speculated that
at some time in the past genes encoding PBPs fused with those specifying
integral membrane proteins resulting in the formation of Class C GPCRs.

12.2 Study of Rhodopsin GPCR with Cryoelectron

Microscopy and X-Ray Crystallography
Cryoelectron microscopy and X-ray crystallography studies of rhodopsin
have provided detailed information on how GPCR helix bundles are organized and how the GPCR activates its heterotrimeric G proteins. As shown
in Figure 12.2 the seven alpha helices in rhodopsin are arranged sequen-

Figure 12.2. Structure of rhodopsin determined by means of X-ray crystallography:

The rhodopsin molecule contains seven
plasma membrane spanning helices (H1
through H7) plus a small C-terminus
loop H8. The helices are connected to
each other by extracellular loops E1E3
and cytosolic loops C1C3. The gure
was prepared using the Protein Explorer
and atomic coordinates deposited in the
Brookhaven PDB under accession code
1F88.

12.3 Subunits of Heterotrimeric G Proteins

279

tially in a clockwise manner when viewed from the intracellular side.

Helices 1, 2, 3, and 6 are tilted. That is, their axes are inclined by about 25
degrees relative to an axis drawn perpendicular to the surface. Helices 4
and 7 are nearly perpendicular to the membrane bilayer. Helix 6 is bent;
one part is nearly perpendicular to the plane of the membrane and the other
part is inclined by about 25 degrees (helix 5 is also bent, but to a lesser
extent). In the electron density plot of rhodopsin, the four tilted helices
form a central band. Helix 4 lies on one side of the band and helices 6 and
7 lie on the other side.
The rhodopsin molecule operates in a switchlike manner to activate the
G protein. Two of the helices, H3 and H6, project into the cytoplasm further
than the others. Ligand binding, or photoisomerization in the case of
rhodopsin, stabilizes the receptor in an alternative conformation in which
there is a shift in the relative positions of helices H3 and H6. When activated by ligand binding the GPCR acts as a guanine nuclcotide exchange
factor, or GEF, for its G protein. In its active shifted conformation, the
rhodopsin GPCR is able to catalyze the release of GDP from the alpha
subunit of the G protein leading to its binding GTP. The GDP-GTP
exchange triggers the dissociation of Gabg into Ga and Gbg subunits and the
subunits migration towards their effectors. The switch is reset by the dissociation of the ligand from the GPCR.

12.3 Subunits of Heterotrimeric G Proteins

As the name implies, heterotrimeric G proteins are assembled from three
distinct subunits. These subunits are designated as Ga, Gb, and Gg. There
are 20 different Ga subunits, 6 known Gb subunits and 12 distinct Gg subunits. The Ga subunits function as GTPases. Like the Ras GTPase discussed
in the last chapter the signaling activity of a Ga subunit is turned off when
it is GDP-bound and switched on when it is GTP-bound. The GPCR provides the activation signal and also serves as the GEF, catalyzing the dissociation of bound GDP from Ga. A family of proteins called regulators of
G protein signaling (RGS proteins) function as GAPs for the Ga subunits.
They catalyze the hydrolysis of GTP by the Ga subunits, thereby rapidly
switching off Ga signaling.
There are four kinds of G protein alpha subunits. For most GPCRs
one type of GPCR couples to and activates only one of the four kinds of
alpha subunits. However, in some cases, the coupling is richer and the GPCR
can switch from one kind of subunit to another. Several portions of the
GPCR contribute to the G protein-specic binding. Regions at the ends
of helices H3, H5, and H6, loops C2 and especially C3, and the short helix
(H8) located in the C-terminal region just after helix H7 are all involved in
coupling and activating the various members of the heterotrimeric G protein
family.

280

12. Signaling in the Endocrine and Nervous Systems Through GPCRs

Once activated the Ga and Gbg subunits are free to diffuse laterally along
the cytoplasmic surface of the plasma membrane, and bind nearby signaling targets, or effectors. The cycle of activation and signaling is completed
with hydrolysis and reassociation of the Ga and Gbg subunits. Upon binding
Ga, the Gbg induces substantial conformational changes and increases the
afnity of Ga for GDP. When bound to Ga, the Gbg subunit cannot bind its
effectors and signal because the binding sites on Gbg for its effectors overlap
that for Ga.

12.4 The Four Families of Ga Subunits

Four families of Ga subunits are presented in Table 12.2. As shown in
the table, Gs subunits bind to and stimulate adenylyl cyclases, while Gi
subunits inhibit these effectors. Gq subunits stimulate phospholipase C, and
the effectors of G12 subunits are as yet unidentied. Each Ga family
contains several variants. Some variants and groups of variants are found
in certain tissues while other are more broadly distributed. For example, G0
subunits are brain-specic and the Gi family is sometimes designated as Gi/0
to reect the inclusion of G0 subunits in this family. Figure 12.3 illustrates
how the Gs alpha subunit binds to the C1 and C2 domains of adenylyl
cyclase.
Gbg subunits target many of the same second messenger generators as
the Ga subunits. Like Ga subunits, some Gbg subunits stimulate adenylyl

Table 12.2. Mammalian Ga subunits: G proteins consist of a Ga subunit that is a

GTPase, and Gb and Gg subunits that remain attached and function as a unied
Gbg subunit. The four different types of G protein alpha subunits are presented in
column 1. Different subtypes are listed in column 2 and their effectors (substrates)
are given in column 3. Tissue-specic G proteins are noted in column 4. The actions
of the subunits on their effectors are given by the plus and minus signs. Plus signs
denote stimulation and minus signs indicate inhibition. Abbreviations: adenylyl
cyclase (AC), cyclic guanosine monophosphate (cGMP), phosphodiesterase (PDE),
phospholipase A2 (PLA2), and phospholipase C (PLC).
Family
Gs
Gi

Gq
G12

Gene variants

Effectors

as(S), as(L)
aolf
ai1, ai2, ai3
a0a, a0b
at1, at2
ag
az
aq, a11, a14, a15, a16
a12, a13

+AC
+AC
-AC
+PLC, +PLA2
+cGMP, PDE
+PLC
-AC
+PLC

Association
Olfactory
Brain
Retina
Gustatory

12.5 Adenylyl Cyclases and Phosphodiesterases

281

cyclases while others inhibit them. Still other Gbg subunits stimulate
phosopholipase C. Thus, Ga and Gbg subunits jointly determine the overall
action of a G protein on second messenger generators.

12.5 Adenylyl Cyclases and Phosphodiesterases Key to

Second Messenger Signaling
There are nine types of adenylyl cyclases. These isoforms are designated as
types I to IX. All of these isoforms can be stimulated by Gs subunits.
However, such is not the case for the Gi subunits. Adenylyl cyclase isoforms
I, V, and VI are the only ones inhibited by Gi subunits; the others are not
affected. The isoforms also differ in their responses to Gbg subunits, and to
the intracellular Ca2+ concentration [Ca2+]i. These response properties are
summarized in Table 12.3. As can be seen in Table 12.3, ve of the AC isoforms can be regulated by calcium. Thus, the two major second messenger
systems are tightly coupled to one another. This coupling can produce a
variety of effects including synchronized oscillations in the intracellular
cAMP and Ca2+ concentrations.
The magnitude and duration of cyclic nucleotide second messenger signaling is regulated by nucleotide phosphodiesterases (PDEs). Recall that
cAMP has a phosphate group attached to both the 3 carbon and 5 carbon
of the sugar ribose. PDEs are enzymes that catalyze the hydrolytic cleavage of 3 phosphodiester bonds in cAMP resulting in its degradation to inert
5AMP. They also carry out the same operation in cGMP to yield inert

Table 12.3. Response properties of adenylyl cyclases

to different regulators: The nine isoforms of adenylyl
cyclase (AC) are listed in column 1. Columns 25 show
how the four different kinds of regulators inuence
them. Stimulatory effects are denoted by plus (+) signs
and inhibitory inuences by minus (-) signs.
AC Isoform

Gs

Gi

Gbg

Ca2+

I
II
III
IV
V
VI
VII
VIII
IX

+
+
+
+
+
+
+
+
+

+
-

+
-

+
+

282

12. Signaling in the Endocrine and Nervous Systems Through GPCRs

Figure 12.3. Complex of the Gs alpha subunit bound to the C1 catalytic domain of
adenylyl cyclase V and the C2 domain of adenylyl cyclase II: Shown is a view of the
complex looking up from inside the cell towards the plasma membrane. The main
features of the interface between the G alpha subunit and the adenylyl cyclase
catalytic units consist of Sw I and Sw II of the Ras-like domain of G alpha that
protrude into a cleft formed by the alpha 1 to 3 helices of the C2 domain. The
gure was prepared using Protein Explorer with atomic coordinates deposited in
the Brookhaven Protein Data Bank under accession code 1AZS.

5GMP, thereby terminating second messenger signaling. They modulate

these signals with regard to their amplitude and duration, and through rapid
degradation restrict their spread to other compartments in the cell.

12.6 Desensitization Strategy of G Proteins to Maintain

Responsiveness to Environment
In order for a cell to maintain its responsiveness to future changes in environmental conditions as relayed through GPCR signaling, it must terminate
current GPCR responsiveness to persistent ligands in a timely fashion. The
process whereby a GPCR, or any other signaling entity, loses responsiveness to binding by its ligand is called desensitization. The turning off of the
GPCR is accomplished in a sequential manner by G protein-coupled receptor kinases (GRKs) and arrestins.
The domain structure of GRK2 and b-arrestin 2 are presented in Figure
12.4. Recall from Chapter 6 and Table 6.1 that G protein-coupled receptor
kinases are members of the AGC family of serine/threonine kinases. As
shown in Figure 12.4a, the GRK2 member of this family has a central kinase
domain plus two anking regulatory domains. The N-terminal domain contains an RGS homology (RH) domain that characterizes the family. In addi-

12.6 Desensitization Strategy of G Proteins to Maintain Responsiveness

283

Figure 12.4. GRK2 and b-arrestin 2 regulators of GPCR signaling: (a) G proteincoupled receptor kinase 2 (GRK2) consists of three domains. The RGS homology
(RH) and PH domains occupy the major portions of the N-terminal and Cterminal domains, respectively. (b) b-arrestin 2 has two domains, an N-terminal
domain and a C-terminal domain. A pair of regulatory segments, R1 and R2, resides
at the ends of the N- and C-terminals. The R2 domain contains clathrin- and
AP2-binding motifs. Other binding motifs are distributed through the N- and Cterminal domains.

tion to these two domains, GRK2 (but not all GRKs) contains a C-terminal
Plectstrin homology (PH) domain. PH domains bind phospholipids and also
proteins. The GRK2 C-terminal half of this domain together with several
residues lying just outside this domain binds Gbg. The three-dimensional
crystal structure of a Gbg subunit bound to GRK2 is presented in Figure 12.5.
The structure of b-arrestin 2 is presented in Figure 12.4b. b-arrestins are
adapter proteins devoid of any catalytic ability. In place of a catalytic domain,
members of this family possess a number of protein-protein binding domains
and motifs that enable them to function as adapters and scaffolds. The barrestins contain an N-terminal domain and a C-terminal domain. Their
proline-rich region in the N-terminal domain and MAP kinase recognition
domain in the C-terminal region provide the protein with the capability of signaling to Src and MAP kinases. In addition, b-arrestins have a phosphorylation recognition domain that mediates binding to the cytoplasmic tail of the
GPCR following its phosphorylation by the GRKs. They also have clathrin
and AP2 motifs in the C-terminal region that mediate their interactions with
the molecular machinery responsible for endocytosis.
The recruitment and subsequent termination of signaling by the arrestins
is mediated by a negative feedback loop that automatically prevents excessively sustained signaling. Ligand binding activates the GPCRs, leading to
activation of G proteins. The Gbg subunits bind (Figure 12.5) and activate

284

12. Signaling in the Endocrine and Nervous Systems Through GPCRs

Figure 12.5. G protein-coupled receptor kinase (GRK) bound to the Gbg subunit
of a heterotrimeric G protein: The structure of the complex determined by means
of X-ray crystallography. The three main domains of GRKthe N terminal RGS
homology (RH) domain, kinase domain and C-terminal PH domainare labeled
in the gure along with the Gb (dark gray) and Gg subunits (light gray) of the G
protein. The gure was prepared using Protein Explorer with atomic coordinates
deposited in the PDB under accession number 1OMW.

the G protein-coupled receptor kinase, which phosphorylates the receptor.

In response the b-arrestins are recruited to and bind the phosphorylated
residues using their phosphorylation domain. The GPCR is then no longer
able to active the G proteins and signaling ceases through this route. Thus,
signaling through this receptor is automatically shut down after a certain
time has elapsed.
Densensitization of the GPCRs is also promoted by second messengeractivated protein kinases. Protein kinase A activation mediated by Gs subunits and protein kinase C by Gq subunits both contribute to receptor
desensitization. The third intracellular loop and the cytoplasmic tail of the
GPCRs are primary sites of interaction with cytoplasmic proteins. The presence of consensus phosphorylation sites in these regions enables phosphorylation by these kinases. The phosphorylation by PKA of a GPCR to turn
off signaling establishes a negative feedback loop that acts through Gs subunits to stimulate cAMP production resulting in PKA activation that shuts
off further action by Gs subunits.

12.7 GPCRs Are Internalized, and Then Recycled

or Degraded
The GPCRs go through a life cycle of activation, signaling, deactivation, and
internalization resulting in either degradation or recycling. As illustrated in
Figure 12.6, the rst steps in this process are the same as discussed in the
previous section involving Gbg subunits, namely, GRK2 binding and

12.8 Hormone-Sending and Receiving Glands

285

Figure 12.6. Receptor internalization: Activation and G protein signaling is followed by desensitization and signaling that is independent of G proteins. Ligand
binding to a GPCR activates the G protein and signaling via second messengers.
The Gbg subunit recruits a G protein-coupled receptor kinase (GRK) to the GPCR,
which is then phosphorylated by the GRK. This action creates a docking site for barrestin, which upon binding to the GPCR blocks further G protein-mediated signaling. Proteins involved in endocytosis such as clathrin and AP-2 attach to the
b-arrestin, which now acts as a scaffold for assembly for factors required for vesicle
formation and internalization. In the internalization, the ligand-receptor complexes
are dissociated and, depending on the type of GPCR, the receptors are either
degraded or recycled rapidly or slowly.

phosphorylation, and b-arrestin recruitment. The subsequent recruitment

of clathrin and AP-2 enables the packaging of the loaded GPCRs into endocytic vesicles that pinch off from the plasma membrane. The cargo consisting of ligand-bound receptors, b-arrestins, and perhaps additional signaling
proteins such as Src are then delivered to endosomes for recycling to the
cell surface or for shipment to lysosomes for degradation. This kind of life
cycle is not restricted to GPCRs. Receptor tyrosine kinases such as the Trks
discussed in the last chapter are also packaged into endodomal vesicles for
internalization and recycling.

12.8 Hormone-Sending and Receiving Glands

The hypothalamus, anterior pituitary gland, adrenal gland, and the endocrine pancreas send and receive hormones. While polypeptide hormones

286

12. Signaling in the Endocrine and Nervous Systems Through GPCRs

signal through receptor tyrosine kinases, as was discussed in Chapter 11,

other nonlipophilic hormones secreted by cells in the hypothalamus,
anterior pituitary, adrenal gland, and endocrine pancreas signal through
GPCRs.
Hypothalamus. The hypothalamus controls the release of hormones by the
anterior pituitary. The hypothalamus produces two hormones that inhibit
hormonal release by the pituitary, and ve hormones that stimulate the
release of pituitary hormones. One of these ve, dopamine, is a catecholamine, a molecule composed of an aromatic (catechol) part, i.e., a
benzene ring plus two hydroxyl groups, and an amine part. The other hormones are peptides that vary in length from 3 amino acids (TRH) to 44
amino acid residues (GHRH). These neurohormones all bind to GPCRs on
cells of the anterior pituitary.
Anterior pituitary. The anterior pituitary produces hormones that control
hormone release elsewhere in the body. Five types of cells secrete anterior
pituitary hormones. Somatotrophic cells secrete growth hormone and lactotrophic cells produce prolactin. Growth hormone and prolactin are bound
by single chain cytokine receptors, and not by GPCRs. Human growth
hormone (hGH) receptor recognition was discussed in Chapter 8. Growth
hormones circulate through the body and inuence all cell types. Prolactin
inuences mammary gland development. Corticotrophic cells secrete corticotropin and melanocyte-stimulating hormone (MSH). Corticotropin
inuences the adrenal gland while MSH targets melanocytes, melanincontaining skin cells. ACTH and MSH are both derived from the precursor
molecule, pro-opiomelanocortin (POMC). Another molecule derived from
POMC is the neuromodulator beta-endorphin. Gonadotrophic cells secrete
follicle-stimulating hormones (FSH) and lutenizing hormones (LH), and
thyrotrophic cells produce thyroid-stimulating hormone (TSH). TSH, FSH,
and LH are large glycoproteins. The addition of carbohydrate groups to
these hormones extends their lifetime by protecting them from degradation. Gonadotropins stimulate a variety of responses in men and women
including sperm development, androgen release, estrogen synthesis,
and ovulation. These pituitary hormones with the exception of GH and
PRL all bind to GPCRs expressed by cells of the above mentioned
tissues and organs. The hormone sources and acronyms are summarized in
Table 12.4.
Adrenal gland. Chromafn cells in the adrenal gland produce two catecholaminesadrenaline (epinephrin) and noradrenaline (norepinephrin).
These hormones, secreted in response to extreme stress, trigger an increased
blood ow and other physiological ght or ight reactions. They are
derived from tyrosine and bind to adrenoreceptors, adrenergic GPCRs distributed in smooth muscle of the vascular system and gastrointestinal tract,

12.8 Hormone-Sending and Receiving Glands

287

Table 12.4. Hormones secreted by the hypothalamus, adrenal, anterior pituitary,

and endocrine pancreas glands: Listed are the names and either common abbreviations or alternative names of the hormones.
Hypothalamus
Corticotropin-releasing hormone (CRH)
Gonadotropin-releasing hormone (GRH)
Growth hormone-inhibiting hormone (GHIH)
Growth hormone-releasing hormone (GHRH)
Prolactin-inhibiting hormone (PIH)
Prolactin-releasing hormone (PRH)
Thyrotropin-releasing hormone (TRH)
Adrenal gland
Adrenaline (Epinephrin)
Noradrenaline (Norepinephrin)
Steroid hormones

Anterior pituitary
Adrenocorticotropic hormone (ACTH)
Follicle-stimulating hormone (FSH)
Growth hormone (GH)
Lutenizing hormone (LH)
Melanocyte-stimulating hormone (MSH)
Prolactin (PRL)
Thyroid-stimulating hormone (TSH)
Endocrine pancreas
Glucagon
Insulin
Somatostatin

adipose tissue, heart muscle, skeletal muscle, brain, and lungs. The adrenal
medulla is surrounded by the adrenal cortex, a second distinct structure in
the adrenal gland. The adrenal cortex releases a large number of steroid
hormones the most important being aldosterone and cortisol that help
maintain salt balance and water homeostasis in the body.
As mentioned previously, steroid hormones do not bind to receptors on
the cell surface but instead pass through the plasma membrane and bind to
intracellular (nuclear) receptors. Peptide hormones stimulate the synthesis
and release of these hormones. Angiotensin II and III produced in the
kidneys stimulate aldosterone production, ACTH stimulates cortisol production, and the glycoprotein hormone LH stimulates the release of the
gonadotropic steroid hormones progesterone and testosterone.
Endocrine pancreas. The insulin molecule, like ACTH and its siblings, is
generated from a precursor molecule, or prohormone. The nished insulin
molecule, consisting of two chains for a total length of 51 amino acids, is
produced by cleavage from a single chain precursor, proinsulin. Once
secreted by beta cells of the Islets of Langerhans in the pancreas, insulin
binds to receptor tyrosine kinases on target fat, muscle, and red blood cells.
Glucogon, a 29-amino acid protein, is produced by alpha cells of the islets
and by cells distributed throughout the gastrointestinal tract. It binds to a
GPCR. A third hormone, somatostatin is produced by cells in the islets as
well as by cells in the hypothalamus. All three of these hormones, insulin,
glucagons, and somatostatin, inuence the ow of nutrients in the body, and
maintain glucose homeostasis by regulating the storage of glucose and its
transport in and out tissues. Insulin release is stimulated by high blood sugar
levels and triggers the uptake of glucose by its target cells. Glucagon works
in the opposite direction. Glucagon receptors are found in the liver.

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12. Signaling in the Endocrine and Nervous Systems Through GPCRs

Glucagon is secreted by the pancreas in response to low blood sugar levels

and signals the liver to make and secrete glucose.

12.9 Functions of Signaling Molecules

Signaling molecules are often used in more than one way. Some function
as hormones and also as neuromodulators, substances that modify the electrical properties (excitability) of neurons. Other substances function both
as neuromodulators and as neurotransmitters. The distinction between
functioning as a hormone or as a neurotransmitter or as a neuromodulator
is based on how the signaling molecule is being used rather than on its
chemical composition.
Neurotransmitters are secreted in a highly directional manner. They are
released from the pre-synaptic terminal of a neuron and diffuse across the
synaptic cleft to the postsynaptic terminal of a neighboring neuron. There
are two kinds of receptors in the nervous system for neurotransmitters,
ionotropic and metabotropic. Ionotropic receptors are referred to as ion
channels. They respond to a binding event by transiently opening a channel,
an aqueous pore through the plasma membrane for the passage of ions
usually Na+, K+, Ca2+ or Cl-, depending on the type of neurotransmitter and
receptorin and out of the cell. This type of signaling is rapid and direct.
Signaling through metabotropic receptors, another term for GPCRs, is
slower and less direct.
Some of the most prominent neurotransmitters can signal through both
ionotropic and metabotropic receptors. Glutamate and g-aminobutyric acid
(GABA) are two of the most often encountered neurotransmitters in the
brain. There are ionotropic glutamate receptors and metabotropic glutamate receptors. Similarly there are ionotropic GABA receptors and
metabotropic GABA receptors; the former are referred to as GABAA
receptors and the latter as GABAB receptors. Another prominent neurotransmitter is acetylcholine. This neurotransmitter can bind to ionotropic
(nicotinic) receptors and to G protein-coupled (muscarinic) receptors.
The four most common neurotransmitters found in the brain are listed in
Table 12.5.The terms excitatory and inhibitory refer to the effects the neuro-

Table 12.5. Excitatory and inhibitory neurotransmitters used in the central nervous system: Common
abbreviations for the neurotransmitters are shown in
parentheses.
Excitatory
Acetylcholine (ACh)
Glutamate (Glu)

Inhibitory
g-Aminobutyric Acid (GABA)
Glycine (Gly)

12.10 Neuromodulators Inuence Emotions, Cognition, and Pain

289

transmitters have on membrane excitability. Inhibitory neurotransmitters

bind to anionic ion channels and decrease excitability, while excitatory neurotransmitters bind to cationic ion channels and increase excitability. The
subject of electrical excitability will be explored in Chapter 19.
Neuromodulators are secreted in a broader manner than neurotransmitters, and this kind of release allows them to inuence a large number of
cells. They bind to GPCRs and modify the excitability of the target cells by
regulating the activities of their ion channels mostly through inhibitory
mechanisms. Neuromodulators acting through GPCRs provide a means
whereby information from cells inuenced by a population of excitable cells
can be fed back to the originating population thereby regulating their electrical activities.
There are several routes of G protein regulation of ion channels. There
is a direct one where Ga and Gbg subunits directly bind and regulate ion
channels, and a number of indirect ones that operate through the second
messenger-binding and second messenger-activated protein kinases. The
direct route provides an efcient means for the regulation of electrical
activity in excitable cells by chemical messages since GPCRs, G proteins,
and ion channels are all membrane associated. A striking example of this
form of regulation is the gating of cardiac potassium channels by Gbg subunits. The specic K+ channels regulated in this manner are known as G
protein-linked inward rectied K+ channels (GIRKs). Binding of acetylcholine to the muscarinic (acetylcholine) GPCR leads to the activation of
the GIRKs by Gbg subunits in cardiac pacemaker cells producing a slowing
of the heart rate. This process is not restricted to the heart; GIRKs are also
found in the brain and pancreas. A classic example of indirect regulation is
phototransduction by rhodopsin and its retinal ligand.
The most common form of neuromodulation is through the indirect,
second messenger-mediated regulation of ion channels. In this process, Gs,
Gi, and Gq alpha subunits stimulate or suppress second messengers and
second messenger-dependent protein kinases that phosphorylate Ca2+, Na+,
and K+ channels, thereby leading to changes in the ion channels structural
and kinetic properties. Because of the amplication inherent in second messenger systems the neuromodulatory signals are able to modulate the activities of many ion channels at the same time. This indirect method adjusts
the overall ring properties of the excitable cells, and in many cases alters
the way the neural circuit operates.

12.10 Neuromodulators Inuence Emotions, Cognition,

Pain, and Feeling Well
Neuromodulators produce changes in the electrical excitability of neurons
and neural circuits that can last for hours and days. Some neuromodulators
are peptides; others are nucleotides or amines. When acting in the brain

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12. Signaling in the Endocrine and Nervous Systems Through GPCRs

Table 12.6. Different categories of neuromodulators:
The neuromodulators are classied according to their
chemical derivation.
Amines
Adrenaline
Dopamine
Histamine
Noradrenaline
Serotonin

Peptides
Angiotensins
Neurokinins
Oxytocin
Substance P
Vasopressin

Nucleotides

Endogenous opioids

Adenosine
ADP, ATP
GDP, GTP

Dynorphins
Endorphins
Enkephalins

these neuromodulators inuence cognition and emotions. Listed in Table

12.6 are a number of the most prominent neuromodulators that act in the
brain, such as adenosine, serotonin, and dopamine. Serotonin is distributed at many places in the brain. It is synthesized from the amino acid Ltryptophan, and it is often referred to as 5-hydroxytryptamine (5-HT).
Another aminehistaminewas discussed earlier as a mediator of inammatory responses in the immune system. When secreted by mast cells it can
bind to receptors expressed on peripheral nerve endings and serve as a
neurmodulator.
Endorphins and tachykinins are examples of peptide neuromodulators.
Endorphinsendogenous morphinealong with many other opiates inuence perceptions of pain and pleasure. These substances bind to the opioid
family of GPCRs expressed on the surface of neurons in the brain and
spinal cord. Neuromodulators binding to opioid receptors have been placed
under a separate heading in Table 12.6 reecting their binding properties
and actions. Endorphins have analgesic properties, and are also involved in
maintaining water balance and other endocrine functions. Members of the
endorphinlike family of opiates include beta-endorphin, enkephalins, and
dynorphins. Beta-endorphins are synthesized in the pituitary as mentioned
earlier, and enkephalins are produced in the adrenal medulla by chromafn cells. These neuromodulators along with dynorphins are broadly distributed in several specic areas of the brain and spinal cord where they
bind to their cognate opioid receptors. The tachykinins are another family
of neuromodulators involved in the perception of pain. Mammalian
members of this family, the neurokinins, include substance P (SP), neurokinin A (NKA) and neurokinin B (NKB).
Changes in the amount of neuromodulators in sensitive parts of the body
produce sensations leading to alterations in behavior. The angiotensins are
a family of regulators of blood pressure, blood volume, and cardiac vascu-

12.11 Ill Effects of Improper Dopamine Levels

291

lar function. Production of angiotensins is stimulated by angiotensinconverting enzyme (ACE) and renin, a glycoprotein hormone produced
by the kidney and other places in the body including the brain. An infusion
of angiotensin II, the primary active angiotensin, into the brain of mammalian test subjects triggers the sensation of thirst and a craving for sodium
(salt). Laboratory animals stop what they are doing and immediately begin
drinking, and they exhibit an increased appetite for sodium when so
infused.

12.11 Ill Effects of Improper Dopamine Levels

As mentioned earlier, G-protein coupled receptors and the signaling molecules that activate them are involved in a host of neurological disorders
and are principal target of therapeutic drugs. Dopamergic neurons are those
neurons that synthesize and secrete dopamine. Improper dopamine levels
contribute to Parkinsons disease, ADHD, schizophrenia, and other disorders. There are three main groups of dopamine secreting cells: one group
located in an area of the midbrain called the substantia nigra (SN), another
in the ventral tegmental area (VTA), and a third in the hypothalamic nuclei.
These cells, along with several other smaller groups of dopamine-secreting
cells, are referred to as the dopamine system. This system of cells is involved
in drug addiction, Parkinsons disease, Tourette syndrome, schizophrenia,
and attention-decit hyperactivity disorder (ADHD). In Parkinsons disease, cells in the SN that secrete dopamine die leading to a deciency in
dopamine in the brain. In schizophrenia, antagonists, drugs that inhibit
dopamine signaling, are used to suppress an overactive dopamine system.
The most common feature of drug addiction is an elevation in dopamine
signaling.
Cells in these three areas send out dopamergic signals to a large number
of brain areas. The cells receiving these signals express two kinds of
dopamine receptors. Receptors of the D1 type (D1 and D5) act through Gs
subunits to activate adenylyl cyclase. Receptors of the D2 type (D2, D3, and
D4) exert their inuences through Gi subunits to suppress adenylyl cyclase
and activate potassium channels.
Children and adults with ADHD exhibit behavior that is inattentive,
impulsive, and hyperactive. In many of these patients there is reduction in
size of the basal ganglia and frontal lobe responsible for controlling these
behavioral responses. It appears that the level of dopamine signaling is too
low, either because of the presence of too many dopamine transporters that
remove dopamine from the extracellular milieu, or because of inadequate
amounts of dopamine released from presynaptic terminals. These decits
are countered through the application of drugs such as methylphenidate
[Ritalin] resulting in increased levels of extracellular dopamine in the
brain.

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12. Signaling in the Endocrine and Nervous Systems Through GPCRs

12.12 Inadequate Serotonin Levels Underlie

Mood Disorders
The serotonergic system has, as its serotonin-producing core, cells in the
midbrain and pons close to where the brain and spinal cord join. The
serotonin-producing cells are organized into clusters called Raphe nuclei.
The neurons in the nuclei are large and send out their processes to almost
every locale in the brain. Serotonin receptors can be grouped into seven
classes, designated as 5-HT1 through 5-HT7. All but the 5-HT3 receptors are
GPCRs. The 5-HT3 receptors are ligand-gated ion channels (discussed
in Chapter 19). The best-studied GPCR classes are the 5-HT1 and 5-HT2
receptors. Members of the 5-HT1 grouping act through Gi and G0 types
of G-proteins alpha subunits. They inhibit adenylyl cyclase activity and
stimulate the opening of hyperpolarization-producing potassium channels.
Serotonin 5-HT2 receptors work through Gq subunits to increase IP3 and
intracellular calcium levels, and depolarize neurons by closing potassium
channels.
Inadequate serotonin levels are responsible in a variety of mood disorders. Serotonin levels are increased in several ways to treat depression and
panic attacks as well as other anxiety disorders. Drugs that increase serotonin levels by inhibiting the reuptake of serotonin are especially popular.
Prozac (uoxitine) is a selective serotonin reuptake inhibitor (SSRI), as
are other popular drugs such as Paxil and Zoloft. Serotonin and noradrenaline are monoamines. Another class of drugs that elevate serotonin
levels is the monoamine oxidase inhibitors, or MAOIs. Monoamine oxidase
is an enzyme that degrades serotonin and noradrenaline. The MAOIs work
by inhibiting the actions of monoamine oxidase. Yet another group of
serotonin-increasing drugs are the tricyclic antidepressants (TCAs). These
too prevent serotonin reuptake.

12.13 GPCRs Role in the Somatosensory System

Responsible for Sense of Touch and
Nociception
G protein-coupled receptors play a central role in the sensing and transmission of messages of a warning nature in the somatosensory system. The
somatosensory system handles four categories of touch information:
proprioception, simple touch (pressure, texture, and vibration sensations),
temperature (heat sensations), and pain. The term proprioception refers
to body and body part position, orientation, and location. This faculty utilizes mechanoreceptors that sense forces in muscles, tendons, and joints.
Simple touches such as pressure are sensed by special receptors in the skin.
Sensations of a warning natureof temperature and painare mediated
by free peripheral nerve endings, called nociceptors, which extend through-

12.14 Substances that Regulate Pain and Fever Responses

293

Table 12.7. Hormonal signaling

during injury and pain.
Local hormone
Adenosine
ADP, ATP
Bradykinin
Histamine
Prostanoids
Serotonin
Substance P

out the body and use a variety of G protein-coupled receptors and ion channels to sense and transduce signals.
Nociceptors are sensitive to extracellular chemical, mechanical, and
thermal environments. In response to inappropriate conditions, they transmit signals through spinal neurons to brain receptors resulting in the perception of pain. The pain pathway is the general term given to the
signaling route from the peripheral neurons located in places such as the
skin to the spinal cord to the brain. Nociceptors are distributed throughout
the body, but are generally absent in the brain, deep tissues, and visceral
organs such as the liver, spleen, and lungs. The cell bodies of the free nerves
are located in dorsal root ganglia (DRG). Signals travel from the DRG to
the dorsal horn of the spinal cord and from there to the brain. There are
two kinds of free nerve bers, Ad and C. Ad bers are thin and myelinated,
and rapidly conduct sharp pain signals. C bers are unmyelinated and
slowly conduct aching, itching, and burning signals. The C bers contain
receptors for chemical signals sent by injured cells and by cells of the
immune system. (Note: Myelinated bers are bers, or axons, that are
sheathed in myelin, a fatty protein material that insulates the axons and
promotes fast conduction of nerve pulses.)
A variety of chemical signals associated with injury and inammation are
exchanged between the nervous and immune systems. Injured cells release
some of these warning chemicals, and cells of the immune systems such as
mast cells secrete others. These messages (Table 12.7) act as local hormones
that bind to receptors on peripheral nerve cells. Some of these signaling
molecules and their receptors were discussed earlier. Those not yet examined include prostanoids, bradykinin, and purines such as adenosine. These
are discussed next.

12.14 Substances that Regulate Pain and

Fever Responses
Nonsteroidal anti-inammatory drugs (NSAIDs) have been relied on for
reducing inammatory responses including pain and fever for 3500 years.
Early medicinal extracts from willow bark led to the rst commercial

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12. Signaling in the Endocrine and Nervous Systems Through GPCRs

Figure 12.7. Generation of fever and pain signals: Pro-inammatory signals activate phospholipase A2, which hydrolyzes membrane phospholipids (here depicted
as phospholipids in the plasma membrane) resulting in arachidonic acid.A sequence
of enzymes, beginning with COX, produces prostanoids from the arachidonic acid.
The prostanoids are short lived with half lives on the order of a minute or so and
are immediately secreted from the cell. Prostanoid receptors on peripheral nerve
endings bind the prostanoids, and the neurons convey the pain and fever signals to
the brain.

product aspirin one hundred years ago and to a host of more recent
NSAIDs such as ibuprofen and naproxen. All of the NSAIDs work the
same waythey inhibit the catalytic actions of a set of enzymes variously
called endoperoxide H synthases (PGHSs) or cyclo-oxygenases (COXs).
Two COX isoforms are targeted by anti-inammatory NSAIDs. The rst of
these, COX-1, is constitutively active and is found primarily in the stomach
and kidneys. The second, COX-2, is induced in many different cell types in
response to inammatory signals conveyed by cytokines, growth factors, and
hormones. Inhibition of COX-1 can be harmful and the aim in drug design
is to preferentially target the COX-2 isoform.
Cells release prostanoids (prostaglandins) when injured by chemical,
thermal, or mechanical agents. Prostanoids are fatty acid derivatives of
membrane lipids. As depicted in Figure 12.7, the rst step in their production is the hydrolysis of membrane lipids by phospholipase A2 (PLA2)
resulting in the release of arachidonic acid (AA). The AA intermediate is
then converted to prostanoids through the sequential actions of the COXs
and several other enzymes. The catalytic activities of PLA2 are stepped
up in response to the cytokine, growth factor, and hormonal signals. In
response the cells produce and then secrete prostanoids. During the inammatory response G protein-coupled prostanoid receptors expressed on
peripheral neurons bind prostaglandin. Signals sent on to thermoregulators

12.15 Composition of Rhodopsin Photoreceptor

295

in the brain trigger an increase in body temperature. Aspirin and the other
NSAIDs act to lower temperature and reduce pain by inhibiting the COXs,
thereby preventing formation of and signaling by the prostanoids. The
prostanoid signals are conveyed in a paracrine fashion to their cellular
targets. For this reason prostanoids and other hormones acting in a
paracrine manner are termed local hormones.
Bradykinin is a nine-amino acid residue peptide rapidly produced
after tissue injury occurs. It is a central element in the inammatory
response. It is involved in regulating blood ow (vascular dilation), increasing vascular permeability, smooth muscle contractions, stimulating
the release of prostaglandins and other inammatory mediators, and
pain signaling. Bradykinins bind to two kinds of receptors, B1 and B2. B2
receptors are constitutive in neurons and smooth muscle cells; B1 receptors,
in contrast, are rapidly upregulated when there is tissue injury. B1 and
B2 receptors are found in free nerve endings and directly mediate pain
signaling.
Adenosine, ATP, and ADP serve as neurotransmitters and neuromodulators in the central and peripheral nervous systems. When released into
extracellular spaces these molecules bind to purine receptors. Purine receptors are divided into two families, P1 and P2, each containing a number of
subtypes (Table 12.1). Adenosine attaches to P1 receptors while ATP, ADP,
and the pyrimidines UTP and UDP bind to P2 receptors. Adenosine and
the other nucleotides carry out a large number of signaling tasks in the
body. In heart muscle, adenosine decreases heart rate and lowers blood
pressure. Adenosine is associated with sleep; during sleep deprivation
adenosine levels build up in the brain. When bound to adenosine receptors
in the brain, adenosine decreases neural activity leading to sleep. Caffeine
is structurally similar to adenosine, and is an adenosine antagonist acting
on adenosine A1 and A2A receptors. Adenosine and ATP are released into
the extracellular spaces during injury and inammation. Mast cells,
neutrophils, and free nerve endings express P1 and P2 receptors, and
adenosine helps mediate communication between the immune and nervous systems, and contributes to the pain response.

12.15 Composition of Rhodopsin Photoreceptor

The rhodopsin photoreceptor is composed of an opsin GPCR and its ligand,
11-cis-retinal. G protein-coupled receptors function as sensors and
transducers of information about the external and internal environments.
GPCRs are involved in sight, taste, smell, touch, proprioception, and pain.
Touch, proprioception, and pain were discussed in the previous sections.
In the remaining sections of this chapter, the focus will be on how signals
conveyed by exogenous ligands such as light are transduced into cellular
responses. The starting point will be how light, or electromagnetic energy,

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12. Signaling in the Endocrine and Nervous Systems Through GPCRs

Figure 12.8. Retinal photoisomerization: A Schiff base is an organic compound

formed by the double bonding of a nitrogen atom of an amino group to a carbon atom.
Schiff bases are formed in rhodopsin by the bonding of nitrogen of the NH3+ group on
a lysine residue to retinal containing a CHO group with the accompanying release of
a water molecule. Photoisomerization is the process whereby light impinges on and is
absorbed by the double bonded structure resulting in the shifting and twisting of the
bonds so that a number of atoms assume a different spatial orientation. (a) The retinal
molecule. (b) Retinal in its 11-cis form covalently attached to a Lys296 side chain by a
Schiff base. (c) The all-trans form of the covalently attached retinal molecule derived
from the 11-cis form by photoisomerization.

striking a retinal molecule in converted into helix movements, a mechanical form of energy.
The retinal molecule, unlike most other ligands, is covalently linked to
opsin and serves as a light-responsive chromophore. The retinal ligand is
buried in a pocket that lies deep in the opsin protein. When a photon strikes
the retinal chromophore it triggers a conformational change (retinal isomerization) from an 11-cis to an all-trans conguration, as shown in Figure
12.8. The retinal molecule is attached to the side chain of Lys296 situated in
the H7 helix. The movement associated with the conformational change is
appreciablewhen stabilizing the alternative all-trans conguration there
is a 4.5-movement that is reected in the positions of the cytoplasmic
portions of the transmembrane helices. In its altered conformation
rhodopsin activates the G-protein and initiates signaling in the vision
pathway. The retinal is hydrolyzed and dissociated from the opsin after
about a minute, enabling a new cycle of 11-cis-retinal attachment and
activation.

12.17 GPCRs Transduce Signals Conveyed by Odorants

297

12.16 How G Proteins Regulate Ion Channels

G proteins activated in response to light absorption regulate ion channels.
Light absorption stimulates the GEF activity of the GPCR, enabling it to
catalyze GDP dissociation from the Ga. The next set of steps used by vertebrates differs from those employed by invertebrates, but both are fairly
typical of GPCR signaling. In vertebrates, the Ga subunits act on phosphodiesterases (PDEs), but in invertebrates such as Drosophila they target
phospholipase Cb. The ultimate effect of both kinds of second messenger
signaling is to trigger changes in ion channel activities. Cyclic GMP is less
frequently encountered as a second messenger than cAMP. One place
where it has a prominent role is in phototransduction. In vertebrates, rod
cells possess cGMP-gated ion channels. In the absence of stimulation by
photons these channels are open resulting in membrane depolarization and
transmitter release from the rod cells. Gt subunits, called transducins, are
found in the retina where they couple to the phototransducer rhodopsin.
When light strikes rhodopsin the transducins are activated. These subunits
activate cGMP phosphodiesterases that hydrolyze cytosolic cGMP to GMP,
thereby reducing its concentration. The cGMP-gated ion channels close,
leading to reductions in intracellular calcium levels and shifts in the membrane potential towards more negative values (Figure 12.9b).
In Drosophila, phospholipase C acts on PIP2 to generate IP3 and DAG
leading to the opening of members of the transient receptor potential (Trp)
family of cation channels. A scaffolding protein called inactivation no afterpotential (InaD) helps organize the signaling events that follow Ga separation. This scaffolding protein comprises ve PDZ domains. Recall from the
last chapter that PDZ domains mediate several different kinds of proteinprotein interactions. In Drosoplila, InaD binds to the Trps and their regulators, serving as a platform for assembly of signaling complexes (Figure
12.9c). Similar proteins are found in vertebrate nerve terminals where they,
too, help organize signaling complexes formed about ion channels.

12.17 GPCRs Transduce Signals Conveyed

by Odorants
Olfactory neurons situated in the nasal cavity express receptors for an enormously wide range of chemical signals. The chemical compounds that can
be sensed, or odorants, number in the thousands. They include aromatic and
alipathic alcohols, aldehydes, esters, ethers, and ketones; aromatic hydrocarbons; and alipathic acids, alkanes, and amines. Tiny changes in structure
can be sensed and converted into different odor precepts. The olfactory
receptors that function as chemical sensors in these neurons belong to a
large family of evolutionarily ancient Group A GPCRs. Many of these
receptors have little sequence homology to one another, especially in

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12. Signaling in the Endocrine and Nervous Systems Through GPCRs

Figure 12.9. Phototransduction in vertebrates and invertebrates: (a) Light absorbed

by the retinal chromophore results in photoisomerization and GPCR conformational
changes leading to activation of heterotrimeric G proteins. (b) Vertebrate signal
transduction in which transducin stimulates phosphodiesterase (PDE) activity
leading to hydrolysis of cGMP and the closing of cGMP-gated ion channels.The insert
depicts the 6-pass transmembrane topology of the subunits of both the cGMP-gated
and Trp family ion channels. (c) Invertebrate signal transduction that targets phospholipase Cb, leading to activation of phospholipid and calcium second messengers,
and assembly of signaling complexes organized by InaD scaffolding proteins.

transmembrane regions H3 through H6, a fact consistent with the role of

these regions in forming the ligand-binding pocket.
Olfactory signal transduction is depicted in Figure 12.10. Cyclic AMP is
now used as a second messenger in place of cGMP. Binding of an odorant
activates the G protein signaling to adenylyl cyclase type III, which catalyzes the production of cAMP from ATP. The cAMP molecules bind to the
cyclic nucleotide-gated (CNG) ion channels resulting in their opening.
The initial signal is then amplied. Calcium ions entering the cell through
the CNG channels bind and activate chloride channels so that not only does
positive change enter the cell but negative change leaves as well.
The olfactory system uses a combinatorial coding scheme to distinguish
between different odorants. A given olfactory neuron expresses a single
type of odor receptor (OR) gene on its surface. Each OR can recognize
multiple orodant ligands and each kind of odor stimulates a number of

12.18 GPCRs and Ion Channels Respond to Tastants

299

Figure 12.10. Olfactory signal transduction: Binding of an odorant activates the G

protein signaling to adenylyl cyclase type III that catalyzes the production of cAMP
from ATP. The cAMP molecules bind to the cyclic nucleotide-gated (CNG) ion
channels resulting in their opening. Calcium ions entering the cell through the CNG
channels bind and activate chloride channels. Increased production of calcium and
cAMP second messengers activate protein kinases such as protein kinase A (not
shown) that phosphorylate the GPCR, leading to its desensitization.

different odorant receptors. Neurons expressing a specic OR gene are

dispersed throughout one of four regions in the nasal cavity. The range of
ligands recognized by different ORs contains overlaps, and a particular
odor is coded by a combination, or pattern, of ORs situated on different
cells. The odorant signals are sent from the nose to the olfactory bulb and
from there to the piriform cortex. The organization and operation of the
olfactory system is discussed in greater detail in Chapter 20.
There are two other families of olfactory receptors besides the odorant
receptors. These are expressed in a second olfactory structure, the
vomeronasal organ. The two families are known as the V1R family with
about 35 members and the V2R family with approximately 150 members.
These receptors may function as receptors for mammalian pheremones.
Interestingly, V1Rs are Class A GPCRs and V2Rs are Class C GPCRs,
paralleling the situation for taste where two families of receptors are
found, one (T1Rs) belonging to Class A and a second larger one (T2Rs)
to Class C.

12.18 GPCRs and Ion Channels Respond to Tastants

There are ve taste modalitiessalty, sweet, sour, bitter, and umami. Two
of these, salty and sour (acidic), are sensed through interactions of salts and
acids with specialized ion channels. These ion channels allow for the direct
entry of H+, K+, and Na+ ions into cells localized in taste buds in the tongue.
The inux of these ions triggers neurotransmitter release leading to the
excitation of other sensory cells resulting in the perceptions of salty and
sour. Sweet, bitter, and umami are more complex. These taste modalities
are sensed through G protein-coupled receptors.

300

12. Signaling in the Endocrine and Nervous Systems Through GPCRs

Umami is the sensation produced by food additive monosodium glutamate

(MSG). Glutamate is found in many protein-rich foods such as meat, milk
products, and seafood, and is an important nutrient. Umami is sensed by a
GPCR that is derived from mGluR4, a metabotropic glutamate receptor
belonging to Class C GPCRs.The taste receptor differs from the neurotransmitter-detecting form in that it is missing 50% of the extracellular domain.
This modication converts the receptor from a high afnity glutamate detector to a low afnity form suited for sensing amino acid and sweet tastants.
There are three receptors in this family of Class A GPCRs. They are designated as T1R1, T1R2, and T1R3. They form heterodimers with one another
that transduce sweet (T1R2/T1R3) and umami (T1R1/T1R3) tastants.
Bitter is an exceptionally important modality since it can signal the presence of alkaloids and other potentially harmful toxins. A separate family of
GPCRs transduces bitter signals. This family, consisting of 30 or more Class
C receptors, is referred to as the T2Rs. The T2Rs are coexpressed with G
protein alpha subunits known as gustducins (Gg). Gustducin is closely
related to transducin (Gt) and is part of the pathway that conveys bitter
signals within the cell. The distribution of receptors for taste differs from
that for olfaction. The goal in olfaction is not only to recognize a wide range
of odors but to discriminate among them as well. As noted above, each
neuron expresses one type of olfaction receptor. Bitter tastes serve as warnings of potentially dangerous substances, and it is not necessary for the body
to discriminate among the different sensations of bitter. Many types of
bitter receptors are coexpressed in each cell thus maximizing sensitivity at
the expense of specicity.

References and Further Reading

G Protein-Coupled Receptors
Baldwin JM, Schertler GFX, and Unger VM [1997]. An alpha carbon template for
the transmembrane helices in the rhodopsin family of G protein-coupled receptors. J. Mol. Biol., 272: 144164.
Bockaert J, and Pin JP [1999]. Molecular tinkering of G protein-coupled receptors:
An evolutionary success. EMBO J., 18: 17231729.
Gether U [2000]. Uncovering molecular mechanisms involved in activation of G
protein-coupled receptors. Endocr. Rev., 21: 90113.
Gether U, and Kobilka BK [1998]. G protein-coupled receptors II: Mechanism of
agonist activation. J. Biol. Chem., 273: 1797917982.
Ji TH, Grossmann M, and Ji I [1998]. G protein-coupled receptors I: Diversity of
receptor-ligand interactions. J. Biol. Chem., 273: 1729917302.
Palczewski K, et al. [2000]. Crystal structure of rhodopsin: A G protein-coupled
receptor. Science, 289: 739745.

GPCR Regulation
Bockaert J, et al. [2003]. The m
agic tail of G protein-coupled receptors: An anchorage for functional protein networks. FEBS Lett., 546: 6572.

References and Further Reading

301

Grimes ML, and Miettinen HM [2003]. Receptor tyrosine kinase and G proteincoupled receptor signaling and sorting within endosomes. J. Neurochem., 84:
905918.
Hall RA, and Lefkowitz RJ [2002]. Regulation of G protein-coupled receptor signaling by scaffold proteins. Circ. Res., 91: 672680.
Hall RA, Premont RT, and Lefkowitz RJ [1999]. Heptahelical receptor signaling:
Beyond the G protein paradigm. J. Cell Biol., 145: 927932.
Koenig JA, and Edwardson JM [1997]. Endocytosis and recycling of G proteincoupled receptors. Trends Pharmacol. Sci., 18: 276287.
Lefkowitz RJ [1998]. G protein-coupled receptors III: New roles for receptor
kinases and arrestins in receptor signaling and desensitization. J. Biol. Chem., 273:
1867718680.
Lodowski DT, et al. [2003]. Keeping G proteins at bay: A complex between G
protein-coupled receptor kinase 2 and Gbg. Science 300: 12561262.
Luttrell LM, and Lefkowitz RJ [2002]. The role of b-arrestins in the termination and
transduction of G protein-coupled receptor signals. J. Cell Sci., 115: 455465.

G Proteins and Their Effectors

Hamm HE [1998]. The many faces of G protein signaling. J. Biol. Chem., 273:
669672.
Wedegaertner PB, Wilson PT, and Bourne HR [1995]. Lipid modications of
trimeric G proteins. J. Biol. Chem., 270: 503506.

Adenylyl Cyclases and Nucleotide Phosphodiesterases

Beavo JA [1995]. Cyclic nucleotide phosphodiesterases: Functional implications of
multiple isoforms. Physiol. Rev., 75: 725748.
Cooper DMF, Mons N, and Karpen JW [1995]. Adenylyl cyclases and the interaction between calcium and cAMP signaling. Nature, 374: 421424.
Houslay MD, and Milligan G [1997]. Tailoring cAMP-signaling responses through
isoform multiplicity. Trends Biochem. Sci., 22: 217224.
Taussig R, and Gilman AG [1995]. Mammalian membrane-bound adenylyl cyclases.
J. Biol. Chem., 270: 14.

The Somatosensory System and Nociception

Marceau F, Hess JF, and Bachvarov DR [1998]. The B1 receptors for kinins.
Pharmacol. Rev., 50: 357386.
Negishi M, Sugimoto Y, and Ichikawa A [1995]. Molecular mechanisms of diverse
actions of prostanoid receptors. Biochem. Biophys. Acta, 1259: 109120.
Smith WL, Garavito RM, and DeWitt DL [1996]. Prostaglandin endoperoxide H
synthases (cyclooxygenases)-1 and -2. J. Biol. Chem., 271: 3315733160.

Phototransduction
Baylor D [1996]. How photons start vision. Proc. Natl. Acad. Sci. USA, 93: 560565.
Clapham DE, Runnels LW, and Str
bing C [2001]. The Trp ion channel family.
Nature Rev. Neurosci., 2: 387396.
Hardie RC, and Raghu P [2001]. Visual transduction in Drosophila. Nature, 413:
186193.

302

12. Signaling in the Endocrine and Nervous Systems Through GPCRs

Kramer RH, and Molokanova E [2001]. Modulation of cyclic-nucleotide-gated

channels and regulation of vertebrate phototransduction. J. Exp. Biol., 204:
29212931.

Odorants and Pheromones

Buck LB [2000]. The molecular architecture of odor and pheromone sensing in
mammals. Cell, 100: 611618.
Clyne PJ, et al. [1999]. A novel family of divergent seven-transmembrane proteins:
Candidate ororant receptors in Drosophila. Neuron, 22: 327338.
Firestein S [2001]. How the olfactory system makes sense of scents. Nature, 413:
211218.
Malnic B, et al. [1999]. Combinatorial receptor codes for odors. Cell, 96: 713723.
Zhao HQ, et al. [1998]. Functional expression of a mammalian odorant receptor.
Science, 279: 237242.

Tastants
Adler E, et al. [2000]. A novel family of mammalian taste receptors. Cell, 100:
693702.
Chandrashekar J, et al. [2000]. T2Rs function as bitter taste receptors. Cell, 100:
703711.
Chaudhari N, Landin AM, and Roper SD [2000]. A metabotropic glutamate receptor variant functions as a taste receptor. Nature Neurosci., 3: 113119.
Lindemann B [2001]. Receptors and transduction in taste. Nature, 413: 219225.
Margolskee RF [2002]. Molecular mechanisms of bitter and sweet taste transduction. J. Biol. Chem., 277: 14.
Nelson G, et al. [2001]. Mammalian sweet taste receptors. Cell, 106: 381390.

Problems
12.1 The b-arrestins can function as switches that rst shut down signaling
through the G proteins and then turn on signaling through growth
pathways. When switching to non-G protein modes of signaling, the
arrestin molecule and the GPCR serve as a platform for the assembly
of Src and other signaling proteins. The essential features that mediate
these associations are the presence of peptide sequences that bind to
SH2, SH3, and PDZ domains, either in the third intracellular loop or
in the cytoplasmic COOH-terminal tail, and the Src non-receptor tyrosine kinase that can bind to the proline rich motifs of the b-arrestins
by means of its SH3 domain. Construct a growth pathway leading to
the nucleus that begins at the GPCR and is routed through Src that
binds to the b-arrestins.
12.2 One of the G protein alpha subunits, Gs, stimulates adenylyl cyclases
to produce cAMP, which activates protein kinase A. In b2 adrenergic
GPCR (b2AR) signaling, protein kinase A phosphorylates the GPCR,
and this negative feedback loop not only desensitizes the receptor but
also switches its G protein preference from Gs to Gi. What are two,

Problems

303

more conventional targets of protein kinase A signaling? Draw the

routes starting from ligand-receptor binding. Using information presented in Tables 12.2 and 12.3, diagram some the routes of activation
of protein kinase B and protein kinase C by the G proteins.

13
Cell Fate and Polarity

There are approximately 1014 cells in the human body. Some of these are
heart cells; others are liver, kidney, nerve, or muscle cells. During development sets of identical progenitor cells undergo different cells fates. They
diverge at the correct time to form different cell types along the appropriate body axes and boundaries. Mutual signaling between these cells drives
many of these decisions so that each cell knows what kind of cell to become.
These signals are transmitted from cell to cell, transduced across the plasma
membrane, and sent on to the nucleus where they activate specic sets of
developmental genes. Some genes are turned on and others are turned off
in response to these signals.
A small number of signaling pathways guide embryonic development. In
the rst part of this chapter, four pathways of particular importance with
respect to developmentNotch, transforming growth factor-b (TGF-b),
Wnt, and hedgehogwill be examined. These pathways regulate the programs of gene expression so that at the correct time in the right place cells with
the same propensity for a particular cell fate give rise to daughters exhibiting
differences in morphology and the mix of proteins being expressed.
A variety of stratagems are used to achieve the developmental goals. One
of these is to lay down gradients of signaling proteins either on cell surfaces
or in extracellular spaces that help determine cell fate when they activate
receptors on a cell. These signaling proteins are known as morphogens.
Another stratagem is to utilize hierarchical sequences of gene expression
so that over time different progeny will become different kinds of cells. The
focus in the rst part of the chapter will be on how signals are sent from
the cell surface to the nucleus through the four pathways. In the second part
of the chapter, the goal will be to see how morphogen gradients and hierarchical patterns of gene expression guide cell fate decisions.
The Notch, TGF-b, Wnt, and Hedgehog signaling pathways are named
either for the transmembrane receptor or for the molecules that serve
as ligands for the receptors. These four pathways are highly conserved in
multicellular organisms. They have been studied extensively in the y
(Drosophila), worms (C. elegans), and vertebrates. A variety of rather color305

306

13. Cell Fate and Polarity

ful names have been given to signaling proteins belonging to these pathways.
The names are usually derived from the types of developmental defects seen
in Drosophila when the genes encoding the proteins suffer a mutation, usually of the loss-of-function type. For example, when the Hedgehog gene is
mutated, a spiky process called denticles is seen, and hence the name hedgehog was given to the protein. In the Notch pathway, partial loss-of-function
defects in the Notch receptor produced notches in the wing. Defects in the
Groucho protein, a downstream-acting element that participates in several
pathways, result in the production of bristles around the eye. These aberrant
structures resemble the eyebrows of the well-known comedian and hence the
name Groucho was given to the gene and its protein product.

13.1 Notch Signaling Mediates Cell Fate Decision

The Notch pathway mediates numerous cell fate decisions. The pathway has
a central role in determining which cells become neurons and which do not
during the early stages of development. This same pathway is utilized in a
variety of cellular contexts to generate a broad spectrum of cell fate decisions. Depending on cellular context Notch pathway mediates patterning,
terminal differentiation, mitosis, and apoptosis fates.
There are three core components of the Notch signaling pathway: ligands,
receptors, and effectors functioning as transcription factors. Like the other
central signaling pathways, these components are highly conserved across
phyla, and corresponding members of the pathway for vertebrates, y,
and worm are listed in Table 13.1. Notch signals through a juxtacrine
mechanism in which a Notch receptor expressed on the surface of one
cell binds to a Delta/Serrate/Lin (DSL) ligand (Table 13.1) expressed
on the surface of an adjacent cell. Notch molecules are 300-kDa transmembrane receptors. They possess large extracellular domain containing
2936 tandem epidermal growth factor (EGF) repeats, and three cysteinerich Lin/Notch repeats (LNRs). The extracellular EGF and LNR repeats
mediate DSL ligand binding and Notch activation. The intracellular domain
contains an NLS, 6 Cdc10/ankyrin repeats and a PEST motif, the latter a
region rich in prolinc (P), glatamic acid (E), serine (S), and threonine (T)
residues.

Table 13.1. The Notch signaling pathway: AbbreviationsSuppressor of Hairless

[Su(H)].
Ligands
Ligands
Receptors
Transcription factors

Vertebrates

Drosophila

C. elegans

Delta1, 2, Jagged1, 2
Notch14
CBF-1

Delta, Serrate
Notch, LIN-12
Su(H)

LAG-2, Apx-1
GLP-1
LAG-1

13.2 How Cell Fate Decisions Are Mediated

307

Figure 13.1. Structure of the Notch protein:

Shown are the 180 kDa extracellular chain
and the 120 kDa transmembrane/intracellular chain. Three cleavage sites are indicated
in the gure; these are labeled by the corresponding proteolytic enzymes.

Notch is synthesized as a 300-kDa precursor molecule. This primary

transcript is cleaved in two in the trans-Golgi network. The two fragments
remain associated with one another during translocation and insertion in
the plasma membrane resulting in the formation of a heterodimer. The
heterodimer consists of an N-terminal 180-kDa molecule containing the
extracellular EGFs and LNRs, and a smaller C-terminal 120-kDa molecule
possessing a short extracellular segment, the transmembrane sequence and
the cytosolic region (Figure 13.1).
The 120-kDa C-terminal chain is cleaved at several locations to create
the Notch intracellular domain (NICD), a fragment that is released from
the membrane and can move to the nucleus where it forms a complex with
several other proteins. Su(H) binds the NICD and together the two proteins enter the nucleus (Figure 13.2). They act as transcription factors
to active genes belonging to the enhancer of a split cluster, which acts to
suppress neural development. In more detail, Notch and Su(H), more
generally, CSL (CBF1, Su(H), Lag-1), work together to stimulate the
transcription of genes belonging to the enhancer of split E(spl) cluster. The
E(spl) gene products, in turn, inhibit transcription of a cluster of proneural
genes referred to as the achaete-scute complex. Since these genes are not
transcribed, cells transducing the Notch signals in response to Delta ligand
binding are inhibited from adopting a neural cell fate. A positive feedback
loop operates to help drive unambiguous decisions and the overall process
is referred to as lateral inhibition.

13.2 How Cell Fate Decisions Are Mediated

Lateral inhibition and positive feedback mediates cell fate decisions. In the
absence of Notch signaling most cells in an equivalence group will adopt
the same primary fate. Notch signaling restricts the number of cells travel-

308

13. Cell Fate and Polarity

Figure 13.2. Signaling through the Notch pathway: The sending cell expresses the
Notch ligand Delta or Jagged, while the receiving cell expresses the Notch receptor. The extracellular chain of Notch binds the ligand and in response the transmembrane segment is cleaved at several places to form the NICD. The NICD,
together with Su(H), translocates to the nucleus where they interact with several
other proteins to activate gene transcription. As depicted in the gure, they may
promote gene transcription by displacing corepressors.

ing along this fate pathway by promoting either a different pathway or by

maintaining the cells in an uncommitted state. The general mechanism for
restricting cell fate decisions is called lateral inhibition because a cell adopting a particular cell fate inhibits its neighbors from adopting the same fate.
A key factor that promotes cell fate decisions is the use of a positive
feedback loop where small stochastic differences in expression levels are
amplied and drive the decision. Lateral inhibition works in the following
manner (Figure 13.3). Cells express both Notch and Delta on their surface.
Notch receptors on one cell bind to Delta ligands on the other. When a
cells Notch receptor binds a Delta ligand on an opposing cell it develops
a diminished capacity for expressing Delta ligands on its own surface. Cells
expressing Notch more strongly will receive a stronger inhibitory signal and
inhibit its neighbor less strongly. This generates a feedback loop that amplies initially small stochastic differences in receptor expression.

13.3 Proteolytic Processing of Key

Signaling Elements
Proteolytic processing plays a key role in many signaling pathways. Notch
is cleaved after ligand binding, and the cytosolic fragment translocates to
the nucleus where it functions as a transcription factor. Thus, proteolytic

13.3 Proteolytic Processing of Key Signaling Elements

309

Figure 13.3. Lateral inhibition through cell-to-cell signaling and positive feedback:
(a) Because of stochastic uctuations there are more Notch receptors on the B cell
than on the A cell and, other things being equal (i.e., the density of Delta ligands
on the two cells being the same), signaling into the B cell is stronger than in the A
cell. (b) In the B cell, Notch signal transduction upregulates Notch receptor expression and reduces Delta ligand expression. On the A cell the strength of Notch signaling fails to compensate for the decay of Notch receptors over time. Thus on the
B cell Delta ligand expression goes down while on the A cell Notch receptor expression declines. (c) This tendency is amplied over time leading to an A cell expressing Delta ligands and a B cell expressing the Notch receptors.

processing enables a single gene product to function rst as a receptor and

then as a transcription factor. As noted earlier proteolytic processing of the
300-kDa precursor takes place in a trans-Golgi network resulting in formation of the two-chain receptor.
Proteolytic processing plays key role in signaling through the Hedgehog
and Wnt pathways, too. As will be seen shortly in Sections 13.6 and 13.7
these processes are, in turn, regulated by protein phosphorylation. In these
pathways, these two forms of posttranslational modication work together
to activate elements of the signaling pathways located within the cell, in the
cytosol, in response to ligand binding at the plasma membrane.
Several families of enzymes participate in cleaving the transmembrane
form of the Notch receptor leading to release of the NICD. One of the families of proteolytic enzymes is the Presenilins. These enzymes have been the
subjects of intensive study in the past few years due to their similar involve-

310

13. Cell Fate and Polarity

Figure 13.4. Components of the g-secretase: (a) PresenilinThe small squares

denote the location of a pair of aspartate residues crucial for the catalytic activities
of presenilin; (b) NicastrinOvals located on extracellular part of the chain denote
essential glycosylation sites. (c) Aph-1. (d) Pen-2.

ment in cleaving the amyloid precursor protein (APP) leading to the release
of the Ab amyloid protein, an important step in the onset of Alzheimers
disease. Like the Notch process depicted in Figure 13.1 the APP undergoes
several cleavages. These are mediated by enzymatic complexes referred to
as a-secretases, b-secretases, and g-secretases. The Presenilins are part of
the g-secretase complex, which has three other membersNicastrin, Aph1 and Pen-2. As illustrated in Figure 13.4 the Presenilins are 8-pass transmembrane (TM) proteins proteolytically cleaved into two chains. A pair of
aspartate residues positioned in opposition to one another is crucial for Presenilins g-secretase actions. Notch and APP pass through the gap formed
by the TM segments containing the aspartates, and are cleaved. The Presenilins carry out their catalytic activities together with the single-pass Nicrastrin protein, the 7-pass Aph-1 protein, and the 2-pass Pen-2 protein, which
help to assemble and stabilize the complex.
The APP is cleaved twice, one in its extracellular domain just outside the
membrane and the other near the middle of the intermembrane region. In
the rst step, the APP proteins are cleaved at one of two alternative extracellular locations termed the a- and b-sites. The g-secretases are responsible for subsequent cleavages within the transmembrane segment. In the
case of cleavage at b-sites, but not a-sites, the products are 40- and 42-amino
acid residue forms of the Ab amyloid protein. The 42-residue form, favored
by the mutated Presenilins, is especially prone to form clumps due to
exposure of hydrophobic patches, as was discussed in Chapter 5. The asecretases and b-secretases responsible for cleaving the APPs at the aand b-sites are members of two other families of proteases. One or more
members of the a disintegrin and metalloprotease (ADAM) family are
the a-secretases, while aspartyl proteases belonging to the membraneassociated aspartyl protease (memapsin) family serve as the b-secretases.
The ADAM enzymes are members of a large group of multidomain
enzymes that possess not only metalloproteinase domains, but also integrinbinding regions and a cytoplasmic domain that bind a variety of signaling

13.4 Three Components of TGF-b Signaling

311

proteins. Members of the ADAM family of proteases degrade collagens and

other extracellular matrix proteins. This activity is particularly important
during embryonic development. During that time the ECM has to be continuously remodeled to accommodate new growth and emerging organs
and appendages. The ADAM proteins also promote the creation of soluble
signaling proteins from membrane-bound forms in another developmentally important process called ectodomain shedding. In this process,
membrane-bound proteins are cleaved at sites just outside the plasma
membrane, thereby converting ligands and receptors that signal in a short
range, juxtacrine manner into soluble signaling proteins that can signal in
a longer-range, paracrine manner. One of the ADAM family members,
a protein called tumor necrosis factor-a converting enzyme (TACE) can
cleave the TNF-a ligand, as its name suggests, and functions as the asecretase for Notch and the APP perhaps along with another protein called
ADAM10 in mammals and Kuzbanian in Drosophila. Lastly, degradation
of ECM molecules is necessary for cell migration. The upregulation of
metalloproteases (metalloproteinases) accompanies the downregulation of
cell adhesion molecules, increased angiogenesis, and increased motility in
the progression of cancer to metastasis.

13.4 Three Components of TGF-b Signaling

The TGF-b signaling pathway has three main components: ligands, receptors and Smad signal transducers. The transforming growth factor-b
pathway, like the Notch pathway, has many different effects on cell fate
depending on the stage of development. Among the processes triggered
through this pathway are patterning, wound healing, bone formation, ECM
production, and homeostasis. There are three core components in a TGF-b
pathwaya ligand, a cell surface receptor complex, and a set of cytoplasmic signal transducers called Smads. The TGF-b pathway is named for
the TGF-b subfamily of diffusible polypeptide ligands. Other prominent
ligands involved in signaling though the TGF-b pathway are the bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs),
members of the Activin subfamily, and Nodal.
TGF-bs, BMPs, GDFs, and related ligands signal through receptors
belonging to the TGF-b receptor family. These receptors are transmembrane glycoproteins possessing cytoplasmic domains that phosphorylate
serine and threonine residues of target proteins. Thus they belong to the
family of receptor serine/threonine kinases. A sequence of events involving
one or more members of two distinct TGF-b receptor groups is required in
order to signal across the plasma membrane. The two receptors groups are
designated Type I and Type II receptors, abbreviated as TbRI and TbRII.
Members of each group possess an extracellular ligand-binding domain and
a cytoplasmic kinase domain. Type I receptors have an additional cytoplas-

312

13. Cell Fate and Polarity

Figure 13.5. Signaling through the TGF-b pathway: Binding of a ligand (TGF-b)
dimer to a TbRII-TbRI receptor complex leads to phosphorylation of serine/threonine residues in the GS region of the TbRIs resulting in the activation of the kinase
domain. The activated receptors phosphorylate the Smad3 proteins leading to the
latters dissocation from the SARA anchors. They form a heterotrimeric complex
with Smad4, and in that form translocate to the nucleus where they form a scaffold
for assembly of transcriptional activators and cofactors such as CPB/p300 and Ski
to induce the transcription of target genes.

mic domain referred to as a GS domain that functions as a key regulatory

region.
Two different mechanisms of ligand-receptor binding are utilized by
TGF-b receptors. BMP ligands have a high afnity for Type I receptors and
a low one for Type IIs. Once BMP ligands bind the Type Is, these receptors have a high afnity for Type II receptors. The assembly setups are, as
follows. First, a complex is formed containing a BMP ligand dimer bound
to a Type I receptor dimer. This assembly then binds to two Type II receptors to make a receptor foursome, as shown in Figure 13.5. TGF-b and
Activin utilize a different assembly method. These ligands prefer to bind to
Type II receptors. Their rst step is the binding of ligands to Type II receptor molecules. These binding events serve to recruit Type I receptor
molecules to the assemblage, resulting again in the formation of a complex
consisting of a ligand dimer bound to a receptor foursome. As a consequence of either series of binding events the cytoplasmic domains of the
Type II and Type I proteins are brought into close proximity and stabilized.
The next step in transducing the signal into the cell is transphosphorylation of serine and threonine residues in the GS domains of the Type I receptors, assisted by the Type II receptors. This step creates docking sites for a

13.5 Smad Proteins Convey TGF-b Signals into the Nucleus

313

number of different adapter proteins. One of these, the Smad anchor for
receptor activation (SARA), couples Smad2/3 to the activated receptor
complex. The SARA protein possesses a lipid-binding FYVE domain that
enables it to tether to the plasma membrane. Two other domains permit it
to bind simultaneously to the receptor and to the Smad protein. Once the
Smad proteins have been recruited to the receptor complex they are phosphorylated, triggering their release into the cytosol (Figure 13.5).

13.5 Smad Proteins Convey TGF-b Signals

into the Nucleus
The Smad proteins convey messages from the cell surface to the nucleus.
The name Smad is a contraction of the names of the rst two Smad-type
proteins to be discoveredthe C. elegans Sma protein and the Drosophila
mothers against dpp, or Mad, protein. There are at least eight known Smads,
designated Smad1 through Smad8, and they fall into three categories
(Table 13.2). Five of the Smads are receptor-activated and they are called
R-Smads. Smad4 is required for signaling through all pathways and is called
a common Smad, or co-Smad. The other two Smads, Smad6 and Smad7 are
inhibitory. They turn off signaling and form an anti-Smad grouping. Smad
6 inhibits signaling through the BMP branch while Smad7 inhibits signaling through all of the branches of the TGF-b signaling pathway.
Smad proteins contain a pair of globular signaling domains connected by
a linker region. The N-terminal Mad Homology-1 (MH1) and C-terminal

Table 13.2. The TGF-b signaling pathway: As indicated in the table, the TGF-b
pathway branches at the Type I receptors (Type I Rs) level into an Activin-like
branch and a BMP-like branch. AbbreviationsActivin receptor-like kinase
(ALK); bone morphogenetic protein (BMP); transforming growth factor (TGF);
receptor (R); inhibitory (I).
Activin

TGF-b

TGF-b

BMP

Ligands

Activins

TGF-bs

TGF-bs

BMPs

Type II Rs

ActII/IIB

TbRII

TbRII

BMPRII,
ActRII/IIB,
MISRII

Type I Rs

ALK4

ALK5

ALK1

ALK3, ALK6,
ALK2

R-Smad

Smad2, Smad3

Smad2, Smad3

Smad1, Smad5,
Smad8

Smad1, Smad5,
Smad8

Co-Smad

Smad4

Smad4

Smad4

Smad4

I-Smad

Smad7

Smad7

Smad6, Smad7

Smad6, Smad7

314

13. Cell Fate and Polarity

Mad Homology-2 (MH2) domains mediate protein-DNA (MH1) and

protein-protein (MH2) interactions. In addition, the R-Smad proteins
contain a phosphorylation site near their C-terminal. Once activated by
TbRI, the R-Smads translocate to the nucleus. On the way to the nucleus
the R-Smads associate with the co-Smad (Smad4) as illustrated in Figure
13.5 for the case of Smad3.
The specic cell fate decision arrived at through the TGF-b pathway
depends on context. The steps outlined above are general and are neither
cell-specic or developmental stage-dependent. Partner and accessory
signaling molecules that combine with the Smads to form the active transcriptional complexes supply the contextural information. Coactivators,
corepressors and other partners provide cell type and developmental stage
inputs, and they x parameters such as the duration of transcription. A few
of these cofactors are presented in Figure 13.5. The p300 and CBP proteins
are coactivators. A number of corepressors associate with the Smad proteins. Examples of these additional factors are the Sloan-Kettering Institute
proto-oncogene (Ski), included in the gure, and the Ski-related novel gene
N (SnoN) and the TG3-interacting factor (TGIF), not included.
Smads are regulated by ubiquitination. Recall that ubiquitination prepares substrate proteins for proteolytic destruction by the proteosome. It
involves sequential operations by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase enzyme. As is the case
for all four of the pathways discussed in this chapter, selective, regulated
proteolysis is an important mechanism for controlling what signals are sent
on to the nucleus. In the TGF-b signaling pathway, proteolytic regulation
of the Smads takes place both in the cytosol and in the nucleus. Members
of the Smurf family of E3 ligases are recruited to the Smad scaffolds. The
WW domains of the Smurfs bind PPXY motifs in the linker region connecting the MH1 and MH2 domains of the Smads. Members of the UbcH5
family of E2s are present, as well. These operations are represented in
Figure 13.5 by the presence of a generic Smurf protein.

13.6 Multiple Wnt Signaling Pathways Guide

Embryonic Development
The Wnt pathway is named for the Wnt family of secreted glycoproteins,
which function as morphogens during development in vertebrates and
invertebrates. Wnt ligands are typically 350 to 400 amino acid residues in
size and are characterized by a highly conserved cysteine-rich domain
(CRD), a pattern of 23 cysteine residues. A large number of Wnt proteins have been identied. There are at least 25 vertebrate Wnt genes,
7 Drosophila genes, and 5 C. elegans genes. The most prominent of
the Drosophila Wnt family members is called Wingless (Wg), and is the
homolog to the Wnt-1 protein found in humans. This ligand is responsible

13.6 Multiple Wnt Signaling Pathways Guide Embryonic Development

315

Figure 13.6. Wnt signal receptors and transducers: (a) LRP coreceptor. (b) Frizzled receptor. (c) Strabismus co-receptor. The rectangle attached to the cytoplasmic
C-terminal tail of the receptor denotes a PDZ binding domain. (d) Dishevelled
adapter. AbbreviationsDishevelled, Egl-10, and Pleckstrin (DEP); Dishevelled
and axin (DIX). The DIX domain mediates attachment to the cytoskeleton, and the
DEP domain promotes binding to the plasma membrane.

for a well-studied anterior/posterior decision that denes tissue polarity.

(Note: Wnt is a contraction of wingless (Wg), and the murine, or mouse,
homolog Int.)
Wnt ligands bind to Frizzled receptors. These receptors are named for the
Drosophila frizzled gene involved in the tissue polarity decisions. Frizzled
proteins have a large extracellular CRD responsible for ligand binding, a
7-pass (serpentine) transmembrane region and a short cytoplasmic Cterminal region. Several coreceptors operate in conjunction with the Frizzled
receptors to transduce signals into the cell following ligand binding. The
chain topology of Frizzled and two of its coreceptors, LRP and Strabismus,
are shown in Figure 13.6. As shown in the gure, LRP is a single-pass receptor while Strabismus passes back and forth through the plasma membrane
four times. The rst cytoplasmic step following ligand binding in some Wnt
pathways is the recruitment of an adapter protein, Dishevelled, to the
plasma membrane. Coreceptors such as Strabismus exert their inuences
by interacting with this adapter protein.
The Wnt signaling pathway has several branches. One of these is referred
to as the canonical or b-catenin pathway. Another is termed the planar cell
polarity (PCP) pathway. As depicted in Figure 13.7, this pair of pathways
splits when signals reach the cytoplasmic adapter protein Dishevelled. The
decision of whether to activate the b-catenin or the planar polarity pathway
is determined by interactions between the coreceptors and this adapter.
The Strabismus coreceptor interacts with Dishevelled through their PDZ
domains. It shifts the routing of the signals away from the b-catenin pathway
and towards the PCP pathway by isolating the adapter from the Frizzed
receptor, while the LRP coreceptor assists in signaling through the canonical pathway.
The canonical Wnt pathway leads through b-catenin. This protein is a
transcription activator, but in the absence of Wnt signaling is prevented
from performing this function. It forms a complex in the cytoplasm with

316

13. Cell Fate and Polarity

Figure 13.7. Signaling through the Wnt b-catenin and PCP pathways: Ligand
binding activates either the canonical b-catenin pathway or the planar cell polarity
pathway, depending on the actions at the Dishevelled switching point. In the canonical pathway, Dishevelled acts through the CKI kinase to inuence the decision
whether LEF/TCF gene transcription is repressed or activated. Alternatively,
Dishevelled acts through the small GTPase protein RhoA and downstream MAP
kinases such as JNK to promote cytoskeleton polarization and gene expression.

axin, the adenomatous polyposis coli (APC) protein, and two serine/threonine kinasesGSK3 and CKI. This module works in the following way. The
kinases phosphorylate the other members of the complex. Phosphorylation
of axin stabilizes it; phosphorylation of APC enhances its interactions with
b-catenin. b-catenin cannot signal because phosphorylation tags it for proteolytic destruction. As a consequence of the phosphorylations, b-catenin
does not translocate to the nucleus and stimulate transcription. Dishevelled
acting through CKI destabilizes the complex, and leads to dephosphorylation of b-catenin. The number of mobile and untagged b-catenin molecules
increases, and they are able to translocate to the nucleus and stimulate transcription of TCF/LEF genes (Figure 13.7).
The presence of two serine/threonine kinases in the complex leads to
a two-step phosphorylation/degradation process. CKI acts rst to phosphorylate substrate residues within the complex. This action primes the
system for subsequent phosphorylation by GSK3, which is then followed
by the recruitment of proteolytic elements to b-catenin. In more detail, CKI
phosphorylates b-catenin at Ser45. This action enables GSK3 to phosphorylate b-catenin at three neighboring sites (Ser33/Ser37/Thr41). In the
absence of the priming CKI phosphorylation, GSK3 cannot phosphorylate
b-catenin at the aforementioned sites.

13.8 Hedgehog Signaling Role During Development

317

13.7 Role of Noncanonical Wnt Pathway

The noncanonical Wnt pathway guides the development of planar cell
polarity and convergent extension. Planar cell polarity (PCP) is the term
used to describe coordinated patterns of polarization of cells lying within
the planar epithelium or sheet. The two best-known examples of PCP are
in the Drosophila wing and compound eye. In the wing, hairs produced by
epithelial cells all point in the same direction, towards the distal tip. In the
compound eye, oriented hexagonal arrays of photoreceptor cell clusters
(ommatidia) are formed. In vertebrates such as frogs and sh, cells in a
tissue shift about and change shape so that their distribution becomes narrower along one axis and longer about an axis perpendicular to the rst
one. These developmental rearrangements are referred to as convergent
extension. They, too, are guided by signaling through the PCP pathway.
Signals sent through the planar cell polarity pathway inuence the organization of the cytoskeleton and gene expression. Whereas signals were
routed by Dishevelled to the CKI and GSK3 kinases and to the b-catenin
module in the b-catenin pathway, they are routed by Dishevelled to the
RhoA GTPase and then to JNK family of MAP kinases in the PCP pathway
resulting in activation of c-Jun and AP-1 dependent transcription (Figure
13.7). A third route, which may actually be a branch of the PCP pathway
since it appears to involve Dishevelled and Strabismus, utilizes G proteins,
increased Ca2+ second messenger production, and activation of calciumdependent serine/threonine kinases and phosphatases such as CaMKII,
PKC, and calcineurin.
One of the crucial steps in generating planar cell polarity is symmetry
breakingthe creation of asymmetries in the cell population using components that are initially distributed uniformly about each cell. Several proteins working in conjunction with Strabismus and Dishevelled accomplish
this task. One of these proteins is an adapter protein called Prickle. This
protein binds to Strabismus and sequesters Dishevelled near the coreceptor and away from Frizzled. A feedback loop amplies initial random and
small differences in Prickle concentration to produce sharp asymmetries
between cells that signal to the nucleus through the PCP pathway and those
for which this pathway is shut down.

13.8 Hedgehog Signaling Role During Development

The fourth signaling pathway to be discussed in this chapter is the signaling pathway named for the Hedgehog (Hh) family of diffusible cell-to-cell
signaling molecules. Members of the Hh family include Hedgehog in
Drosophila and at least seven vertebrate Hedgehogs, the most prominent
of which are Sonic hedgehog (Shh), Desert hedgehog (Dhh), and Indian
hedgehog (Ihh). This family regulates a wide spectrum of developmental

318

13. Cell Fate and Polarity

Figure 13.8. Hedgehog receptors Patched and Smoothened: (a) Patched receptor.
(b) Smoothened receptor in a closed conformation. (c) Smoothened receptor in an
open conguration.

events. For example, the Hh pathway is responsible for body, wing, eye,
genitals, and leg patterning in Drosophila while the Shh pathway regulates
eye, portions of the brain, hair, lung, gut, bladder, and urethra patterning in
mammals.
The Patched receptor is a 12-pass transmembrane protein (Figure 13.8).
It functions together with a partner receptor called Smoothened. The
Smoothened protein is a 7-pass transmembrane receptor similar to the
Wnt receptor Frizzled. Patched is responsible for ligand binding and
Smoothened is responsible for transducing the signal across the plasma
membrane. Two different conformations of the Smoothened receptor are
depicted in Figure 13.8. In the rst, (b), the cytoplasmic tail cannot bind to
the downstream signaling partners, but, in the second, (c), binding sites
along the cytoplasmic tail become available. In the absence of ligand
binding, Patched acts catalytically to maintain Smoothened in its closed
conformation, but ceases to do so when bound to Hedgehog.
The cytoplasmic tail (CT) of Smoothened in its open conformation interacts with two cytoplasmic proteinsCostal-2 (Cos2) and Fused (Fu). The
rst of these, Cos2, is a kinesin-like protein that binds to microtubules while
the other, Fu, is a serine/threonine kinase. The Cos2 protein functions as a
scaffold for assembly of several signaling elements. These include, besides
Smoothened, CT and Fused, Suppressor of Fused (Su(Fu)) and a downstream-acting transcription factor called Cubitus interruptus (Ci). Two
additional serine/threonine kinasesPKA and GSK3complete the
specication of this Hedgehog signaling node (Figure 13.9).

13.9 Gli Receives Hh Signals

The primary recipients of Hh signals are Gli transcription factors. Glis (so
named because of their involvement in malignant gliomas) are zinc nger,
DNA-binding proteins. They have ve zinc ngers, three of which grip the
DNA molecule, a CBP binding domain, and numerous phosphorylation
sites (Figure 13.10). The best studied of these proteins is the Drosophila
Cubitus interruptus protein. As just described this protein forms a complex

13.9 Gli Receives Hh Signals

319

Figure 13.9. Signaling through the Hedgehog pathway: In the absence of ligand
binding, Patched (Ptc) represses Smoothened (Smo), depicted in the gure as acting
through a small molecular (Sm mol) intermediary. In response to ligand binding,
the repression is relieved and Smo undergoes a conformational change from a
closed conguration to an open one that can bind Cos2 and Fu. This binding action
prevents phosphorylation of Ci by PKA and GSK3. The complex dissociates and
the full length 155 kDa CiA translocates to the nucleus where it promotes transcription. In the absence of ligand binding, the Ci protein is cleaved following phosphorylation, and the 75 kDa CiR protein translocates to the nucleus where it
represses transcription.

Figure 13.10. Organization of the Drosophila Cubitus interruptus (Ci) protein: Five
tandem zinc ngers situated in the N-terminal half of the protein are responsible
for binding DNA. A CBP binding domain located in the C-terminal portion of Ci
binds a CBP cofactor. Cleavage of the protein produces the CiR protein containing
the N-terminal portion and acting as a transcriptional repressor. Three of the many
S/T phosphorylation sites (pSer) are shown. The sites indicated are the priming sites
phosphorylated by protein kinase A (PKA). Sites located both N-terminal and
C-terminal to these sites are phosphorylated by GSK3.

320

13. Cell Fate and Polarity

with Cos2, Fu, and Su(Fu) that is bound to microtubules. In the absence of
Hh signaling, another member of the signaling pathway, protein kinase A
(PKA) phosphorylates Ci. This phosphorylation event tags Ci for proteolysis. The result of this process is the formation of a 75-kDa CiR protein
(Figure 13.9), which translocates to the nucleus where it functions as a
repressor of the transcription of several genes. When Hh is present, phosphorylation of Ci by PKA is blocked, the complex dissociates, and the fulllength 155-kDa protein is left intact and free to move into the nucleus. This
intact CiA protein functions as an activator of transcription of a number of
genes including patched, dpp, and wg.

13.10 Stages of Embryonic Development

Use Morphogens
The fertilized egg of multicellular animals goes through a sequence of
developmental stages. The egg rst goes through a cleavage stage where it
divides mitotically several times to form a ball of smaller cells, or blastomeres. In the next phase, the cells move to the outside forming an epithelial sheet that encloses a uid-lled chamber. In the third stage, gastrulation,
the single-layered blastula develops into a gastrula consisting of three layers
of cellsthe endoderm, mesoderm, and ectoderm, roughly corresponding
to gut, connective tissue and muscles, and epidermis (respectively) of the
adult organism.
In more detail, the lungs, components of the digestive system such as
stomach and liver, and associated glands and structures, develop from the
endoderm. In the developing mesoderm, left and right sides of the body are
delineated by a notocord that denes the central axis of the body. Sections
of mesodermal cells progressively bud off on both sides of the notocord to
form somites, which then develop further into the individual vertebra and
muscle groups. The vascular system, including the heart, bone, and cartilage,
develop from the mesoderm, while the nervous system and associated
sensory organs develop from the ectoderm in a developmental stage called
neurulation.
The term morphogen was introduced at the beginning of the chapter.
Morphogens are signaling proteins that are expressed either on cell surfaces or secreted into the extracellular spaces in the form of concentration
gradients. These gradients are subsequently read by cells to determine
their developmental fate. Cells adopt different cell fates according to their
position in the gradient relative to the signal source.
Cells in organizing centers, at boundaries between different layers or
regions, and within regions, in the embryo secrete morphogens during development. Organizing centers are localized groupings of one or more kinds
of cells that secrete morphogens in order to impart patterns of cell fates to
elds of progenitor cells. The morphogens are secreted not only at specic

13.11 Gene Family Hierarchy of Cell Fate Determinants in Drosophila

321

locations but also at specic times during embryonic development. These

morphogens and associated signaling proteins are expressed sequentially;
that is, through a hierarchical pattern of gene expression with each family of
gene products preparing the way for the next family of gene products.

13.11 Gene Family Hierarchy of Cell Fate

Determinants in Drosophila
In Drosophila, ve sets of gene products have major roles in determining
cell fate. The gene families form a hierarchy with one set of gene products
preparing the way for the next set of genes and their protein products. Each
set contributes to the emergence of the body plan, producing a succession
of progressively ner partitions of the embryonic body into segments and
compartments that eventually become adult body parts such as head,
thorax, abdomen, wings, and legs. Although the details differ from phylum
to phylum these families are highly conserved among multicellular organisms up to an including vertebrates. These families, their functions, and
members are presented in Table 13.3.
The earliest set of gene products is the maternal effect genes. As their
name indicates, they are supplied maternally. Some of these gene products
function as morphogens, while others assist in morphogen localization. The
morphogen gradients are generated internally within the single cell, the egg,
rather than externally by many cells, and contribute to the establishment of
cell polarity and asymmetric cell division. Maternal effect genes prepare the
cell for expression of the gap genes. These are distributed in broad bands

Table 13.3. Hierarchy of Drosophila patterning genes.

Gene family

Function

Members

Maternal effect

Establish anterior-posterior and

dorsal-ventral body axes;
regulate gap and pair rule
gene expression pattern

Gap

Partition body into three broad

regions; regulate pair rule
gene expression
Partition body into bands;
regulate segmentation gene
expression
Establish anterior-posterior
axis within each
compartment
Establish body part identity

A/P axis: Bicoid, caudal,

hunchback, nanos, oskar,
stauffen; D/V axis: cactus,
dorsal, pelle, sp
tzle, toll,
tube
Zygotic caudal, giant, zygotic
hunchback, huckebein,
knirps, kr
ppel, tailless
Even-skipped, fushi tarazu, hairy,
odd-paired, odd-skipped,
paired, runt, sloppy paired
Armadillo, cubitus interruptus,
engrailed, frizzled, fused,
hedgehog, patched, wingless
Abd-A, Abd-B, Antp, Dfd, Lab,
Pb, Scr, Ubx

Pair rule

Segment
polarity
Hox cluster

322

13. Cell Fate and Polarity

to anterior, middle, and posterior regions of the embryo. If a gap gene is

missing the corresponding portion of the body does not develop, producing a gap, and hence the name. These genes prepare the way for the expression of pair rule genes. The pair rule genes further partition the broad
segments produced by the gap genes into seven embryonic segments, or
parasegments. If one of these genes is missing every other segment in
the developing larva is absent, and hence the name pair rule. The next
set of genes, segment polarity genes, specify the polarity within each
parasegement; that is, they dene the anterior-posterior axes of each of the
parasegments. Finally, the Hox genes help delineate the various adult body
parts.

13.12 Egg Development in D. Melanogaster

The establishment of asymmetry and polarity in D. melanogaster begins
prior to fertilization, during the development of the egg. In the egg development (oogenesis) stage, an initial germ cell divides four times, producing
a single egg cell and 15 nurse cells. The nurse cells are connected to the egg
by cytoplasmic bridges that permit mRNAs to ow into the egg cell from
the nurse cells. Somatic follicle cells surround and nurture the resulting
assemblage. Whereas the point of entry of the sperm into the egg determines the polarity of the C. elegans zygote, the orientation of the Drosophila
oocyte is determined by signals exchanged between it and the most posterior follicle cell. The Gurken protein plays a key role in this signaling
pathway, which involves two-way signaling between the egg cell and its surrounding cells in which directional information is encoded through a polarized microtubule cytoskeleton.
Once an initial orientation is specied through communication with the
neighboring cells, distributions of mRNAs and proteins are set up in regions
of the egg and zygote delineated by anterior/posterior and dorsal/ventral
axes. Some mRNAs and proteins are localized either to the anterior or posterior regions of the cell, while others are localized in a dorsal/ventral
manner.The proteins being localized belong to the signal transduction pathways and include most importantly a considerable number of transcription
factors. When the cells divide, the programs of gene expression in the
daughters differ from one another because of the asymmetric distributions
of transcription factors, and the daughters have different cell fates.
Two of the proteins involved in delineating the anterior and posterior
regions are Bicoid and Oskar. Bicoid localizes to the anterior pole and
Oskar protein collects near the posterior pole (Figure 13.11). These proteins, along with several others, guide the formation of graded distributions
of mRNA for Hunchback and Caudal in the anterior and posterior regions,
respectively. These last-named proteins are intracellular morphogens and
their graded distributions help determine cell fate. Genes expressed in the

13.13 Gap Genes Help Partition the Body into Bands

323

Figure 13.11. Schematic depiction of a Drosophila oocyte: Shown in the gure are
the polynuclear nurse cells, oocyte, and posterior array of follicular cells. Bicoid
mRNAs are laid down in a strip in the anterior end and diffuse towards the posterior pole to form a concentration gradient. Oskar mRNAs and proteins are laid
down at the posterior pole and diffuse out to form a countergradient.

oocyte, acting as they do in the absence of paternal gene products, are

referred to as maternal effect genes. The bicoid and oskar genes are
maternal effect genes acting to establish cell polarity. Bicoid and Nanos,
another maternal effect gene product, act as morphogens. Bicoid is laid
down to form a gradient peaked at the anterior pole, while Nanos is laid
down opposite way to Bicoid, having its highest concentration in the posterior pole.
Maternal effect gene products belonging to the Toll pathway are involved
in delineating the dorsal/ventral distributions. Recall that the Toll pathway
is an evolutionary ancient pathway that mediates innate immune responses.
Drosophila counterparts to the mammalian Toll gene products are involved
in establishing the early dorsal/ventral patterning. Sp
tzle is the Drosophila
Toll ligand; Cactus is the Drosophila counterpart to the IkB inhibitor, and
Dorsal the counterpart to the NF-kB transcription factor. After fertilization
occurs, the Toll signal cascade is initiated resulting in activation of Dorsal
in a graded fashion due to ventral restriction of Spatzle signaling. Tube and
Pelle are intermediaries in the Toll signaling pathway.

13.13 Gap Genes Help Partition the Body into Bands

Gap genes are expressed in broad regions of the developing Drosophila
embryo. Maternal effect gene products initiate the expression of these
genes.Working in concert the Hunchback, Knirps, Giant, and Kr
ppel genes
products subdivide the embryo into regions along the anterior posterior
axis. As shown in Figure 13.12, Kr
ppel is expressed in a wide band in the

324

13. Cell Fate and Polarity

Figure 13.12. Patterns of distributions of developmental genes in the Drosophila

embryo: (a) Distribution of gap gene products Kr
ppel, Giant, and Knirps in broad
bands. (b) Distribution of pair rule gene product Even-skipped (Eve) in the
Drosophila embryo. Shown is the 7-striped zebra pattern of eve gene expression.
Stripes are numbered from the anterior end to the posterior end.

center of the embryo. Knirps is expressed the in a broad posterior region

adjacent to the central Kr
ppel region, and Giant genes are expressed on
the two outer sides of the centrally situated Kr
ppel and Knirps regions. A
second, small Knirps region is situated near the anterior pole (not shown
in the gure).
Tailless and huckebein are terminal gap genes.They are expressed at both
the anterior pole and posterior pole to complete the A/P partitioning of the
embryo. Hunchback inhibits expression of posterior gap genes knirps and
giant in the anterior part of the embryo. If hunchback is not expressed the
head will be missing; if Knirps is missing the abdomen will be missing, and
if Kr
ppel is missing the thorax and abdomen will not appear. Thus, the
proteins encoded by these genes are cell fate determinants that divide the
growing embryo into head, abdomen, and terminal bands.

13.14 Pair-Rule Genes Partition the Body

into Segments
Like the gap genes, the pair rule genes are transcription factors. Three of
themeven-skipped, hairy and runtare regarded as primary pair rule
genes. The other pair rule genes are referred to as secondary pair rule genes;
they are expressed later and are governed by the expression patterns of the
primary pair rule genes. The pair rule genes specify segment boundaries;
they are laid down in zebra-like patterns giving rise to the 7-segment striped

13.15 Segment Polarity Genes Guide Parasegment Development

325

body of the developing larva. As is the case for the other primary pair rule
genes, the regulatory region for even-skipped (eve) contains a set of special
binding sites, called enhancer elements, for transcription factors, one for
each stripe. These enhancers correspond in a unique way to the gradient
information in the region of the stripes.
Gap and maternal effect genes both regulate the pair rule gene expression patterns.The gene regulatory region (enhancer) for eve in stripe 2 illustrates the general theme. Bicoid and Hunchback bind to sites in the
enhancer and function as activators of gene expression. Giant and Kr
ppel
also bind to sites in the enhancer and operate as repressors of gene expression. Since Giant is expressed at the anterior end and Kr
ppel in the middle,
stripe 2 is inhibited in those regions but is encouraged by Bicoid and
Hunchback in a stripe in the anterior region located in between the two
repressed regions. Similarly, enhancers of the other stripes reect the gradients of the corresponding sets of maternal effect and gap genes in the
regions where the other stripes are to be sited. The net result is the emergence of seven stripes of expression for eve, and similarly, seven stripes for
hairy and seven stripes for runt. The gene products appear in regions that
partially overlap one another and so they operate in combinatorial manner
to delineate the parasegments.

13.15 Segment Polarity Genes Guide

Parasegment Development
The development of the Drosophila larva continues with the expression of
segment polarity genes. The expression of gap and pair rule genes occurs
during the precellular blastomere stage of embryo development. The blastomere stage ends with cellularization where movements of the membrane
about the nuclei take place, separating the nuclei and forming distinct cells.
The segment polarity genes are expressed at the start of gastrulation.
Whereas the gap and pair rule gene products function as transcription
factors, the segment polarity gene products include both transcription
factors and signaling proteins that mediate communication between the
newly formed cells and coordinate their programs of gene expression.
Genes that encode several receptor-ligand combinations appear in the
list of segment polarity genes. Among the entries in Table 13.3 are patched
and hedgehog, and frizzled and the gene encoding the Wnt ligand Wingless.
Downstream signal transducers such as Cubitus interruptus are expressed;
as well. As this stage begins Engrailed, a transcription factor, is expressed
along with two secreted signaling proteinsWingless and Hedgehog. Pair
rule gene products such as Eve and Ftz establish patterns of Wg, En, and
Hh gene expression, and cell-to-cell signaling sustains them. The result of
this activity is the formation of 14 stripes of Engrailed/Hh gene expression
and 14 stripes of Wingless gene expression.

326

13. Cell Fate and Polarity

The boundary between cells expressing Engrailed/Hedgehog and those

expressing Wingless is the parasegment boundary, and the regions between
pairs of boundaries are the parasegments. Cells residing in the anterior half
of each parasegment express Wg and those situated in the posterior compartments express En/Hh. The cell fates in the two compartments differ:
cells in the anterior compartment become A (anterior) cells while those
in the posterior compartment become P (posterior) cells.

13.16 Hox Genes Guide Patterning in Axially

Symmetric Animals
Bilaterally symmetric animals such as humans express a family of highly
conserved regulatory genes, called Hox genes. These genes play an important role in specifying which cells in the mesoderm become which body
parts. In vertebrates, these genes guide the emergence of the axial skeleton
(for example, the breastbone, ribcage, and spine), voluntary muscles, and
dermis of the back from the somites mentioned earlier in the chapter, that
is, from the repeated, identical segments of cells belonging to the mesoderm.
These genes have been studied extensively in the y and other insects,
the nematode, chick, and mouse. In all organisms studied, the genes appear
as a cluster, one gene after the other. In vertebrates there are four clusters
of genes, each cluster containing from 9 to 11 genes. In insects and the nematode there is a single gene cluster. The members of the Drosophila Hox
cluster are listed in Table 13.3. Three of the Hox genesDfd, Lab, and Pb
delineate the head. Two gene productsAntp and Scrspecify the thorax,
and the remaining threeAbd-A, Abd-B and Ubxspecify the abdomen.
In many metazoans, a small number of regulatory genes control the
development of organs and morphological structures. These genes, called
selector genes, have been intensively studied in Drosophila, where they
control the formation of body parts and bilateral partitioning. These regulatory genes, of which Hox genes are prominent members, function as transcription factors. Selector genes do not work alone. Rather, they work
together with other regulatory components in a combinatorial fashion to
create specic patterns of gene expression, leading to the development and
arrangement of body parts. The segment polarity gene, engrailed, discussed
previously, is a good example of a selector gene. It works in concert with
other segment polarity genes to determine which cells become A cells and
which ones become P cells.
Hox genes are activated in a spatially sequential manner from anterior
(head) to posterior (tail) end. Cells progressively bud off from the main axis
to form the somites. The differentiation of the cells within the somites to
form vertebrae and muscles takes the form of a wave of activity whose
leading edge moves down the A/P axis, with presomitic mesoderm in front
of the wave and somites behind it. The movement of this wave must be care-

References and Further Reading

327

fully timed. The timing activity keeps the movement of the wave in phase
with the time needed for development of the somites.

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De Strooper B [2003].Aph-1, Pen-2, and Nicastrin with Presenilin generate an active
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Wnt Signaling Pathway

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Kalderon D [2002]. Similarities between the Hedgehog and Wnt signaling pathways.
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Mlodzik M [1999]. Planar polarity in the Drosophila eye: A multifaceted view of
signaling specicity and cross-talk. EMBO J., 18: 68736879.
Moon RT, Brown JD, and Torres M [1997]. Wnts modulate cell fate and behavior
during vertebrate development. Trends Genet., 13: 157162.
Wallingford JB, Fraser SE, and Harland RM [2002]. Convergent extension: The
molecular control of polarized cell movement during embryonic development.
Dev. Cell, 2: 695706.

Hedgehog Signaling
Collins RT,and Cohen SM [2003].The secret life of Smoothened.Dev.Cell,5:823824.
Hammerschmidt M, Brook A, and McMahon AP [1997]. The world according to
Hedgehog. Trends Genet., 13: 1421.
Ingham PW [1998]. Transducing Hedgehog: The story so far. EMBO J., 17:
35053511.
Ingham PW, and McMahon AP [2001]. Hedgehog signaling in animal development:
Paradigms and principles. Genes Dev., 15: 30593087.
Taipale J, et al. [2002]. Patched acts catalytically to suppress the activity of
Smoothened. Nature, 418: 892897.

Morphogens
Lawrence PA, and Struhl G [1996]. Morphogens, compartments, and pattern:
Lessons from Drosophila? Cell, 85: 951961.
Teleman AA, Strigini M, and Cohen SM [2001]. Shaping morphogen gradients. Cell,
105: 559562.

Vertebrate Organizers and Body Plan

Lemaire P, and Kodjabachian L [1996]. The vertebrate organizer: Structure and
molecules. Trends Genet., 12: 525531.
Schier AF, and Shen MM [2000]. Nodal signaling in vertebrate development. Nature,
403: 385389.
Sokol SY [1999]. Wnt signaling and dorsal-ventral axis specication in vertebrates.
Curr. Opin. Genet. Dev., 9: 405410.

Feedback Loops in Development

Eldar A, et al. [2002]. Robustness of the Bmp morphogen gradient in Drosophila
embryonic patterning. Nature, 419: 304308.
Freeman M [2000]. Feedback control of intercellular signalling in development.
Nature, 408: 313319.

Selector Genes, Gene Regulatory Networks, and the Segmentation Clock

Curtiss J, Halder G, and Mlodzik [2002]. Selector and signaling molecules cooperate in organ patterning. Nature Cell Biol., 4: E48E51.
Davidson EH, et al. [2002].A genomic regulatory network for development. Science,
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Problems

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Guss KA, et al. [2001]. Control of a genetic regulatory network by a selector gene.
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by a negative feedback loop. Science, 298: 840843.
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Problems
13.1 The core components and basic features of the four primary developmental pathways have now been introduced. The underlying picture
is a fairly simple one in which ligand binding at the plasma membrane
triggers a sequence of signaling events culminating in the activation
of gene transcription in the nucleus. The signaling pathways possess a
number of characteristics that enable them to guide the development
of complex metazoans possessing a large variety of different cell types
organized into tissues and organs. The rst of these characteristics is
that signaling is context dependent. The term context refers to the
mix of proteins being expressed. How a cell responds to a signal
depends on what other signaling events are taking place at the same
time and in previous times that have altered the mix of proteins being
expressed. How might context dependence come into play at the
plasma membrane top inuence signaling?
13.2 Control points are situated at the plasma membrane, in the cytoplasm,
and in the nucleus. Examples of cytoplasmic control points are the
beta catenin complex formed in the Wnt signaling pathway and the Ci
signaling complex formed in the Hedgehog signaling pathway. These
along with MAP kinase and NF-kB modules function as major signaling nodes. Give some examples of how cellular context may inuence what happens at these nodes in the signaling pathway.
13.3 Two kinds of posttranslational modicationsphosphorylation and
proteolysis are especially prominent in the four developmental pathways. These modications operate individually and jointly in these
pathways. List the various ways these modications inuence signaling.
13.4 Despite the presence of different proteins, the developmental pathways have many features in common. In response to ligand binding, a
series of signaling events takes place resulting in the translocation
from the cytoplasm to the nucleus of a transcriptional factor. In these
pathways downstream signaling elements can often function both as
activators of transcription or as repressors, with the choice depending
on cellular context. How does context come into play in the nucleus?
How was this dual capability used in the pathways discussed in this
chapter?

14
Cancer

Cancers arise from malfunctions in the cell control layer that lead to the
unregulated proliferation of cells. The underlying causes of cancers are
mutations and other alterations in DNA, and attendant inappropriate
expression levels, of genes encoding proteins that either promote growth or
restrain it, or direct the apoptosis machinery, or are responsible for DNA
damage repair and signaling, and chromatin remodeling.The mutations may
be heritable, that is, they may be present in the germ cells, or they may be
produced in somatic cells.
DNA can be damaged in several ways. Hydrolytic processes and oxidative byproducts of normal cellular metabolism can damage DNA. Ionizing
radiation from cosmic rays and from natural-occurring radioactive materials in the soil, water, and air such as uranium, thorium, and radon can
damage DNA. In addition, ultraviolet (UV) radiation from the sun can
damage DNA. The main step leading to DNA damage by environmental
and endogenous stimuli is the generation of oxidative free radicals near the
DNA. Free radicals are molecular species with unpaired electrons, making
them highly reactive. The most damaging of these reactive oxidative species
(ROS) is the hydroxyl radical. When produced in the vicinity of a DNA
molecule the hydroxyl radicals and other ROS attack sugars and bases producing single strand breaks, base losses, and modied bases. In addition to
this ROS mechanism, ionizing radiation such as X-rays and gamma irradiation can directly generate double strand breaks in DNA. When a cell that
has been damaged divides without having the damage repaired the changes
are carried on to its daughter cells and become a mutation.
In metastasis, malignant cancer cells break away from where they are
immobilized, enter the circulatory system, and invade other organs. They
form colonies, or secondary tumors, at the new locations and cause damage
to their neighbors by appropriating their sources of nutrition. Cancers
derived from epithelial cells are the most common type of cancer. In order
for the cancerous epithelial cells to break away from their initial location
they must detach from other epithelial cells and from the ECM. To do so
they must disrupt the cell-to-cell adhesive contact established by E331

332

14. Cancer

Figure 14.1. Matrix metalloproteinase structure: Most MMPs contain an Nterminal signal peptide (not shown) that targets the enzyme for secretion, followed
by a prodomain. The catalytic domain contains a zinc-binding domain and all MMPs
except MMP7 and MMP26 contain a Hemopexin domain C-terminal to the catalytic
domain and separated from it by a linker region. Dashed boxes represent domains
present in some MMPs but not others. Included in this set are Furin-like domains,
Type II Fibronectin repeats, and transmembrane + cytoplasmic segments.

cadherins, and cell-to-ECM contacts maintained by the integrins. Once they

have detached from one another and from the ECM they have to pass
through the basement membrane to reach and enter the circulatory system.
This is accomplished using matrix metalloproteinases to degrade matrix
proteins. The cancer cells make and secrete sufcient quantities of these
proteolytic enzymes to weaken the membrane and allow passage of the cells
through it into the blood vessels.
Matrix metalloproteinases (MMPs) are a family of over two dozen
secreted proteolytic enzymes that (i) cleave components of the ECM, and
(ii) cleave signaling proteins associated with the ECM and cell surfaces
resulting in their activation and solubilization. These enzymes are normally
synthesized in small quantities but production is increased in response to
cytokines and stress, and accompanies the transformation of normal cells
to cancerous ones.
As shown in Figure 14.1, MMPs contain a prodomain, which is cleaved
to activate the enzyme, and bronectin, hemopexin, and collagen V (not
shown) domains that promote substrate and inhibitor binding. The furin
domain provides an alternative cleavage site. Members of a family of four
proteins called tissue inhibitors of metalloproteinases, or TIMPs, bind the
hemopexin domain of MMPs, forming MMP-TIMP complexes that regulate the activities of the MMPs. A zinc-binding site is present in the catalytic
domain. Zinc binding (mediated by a trio of histidine residues) is necessary,
and this requirement gives rise to the name metalloproteinase.

14.1 Several Critical Mutations Generate

a Transformed Cell
The transformation of a normal cell into a cancerous one is a multistep
process. Mutations accumulate over time and over cell generations, and the
susceptibility to cancer increases rapidly with age. As the mutations accumulate a variety of genetic modications are produced. These include alterations in chromosome number (aneuploidy) involving the loss or gain of

14.1 Several Critical Mutations Generate a Transformed Cell

333

entire chromosomes, chromosome translocations that can generate a new

gene by fusing two different genes, and gene duplications (gene amplication). One or more of these dramatic changes in chromosome organization
are present in most human tumors.
The genetic targets of mutations that promote cancerous growth can be
grouped into four functional classes. The rst of these are genes that encode
proteins that function in the growth signaling pathways. These gene products
may convey progrowth signals or serve as brakes on growth. Mutated forms
of the receptor tyrosine kinases, GTPases, and nonreceptor tyrosine kinases
discussed in Chapters 10 and 11 are present in many different kinds of
cancer. The developmental pathways discussed in the last chapter are not
simply turned off at the end of embryogenesis, but rather remain active in
one form or another during adult life. Mutated and/or overexpressed elements of these pathways are encountered in cancers as well.
The second set of mutations is to genes that encode proteins that regulate
cell suicide. These target proteins either trigger or inhibit the cellular apoptosis program activated when aberrant conditions are detected. The third
group of crucial mutations is to genes that encode cellular caretakers. These
proteins carry out DNA repair and maintain chromosome integrity. The
fourth and last set of crucial gene products consists of the central regulators of growth, repair, and death. These gene products are referred to as
controller proteins. They are responsible for ensuring an orderly progression through the cell cycle, halting or advancing it when necessary and signaling to the apoptosis machinery when that outcome in required. Listed
in Table 14.1 are a number of prominent members of each of these classes
of proteins.
Oncoproteins are proteins that have been structurally altered by mutations in the genes that encode them. These proteins operate in the signal
Table 14.1. Protein with mutated and altered forms associated with cancer:
AbbreviationsOncoprotein (o); tumor suppressor (ts); DNA caretaker (c).
Protein
Ras
Src
Abl
APC
b-catenin
Myc
Bax
Bcl-2
hMLH1
hMSH2
ATM
NBS
BRCA1,2
p53
pRb

Function/pathway

Class

Cancer role

Growth
Growth
Growth
Growth
Growth
Growth
Apoptosis
Apoptosis
Repair
Repair
Repair
Repair
Repair
Controller
Controller

o
o
o
ts
o
o
ts
o
c
c
c
c
c
ts
ts

Many cancers
Sarcomas
Leukemias
Colorectal cancers
Colorectal cancers
Many cancers
Many cancers
Many cancers
Colon cancers
Colon cancers
Ataxia telangiectasia
Nijmegen breakage syndrome
Breast/ovarian cancers
Most cancers
Most cancers

334

14. Cancer

transduction, integration, and regulatory pathways involved in cellular

growth, multiplication, differentiation, and death. As a result of the structural alterations these proteins do not function normally, but instead are
changed in a manner that stimulates unregulated cell growth and proliferation thus promoting the development of cancer. Tumor suppressors are
similar to oncoproteins except that they normally act as brakes on growth.
When they suffer critical mutations these brakes on growth are removed.

14.2 Ras Switch Sticks to On Under

Certain Mutations
The rst group of entries in Table 14.1 consists of proteins that relay growth
signals from the plasma membrane to target sites in the cell interior. Ras
and Src are prominent members of this group. Src is implicated in about
80% of human colon cancers. Ras oncoproteins are even more widespread
in their cancer occurrences. They are present in 30 to 40% of human
cancers. Not all mutations are equally important. Recall that a codon is a
sequence of three nucleotide bases that encodes an amino acid. In Ras
mutations, codons 12, 13, and 61 serve as hot spots for oncogene activity.
The mutations occurring at these sites are point mutations that change one
of the base pairs in the codons encoding glycine (12 and 13) and glutamine
(61) into those encoding a different amino acid.
Ras is a crucial relay operating in the pathway that relays growth signals
to downstream targets. It functions as a binary switch. Like all GTPases it
is turned on by a GEF and turned off by a GAP. In the absence of growth
signals, the switch is in its off position, but turns on in response to the appropriate signals. The mutations leading to cancer result in the switch being
stuck in the on position, unable to turn off, and continually sending growth
signals into the cell.
X-ray crystallography of Ras in complexes with its GEF (Figure 14.2) and
GAP (Figure 14.3) provide insights into how the binary switch operates and
how certain mutations leave Ras stuck in its on position. As can be seen in
Figure 15.1a the Sos protein is organized into two domains, an N-terminal
domain that is largely structural in nature, and a C-terminal that catalyzes
the release of GDP. The catalytic domain forms a bowl about Ras. The Ras
protein has two switch regions, called Sw 1 and Sw 2. These regions create
a cavity within which GTP and GDP along with Mg2+ bind and release.
The Ras switch operates in the following manner. In the absence of
growth signals, Ras is in its off position with GDP bound rmly in the
pocket. When growth signals are present Sos is recruited to the plasma
membrane and binds to the Ras-GDP complex. The aH helix of Sos
engages the switches and ips Sw 1 to an open position in which it has
rotated away from Sw 2. The GDP molecule dissociates from the
complex, followed shortly thereafter by the dissociation of Sos. The GTP

14.2 Ras Switch Sticks to On Under Certain Mutations

335

Figure 14.2. Structure of Ras in a complex with its Sos GEF as determined using
X-ray crystallography: Ras is shown in light gray while Sos is depicted in black. Small
lled circles and squares superimposed on the gure serve to outline Sw 1 and Sw
2, respectively. The gure was generated using Protein Explorer with atomic coordinates deposited in the Brookhaven Protein Data Bank (PDB) under accession
code 1BKD.

Figure 14.3. Structure of Ras in a complex with its GAP as determined using Xray crystallography: Ras is shown in light gray while the RasGAP is depicted in
black. Small lled circles and squares superimposed on the gure serve to outline
Sw 1 and Sw 2, respectively. The Gly12 residue is located at the tip of the Ras Ploop while a second crucial residue, Glu61, located in the Ras Sw 2 region in close
opposition to the arginine nger loop of the RasGAP. The gure was generated
using Protein Explorer with atomic coordinates deposited in the Brookhaven PDB
under accession code 1WQ1.

336

14. Cancer

molecule is plentiful and binds in the pocket that was vacated by the GDP
molecule.
Mutations of the glycine residue at position 12 in the Ras chain convert Ras
into a form that is active all the time.That is, the RasGAP is unable to turn off
Ras.The reason for this can be discerned from the crystal structure exhibited
in Figure 14.3. The Gly12 residue is positioned at a crucial place at the very
end of the P loop.Because of its small size,replacement of glycine by any other
residue blocks the arginine in the nger from interacting with the ATP
molecule bound in the cleft, and hydrolysis is consequently impeded.

14.3 Crucial Regulatory Sequence Missing in

Oncogenic Forms of Src
Both Src and Abl are nonreceptor tyrosine kinases and were discussed in
Chapter 11. The Src gene was rst identied in retroviruses. These are
viruses that use RNA as their genetic coding medium rather than DNA.
When a retrovirus invades a cell the viral RNA is transformed into DNA
and integrated into the host genome. Retroviruses sometimes acquire genes
with oncogenic capabilities from an early host and deliver these into later
hosts. These genes are referred to viral oncogenes. The Src oncogene was
rst identied in the chicken Rous sarcoma virus, and then a cellular counterpart to the viral oncogene was found in normal cells in the chicken and
then in humans. The viral forms of Src and other viral oncogenes usually
differ in some way from their cellular counterparts. For that reason the viral
form of Src is denoted as v-Src while its cellular form is designated as c-Src,
and a similar situation obtains for other oncogenes.
Recall from Chapter 11 that a critical residue Tyr527 located in its COOH
tail controls the catalytic activity of c-Src. Phosphorylation of this residue
by Csk deactivates c-Src, and the Tyr527 phosphorylation site is required
for proper function of the kinase. In v-Src, the tail region containing Tyr527
is missing, and the truncated protein cannot be turned off. The result is that
the v-Src protein is constitutively active sending uncontrolled cell growth
and proliferation signals to the nucleus. The cellular form of Src can be
mutated in several ways to generate oncogenic forms. Point mutations in
the codon for Tyr527 converting this amino acid to phenylalanine can transform Src as can specic mutations that disrupt the ability of the SH2 and
SH3 domains to cooperate with the COOH tail in inhibiting Src activation.

14.4 Overexpressed GFRs Spontaneously

Dimerize in Many Cancers
Growth factors and growth factor receptors (GFRs) support the development of malignant tumors in several ways. One prominent contributor to
the onset of malignancy is the vascular endothelial growth factor (VEGF).

14.5 GFRs & Adhesion Molecules Cooperate to Promote Tumor Growth

337

In order for a solid tumor to grow and thrive it must have an adequate
blood supply. In response to this need for vascular expansion, tumor angiogenesis takes place. The expression of messenger RNAs for VEGF ligands
is enhanced in most human tumor cells. Increased VEGF mRNAs are
present in rapidly growing glioblastoma multiform brain tumors, and in
cancers of the lung, breast, gastrointestinal tract, female reproductive
organs, thyroid gland, and urinary tract.
Growth factor receptor dimerization is a critical step in relaying signals
conveyed by polypeptide growth factors into the cell interior.As discussed in
Chapter 11, receptor tyrosine kinases are brought into close physical proximity through ligand binding, resulting in the formation of receptor dimers or
oligomers. Autophosphorylation in the activation loop occurs next followed
by recruitment of cytoplasmic signaling molecules. In the absence of a ligand
the receptors do not dimerize and there is no signaling across the plasma
membrane. In contrast to this normal situation, spontaneous dimerization
occurs in many cancers. In these abnormal situations the receptors dimerize
in the absence of ligand binding. Ligand-free dimerization can be produced
in several different ways. Most often it is generated through overexpression
of receptors arising as a consequence of gene amplication.It can also be generated through point mutations and exon deletions.
Epidermal growth factor receptors (EGFRs) undergo spontaneous
dimerization in many cancers including those of the breast, lung and ovarian
cancers and gliomas, and brain tumors of glial origin. Amplication of the
EGFR (ErbB1) gene occurs in about 40% of gliomas, and the amplication of the ErbB2 gene takes place in about 30% of breast cancers. In many
of the brain tumors, gene rearrangements accompany gene amplication
and these alterations often involving truncations of portions of the molecule. The main effect of spontaneous dimerization is to activate a pathway
that sends inappropriate growth/proliferation signals to the nucleus.

14.5 GFRs and Adhesion Molecules Cooperate to

Promote Tumor Growth
Alterations in the mix of cell adhesion molecules being expressed accompany tumor progression. The altered expression patterns occur not only
during metastasis but also during solid tumor growth. Most cancers develop
from epithelial cells, and loss of E-cadherins is a common occurrence. Recall
that E-cadherins help maintain tight adhesive contacts in populations of
these cells. Loss of adhesive junctions and changes in cytoskeleton organization accompanies the transformation to malignancy. Among the changes
in expression patterns are the upregulation of aVb3 and a6b4 integrins, and
the switching from N-cadherins to E-cadherins and back when adhesive
contacts are again needed.
Integrins along with cadherins and Ig superfamily cell adhesion molecules form complexes with growth factor receptors. Examples of coopera-

338

14. Cancer

tivity between adhesion and growth factor receptors are a6b1 and a6b4 and
EGFRs, NCAM, and N-cadherins with FGFRs, and VE-cadherins with
VEGF receptors. One result of this form of association is the ability of integrins and/or growth factor receptors to convey signals into the cell without
having to engage their natural ligands. Clustering brings the receptors into
close proximity with one another, and promotes phosphorylation and the
recruitment of cytoplasmic signaling transducers, thereby alleviating the
need for ligand engagement.
A second consequence of the growth factor receptoradhesion molecule
clustering is the strengthening of signals that would otherwise be too weak
to elicit a cellular response if conveyed by one or the other alone. An
example of this form of cooperativity is that which occurs between Met and
a6b4 integrins. Recall from Chapter 10 that Met is the receptor for HGF/SF,
a set of diffusible ligands that are central participants in invasive growth
and branching morphogenesis. SF does not stimulate growth, but rather
triggers the dissociation, or scattering, of cells. By forming clusters the cytoplasmic segments of the Met receptors and integrins come into close
contact; they are able to promote phosphorylation and provide multiple
docking sites for adapters and other cytoplasmic signaling elements. In this
second signaling role, the a6b4 acts as an amplier to increase the magnitude of the cellular response to a growth signal and promote invasive
growth independent of binding to the ECM.

14.6 Role of Mutated Forms of Proteins in

Cancer Development
The likelihood of getting a colorectal tumor exceeds 50% by age 70. Most of
these tumors do not progress to a lethal stage, but nevertheless colorectal
tumors are the second leading cause of cancer death in the United States.
Mutated forms of several gene products contribute to the onset of colorectal
tumors.Two of these, the APC protein and b-catenin will be discussed in this
section,while mutations in another pair of gene products,the mismatch repair
proteins hMLH1 and hMSH2, will be examined in a later section.
The adenomatous polyposis coli (APC) protein and b-catenin participate
in the Wnt signaling pathway. The Wnt signaling system is thought of as a
developmental pathway, and was discussed in the last chapter. APC is localized in the basolateral compartment of epithelial cells along with glycogen
synthase kinase (GSK) that regulates its signaling activity. Recall that in the
absence of a Wnt signal, GSK phosphorylates b-catenin thereby tagging
that molecule for destruction. In the presence of Wnt signaling GSK is
antagonized and does not tag b-catenin. The latter is stabilized as a cytoplasmic monomer and can then translocate to the nucleus. APC forms a
complex with GSK and b-catenin. If APC is absent or is mutated, b-catenin
is not properly regulated by GSK. Instead, the b-catenin levels are raised

14.7 Translocated and Fused Genes Are Present in Leukemias

339

mimicking the effect of active Wnt signaling. Similarly, certain mutations to

b-catenin render that molecule insensitive to APC/GSK regulation leading
to the same result. Mutations in APC or b-catenin are encountered in many
colorectal tumors.
One of the main endpoints of signaling through APC/GSK/b-catenin
is the transcription machinery in the nucleus. Upon translocation to the
nucleus, b-catenin binds to the transcription factor Tcf-4/LEF to form a
dimeric complex. Thus, the program of gene expression is altered when bcatenin is not properly regulated. A second end point of signaling through
APC is the cytoskeleton. During cell division APC interacts with proteins
in the ends of microtubules that form the mitotic spindles. APC contributes
to the linking of microtubules to kinetochores, attachment sites on the chromosomes, and it contributes to signaling that the correct attachments are
made. Cells containing mutated forms of APC exhibit chromosome instability, that is, they have incorrect numbers of chromosomes.
The Wnt pathway is not the only developmental pathway whose components can contribute to cancer. Aberrant signaling in the Hedgehog and
TGFb pathways can promote cancer as well. Mutated forms of Patched and
Smoothened, the two receptors that operate in the Hedgehog pathway, are
encountered in basal cell carcinoma, the most common human cancer.
Recall from the last chapter that Patched normally binds and sequesters
Hh, and thus restricts its growth-promoting effects. It is therefore a tumor
suppressor, while Smoothened, which transduces growth signals into the
cell, acts as a growth stimulator. Gli transcription factors acting downstream
from the aforementioned receptors in the Hedgehog pathway convey the
growth signals to the nucleus. Raised levels of Gli activity are found in these
cancers as a result of Gli mutations and aberrant receptor behavior.
As noted in the last chapter, Smads, acting as downstream signal transducers in the TGFb pathway, can promote or inhibit growth. In their capacity as growth inhibitors they inhibit G1 cyclin-dependent kinases Cdk4
and Cdk2 that act during the G1 phase of the cell cycle. (These kinases will
be discussed later in the chapter.) The Smads stimulate production of
p15Ink4b, a protein that binds to and inhibits these cdks, and prevents
assembly of Cyclin D-cdk4 and Cyclin E-Cdk2 complexes. Mutations in
components of the TGFb pathwayligands, receptor, Smads (Smad2 or
Smad4) and cofactorsare present in the majority of colon and pancreatic
cancers.

14.7 Translocated and Fused Genes Are Present

in Leukemias
Member of the Bcl-2 family of proteins are central regulators of apoptosis.
When these proteins are not expressed at the proper levels, the option of
triggering apoptosis to remove cells that are either damaged or infected or

340

14. Cancer

growing out of control is no longer possible. As will be discussed in the next

chapter, there are several types of Bcl-2 proteins. Some promote apoptosis
while others inhibit it. Bcl-2 is a prominent member of the antiapoptosis
group. Aberrant bcl-2 genes are present in 90% of follicular lymphomas or
B-cell lymphomas for which the Bcl-2 protein is named. In these cells, a
translocation has occurred. The gene encoding Bcl-2 located on chromosome 18 has been translocated and fused with an immunoglobulin heavy
chain (IgH) gene located on chromosome 14. The result of this translocation, designated symbolically as t(14 : 18), is that the bcl-2 gene is positioned
next to the enhancer for the antibody gene. Antibody genes are vigorously
expressed and as a result of the translocation the Bcl-2 gene is continually
overexpressed. The decision between proliferation and apoptosis is shifted
towards the former, and the B-cells do not die off, as they should when
they age. Furthermore, the B cells are resistant to radiation therapy and
chemotherapy, since these forms of treatment work at least in part by stimulating apoptosis.
Several other fusion products play prominent roles in leukemias.
Burkitts lymphoma is characterized by the translocation t(8 : 14) and subsequent fusion of the c-Myc gene with an IgH gene. Chronic myelogenous
leukemia and acute lymphocytic leukemia involve the translocation of the
abl gene from chromosone 9 to chromosome 22, where it fuses with the
BCR gene. The altered form of chromosome 22 is known as the Philadelphia chromosome. All of these examples involve the joining of a gene with
oncogenic capabilities (Bcl-2, c-Myc, and Abl) with a strongly expressed
gene (BCR and IgH) leading to the continual overexpression of the
oncogene.

14.8 Repair of DNA Damage

DNA repair systems are responsible for maintaining the integrity of the
genome throughout the life of the cell. There are about 130 known genes
that encode DNA repair proteins. These are organized into ve DNA
damage repair systems (Table 14.2). The base excision repair (BER) and
nucleotide excision repair (NER) systems treat single strand damage.
Another, the mismatch repair (MMR) system, corrects for mismatched base
pairings generated during DNA replication and recombination. Finally, two
interlocking systems, the homologous recombination (HR) and nonhomologous end joining (NHEJ) systems, handle double-strand breaks. Each of
these systems contains an ensemble of enzymes and regulatory molecules
that repair and rejoin DNA strands through sequences of chemomechanical operations.
As noted in Table 14.2, the base excision repair system removes bases
damaged by UV radiation and X-rays, and by endogenous oxygen radicals
and alkylating chemicals. The nucleotide excision repair system handles

14.8 Repair of DNA Damage

341

Table 14.2. The four kinds of DNA repair.

Type of damage repair

System(s)

Base excision repair

BER

Nucleotide excision repair

NER

Mismatch repair

MMR

Double-strand break repair

HR, NHEJ

Description
Removes bases damaged by UV, X-rays,
oxygen radicals, and alkylating agents
Removes DNA lesions brought on by
environmental agents
Repairs damage occurring during DNA
replication and meiotic recombination
Repairs double-strand breaks caused by
ionizing radiation, oxidative stresses, and
other environmental factors

bulky DNA lesions and damage arising from environmental agents such as
polycyclic aromatic hydrocarbons compounds contained in cigarette
smoke. Portions of chromosomes bearing genes that are actively undergoing transcription receive greater NER attention than DNA segments containing genes that are only rarely transcribed. Several disorders including
skin cancers and Cockaynes syndrome are associated with mutations in
genes encoding members of the NER system.
The mismatch repair system monitors and treats damage occurring
during DNA replication and meiotic recombination. Among AGTC combinations corrects A-G and T-C mismatches, as well as improper sequence
insertions and deletions. Mutations in this repair systems lead to increases
in the mutation rate and to cancer development. Mutations in two members
of the MMR system, hMLH1 and hMSH2, are prominently linked to colorectal and other cancers.
The machinery for repairing double strand breaks is utilized for several
purposes in the cell. It is central to V(D)J recombination in lymphocytes.
DNA undergoing replication is subject to breaks and the machinery for
repairing DNA participates in remedying broken replication forks. This
machinery is involved in genetic recombination, the process whereby segments of DNA are exchanged between chromosomes. It is involved in both
mitotic recombination, involving sister chromatids, and meiotic recombination, involving homologous chromosomes. Homologous recombination and
nonhomologous end joining are responsible for repairing DNA doublestrand breaks. HR utilizes regions of DNA sequence homology from the
sister chromatid to repair damaged DNA in an error-free manner. NHEJ
does not utilize extensive regions of sequence homology to effect repairs
and is not necessarily error-free.
Double-strand breaks (DSBs) can be generated endogeneously by oxidative agents and environmentally by ionizing radiation. This form of damage,
while not as common as the other forms of DNA injury, can be extremely
harmful. DSBs can lead to chromosomal translocations, deletions, and fragmentation. Mutations in several members of the machinery that repair

342

14. Cancer

double-strand breaks are associated with a variety of different cancers. This

machinery will now be looked at in more detail.

14.9 Double-Strand-Break Repair Machinery

Proteins belonging to the DSB repair machinery form a number of distinct
repair complexes. The protein responsible for repairing and rejoining
broken strands of DNA are organized into several complexes, each consisting of proteins that come into physical contact with one another to carry
out their repair functions. Proteins comprising the modules have been
grouped together in Table 14.3. One of the key ndings in studies of the
DSB repair machinery is that some of the proteins are centrally involved
in a number of rare, inherited genetic disorders whose study reveals important details. These participants in DSB repair are named for the disorders
in which they were discovered. Examples of this naming convention include
Ataxia-telangiectasia mutated (ATM) and Nijmegen breakage syndrome
(NBS) proteins. Other rare, cancer-associated diseases involving DSB proteins are Fanconi anemia and Bloom syndrome.
The rst entry in the table is ATM/ATR, two proteins that function as
sensors and as signaling elements that convey damage signals to the p53
cellular controller and to the regulatory units of the repair modules. The
ATM/ATR proteins are kinases and convey their signals by catalyzing the
transfer of phosphoryl groups to their substrates. Three protein complexes
follow the ATM/ATR entry. Each complex is made up of proteins, some
Table 14.3. Double-strand-break sensing, repair, and signaling.
Component

Function

ATM/ATR

Signaling

Rad50

HR, NHEJ

Mre11
NBS1
Rad51

HR, NHEJ
HR, NHEJ
HR

Rad52
Rad54
BRCA1,2
Ku70

HR
HR
HR
NHEJ

Ku80
DNA-PKcs
XRCC4
DNA ligase IV

NHEJ
NHEJ
NHEJ
NHEJ

Description
Phosphorylates p53, NBS1, and BRCA1 in
response to DSBs
Forms an end-processing complex with Mre11 and
NBS1
Endonuclease activity
Regulatory
Searches for DNA homology; forms a complex
with Rad52, Rad 54 and BRCA1,2
Stimulates Rad51 activity
Chromatin remodeling
Regulatory; chromatin remodeling
Forms a heterodimer with Ku80; recruits and
activates DNA-PKcs
KU70 and KU80 bind to the free ends of the DSB
Regulatory; signals p53
Forms a complex with DNA ligase IV
Ends reattachment

14.9 Double-Strand-Break Repair Machinery

343

Figure 14.4. Onset of double-strand-break repair: The ATM proteins senses

double-strand breaks and conveys damage signals to the NBS1 protein in the
Rad50/Mre11/NBS1 complex and to the BRCA1 protein in the Rad51/Rad52/
Rad54/BRCA1,2 complex. Damage signals are also sent to the Fanconi anemia (FA)
complex, which includes the Bloom syndrome helicase BLM. In response, the FA
ubiquitin ligase FANCL (L) activates the BRCA2 protein, which then joins the
other DSB repair proteins at the repair site.

with regulatory functions and others with repair functions. The modules
listed in Table 14.3 work synergistically and sequentially to make the repairs
to the DNA. The early steps in operation of this repair system are depicted
in Figure 14.4.
The rst module, referred to as the Mre11 complex, is composed of the
Rad50, Mre11, and NBS1 proteins. This module participates in both homologous recombination and nonhomologous end joining along with several
other DNA strand manipulations involved in mitosis and meiosis. The key
function of this complex is to form exible bridges between ends of broken
DNA strands. The Rad50 proteins contain hooks, which join opposing
Rad50 proteins protruding from the ends of broken DNA strands. The
hooks join pairs of Rad50 proteins in the middle of the break while the
ends of the Rad50s remain attached to the DNA strands via the Mre11
proteins.
The next module is the Rad52 group. The Rad52 family of proteins is
responsible for homologous recombination. The rst step in HR is the
coating of the single strand DNA (ssDNA) with molecules of replication
protein A (RPA). In the next step, molecules of Rad51 and BRCA2 are
loaded onto the strands so that Rad51 displaces RPA. Both RPA and Rad51
remove secondary structure from the DNA to facilitate the pairing of sister

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14. Cancer

chromatids. As indicated in Table 14.3, Rad52 facilitates the replacement

of RPA by Rad51. The Rad51 and BRCA2 proteins form nucleoprotein
lamentsBRCA2 anchors the laments to the DNA and Rad51 proteins form the body of the laments.The pairing of sister chromatids follows
growth of the laments.
The nonhomologous end joining (NHEJ) proteins complete the list. The
last two entries in the table, XRCC4 and DNA Ligase IV, are needed in the
nal steps of DSB repair. These proteins, operating synergistically with
members of the Ku module, rejoin the two ends of the DNA to complete
V(D)J recombination and NHEJ.

14.10 How Breast Cancer (BRCA) Proteins Interact

with DNA
The Ataxia-telangiectasia mutated, Nijmegen breakage syndrome, and
breast cancer proteins belong to two families of protein that operate in
DNA damage detection, checkpoint signaling, and repair. One of these is a
large family of proteins operating in DNA damage-signaling, and characterized by the presence of one or more BRCT (dened below) repeats.
The second family is characterized by the presence of a COOH terminal
phosphoinositide-3 kinase- (PI3K) like domain.
The BRCT proteins are characterized by the presence of a motif consisting of about 95 amino acid residues, sometimes repeated several times.
This domain may be best thought of as an adapter module that enables
members of this family of proteins to interact and participate with a variety
of other proteins in DNA repair and recombination, transcription regulation, and cell cycle control. First discovered in the breast cancer 1 (BRCA1)
protein, this motif is called a BRCA1 C-terminal (BRCT) domain. Its secondary structure consists of four beta strands plus two alpha helices.
BRCA1 (1863 amino acids) and BRCA2 (3418 amino acids) along with
other members of the BRCT family are large multidomain proteins.
BRCA1 contains a RING nger domain, a pair of nuclear localization
signal sequences, and several BRCT repeats in its transcriptional activation
domain. BRCA2 possesses several BRC repeats, an NLS and a transcriptional activation domain. Its BRCT region is followed by a COOH
terminal region containing multiple single strand and double strand
DNA-binding domains (Figure 14.5). Its tower domain is the site of several
cancer-inducing mutations.
Inherited mutations in BRCA1 and BRCA2 genes predispose women
to breast and ovarian cancer. Cancer cells having defective BRCA1 or
BRCA2 proteins exhibit several kinds of chromosome instability.They have
chromosome breaks and incorrect chromosome numbers and abnormal
centrosomes. These instabilities are produced by defective transcriptioncoupled DSB repair, failures in checkpointing, and improper regulation

14.11 PI3K Superfamily Members that Recognize Double-Strand Breaks

345

Figure 14.5. Crystal structure of the COOH terminal portion of the BRCA2 tumor
suppressor protein: Shown are the main secondary structure elements helices
(corkscrew-shaped ribbons) and strands (at arrow-shaped ribbons), and the overall
organization of the COOH terminal portion of the molecule into ve domains. The
tower domain binds DNA and is the locus of a large number of cancer-inducing
mutations. The oligonucleotide/oligosaccharide binding (OB) domains bind ssDNA.
Two fragments of a DSS1 protein found in association with BRCA2, and needed
for crystallization, are included in the gure. The gure was generated using Protein
Explorer. The Brookhaven PDB accession number is 1MIU.

of centrosome duplication. The ensuring genetic instability then leads to

further mutations and to tumor formation.

14.11 PI3K Superfamily Members that Recognize

Double-Strand Breaks
ATM, ATR, and DNA-PKcs are members of the phosphatidylinositol-3
kinase (PI3K) superfamily that recognize DNA double-strand breaks, and
are involved in their repair. Members of this grouping, which includes ATM,
ATR, and DNA-PKcs, have a characteristic PI3K homologous domain in
their COOH terminal region. The PI3K-like domain of these molecules
does not appear to possess any lipid kinase activity. For that reason these
proteins are called PIK-related kinases. They are large serine-threonine
kinases; the ATM gene product is a 350-kDa protein, and DNA-dependent
protein kinase (DNA-PK) is even larger. The DNA-PK molecule contains
a Ku heterodimer consisting of 70-kDa and 80-kDa Ku subunits, plus a
460 kDa catalytic subunit, DNA-PKcs. The Ku subunits are named after the

346

14. Cancer

rst two letters of a patient found to be suffering from an autoimmune disorder associated with the Ku proteins.
DNA-PKcs and ATM are structurally similar, and both molecules are
capable of binding DNA. DNA-PK not only participates in repair of DSBs
caused by ionizing radiation but also is involved in V(D)J recombination
used to generate diversity in cells of the immune system. A mutation in
the kinase domain produces severe combined immunodeciency (Scid)
in mice and defects lead to ionizing radiation hypersensitivity in humans.
ATM and ATR are found on meiotic chromosomes and have a role in
processing double-strand breaks generated during that process. These
molecules serve as sensors of ionizing radiation-generated DSBs. They not
only function as damage sensors but also as signaling molecules that relay
damage signals to regulatory proteins that halt the cell cycle progression
while repairs are made.
The ATM protein functions as a sensor of DNA double-strand breaks in
the following manner. In the absence of DSBs, the ATM proteins exist as
inactive dimers. Double strand breaks produced by, for example, ionizing
radiation, induce alterations in chromatin structure. The alterations in chromatin structure trigger the dissociation of the ATM dimers resulting in
autophosphorylation and activation. Once activated, ATM migrates over to
p53, to NBS1 and BRCA1, and to cell cycle checkpoint proteins, and phosphorylates them at one or more sites.

14.12 Checkpoints Regulate Transition Events

in a Network
The cycle of cell growth and division passes through four stages (Figure
14.6). The rst of these stages (G1) is a cellular growth stage that prepares
the way either for entry into a cell division series of stages (S, G2, and M)
or to growth arrest (G0), or to apoptosis. Cells that do not undergo growth
arrest or apoptosis enter a synthesis (S) stage where the DNA is replicated
in preparation for mitosis. This stage is followed by a further mitosis
preparatory stage (G2) where RNAs and proteins required for mitosis are
synthesized. Finally, the cell enters into mitosis (M) where the cell divides

Figure 14.6. The cell cycle: Chevrons mark the divisions of the cell cycle into its four stages. The slash
placed late in G1 phase denotes the restriction (R)
point and the second slash placed in mitosis represents the spindle checkpoints. The three nonmitosis
stages are commonly referred to as interphase.

14.14 pRb Regulates Cell Cycle in Response to Mitogenic Signals

347

to produce two offspring. The three stages preceding mitosis are collectively
referred to as interphase.
Checkpoints are signaling pathways that ensure that a process does not
begin before a prior process is completed.They control the order and timing
of transition events in a network. DNA damage checkpoints, for example,
sense the presence of damage, producing signals that halt progression
through the cell cycle while the damage is repaired, and stimulate the transcription of repair genes to deal with it. The cell cycle includes several
checkpoints. The decision to undergo cell division or not occurs late in G1
phase. This point is called the G1/S checkpoint, or alternatively, START in
yeast and the restriction (R) point in mammals. Growth conditions signied by receipt of growth (mitogenic) signals from outside the cell are
evaluated, DNA is checked for damage, and a decision whether to proceed
through the cell division cycle is made. A second halt for checkpointing
occurs during S phase and the third takes place late in G2 phase and is
known as the G2 checkpoint. Checkpointing takes place during mitosis, as
well. The checkpoints halt mitosis if the spindle is damaged or if the chromosomes are not properly attached to the microtubules.

14.13 Cyclin-Dependent Kinases Form the Core of

Cell-Cycle Control System
The timing and duration of events occurring during the cell cycle are tightly
regulated by a control system containing at its core a family of serine/
threonine kinases called cyclin-dependent kinases (cdks) and a set of regulatory subunits called cyclins. Cyclin-dependent kinases are present
throughout the cell cycle but must bind to their regulatory cyclin to become
active. There are several different kinds of cyclin-dependent kinases, each
kind is associated with a particular cyclin subunit (Figure 14.7).
The concentrations of the different cyclins and cyclin-dependent kinases
oscillate with the cell cycle, and different cdk-cyclin pairs are active different times in the cell cycle. The correspondence between cdks-cyclins
and the phases of the cell cycle are presented in Table 14.4. The cyclindependent kinases carry out their regulatory roles by phosphorylating the
retinoblastoma protein pRb, which along with p53 lie at the heart of the
cells control system.

14.14 pRb Regulates Cell Cycle in Response to

Mitogenic Signals
In multicellular organisms, neighboring cells send mitogenic signals that
coordinate differentiation during development and trigger growth and proliferation. These events must be coordinated in order for the cells to work

348

14. Cancer

Figure 14.7. Crystal structure of the Cdk2Cyclin A complex: Binding of Cyclin

A to the kinase results in partial activation. Binding of the cyclin results in the extension of the catalytically important T loop. A further extension and full activation of
the cdk is achieved through phosphorylation by a separate kinase. The gure was
generated using Protein Explorer. The Brookhaven PDB accession number is 1FIN.

Table 14.4. Cyclins and cyclin-dependent kinases

(cdks).
Cyclin
Cyclins D1 to D3
Cyclin E
Cyclin A
Cyclin A
Cyclin B

Protein kinase

Cell-cycle phase

Cdk4, Cdk6
Cdk2
Cdk2
Cdk1 (Cdc2)
Cdk1 (Cdc2)

Late G1 phase
G1/S phase transition
S-phase
S-phase, G2-phase
G2-phase, M-phase

together in a tissue or organ. The retinoblastoma protein, pRb, functions

in a checkpoint role by inhibiting activation of an array of transcription
factors, collectively designated as E2Fs, required for cell cycle progression.
It keeps the gate shut at the G1 checkpoint where most of the decisions
are made whether to proceed to mitosis. The pRb gatekeeper integrates a
variety of positive and negative mitogenic signals conveyed as phosphorylation events and if conditions are propitious it opens the gate and enables
progression through to mitosis.
The retinoblastoma protein binds to members of the E2F family of transcription factors. During G0 and G1 stages of the cell cycle the E2Fs are
maintained in an inactive state by their pRb binding. During this time,
pRb is underphosphorylated (hypophosphorylated), and is able to form
stable complexes with these transcription factors. Its phosphorylation
state changes near the G1/S boundary when the protein becomes hyperphosphorylated. Cyclins and their associated cyclin-dependent kinases play

14.15 p53 Halts Cell Cycle While DNA Repairs Are Made

349

Figure 14.8. Growth signal stimulation of the transition from G1 phase to S phase:
Activated Cdk4s and Cdk6s along with kinases activated by growth signals hyperphosphorylate the retinoblastoma protein (pRb). In response, the E2Fs dissociate
from pRb and stimulate transcription of genes required for S-phase. Activation of
cyclin E is associated with the passage to S phase from G1 phase. The transcription
of cyclin E along with cyclin A and DNA polymerase is stimulated by the E2Fs.
These actions are terminated when Cyclin A-Cdk2 build up and turn off the E2Fs
transcriptional activities.

a key role in inactivating pRb. Phosphorylation of pRb by these cell cycle

regulators and by the growth factor-activated kinases frees the E2Fs
from their pRb inhibition and they can then carry out their cell cycleprogression-driving transcriptional activities (Figure 14.8).
Two noncontiguous domains, referred to as the A and B domains,
collectively form a binding locus known as an A/B pocket that binds many
proteins to pRb (Figure 14.9). For that reason, pRb and other members
of its family are known as A/B pocket proteins. This importance of this
interaction manifests itself by the occurrence of cancer-causing mutations
in residues located in the A/B pocket. The cyclin cell cycle regulators are
among the key proteins that bind to the A/B pocket. The cyclins, most
notably cyclins D and E, recruit cyclin-dependent kinases to the A/B pocket
and to a docking site located in the C terminus.

14.15 p53 Halts Cell Cycle While DNA Repairs

Are Made
The integration of the G1/S DNA damage checkpoint pathway into the
cell cycle occurs in the following way. Damage to DNA is sensed by the
ATM/ATM and Rad3-related (ATR) kinases. When they detect damage to

350

14. Cancer

Figure 14.9. Binding of an E2F peptide fragment to the pRb pocket: Shown in the
gure is a 18-residue fragment derived from the transactivation domain of E2F
(residues 410427) in a complex with the A/B pocket domain of pRb. The gure
was generated using Protein Explorer with atomic coordinates deposited in the
Brookhaven PDB under accession number 1N4M.

DNA they phosphorylate a pair of kinases called checkpoint 1 (Chk1) and

checkpoint 2 (Chk2). The Chk1 and Chk2 kinases phosphorylate a protein
phosphatase known as cell division cycle 25 (Cdc25). The Cdc25 protein
phosphatase is a cyclin-cdk activator. It is active when it is in a dephosphorylated state and is inactivated and degraded when it is phosphorylated.
Phosphorylation of Cdc25 by the checkpoint kinases inactivates the phosphatase and tags it for proteolysis, leading to phosphorylation and inactivation (arrest) of the cyclin-cdks through actions of another protein kinase
called Wee1.
When damage to the cellular DNA is sensed, ATM, ATR, and also DNAPKcs convey that information to p53 by phosphorylating the molecule at
sites specic to the type of damage sensed. In response to these signals, p53
acts as a transcription factor and upregulates a number of proteins. One of
these, p21, halts the progression through the cell cycle until DNA repairs
can be made. The p21 protein upregulated by p53 is a universal regulatory
subunit of cyclin-cdks. When p21 is activated it binds to the Cyclin E/Cdk2
complex, resulting in Cdk2 inactivation. The Cdk2s cannot phosphorylate
pRb, which then becomes hypophosphorylated and inhibits the E2Fs
(Figure 14.10) thereby halting progression from G1 to S phase. If the
damage is regarded as unrepairable, p53 functions in a second different way.
It triggers an apoptosis circuit that targets the cell for suicide.

14.16 p53 and pRb Controllers Central to Metazoan

Cancer Prevention Program
The p53, pRb, and connecting signaling proteins and other key regulators
form a circuit that regulates the progression through the cell cycle. Proper
operation of this circuit is central to a cells cancer prevention program. As

14.16 p53 and pRb Controllers

351

Figure 14.10. Cell cycle arrest triggered by DNA damage checkpoint signals: ATR
and ATM phosphorylate Chk1 and Chk2, which then phosphorylate the protein
phosphatase Cdc25 thereby inactivating it. Cdk2 is phosphorylated by the protein
kinase Wee1 and its actions are arrested. ATM signals through p53 to activate p21,
which arrests entry into S phase by inhibiting the transcriptional activities of the
E2Fs.

Figure 14.11. Controller circuit coordinating p53 and pRB activities: Mitogenic
signals are relayed into the cell via Ras, Ink4a and c-Myc. Pointed arrows denote
stimulatory inuences and at-headed arrows denote repressive inuences.

shown in Table 14.1, mutations in p53 or pRb leading to malfunctions in the

circuitry are found in most human cancers. A simplied representation of
this circuitry highlighting some of the most crucial protein-protein interactions is depicted in Figure 14.11. INK4a, Ras and c-Myc relay mitogenic
(growth and proliferation) signals. These signals converge upon ARF which
sits just upstream of p53. As shown in the gure, when it is activated, ARF
stimulates p53 activity. It does so by disrupting mdm2s inhibition of p53.
Because of its key location, ARF mutations are encountered in a host of
human cancers. Another prominent gene encountered in many cancers is
the adenovirus early region 1A (E1A) gene. This gene product interacts
with a variety of control proteins including pRb. When E1A binds pRb, it

352

14. Cancer

disrupts formation of pRb-E2F heterodimers and thus promotes establishment of cancer cell cycles resembling those produced by mutations in pRb
itself.
The pRb and p53 work together. Early in the cell cycle, pRb inhibits the
E2Fs, and Mdm2 keeps p53 at a low level of activity. When pRb becomes
hyperphosphorylated its block on the E2F is relieved, ARF is stimulated
and it, in turn, stimulates p53 so that as the cell enters S phase, p53s surveillance activities are stepped up. If p53 detects DNA damage it signals
p21, which blocks the cyclin/cdk complexes from hyperphosphorylating
pRb. The retinoblastoma protein then inhibits the E2Fs while the repairs
to DNA are carried out.
The ATM/ATR and DNA-PKcs proteins activate p53 by stimulating
proteolysis of the Mdm2 inhibitor. Under normal cellular conditions, the
Mdm2 protein represses the checkpointing and apoptosis promoting activities of p53. The Mdm2 protein acts as a p53-specic, E3 ubiquitin ligase.
It suppresses the transcriptional actions of p53 by tagging it for proteolytic
destruction and keeping its expression levels low. One of the genes turned
on by p53 is the Mdm2 gene. This activity leads to the formation of an
autoregulatory loop in which p53 regulates Mdm2 at the level of transcription, and Mdm2 regulates p53 at the level of its activity. The Mdm2p53 system is balanced through these mutual dependencies so that the
appropriate signals can activate p53. These signals dislodge Mdm2 from
p53, leading to the transcriptional activation of p53.

14.17 p53 Structure Supports Its Role as a

Central Controller
As might be expected from the appellation controller, the p53 and pRb
proteins receive input signals from many proteins and in turn inuence
many other proteins. The rst of these, p53, functions as a transcription
factor while the second protein, pRb, acts through the E2F family of transcription factors. In the language of networks, p53 and pRb operate as
highly connected nodes, and therefore it is not surprising that disabling
these proteins through mutations has such strong negative consequences.
The signals convergent upon p53 take the form of phosphorylations and
acetylations of specic residues. Some of the best characterized of the sites
of phosphorylation and acetylation are depicted in Figure 14.12.
The exibility in the chain connecting the core unit to the DNA-binding
domain allows the p53 molecule to contact the DNA in any of a number of
ways. This property is illustrated in Figure 14.13 where three DNA-binding
domains bind the DNA molecule, each in a slightly different way. The most
frequent mutations in p53 found in cancer cells are all situated in the DNAbinding domain. Of these, ve involved arginine residues and one a glycine
residue. The leading mutation is to Arg248, which mediates the direct

14.17 p53 Structure Supports Its Role as a Central Controller

353

Figure 14.12. Organization of the p53 protein: Shown in the upper part of the gure
is the domain structure of the p53 monomer. It contains an N-terminal transactivation (TA) domain, a proline-rich (PR) region, a DNA-binding domain, and in its Cterminal region a nuclear export sequence (NES), tetramerization (Tetra) domain
and a negative regulatory (NR) domain. Expanded depictions of some of the key
regulatory regions are shown in the lower part of the gure. The specic amino acid
residues that are phosphorylated (P), acetylated (A) or sumoylated (S) are shown.
Abbreviations: serine (S), threonine (T), lysine (K).

Figure 14.13. p53-DNA binding: The DNA-binding domains of the p53 protein are
depicted as ribbons while the dsDNA molecule is shown in a space-lled model.
The gure was generated using Protein Explorer with atomic coordinates deposited
in the Brookhaven PDB under accession numbers 1TUP and 1TSR.

354

14. Cancer

binding of the p53 molecule to the minor groove of DNA. Another often
mutated residue, Arg273, makes contact with the backbone phosphate.
The other residues help stabilize the interface between the p53 and DNA
surfaces.

14.18 Telomerase Production in Cancer Cells

The ends of chromosomes are capped by telomeres, a series of TTAGGG
repeats and associated proteins, and terminated at the very end by a lassolike structure called a T-loop. Telomeres are shortened by about 100 base
pairs every cell division. When the capping structure is degraded sufciently
the cells DNA repair machinery is able to sense the ends of the DNA molecules and interprets the ends as double strand breaks. If the cell keeps
dividing several negative outcomes become possible. These include degradation, chromosome recombination leading to loss of genetic information,
aberrant rearrangements of chromosomes, and genomic instability. To
avoid such dangerous situations the cell ceases to divide after several kilo
base pairs of telomere are lost and instead enters a nondividing stage called
senescence.
Several proteins associate with the telomeres. One of these is the
(human) telomerase reverse transcriptase (hTERT), which catalyzes the
addition of multiple TTAGGG repeats at the ends of chromosomes and
protects them from the DSB repair machinery. Among the others are two
proteins, named telomeric repeat binding factors 1 and 2 (TRF1 and TRF2),
that bind the TTAGGG repeats. In addition, a number of double strand
break repair proteins form complexes with the TRFs and the telomerase
components (the hTERT catalytic subunit and a human telomerase RNA
(hTR) that contains the template for adding telomeric repeats). This grouping includes members of the Rad50/Mre11/NBS1 double strand break
repair complex, and Ku86 and DNA-PKcs involved in NHEJ. Members of
these complexes are thought to convey prosenescence signals to the p53 circuitry when telomeres become shortened.
Signals sent through p53 and pRb ensure that the cell ceases to divide.
In response to critical telomere shortening, production of regulators of p53
and pRb activity such as ARF, p21, and Ink4a is increased. The pRb protein
becomes hypophosphorylated and binds E2F thereby suppressing proliferation. In more detail, ARF suppresses Mdm2 leading to the release of p53
from Mdm2 inhibition; p53 stimulates p21, which contributes to hypophosphorylation of pRb. The Ink4a protein contributes to pRb hypophosphorylation by suppressing the Ckd4/6 inhibition of pRb.
Cancer cells avoid senescence by increasing the production of the telomerase chromosome-capping enzyme. This activity greatly expands the
number of cell divisions possible, effectively immortalizing the cells. The
cells avoid passing into senescence, and instead continue to divide and

References and Further Reading

355

progress towards more lethal states. Mutations in the p53 and pRb that
disable these proteins can contribute to the immortalization by disrupting
the conveyance of prosenescence signals.

References and Further Reading

MMPs
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Stamenkovic I [2003]. Extracellular matrix remodeling: The role of matrix metalloproteinases. J. Pathol., 200: 448464.

Growth Factor Signaling

Birchmeier C, et al. [2003]. MET, metastasis, motility and more. Nature Rev. Mol.
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Blume-Jensen P, and Hunter T [2001]. Oncogenic kinase signaling. Nature, 411:
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Growth Factor Receptor, and Adhesion Molecule Cooperativity

Comoglio PM, Boccaccio C, and Trusolino L [2003]. Interactions between growth
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Conacci-Sorrell M, Zhurinsky J, and Ben-Zeev A [2002]. The cadherin-catenin
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Developmental Pathways
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Peifer M, and Polakis P [2000]. Wnt signaling in oncogenesis and embryogenesis
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Taipale J, and Beachy PA [2001]. The Hedgehog and Wnt signalling pathways in
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DNA Repair Mechanisms

Critchlow SE, and Jackson SP [1998]. DNA end-joining: From yeast to man. Trends
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De Laat WL, Jaspers NGL, and Hoeijmakers JHJ [1999]. Molecular mechanisms of
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Yang W [2000]. Structure and function of mismatch repair proteins. Mutation Res.
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Double-Strand-Break Repair
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DAndrea AD, and Grompe M [2003]. The Fanconi anaemia/BRCA pathway.
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The ATM Protein

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The Cell Cycle, E2Fs, and the Retinoblastoma Protein

Dyson N [1998]. The regulation of E2F by pRb-family proteins. Genes Dev., 12:
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Problems

357

The p53 Protein

Espinosa JM, and Emerson BM [2001]. Transcriptional regulation by p53 through
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Sherr CJ [2001]. The INK4a/ARF network in tumor suppression. Nature Rev. Mol.
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Telomere Maintenance
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Problems
14.1 Make a list of the ways a cancer cell differs from a normal one. Identify the signaling proteins that promote the altered response and how
these proteins are altered to promote cancer for each entry in the list.
For example, mutations in the Ras proteins that leave the GTPase
stuck in the on position contribute to aberrant growth signaling that
cannot be turned off.
14.2 Cancer is a multistep process. Arrange the entries in the list generated
in Problem 14.1 in a temporal order with early acting changes at the
top and late acting changes at the bottom. Which kinds of alterations
promote metastasis? Which ones help generate genome instabilities
and chromosome abnormalities?

15
Apoptosis

Kerr, Wyllie, and Currie coined the term apoptosis in an article that
appeared in the British Journal of Cancer in 1972. The term was taken from
the Greek and has the meaning of falling off as in the dropping of petals
from owers, and leaves from trees, which occur in a programmed manner
every autumn. Apoptosis, or programmed cell death, is widespread during
animal development where it is used to sculpt and rene tissues and organs.
Apoptosis makes possible the regression of tadpole tails, permitting the
emergence of an adult tailless form. It is used to remove larval organs in
insects, and to sculpt digitsngers and toesfrom undelineated limb buds
in mammals.
Apoptosis is widely encountered during development of the nervous
system. Some neurons are only transiently formed, and once their task is
completed they are removed. In many parts of the nervous system, cells are
initially overproduced. This overproduction ensures that there is adequate
input to target neurons during the initial phase of the nervous system connection. Excess cells are removed by numerically matching pre- and postsynaptic structures. The initial set of neural connections is rened, and cells
that are inappropriately wired are removed. In some neural populations,
most of the cells are removed in order to produce a set of precise neural
connections.
Apoptosis is an essential ingredient in the operation of the immune
system. Cells of the immune system such as B cells die off as they age and
when they are no longer needed. Virally infected cells, and damaged cells
that cannot be repaired, are targeted for apoptosis to prevent their harming
undamaged cells in the tissue or organ where they reside. The same strategy is used by the immune system to rid the body of cancer cells.
Apoptosis is different from necrosis. The plasma membrane does not
rupture in apoptosis as it does in necrosis, but instead cellular components
are degraded and packaged, and then digested. During apoptosis cells
undergo an orderly sequence of morphological changes. These changes
include cell shrinkage, chromatin condensation, DNA fragmentation, and
membrane blebbing, in which membrane-wrapped pieces of cell boil off of
359

360

15. Apoptosis

the cell surface as apoptotic bodies containing fragments of DNA and other
macromolecules.
Malfunctions in the cellular machinery that controls apoptosis are
encountered in many disease situations. Excessive apoptotic cell death
occurs in Alzheimers disease, Parkinsons disease, Huntingtons disease,
and ALS (Lou Gehrigs disease). Too little cell death is a hallmark of
cancer. In B-cell leukemia, for example, key regulators of the decision
circuitry that determines whether a cell survives or dies are overexpressed.
Apoptosis is suppressed and because population control is lost leukemia
develops.

15.1 Caspases and Bcl-2 Proteins Are Key Mediators

of Apoptosis
Apoptosis is largely carried out by caspases, proteolytic enzymes that catalyze the cleavage of specic molecules and groups of molecules in response
to activating signals. Caspases target critical repair, splicing, and replication
components, they cut up membranes and cytoskeleton regulators, and they
destroy cellular DNA. They also stimulate the expression of markers on the
cell surface that tag the cell for orderly destruction and engulfment by
neighboring cells. This orderly disassembly of a cell occurs in a way that
prevents damage due to leakage.
Bcl-2 proteins are a second group of proteins intimately involved in apoptosis. They function as sensors and regulators of the apoptosis program.
They were rst identied in B-cell lymphomas and, since then, mutated
forms have been found in a variety of cancers. These proteins are characterized by the presence of one or more Bcl-2 homology (BH) domains, the
ability of some to form pores in internal membranes, and their propensity
to either promote or inhibit the release of apoptotic signals and agents from
the internal membranes.
Apoptosis can be initiated by death signals sent into the cell from other
cells and by stress signals generated within the cell. Signals sent by other
cells instructing a cell to undergo apoptosis are received by death receptors
belonging to the tumor necrosis factor (TNF) family. When a death ligand
binds the death receptors, a death inducing signaling complex is formed that
initiates the apoptosis process. Death signals are also triggered by cellular
stresses detected internally in organelles such as the endoplasmic reticulum, Golgi, nucleus, and mitochondria. Apoptosis signaling is sent in
response to conditions such as irrevocable DNA damage in the nucleus,
unfolded protein stresses in the ER, and oxidative stresses in the mitochondria. Two loci, one within the mitochondria and the other just outside
that organelle, serve as the main control points for the launching of apoptotic responses.

15.2 Caspases Are Proteolytic Enzymes Synthesized as Inactive Zymogens

361

15.2 Caspases Are Proteolytic Enzymes Synthesized as

Inactive Zymogens
The activity of enzymes that chop up and digest molecules is tightly
controlled in the cell. One common strategy for controlling proteolytic
enzymes, or proteases, is to synthesize them in an inactive form that requires
further processing for their activation. One common kind of inactive form
is a zymogen, a proenzyme containing a prodomain that must be removed
in order to create an active form of the enzyme. Zymogens are converted
to catalytically active forms by their proteolytic cleavage into two or three
pieces followed by assembly of the catalytic subunits into complexes. Examples of proteases synthesized as zymogens include digestive enzymes that
reside in the stomach (pepsin) and pancreas (trypsin), and also include
blood-clotting enzymes (thrombin).
Caspases are cysteine aspartate-specic proteases. They break peptide
bonds after Asp residues, i.e., at Asp-X sites, and possess a highly conserved
cysteine residue in their catalytic site. Caspases are synthesized as zymogens. They contain a prodomain in the amino terminal region that regulates
the proenzyme, followed by a large domain, approximately 20 kDa in size,
and then a small domain, about 10 kDa in size. The proenzyme is activated
by proteolytic cleavage at two Asp-X sites, one situated at the end of the
prodomain and the other separating the large and small domains (Figure
15.1).
The tetramer is the functional (active) form of the caspase. It is constructed in several stages. In the rst stage, two zymogens associate to form
a zymogen homodimer. Adjacent large and small subunits (left hand pair
and right hand pair) are part of the same polypeptide chain with the small
units placed on the inside and the large subunits on the outside. In the next
stage, each of the polypeptide chains making up the caspase zymogen dimer
is cleaved at the Asp-X sites. These cuts induce conformational changes in
the subunits that are part of the caspase activation process because they
bring the caspases closer to conformation supporting catalysis. The similarity in conformations can be seen in Figure 15.2 where zymogen and caspase
homodimers are compared side-by-side. Differences in the crucial loops L1

Figure 15.1. Caspase domain structure: The N-terminal prodomain is followed by

a large subunit, a linker, and a small subunit. The location of the two Asp-X cleavage sites is indicated in the gure by arrows. Following cleavage, two large and two
small subunits associate to form a caspase tetramer with the two small subunits
inside and two large subunits on the outside.

362

15. Apoptosis

Figure 15.2. Structure of caspase-7 homodimers as determined by means of X-ray

diffraction measurements: (a) Procaspase-7 zymogen homodimer, and (b) Caspase7 homodimer. The gures were prepared using Protein Explorer using atomic coordinates deposited in the Brookhaven Protein Data Bank under accession codes
1K88 (a) and 1K86 (b).

to L4 that determine the catalytic cleft are clearly visible in the left and
right hand panels of the gure. The caspases are still not fully activated in
the more open caspase form, but a nal set of changes occurring during substrate binding renders the caspases fully active.

15.3 Caspases Are Initiators and Executioners of

Apoptosis Programs
More than a dozen different kinds of caspases have been identied in
mammals. Those that have been found in humans have been placed in one
of three groups in Table 15.1. Caspases belonging to Group I are associated
with inammatory responses. These caspases were rst named for interleukin-1b converting enzyme (ICE), and then renamed caspases 1, 4, and
5. These enzymes process pro-inammatory cytokines. The remaining two
groups of caspases are specically associated with apoptosis, either as initiators that convey signals through their proteolytic actions or as effectors
that degrade cellular components. Group II caspases are effectors. They
proteolytically degrade a variety of cellular components and are thus the
executioners of the apoptosis program.
Group III caspases are apoptosis initiators. They act upstream of the
effectors and activate them in response to proapoptotic signals and events.
The four caspases appearing as Group III caspases all have large prodomains in which there is either a DED (death effector domain) or a
CARD (capsase recruitment domain) proteinprotein interaction domain.

15.4 There Are Three Kinds of Bcl-2 Proteins

363

Table 15.1. Mammalian caspases and their roles in the cell: Group III initiator
caspases act upstream of the Group II effector caspases.The executioners have small
prodomains and require assistance of the initiators for their activation. Four element
consensus sequences that the caspases recognize and cleave are presented in column
4. The most conserved residue in the consensus sequence is the Asp (D) residue
proximal to the cleavage site while the residue in the fourth position mostly
determines the substrate specicity.
Group
I: ICE

II: Effectors

III: Initiators

Caspase

Consensus
sequence

Prodomain

1
4
5
3
6
7
2
8
9
10

(WL)EHD
(WL)EHD
(WL)EHD
DExD
(ILV)ExD
DExD
DExD
(ILV)ExD
(ILV)ExD
(ILV)ExD

Large, CARD
Large, CARD
Large
Small
Small
Small
Large, CARD
Large, DED
Large, CARD
Large, DED

These regulatory sequences target the procaspases either to adapters bound

to death receptors at the cell surface or to adapters positioned near mitochondria. At these locations the initiator caspases are positioned to respond
to proapoptotic signals. Caspases 8 and 10 contain a pair of DEDs. Caspase
2 and 9 contain CARDs. The effector (Group II) caspases do not have a
large prodomain in their N-terminus but instead possess a small Nterminal peptide. One other initiator caspase has been foundCaspase 12,
a murine (mouse) caspase that is localized to the endoplasmic reticulum.

15.4 There Are Three Kinds of Bcl-2 Proteins

Bcl-2 proteins are central regulators of caspase activity and of the cell decision concerning whether or not to undergo apoptosis. Bcl-2 proteins can be
grouped into three subfamilies according to their domain structure and
their pro- or antiapoptotic activities (Table 15.2). The dening characteristic of the Bcl-2 proteins is the presence of one or more BH domains. The
Bcl-2 and Bax subfamilies contain multiple BH domains while the Bad subfamily only possesses a BH3 domain.
There are four kinds of BH domains, designated as BH1 through BH4.
A typical Bcl-2 subfamily member contains at least three of the BH
domains, namely, BH1, BH2, and BH3. In addition, it has a hydrophobic tail
that anchors the protein to the outer membrane of mitochondria, the endoplasmic reticular membrane, and the outer nuclear envelope. Members of
the Bcl-2 subfamily inhibit apoptosis by restricting membrane permeabil-

364

15. Apoptosis

Table 15.2. Bcl-2 family of apoptosis regulators: Listed are the numbers of amino
acid residues in the proteins.
Bcl-2 subfamily:
Antiapoptotic
A1
Bcl-2
Bcl-xL
Bcl-w
Boo
Mcl-1

size (aa)
172
239
233
193
191
350

Bax subfamily:
Proapoptotic

size (aa)

Bak
Bax
Bcl-xS
Bok

211
192
170
213

Bad subfamily:
Proapoptotic

size (aa)

Bad
Bid
Bik
Bim
Blk
Bmf
Hrk
Noxa
Puma

197
195
160
196
150
186
91
103
193

ity through interactions with mitochondrial membrane components and by

binding to and sequestering members of the proapoptotic Bax group.
The proapoptotic Bax subfamily consists of proteins that possess a BH3
domain, a hydrophobic transmembrane tail, and at least one other BH
domain. Some have a BH4 domain; others have BH1 and BH2 domains and
perhaps a BH4 domain. Their 3-D structure is similar to pore-forming bacterial toxins. The Bax proteins interact with the proteins embedded in the
outer mitochondrial membrane to increase membrane permeability, and
they can form pores by themselves in membranes when they oligomerize.
The pro- and antiapoptotic, multi-BH domain proteins have electrostatic
and structural properties that enable them to not only operate in the cytoplasm but also insert into the membrane to make pores. Their polypeptide
chains are organized into sets of eight a-helices. There are three layers of
two a-helices and a pair of short capping helices at one end of the chain.
The organizaztion of the protein and the correspondence between helices
and BH domains is presented in Figure 15.3. Several structural features
support pore forming. The structure is fairly exible and so can rearrange
itself with little energy penalty. Two of the helicesa5 and a6are able to
span the membrane. There are several disordered, exible regions, and
there are three cavities. Charge-wise, the bottom of the protein is lined with
basic (positively charged) residues that complement the acidic (negatively
charged) membrane surface, and there is a pronounced hydrophobic cleft
surrounded by basic residues. The picture that emerges from examinations
of these structures is that of a5 and a6 along with the corresponding a5
and a6 helices from dimerization partners forming a pore, with a2 to a4
forming a binding groove, and the C-terminus forming an anchor.
The Bad subfamily is referred to as the BH3-only family. Some members
of this group have a transmembrane anchor while others do not. When activated by proapoptotic stimuli, these proteins translocate to the mitochondria and stimulate apoptotic responses. BH3-only proteins function as
cellular sentinels. In unstressed cells Bid, Bim, and Bmf are immobilized in

15.5 How Caspases Are Activated

365

Figure 15.3. Structure of the antiapoptotic protein Bcl-xL: Shown in part (a) of the
gure is a ribbon diagram of the portions of the protein whose structure could be
determined through X-ray crystallography (i.e., highly disordered regions are not
included in the model). Gray-scale shadings highlight the four BH regions. The correspondence between BH regions and the eight a-helices is presented in part (b)
of the gure. The gure was generated using Protein Explorer from Brookhaven
Protein Data Bank entry 1AF3.

the cytoplasm. Bid is a sensor of death signals sent into the cell through the
death receptors, and is localized in the vicinity of the death receptors. Bim
is sequestered at microtubule associated myosin V motors where it awaits
activation by cytokine and other stress signals. Bmf is also immobilized at
myosin V motors where it responds to loss of cell attachment (anoikis)
signals. Two other BH3-only proteins, Bik and Blk, function in the endoplasmic reticulum as sensors of cellular stress. The remaining BH3-only
proteins are regulated at the transcriptional level. Noxa and Puma are
transcribed in a p53-dependent manner and may be regarded as DNA
damage sensors, while Hrk and Bim are upregulated in response to growth
factor deprivation and cytokine withdrawal.

15.5 How Caspases Are Activated

Caspases are activated by external suicide instructions and internal stress
signals. Cells receive death instructions from other cells. These messages are
conveyed by cell-to-cell messengers called death ligands. The messages are
received by death receptors embedded in the plasma membrane. The death
messages are transduced into the cell interior through a multiprotein signaling complex formed by the activated death receptors. This complex is

366

15. Apoptosis

called the death-inducing signaling complex, or DISC. The DISC is the

control point for external signal activation of initiator Caspases 8 and 10
and signal to the BH3-only sensor protein Bid.
The counterpart to DISC for internal stress signals is a signaling complex
called the apoptosome. This multiprotein signaling complex is formed just
outside the mitochondria in response to internal stress signals. Initiator
caspase 9 is activated at this control point in response to the stress-induced
release of proapoptotic factors from the mitochondria. The release of the
mitochondrial factors is triggered by activity at the mitochondrial control
point called the permeability transition pore complex, or PTPC, where
multidomain Bcl-2 proteins are localized and BH3-only sensor proteins
converge when activated.
Several families of positive and negative regulators control the caspase
machinery. The regulatory proteins ensure that apoptosis is not triggered
inappropriately in response to random perturbations and aberrant signals.
External and internal signals are integrated together, and both contribute
to the live or die decision. If strong signals are sent into the cell instructing
it to undergo apoptosis, and strong stress signals are also present within the
cell, the decision is fairly simplecaspases will be activated and the cell will
die. If, as is normally the case, neither external nor internal death signals
are present the cell will live. All other situations are more complex. Cellular context comes into play through adjustments in the expression levels of
the positive and negative regulators that determine the set, balance, or commitment point for apoptosis. The remainder of the chapter is devoted to
exploring how the apoptosis control system works.

15.6 Cell-to-Cell Signals Stimulate Formation

of the DISC
The death receptors that transduce the death messages into the cell belong
to the tumor necrosis factor (TNF) superfamily. The TNF superfamily in
humans includes 19 Type II ligands, single-pass transmembrane proteins
with cytoplasmic N-terminals, and at least 29 receptors, mostly Type I
(extracellular N-terminals). Recall from Chapter 9 that these receptors are
widely expressed in the immune system where they respond to variety of
growth, proliferation, and death signals. Their extracellular region is characterized by the presence of from 2 to 5 repeats of a cysteine-rich motif
containing a number of disulde bridges.Their cytoplasmic portions contain
docking sites for several different kinds of adapters that mediate the
recruitment of key signaling elements to the receptors.
The death receptor family includes the TNF-a, Fas/Apo-1, and TNFrelated apoptosis-inducing ligand (TRAIL) receptors (Table 15.3). The
receptors and ligands operate as homotrimeric proteins. Signaling begins
when a trio of ligands binds to a trio of receptors. This event triggers the

15.7 Death Signals Are Conveyed by the Caspase 8 Pathway

367

Table 15.3. Members of the TNF family of receptors

containing death domains in their cytoplasmic region:
AbbreviationsDeath receptor (DR); TNF-related
apoptosis inducing ligand (TRAIL).
Death receptor
Fas
TNF R1
DR3
TRAIL R1
TRAIL R2
DR6

Alternative name(s)
Apo-1, CD95
CD120a
Apo-3
Apo-2, DR4
DR5

recruitment of the adapters to the cytoplasmic portion of the receptors

leading to the assembly of a DISC. In forming a DISC, the TNF-associated
death domain (DD) proteins are rst recruited to the cytoplasmic portion
of the TNF receptors and bind by means of their death domains. Proteins
with death effector domains (DEDs) bind next, and other binding events
follow. In this manner, a DISC is formed with FADD, TRAF2, TRADD
adapters serving as a starting point for three pathwaysCaspase 8, NF-kB,
and MAP kinase, respectively.

15.7 Death Signals Are Conveyed by the

Caspase 8 Pathway
The Caspase 8 pathway (Figure 15.4) begins when the Fas-associated death
domain (FADD) protein is recruited to the nascent DISC. Zymogens with
large prodomains such as Caspase 2, Caspase 8, and Caspase 10 are brought
into close proximity with one another at the FADDs and as a result can
form zymogen dimers and act on themselves to remove their prodomains.
Several caspase molecules can enter, become activated, and leave, one after
the other. Once activated these enzymes make contact with and activate
the effector caspases such as Caspase-3.
One of the Caspase-3 substrates is the caspase-activated deoxyribonuclease (CAD) protein and its inhibitor ICAD. The CAD and ICAD proteins are the catalytic and regulatory subunits of a protein referred to as
the DNA fragmentation factor, or DFF. The ICAD subunit remains bound
to the CAD subunit in the absence of Caspase 3 activities and inhibits its
enzymatic actions. Caspase 3 cleaves the ICAD thereby freeing the CAD
DNase and allowing it to move into the nucleus where it cleaves chromatin.
The BH3-only protein, Bid, is sited at the DISC. It functions as a sensor
and as part of the circuitry that integrates externally and internally generated signals. As indicated in Figure 15.4, Caspase 8 cleaves the 22-kDa Bid
sensor protein to create the 15-kDa tBid. Once formed, the tBids translo-

368

15. Apoptosis

Figure 15.4. Signaling through the DISC located at the plasma membrane:
Depicted are the main positive-regulating signaling elements. Homotrimeric TNF
ligands bind homotrimeric TNF receptors. In response, several adapter molecules
are recruited to the cytoplasmic portion of the receptors. The FADD adapter mediates recruitment and activation of the Caspase 8 pathway. The TRAF2 and RIP proteins mediate signaling and activation of the IKKs, which disinhibit NF-kB from the
IKKs. The TRADDs also signal to the JNK MAP kinase cascade.

cate to the mitochondrial PTPC where they promote the activities of

proapoptotic Bcl-2 proteins. If the overall mix of BH proteins at the PTPC
favors apoptosis, proapoptotic factors are released from the mitochondria
leading to stimulation of the apoptosome and the sequential activation of
Caspase 3 and then Caspase 6, which further stimulates Caspase 8 activities in situations where the externally driven stimulation is weak.

15.8 How Pro- and Antiapoptotic Signals

Are Relayed
Pro- and antiapoptotic signals are relayed to the nucleus by NF-kB proteins
and MAP kinases. The two other pathways activated by death ligand relay
signals via NF-kB proteins and MAP kinase modules as is usual for other
members of the TNF superfamily. These pathways were discussed earlier in
Chapter 9. Downstream signaling proteins establish contact with receptor
interacting proteins (RIPs) and tumor necrosis factor receptor associated
factor (TRAFs) that are recruited into the DISC. These pathways promote
the expression of both pro- and antiapoptotic genes. The NF-kB module
usually, but not always, acts to promote survival by raising the threshold for

15.9 Bcl-2 Proteins Regulate Mitochondrial Membrane Permeability

369

apoptosis. The IKKs are the key point of convergence of a variety of regulatory signals triggered by cellular stresses. The IKKs are activated when
recruited to and phosphorylated at the DISC. They, in turn, phosphorylate
the IkBs, resulting in the activation of NF-kB. In their prosurvival mode,
the NF-kB dimers translocate to the nucleus where they stimulate transcription of negative regulators of not only DISC signaling but also of mitochondrial proapoptotic signaling elements. As a consequence, the balance
between pro- and antiapoptotic factors is shifted in favor of the antiapoptotic ones and apoptosis is prevented.
Recall from Chapter 9 that MAP signaling pathways convey stress (JNK
and p38) and growth (ERK) signals from the plasma membrane to the
nucleus where they inuence transcription of a different sets of target genes.
As shown in Figures 9.4 and 15.4, the JNK pathway begins in the DISC,
where the TRADDs recruit and activate MEKK1, the rst of the kinases
in the MAP kinase cascade. The last kinase in the cascade is JNK. Once
activated this kinase translocates to the nucleus where it phosphorylates
members of the AP-1 family of transcription factors. A similar set of signaling steps occurs in the p38 pathway.
Transcription factors such as AP-1 family members and NF-kB reect
cellular conditions and prior signaling events in their transcriptional
activities. Depending on the specic mix of coactivators and corepressors
present, subunit composition, and the set of residues that have been phosphorylated (and acetylated), these transcription factors will either promote
or inhibit apoptosis. For instance, the c-Jun transcription factor, an AP-1
family member activated at the end of the MAP kinase cascade, usually
functions as a transcription activator, but can also function as transcription
repressors when associated with corepressors. In NF-kB signaling, exibility of response is provided by variations in subunit composition. Depending on Rel subunit composition, NF-kB will either promote apoptosis by
expressing TRAIL receptors or inhibit apoptosis by expressing antiapoptotic survival factors.

15.9 Bcl-2 Proteins Regulate Mitochondrial

Membrane Permeability
Mitochondria occupy a central place in internal stress-induced apoptosis.
As noted earlier in the chapter, the PTPC located in the mitochondria
serves as a key control point for internal stress responses. The PTPC, or
alternatively, the permeability transition pore (PTP), is formed at points
of contact between the inner and outer mitochondrial membranes. These
complexes are a conduit for the passage of agents such as Cytochrome c
and Smac/DIABLO that trigger apoptosome assembly and activation of
Caspase 9 (Figure 15.5). The PTPC encompasses the crucial inner membrane (IM) and outer membrane (OM) proteins along with key constituents

370

15. Apoptosis

Figure 15.5. Central components of the mitochondrial PTPC: Depicted in the

gure are the central elementsthe ANT located in the inner membrane and the
VDAC situated in the outer membrane. They form a pore through which Cyto c,
Smac/DIABLO, and a number of different effector molecules can diffuse through
and into the cytoplasm. Also shown are a variety of key Bcl-2 family members. A
proapoptotic Bcl-2 protein, for example, Bax, is depicted bound to the ANT/VDAC,
having displaced an antiapoptotic Bcl-2 protein, while several other Bax/Baks form
a pore.

of the intermembrane space and the matrix. It is composed of voltagedependent anion channels (VDACs) located in the outer mitochondrial
membrane and the adenosine nucleotide translocator (ANT) situated in the
inner membrane. The PTPC also includes the peripheral benzodiazepine
receptors, creatine kinases, hexokinase II, and cyclophilin D.
The mitochondrial PTPC is the main target of Bcl-2 signaling and is the
main site of their pro- and antiapoptotic actions. If a molecule can cross a
membrane by means of simple passive diffusion, driven only by a concentration gradient, then that membrane is permeable to that molecule. In
apoptosis, the Bcl-2 proteins regulate the permeability of the mitochondrial membrane to the apoptosis-promoting molecules. Bcl-2 proteins act as
sensors of stress signals and as regulators of mitochondrial membrane permeability. They regulate membrane permeability by binding to and altering
the pores formed by the ANT and VDAC, and by oligomerizing and
forming pores by themselves.
BH3-only proteins stimulate the release from mitochondria of Cytochrome c and other apoptosis-promoting factors. The BH3-only protein
tBid, for example, promotes the permeability-increasing activities of Bax
and Bak proteins, stimulates the remodeling of the mitochondrial cristae,
and triggers the release of Cytochrome c. Members of the anti-apoptotic
branch of the family inhibit apoptosis by sequestering BH3-only proteins
thereby preventing their stimulation of Bax and Bad. There are two kinds
of BH3 protein actions. Bid-like BH3 proteins facilitate the oligomeriza-

15.10 Mitochondria Release Cytochrome c

371

tion of Bax and Bak, while Bad-like BH3 proteins bind Bcl-2s and displace
Bid-like peptides from them.

15.10 Mitochondria Release Cytochrome c in Response

to Oxidative Stresses
Oxidative phosphorylation is carried out in mitochondria. One of the key
agents in this process is Cytochrome c, an evolutionary ancient electron
carrier. It is a small protein, consisting of 104 amino acid residues and a
covalently attached heme group. Cytochrome c is part of the machinery that
transfers electrons through several protein complexes located in the inner
mitochondrial membrane. It carries electrons from the cytochrome reductase complex to the cytochrome oxidase complex. The electron transport
activity leads to the pumping of protons from the matrix side to the cytoplasmic side of the inner mitochondrial membrane. The biochemical
process, oxidative phosphorylation, generates ATP by utilizing the energy
released during the electron transfer from NADH or FADH2 to O2.
Recall that atoms in stable molecules are tied together by bonds consisting
of pairs of electrons, one with spin up and the other with spin down. When
bonds are broken the molecules may end up with one or more unpaired
electrons. Molecules possessing unpaired electrons are referred to as free
radicals. The presence of an unpaired electron renders the molecules highly
reactive. Because of the presence of an electron the molecule has a propensity to steal electrons from other molecules, in many cases breaking bonds
to acquire the electrons. Chain reactions can be produced in the cell, and free
radicals are highly dangerous. Most commonly encountered free radicals in
the cell involve oxygen and these are called reactive oxygen species (ROS).
Mitochondria are the major cellular source of reactive oxygen species. In
mitochondria, a small fraction, perhaps 2 to 5%, of the molecular oxygen
being reduced by the respiratory electron transport chain is converted to
superoxide (O2-) and then to hydrogen peroxide (H2O2) or to the hydroxyl
radical (OH-). The ROS cause cellular (oxidative) stresses because they can
oxidize DNA, proteins, and lipids. The presence of ROS inuences cellular
physiology in many ways and triggers protective reactions involving the
upregulation of antioxidants and detoxifying enzymes. Antioxidants are
free radical scavengers. They supply electrons to the free radicals, allowing
them to form bonds and converting them nonreactive forms.
Cytochrome c is loosely bound to the inner mitochondrial membrane
by cardiophilin and other anionic lipids. When ROS levels rise it disrupts
the binding of Cytochrome c thereby making it easier to release. Once
Cytochrome c is released it stimulates assembly of the apoptosome leading
to activation of Caspase 9 and Caspase 3. Thus, oxidative stress conditions
arising when ROS activity levels in mitochondria become excessive and are
no longer controlled by antioxidant scavengers are signaled by Cytochrome

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15. Apoptosis

c release from the mitochondria. The Cytochrome c molecules act as local

signaling molecules that convey stress messages from the mitochondria to
the apoptosome.

15.11 Mitochondria Release

Apoptosis-Promoting Agents
Several different kinds of molecules are sent out through the PTPC, as indicated in Table 15.4. Cytochrome c and deoxyadenosine triphosphate
(dATP) convey signals that are required for assembly of the apoptosome
and the activation of Caspase 9. Two other regulators of the apoptosome,
Smac/DIABLO and Omi/HtrA2 are also released from the mitochondria.
A number of apoptotic effectors are released in addition to the activators
and regulators of apoptosome and Caspase 9 functions. These include
Apoptosis inducing factor (AIF) and Endonuclease G, which target nuclear
chromatin and degrades it.
Chromatin condensation and fragmentation of nuclear DNA is an important part of apoptosis. A number of proteins fragment DNA and target
chromatin in response to proapoptotic signals. One of these, the CAD
DNase, was discussed in Section 15.7. Two apoptotic enzymes are released
by mitochondria that target chromatin and fragment DNA. One of the
DNA chopping proteins, or DNases, is Endonuclease G. This enzyme is
released from mitochondria in response to stimulation by proapoptotic proteins. Once it is released into the cytosol it translocates to the nucleus where
it fragments DNA into nucleosomal-sized fragments. In the rst nuclear
steps of apoptosis, DNA repair is halted and chromatin condensation
occurs, which turns off DNA transcription. Another apoptosis promoting
agent sent out from the mitochondria is Apoptosis-inducing factor. This
protein is released from mitochondria at an early stage in the apoptosis
process. It is responsible for peripheral chromatin condensation and for
large-scale chromatin fragmentation.
Table 15.4. Agents released by mitochondria: AbbreviationsSecond mitochondrial activator of caspases
(Smac); direct IAP binding protein with low pI
(DIABLO).
Agent
Cytochrome c
dATP
Smac/DIABLO
Omi/HtrA2
AIF
Endonuclease G

Action
Required for assembly of the
apoptosome
Required for assembly of the
apoptosome
Proapoptotic regulator
Proapoptotic regulator
Early acting nuclear factor
Later acting nuclear factor

15.12 Role of Apoptosome in (Mitochondrial Pathway to) Apoptosis

373

15.12 Role of Apoptosome in (Mitochondrial Pathway

to) Apoptosis
The apoptosome is the main control point in the mitochondrial pathway to
apoptosis. When released from the mitochondria, Cytochrome c interacts
with a 130-kDa adapter protein called Apaf-1 located in the cytoplasm just
outside the mitochondria. This adapter contains three domains (Figure
15.6a). It has an N-terminal caspase recruitment domain (CARD), a central
domain that binds dADP/ATP, and a C-terminal domain containing a series
of WD-40 repeats. When Cytochrome c binds to the WD-40 repeats to override the autoinhibition, and deoxyadenosine triphosphate (dATP) hydrolysis occurs, Apaf-1 is able to form oligomers leading to the formation of a
1-MDa apoptosome.
The apoptosome is a sevenfold symmetric platform. It is wheel-shaped
with seven spokes radiating out from a central hub (Figure 15.6b), and functions to activate Procaspase 9 when these zymogens are incorporated
into it. When fully saturated, seven Procaspase 9 molecules are bound to
the seven Apaf-1 CARD domains and seven Cytochrome c molecules are
bound to the Y domains. The formation of an apoptosome containing activated Caspase 9 molecules triggers the apoptosis process by interacting with
and activating Caspase 3.
The apoptosome is a major control point where proapoptotic agents
released by the mitochondria converge and where Caspase 9 and Caspase
3 interact and activate one another.At the apoptosome, Caspase 9 processes
and activates Caspase 3. A feedback loop in which processed Caspase
3 cleaves and activates Caspase 9 amplies the production of activated
caspases. Several other proteins are present, and form complexes with the
caspases.Among these are caspase inhibitors and caspase counterinhibitors.

Figure 15.6. Apaf-1 and the apoptosome: (a) Apaf-1 molecule bound to
Cytochrome c and Procaspase 9Shown are a pair of WD-40 repeats in the Cterminal Y domain that bind Procaspase 9, and a pair of CARD domains in the Nterminal that bind Cyto c. A exible arm containing a CED4 homology motif that
binds dATP/ADP connects the N- and C-terminal regions to one another. (b) Fully
assembled apoptosome illustrating how the individual Apaf-1 molecules associate
into a sevenfold symmetric platform.

374

15. Apoptosis

15.13 Inhibitors of Apoptosis Proteins Regulate

Caspase Activity
Inhibitor of apoptosis proteins (IAPs) bind to and inhibit the activities of
caspases once they have been converted by proteolysis from zymogens to
caspase monomers and homodimers. The presence of these regulators (and
their counterregulators) establishes a second tier of control over the catalytic activities of caspases. The dening feature of the IAPs is the presence
of one or more BIR (baculoviral IAP repeat) domains (Figure 15.7). These
are found in the eight mammalian IAPs discovered to date. A second
prominent feature of the IAPs is the presence of a RING domain in the Cterminal of some but not all IAPs.
Caspases must form homodimers to become catalytically active. The
homodimerization partner supplies a sequence crucial for catalysis, a
sequence that that monomeric form of the caspases lacks. Crystal structure
studies of Caspase 9 in complex with the BIR3 domain of an X-linked AIP
(XIAP) protein show how the IAP proteins inhibit the catalytic activities
of the caspases. The BIR3 domain binds to the homodimerization domain
of monomeric caspase, thereby preventing formation of caspase homodimers. This mechanism differs from that used by the IAPs to inhibit effector caspases such as Caspase 3 and Caspase 7. Rather than preventing
formation of the homodimers, XIAP uses a sequence just forward of its
BIR2 domain to block the active site of the effector caspases.
Inhibitor of apoptosis proteins possess a ubiquitin ligase activity. As
shown in Figure 15.7, IAPs such as XIAP and c-IAP1 and c-IAP2 possess
a RING nger domain in their C-terminus. This motif is often encountered
in E3 ubiquitin ligases. In the case of IAPs bearing this motif, there seem

Figure 15.7. Domain organization of the inhibitor of apoptosis proteins (IAP):

(a) Structure of XIAP. (b) Structure of c-IAP1 and c-IAP2. ILP-2 and Livin resemble XIAP except that they only have one BIR domain (BIR3 in the case of ILP-2
and BIR2 in the case of Livin). The other three mammalian AIP proteinsNAIP,
Survivin, and Apollon/Brucehave from one to three BIR domains but lack a
RING nger domain. Abbreviations: X-linked IAP (XIAP); IAP-like protein (ILP);
neuronal inhibitory apoptosis protein (NAIP); baculoviral IAP repeat (BIR);
caspase recruitment domain (CARD).

15.15 Feedback Loops Coordinate Actions at Various Control Points

375

to be two choices of substrates. In response to proapoptotic signals, the

IAPs trigger their own ubiquitination. Alternatively, in the absence of
proapoptotic signals, the IAPs act in an antiapoptoptic capacity by promoting the ubiquitination of activated Caspases 3 and 7.

15.14 Smac/DIABLO and Omi/HtrA2 Regulate IAPs

Smac/DIABLO is an IAP counterregulator. It is released from mitochondria in response to proapoptosis signals, and once released disrupts the
ability of IAPs to inhibit the caspases. The IAP family member XIAP is one
of its main targets. In the absence of Smac/DIABLO, XIAP binds to the
small subunit of Caspase 9. It does not bind to Caspase 9 in its procaspase
form, but only to the cleaved and assembled Caspase 9 monomer. Smac/
DIABLO disrupts the ability of XIAP to inhibit Caspase 9 by binding to
its BIR3 domain. A sequence of four residues in the N-terminal recognizes
and binds a surface groove in the BIR3 domain.
The protein Omi/HtrA2 is another IAP counterregulator released by
mitochondria in response to proapoptotic signaling. Like Smac/DIABLO
it promotes apoptosis by antagonizing the IAPs ability to bind to and inhibit
activation of Caspases 3, 7, and 9. Upon release from the mitochondria
Omi/HtrA2 forms complexes with XIAP and disrupts the latters caspaseinhibiting functions. Although Omi/HtrA2 binds to the IAP in a manner
resembling that of Smac/DIABLO, its manner of action is different. Unlike
Smac/DIABLO, Omi/HtrA2 is a serine protease and may act in a proteolytic manner to degrade and thus limit the inhibitory activities of the IAPs.

15.15 Feedback Loops Coordinate Actions at Various

Control Points
The apoptosome and the mitochondrial PTPC are major control points for
apoptosis. These control points communicate with each other and with the
DISC in order to arrive at live or die decisions. The communication between
PTPC and apoptosome is summarized in Figure 15.8.In the gure,a stress-generating perturbation causes the loss of inner mitochondrial transmembrane potential Ym, produces ROS, and/or elevates the free calcium
concentration [Ca2+]m in the mitochondrial matrix. In response, the mitochondrial membranes become more permeable to Cytochrome c and
other proapoptotic agents. These agents leave the mitochondria. Some
(cytochrome c and dATP) trigger formation of the apoptosome and others
(Smac/DIABLO and Omi/HtrA2) activate the caspases by binding and
inhibiting the IAPs. Caspase 3 activated at the apoptosome diffuses to and
enters the mitochondria. It then cleaves a number of components of the mitochondrial electron transport chain. These feedback operations trigger the

376

15. Apoptosis

Figure 15.8. Mitochondrial PTPCApoptosome circuit: Perturbations of the mitochondrial permeability transition pore complex (PTPC) stimulates the release of
apoptosis-promoting factors such as Cytochrome c and counter inhibitor of apoptosis proteins (IAPs). These act at the apoptosome to activate Caspases 3 and 9.

stepped-up production of ROS and efux of Cytochrome c, which then acts at

the apoptosome to amplify the amount of activated Caspase 9 and Caspase 3.
Another feedback loop connects events at the apoptosome with those
occurring at the DISC. The connecting link is Caspase 6. Caspase 3 cleaves
and activates Caspase 6, which then migrates to the DISC where it stimulates Caspase 8, thereby amplifying the strength of the apoptotic signal at
the cell surface as was discussed in Section 15.7. The BH3-only protein Bid
completes the circuit by connecting actions taking place at the DISC with
those occurring at the PTPC.
Apoptosis is controlled by a plethora of positive feedback loops that
ensure that once the appropriate thresholds are passed there will be a rm
commitment to apoptosis. The presence of thresholds ensures that random
excursions and perturbations do not unnecessarily commit the cell to apoptosis when it ought not to. An equally important ensemble of negative feedback loops generates the threshold dependences.

15.16 Cells Can Produce Several Different Kinds of

Calcium Signals
Calcium is an important signaling intermediary, or second messenger, and
was introduced as such in Chapter 8. Calcium can also function as a rst
messenger. Calcium is well suited to function as a signaling molecule. It is
able to accommodate from 4 to 12 oxygen atoms in their primary coordination sphere. This property allows the calcium ions to contact multiple

15.17 Excessive [Ca2+] in Mitochondria Can Trigger Apoptosis

377

partners and trigger large conformational changes when binding a protein.

In addition, calcium does not diffuse very far because of the presence of a
large number of calcium buffers in the cytosol.
The normal intracellular calcium ion concentration is about 100 nM. This
level is several orders of magnitude lower than the calcium ion concentrations in the extracellular spaces, which is roughly 2 mM. To maintain the
intracellular calcium concentrations at the resting levels, the Na+/Ca2+
ion exchanger and pumps such as the plasma membrane calcium ATPase
(PMCA), remove calcium from the cell. In addition, the sarco-endoplasmic
reticulum calcium ATPase (SERCA) embedded in the SR/ER ships
calcium from the cytosol into the intracellular stores. Because of the presence of cellular machinery keeping calcium levels low, transient local
increases in calcium concentration can be produced easily and serve as a
signal.
Lipid second messengers relay signals from the plasma membrane to the
intracellular stores. These molecules trigger the release of calcium when
they bind inositol (1,4,5) triphosphate receptors (InsP3Rs). A second kind
of calcium release channel, the ryanodine receptor (RyR), releases calcium
from the intracellular stores, as well. Several different kinds of calcium
signals can be produced. One kind of calcium signal, the calcium wave, is
a global signal that propagates over large distances across the cytosol. The
propagation of these waves over large intracellular distances is made
possible by positive feedback in which calcium released from stores in one
locale diffuses to and triggers the release of calcium from nearby stores that
sets off further releases, thereby generating a wave of calcium. Calcium can
be released from stores in a more localized manner. The local releases of
calcium from intracellular stores are called puffs when produced by
InsP3Rs and sparks when facilitated by RyRs.

15.17 Excessive [Ca2+] in Mitochondria Can

Trigger Apoptosis
Calcium signals are sent to the mitochondria under normal conditions and
also under abnormal conditions. Calcium signals are sent to the mitochondria in response to increased signaling at the plasma membrane in order to
spur increases in metabolic activity to support the elevated signaling load.
This coupling of metabolism and calcium is made possible by the presence
in the mitochondrial matrix of a number of calcium sensitive metabolic
enzymes. Increased calcium signaling to the mitochondria also occurs under
abnormal conditions of cytosolic calcium overload (i.e., disruptions in
calcium homeostasis) and ER stresses. In these situations, the changes in
mitochondrial physiology drive the cell towards apoptosis.
Calcium ions are cycled between the SR/ER and mitochondria. The
membranes of these organelles are in close proximity to one another and

378

15. Apoptosis

highly local and directional signaling similar to that occurring at chemical synapses takes place. One of the early events taking place in stressgenerated apoptosis is an increased permeability of the PTPC to calcium
entry. This event leads to increases in permeability to cytochrome c, resulting in its increased efux from the mitochondria.
Combinations of proapoptotic conditions such as oxidative stresses and
calcium signaling between the ER and mitochondria can initiate apoptotic
responses under condition where one or the other condition by itself
cannot. The cooperation between the two is mediated by a positive feedback loop in which ROS generated in the mitochondria promote increased
Ca2+ release from the ER. The calcium accumulates in the mitochondria
and triggers further increases in ROS production. This process generates
the progressive depolarization of the inner mitochondrial membrane and
increases membrane permeation leading to cell death.
The PTPC is not the only control point where Bcl-2 family proteins exert
their regulatory inuences. They also act at the endoplasmic reticulum
where they inuence the release of Cytochrome c occurs through their
modulation of calcium signaling. They help maintain homeostatic control
over movement of calcium into and between the two organelles (the ER
and the mitochondria), and mobilize calcium release. Proapoptotic Bax and
Bak proteins localize to both the endoplasmic reticulum and mitochondria
where they control calcium trafcking between the two organelles. In
the ER, Bax and Bak help initiate the apoptosis process by contributing
to Caspase 12 activation, and in the mitochondria they help activate
Caspase 7.

15.18 p53 Promotes Cell Death in Response to

Irreparable DNA Damage
In response to DNA damage signals, the p53 protein will either halt the
cell cycle to allow for repairs or promote apoptosis if the damage is
irreparable. The signaling proteins that convey messages to p53 do so
through phosphorylations and acetylations of specic residues. The specic
mix of phosphorylations and acetylations determines in large measure the
response that will be made by p53. For example, phosphorylation of p53 on
Ser46 stimulates transcription of apoptosis-promoting genes, but does not
stimulate transcription of cell cycle genes. A second factor that guides the
p53 response is the mix of cofactors present at the promoter sites. As is the
case for other transcription factors, the cofactors help determine which
genes get transcribed and which ones do not. In the case of p53 two other
p53 family membersp63 and p73work together with p53 to transcribe
proapoptosis genes.
The p53 protein can shift the balance of pro- and antiapoptosis factors
in at least two distinct ways. First, in its role as a transcription factor,
the p53 proteins can step up the transcription of proapoptotic genes. As

15.19 Anti-Cancer Drugs Target the Cells Apoptosis Machinery

379

Figure 15.9. Regulation of apoptosis by p53: DNA damage signals are conveyed to
p53 in the form of posttranslational modications of specic residues such as phosphorylation on Ser46. In response, p53 (along with p63 and p73, not shown) stimulates transcription of apoptosis-promoting proteins acting at the DISC, PTPC, and
apoptosome. It also diffuses to the mitochondria where it interacts with antiapoptotic Bcl-2 family members to increase membrane permeability.

depicted in Figure 15.9, p53 can increase the numbers of propapoptotic proteins. These include death ligands and receptors at the DISC, the Apaf-1
scaffold proteins at the PTPC, and proapoptotic multidomain Bax, and
BH3-only proteins, at the apoptosome. Second, p53 can translocate to the
mitochondria where it can interact with the antiapoptotic Bcl-xL and Bcl-2
proteins to increase the efux of Cytochrome c from the mitochondria.
Recall from the last chapter that some of the most lethal mutations to p53
occur in its DNA binding domain. This domain contains the binding site for
attachment to the Bcl-2 proteins so these mutations not only destroy p53s
ability to bind DNA in the nucleus but also negate its ability to interact
with the Bcl-xL proteins at the mitochondria.

15.19 Anti-Cancer Drugs Target the Cells

Apoptosis Machinery
The machinery involved in regulating apoptosis is a major target of anticancer therapies. The goal of these therapeutic strategies is to induce apoptosis in the malignant tissue while leaving healthy tissue alone. Components
of the key control lociDISC, apoptosome, and PTPCare the primary
targets of many of these strategies. One set of strategies revolve about
causing apoptosis-inducing damage to DNA and other cellular components,
while another set of approaches focuses on causing oxidative damage in the
mitochondria in a way that stimulates the release of proapoptotic factors.

380

15. Apoptosis

The majority of chemotherapeutic approaches to killing tumors is based

on the idea of selectively inducing apoptosis in the diseased cells. Many anticancer drugs target the mitochondria with the aim of producing a decrease
in membrane potential leading to the efux of pro-apoptotic molecules and
the activation of the apoptosome/Caspase9 pathway. There are a number
of ways that a decrease in mitochondrial membrane potential may be
induced. One way is to supply ligands for either the ANT or the VDAC.
Alternatively, drugs may be used that trigger increases in ROS leading to
an increase in membrane permeability.
All of the major components of the apoptosis machinery are tightly controlled. Negative feedback loops ensure that small perturbations and
random releases of Cytochrome c or Smac/DIABLO from the mitochondria do not set off apoptosis. Restorative processes in the form of antioxidant scavenger systems prevent ROS generated within the mitochondria
from causing excessive damage. Calcium homeostasis within the cytosol and
organelles such as the ER and mitochondria are under negative feedback
control. Finally, the inner mitochondrial transmembrane potential Ym is a
quantity with an important role in apoptosis and endowed with restorative
properties. In order to be effective, therapeutic approaches that produce
stresses and perturb the mitochondria must overcome the plethora of protective measures used by the cell to deal with such stresses. These issues
compound the difculties in dealing with machinery that doesnt work quite
right because of mutations in genes that encode its key elements.

References and Further Reading

General References
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apoptosis. Nature Rev. Cancer, 2: 277288.
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genetics and chemotherapy. Cell, 108: 153164.
Mattson MP [2000].Apoptosis in neurodegenerative disorders. Nature Rev. Mol. Cell
Biol., 1: 120129.
Meier P, Finch A, and Evan G [2000]. Apoptosis in development. Nature, 407:
796801.
Vila M, and Przedborski S [2003]. Targeting programmed cell death in neurodegenerative diseases. Nature Rev. Neurosci., 4: 111.

Caspases
Boatright KM, et al. [2003]. A unied model for apical caspase activation. Mol. Cell,
11: 529541.
Chai JJ, et al. [2001]. Crystal structure of a procaspase-7 zymogen: Mechanisms of
activation and substrate binding. Cell, 107: 399407.
Chang DW, et al. [2003]. Interdimer processing mechanism of procaspase-8 activation. EMBO J., 22: 41324142.

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Shi YG [2002]. Mechanisms of caspase activation and inhibition during apoptosis.

Mol. Cell, 9: 459479.
Stennicke HR, and Salvesen GS [2000]. CaspasesControlling intracellular signals
by protease zymogen activation. Biochim. Biophys. Acta, 1477: 299306.

Bcl-2 Proteins
Cheng EHYA, et al. [2001]. Bcl-2, BclxL sequester BH3 domain only molecules
preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol. Cell, 8:
705711.
Cory S, and Adams JM [2002]. The Bcl-2 family: Regulators of the cellular life-ordeath switch. Nature Rev. Cancer, 2: 647656.
Gross A, McDonnell JM, and Korsmeyer SJ [1999]. Bcl-2 family members and the
mitochondria in apoptosis. Genes Dev., 13: 18991911.
Letai A, et al. [2002]. Distinct BH3 only domain either sensitize or activate mitochondrial apoptosis, serving as prototypes cancer therapeutics. Cancer Cell, 2:
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Moreau C, et al. [2003]. Minimal BH3 peptides promote cell death by antagonizing
anti-apoptotic proteins. J. Biol. Chem., 278: 1942619435.
Scorrano L, et al. [2002]. A distinct pathway remodels mitochondrial cristae and
mobilizes cytochrome c during apoptosis. Dev. Cell, 2: 5567.

DISC Signaling
Enari M, et al. [1998]. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature, 391: 4350.
Nagata S [1997]. Apoptosis by death factor. Cell, 88: 355365.
Scafdi C, et al. [1998]. Two CD95(APO-1/Fas) signaling pathways. EMBO J., 17:
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Weber CH, and Vincenz C [2001]. The death domain superfamily: A tale of two
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Regulation of the Apoptotic Signaling Pathways by NF-kB

Karin M, and Lin A [2002]. NF-kB at the crossroads of life and death. Nature
Immunology, 3: 221227.
Mayo CY, et al. [1998]. NF-kB antiapoptosis: Induction of TRAF1 and TRAF2 and
c-IAP1 and c-IAP2 to suppress caspase 8 activation. Science, 281: 16801683.

Permeability Transition Pore Complex

Crompton M [1999]. The mitochondrial permeability transition pore and its role in
cell death. Biochem. J., 341, pt. 2: 233249.
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Apoptosome Assembly
Acehan D, et al. [2002]. Three-dimensional structure of the apoptosome: Implications for assembly, pro-caspase-9 binding, and activation. Mol. Cell, 9: 423432.

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Bratton SB, et al. [2001]. Recruitment, activation and retention of caspase-9 and -3
by Apaf-1 apoptosome and associated XIAP complexes. EMBO J., 20: 9981009.

Inhibitor of Apoptosis Proteins

Huang HK, et al. [2000]. The inhibitor of apoptosis, cIAP2, functions as a ubiquitinprotein ligase and promotes in vitro monoubiquitination of caspases 3 and 7.
J. Biol. Chem., 275: 2666126664.
Salvesen GS, and Duckett CS [2002]. IAP proteins: Blocking the road to deaths
door. Nature Rev. Mol. Cell Biol., 3: 401410.
Suzuki Y, Nakabayashi Y, and Takahashi R [2001]. Ubiquitin-protein ligase activity
of X-linked inhibitor of apoptosis protein promotes proteosomal degradation and
caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death. Proc.
Natl. Acad. Sci. USA, 98: 86628667.
Yang Y, et al. [2000]. Ubiquitin protein ligase activity of IAPs and their degradation
in proteosomes in response to apoptotic stimuli. Science, 288: 874877.

IAP Counterregulators Smac/DIABLO and Omi/HtrA2

Holley CL, et al. [2002]. Reaper eliminates IAP proteins through stimulated IAP
degradation and generalized translational inhibition. Nature Cell Biol., 4: 439444.
Nicholson DW [2002]. Baiting death inhibitors. Nature, 410: 3334.
Srinivasula SM, et al. [2001]. A conserved XIAP-interaction motif in caspase-9
and Smac/DIABLO regulates caspase acytivity and apoptosis. Nature, 410:
112116.
Yoo SJ, et al. [2002]. Hid, Ror and Grim negatively regulate DIAP1 levels through
distinct mechanisms. Nature Cell Biol., 4: 416424.

Mitochondrial Physiology
Duchen MR [2000]. Mitochondria and calcium: From cell signaling to cell death.
J. Physiol., 529: 5768.
Jacobson J, and Duchen MR [2002]. Mitochondrial oxidative stress and cell death
in astrocytesRequirement for stored Ca2+ and sustained opening of the permeability transition pore. J. Cell Sci., 115: 11751188.
Kowaltowski AJ, Castilho RF, and Vercesi AE [2001]. Mitochondrial permeability
transition and oxidative stress. FEBS Lett., 495: 1215.
Marchetti P [1997]. Redox regulation of apoptosis: Impact of thiol oxidation status
on mitochondrial function. Eur. J. Immunol., 27: 289296.
Ricci JE, Gottlieb RA, and Green DR [2003]. Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis. J. Cell
Biol., 160: 6575.

ER and Calcium Signals to the Mitochondria

Boehning D, et al. [2003]. Cytochrome c binds to inositol (1,4,5) triphosphate receptors, amplifying calcium-dependent apoptosis. Nature Cell Biol., 5: 10511061.
Li C, et al. [2002]. Bcl-xL affects Ca2+ homeostasis by altering expression of inositol
1,4,5-triphosphate receptors. Proc. Natl. Acad. Sci. USA, 99: 98309835.
Nutt LK. et al. [2002]. Bax-mediated Ca2+ mobilization promotes cytochrome c
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Problem

383

Orrenius S, Zhivotovsky B, and Nicotera P [2003]. Regulation of cell death: The

calcium-apoptosis link. Nature Rev. Mol. Cell Biol., 4: 552565.
Scorrano L, et al. [2003]. BAX and BAK regulation of endoplasmic reticulum Ca2+:
A control point for apoptosis. Science, 300: 135139.

p53 Regulation
Flores ER [2002]. p63 and p73 are required for p53-dependent apoptosis in response
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Mihara M, et al. [2003]. p53 has a direct apoptogenic role at the mitochondria. Mol.
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Rev. Cancer, 2: 594604.

Cancer Therapy
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genetics and chemotherapy. Cell, 108: 153164.
Kaufmann SH, and Earnshaw WC [2000]. Induction of apoptosis by cancer
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Nature, 407: 810816.

Problem
15.1 Some cell types are more susceptible to apoptosis than others. What
classes of cells are especially responsive to apoptosis signals? What
kinds of cells send out proapoptosis messages as part of their normal
functions? Briey describe some of the ways a cell has of regulating
its own sensitivity to apoptosis.

16
Gene Regulation in Eukaryotes

The nding that metazoan genomes are as small as they are was remarked
upon in Chapter 1. In the introductory chapter, several aspects of eukaryotic cell organization that make this possible were noted. One of the most
important consequences of the changes in going from prokaryotic to
eukaryotic cell organization was creation of many different ways of regulating gene and protein expression, and for ne-tuning and specializing the
functions being performed. As a result of changes in the way the cells are
organized and gene expression and protein function are regulated, large
increases in complexity become possible without requiring large numbers
of additional genes. This central aspect will be examined in detail in this
chapter, which is devoted to the regulation of gene expression and protein
synthesis. Transcription will be discussed rst, followed by alternative splicing and translation.
Transcription in eukaryotes, even in the unicellular yeast, is more
complex than in prokaryotes because of the presence of chromatin. Recall
from Chapter 2 that eukaryotic DNA is highly condensed. Most of the chromatin is transcriptionally silent; that is, the DNA is tightly wound about the
histone core and promoter sites are not accessible. The molecular machinery responsible for transcription not only contacts and manipulates the
DNA molecule but also interacts with and alters the chromatin structure.
The molecular machines involved in modifying the chromatin structure and
transcribing genes are large. They can contain 50 or more subunits and have
molecular masses of several megadaltons. An example of how large these
machines may become is supplied by an examination of yeast cells. A set
of complexes that might be found in a typical yeast cell is presented in
Table 16.1.
The rst kind of complex is the basal (core) transcription machinery,
which includes RNA polymerase II and a set of general transcription
factors. It binds at the transcription start site and catalyzes the polymerization of RNA chains from DNA templates. This complex is commonly
referred to as the preinitiation complex (PIC). The second kind of protein
complex is exemplied in yeast by a set of about 20 proteins collectively
385

386

16. Gene Regulation in Eukaryotes

Table 16.1. Protein complexes involved in transcription in yeast cells.

Number of
subunits

Molecular mass
(MDa)

Basal transcription

>40

Mediator
SWI/SNF complex

20
11

1
2

SAGA

15

Machine

Function
Transcription
driver (PIC)
Signal integrator
Chromatin
remodeler
Histone modier

called the Mediator. Complexes similar to the Mediator have been found in
all metazoans examined. They contain suppressor of RNA polymerase B
(another name for RNA polymerase II) proteins (Srb proteins) and Mediator proteins (Med proteins), and serve as intermediaries between the
transcription factors and the basal transcription machinery. The last
two complexes modify chromatin by chemomechanical means. The rst
(SWI/SNF) breaks and reforms histone-DNA bonds, while the second
(SAGA) alters the chemical afnity of histones for the DNA thereby either
promoting or inhibiting transcription through acetylation of the histone
tails. If all the contributions from the four complexes are combined the
result is a machine with about 90 subunits and a molecular mass of 7 MDa.
(A few subunits are shared by more than one complex but this changes the
additive result only slightly.) These machines and the underlying mechanisms are highly conserved from yeast to man.

16.1 Organization of the Gene Regulatory Region

The molecular machines introduced in the last section carry out their functions at promoters. This is the name given to regions of DNA that contain
binding sites for the transcription control elements and transcription start
sites for RNA polymerase II (RNAP II). The core promoter is a region of
approximately 40 bp that directs the start of transcription by the preinitiation complex. The core promoter contains several short DNA sequences
that are recognized by DNA-binding proteins belonging to the PIC. One of
these is the TATA box, an A/T rich, 8 bp sequence located 25 to 30 bp
upstream of the start site for transcription. A second sequence known as
initiator (Inr) is positioned at the start site. Other important sequences contained within the core promoter include the TFIIB recognition element
(BRE) and the downstream promoter element (DPE).
The core promoter correctly positions and orients RNAP II at the start
site. The TBP subunit of the PIC recognizes the TATA box and the TFIIB
subunit recognizes the BRE. The architecture of the core promoter in yeast
and metazoans is depicted in Figure 16.1. Not all promoters contain TATA

16.2 How Promoters Regulate Genes

387

Figure 16.1. Eukaryotic core promoter: Promoters are major endpoints of signaling pathways and have multiple sites for binding proteins. (a) The core promoter
contains several binding sites for components of the basal transcription machinery
to bind. The TATA box is located 25 to 30 bp upstream from the transcription start
site, Inr, centered about the +1 position. The TFIIB recognition element (BRE) is
located just above the TATA box while the downstream promoter element (DPE)
is located about 30 bp from Inr. (b) The rst event to occur is the binding of the
TATA box binding protein (TBP) to the TATA box, followed by recruitment of
TFIIB to the TBP and the BRE, followed by recruitment of RNA polymerase II
and other elements of the PIC.

boxes. Both the TATA box and the Inr can direct transcription initiation by
RNAP II. TATA box sequences are present in promoters for many abundantly transcribed genes. Housekeeping genes and genes encoding growth
factors and transcription factors often have TATA-less promoters. They
utilize the Inr for correct positioning of RNAP II at the start site. Some
promoters, lacking both the TATA box and the Inr, rely on the DPE for
positioning.

16.2 How Promoters Regulate Genes

One of the most important aspects of gene expression is the presence of
multiple binding sites for regulatory proteins in the promoter. The core promoter was discussed in the last section. There are two other components
the proximal control region and the distal control region. These regions
contain binding sites that are recognized by regulatory proteins in a
sequence-specic manner. Several binding sites are usually found in the
proximal region located just upstream of the core promoter. These binding
sites are referred to as response elements. Proteins that stimulate transcription when they bind at these sites are called activators while those that
impede transcription are called repressors. A third kind of protein, which

388

16. Gene Regulation in Eukaryotes

Figure 16.2. Eukaryotic promoter consisting of the core promoter plus proximal,
distal, and downstream regulatory sequences called responsive elements (REs): The
REs are represented in the gure as dark patches separated from the core promoter
and from one another by spacers. A pair of insulators, one at each end, bounds the
gene regulatory region.

provides sites for attachment of coactivators and corepressors and mediates long range enhancer-promoter interactions, is called a tethering element
(Figure 16.2).
Distal sites can be located either upstream of the core promoter or downstream, in between coding regions and even inside introns. These are the
sites where the long-range interactions between regulatory proteins and the
basal transcription machinery take place. The positively acting proteins that
bind the DNA are called enhancers and those that inhibit transcription are
called silencers. The distal regulatory regions are generally larger than the
proximal ones and supply binding sites for up to a dozen or so proteins.
Besides proximal and distal sites, a third kind of siteone that marks a
boundaryis called an insulator. Insulators are DNA sequences that mark
boundaries between independent sections of DNA. They protect the genes
they delineate from inuences outside the protected regions, serving as
blockers of enhancer functions or as barriers against elements that inactivate chromatin. When situated between an enhancer and a promoter the
insulators are able to block action of that enhancer on the promoter. They
also block the actions of gene silencers that act on the chromatin, thereby
marking the ends of chromatin domains (Figure 16.2).
Promoters are major endpoints of signaling pathways. Transcription
factors (TFs), the signaling elements that relay signals into the nucleus, bind
to responsive elements in promoters in a sequence-specic manner.The TFs
contain DNA-binding motifs or domains. These motifs enable the proteins
to bind to specic DNA sequences just as protein recognition modules
enable one protein to bind to another in an amino acid sequence-specic
manner. A typical promoter contains several responsive elements. This
architecture allows a number of transcription factors to bind and jointly
regulate transcription.
Each gene has its own promoter site consisting of the core promoter and
a number of responsive elements for transcription factors. A signaling
pathway that activates downstream transcription factors can regulate the

16.3 TFs Bind DNA Through Their DNA-Binding Domains

389

expression of multiple genes. The activation process is a differential one

because of differences in promoter architecture from promoter site to promoter site and in the mix of proteins that are present at each. The sites can
differ from one another in the mix of responsive elements, in the presence
or absence of other transcription factors and coactivators (Mediator and
the like), the state of the chromatin, and disposition of chromatin modiers.
This type of regulation, where multiple proteins jointly determine the regulatory outcome, is called combinatorial control.

16.3 TFs Bind DNA Through Their

DNA-Binding Domains
Transcription factors enter the nucleus, bind to DNA in a sequence-specic
manner, and regulate gene expression. Transcription factors, like other elements of the control layer, are highly modular in design. Sequences that
code distinct functions are organized into modules that can be arranged in
different ways to generate a variety of functional proteins. The separable
units in a typical transcription factor are

DNA-binding domains,
transcriptional activation domains,
dimerization domains,
protein-protein interaction domains, and
nuclear localization sequences.

In the above, the protein-protein interaction domains mediate their interactions with upstream elements of the signaling pathways. The nuclear
localization sequences include nuclear import sequences and/or nuclear
export sequences. The DNA-binding domains are modules designed to recognize specic nucleotide sequences. Recall that the edge of each base pair
is exposed on the outside of the DNA helix. The DNA-binding domains
recognize distinctive patterns of hydrogen bond donors and acceptors, as
well as hydrophobic patches along the edges located in the major and minor
grooves. As is the case of protein-protein binding interfaces, electrostatic
surface complementarity is accompanied by geometric complementarity,
that is, by geometric matching of the shape of the DNA helix and protein
surface. Protein-DNA binding interfaces were discussed in general terms in
Chapter 4. It was noted that some DNA-binding proteins bind the major
groove; others bind the minor groove; and some simultaneously contact
both. Besides hydrogen bonds and hydrophobic patches, salt bridges and
van der Waals contacts contribute to the establishment of good surface
complementarity.
There are a number of prominent protein-DNA binding motifs. One of
these is the helix-turn-helix (HTH) motif. Others include the helix-loophelix (HLH), zinc nger, and basic region-leucine zipper (bZIP) domains.

390

16. Gene Regulation in Eukaryotes

Figure 16.3. Lambda repressor in a complex with DNA determined by means of

X-ray crystallography: A pair of lambda repressors makes contact with the DNA
double helix in its major groove. Protein helices are depicted as ribbons and DNA
molecules as balls-and-sticks. The gure was prepared using Protein Explorer with
atomic coordinates deposited in the Brookhaven Protein Data Bank under accession number 1LMB.

The HTH domain consists of two short alpha helices connected by a

sequence of four amino acids. The rst helix executes two or three turns
and the second helix has four turns. The two helices are oriented 120
degrees to each other. The second helix contacts the DNA in the major
groove, and the rst helix assists in the positioning. HTH motifs are
common in bacterial DNA-binding proteins, and are found embedded in
eukaryotic homeodomains. These domains are often found in proteins
needed for Drosophila development, and are widely distributed among
eukaryotes from yeast to man. An example of the helix-turn-helix motif is
the lambda repressor protein shown in Figure 16.3 (The lambda repressor
will be discussed in Chapter 18).
The HLH motif is found in many of the transcription factors involved in
eukaryotic development and regulation of adult physiological function. This
motif consists of an alpha helix followed by a loop that connects the rst
helix to a longer second alpha helix. The loop is highly exible and this
property enables the two helices to fold and pack against one another. This
ability promotes the formation of dimers. When forming a dimer a portion
of the longer helix contacts the DNA, and the shorter helix contacts the
corresponding shorter helix in the HLH motif of its dimerization partner.
The overall result is the formation of a four-helix bundle that grips the DNA
molecule.
Leucine zipper domains are present in many proteins that form dimers.
These proteins consist of a pair of alpha helices. A portion of one of the
helices is enriched in hydrophobic leucine residuesthere is a leucine
residue in every seventh amino acid position. The leucines from one protein

16.3 TFs Bind DNA Through Their DNA-Binding Domains

391

Figure 16.4. CREB bZIP dimer bound to DNA determined using X-ray crystallography: The basic region (residues 285 to 307 straddle and grip the DNA helix
while leucine zipper residues 308 through 336 form a dimerization interface. The
basic residues contact the major groove of the CRE region in the promoter. Protein
helices are depicted as ribbons and DNA molecules as balls-and-sticks. The gure
was prepared using Protein Explorer with atomic coordinates deposited in the
Protein Date Bank under accession number 1DH3.

are interdigitized with the leucines from the partner molecule to form a
short coiled-coil dimerization region, hence the name leucine zipper.
These proteins also contain a region rich in basic residues that interfaces
with the DNA. The basic region begins at a location seven residues Nterminal to the rst leucine in the leucine zipper domain. The overall
domain is referred to as a basic region leucine zipper (bZIP) domain; the
resulting bZIP dimer tightly grips the DNA molecule in the major groove
as illustrated in Figure 16.4 for the CREB transcription factor (CREB transcription factors will be discussed in Chapter 21). A number of transcription factors possess a basic region plus HLH and leucine zipper domains.
These DNA-binding proteins are referred to as bHLH-LZ proteins. The
bHLH-LZ and bZIP proteins form a variety of homeodimers and heterodimers with one another.
The nal category of DNA-binding proteins to be discussed is the zinc
ngers. These proteins utilize one or two zinc atoms to help form compact
domains of about 30 amino acid residues. The zinc nger motif is often
repeated, and each small module is organized about a central zinc atom.
Adjacent zinc nger modules form an overall DNA-binding domain whose
zinc ngers grip the DNA molecule. This motif is widely distributed among
eukaryotic transcription factors. The specifc composition of these modules
is variable but all contain zinc atoms and independently fold into small
compact structures as depicted in Figure 16.5.

392

16. Gene Regulation in Eukaryotes

Figure 16.5. Zinc ngers of the TFIIA protein in contact with DNA determined
using X-ray crystallography: The DNA-binding domain of the transcription factor
TFIIA has six zinc ngers each organized by a central zinc atom (indicated in the
gure by small lled circles). Protein helices are depicted as ribbons and DNA molecules as balls-and-sticks. The gure was prepared using Protein Explorer with
atomic coordinates deposited in the Protein Data Bank under accession number
1TF6.

16.4 Transcriptional Activation Domains

Initiate Transcription
The tight packaging of the DNA into nucleosomes inhibits transcription.
Transcription factors stimulate transcription by: recruiting to transcription
sites components of the transcription machinery; recruiting cofactors such
as the Mediator; and by attracting to these sites molecules and complexes
that remodel and modify chromatin structure.
Transcription factors possessing DNA-binding and dimerization domains
alone cannot trigger transcription. Instead, an activation domain must be
present. Transcription factors utilize several kinds of activation domains.
These domains are enriched in specic amino acids and are classied
according to their composition rather than by any particular secondary or
supersecondary structures. Some are enriched in acidic amino acid residues
and are called acid blobs. Others are enriched in glutamine or proline
residues.
The activation domains of transcription factors are protein-binding
regions. One of the ways the activation regions stimulate transcription is by
recruiting components of the general transcriptional machinery. The transcription activators bind to sites along the DNA several hundred base units
upstream of the transcription start site using their DNA-binding domains.
Components of the transcription machinery are then recruited to and bind
the activation domains, thus seeding the further assembly of transcription
components and, so, stimulating onset of transcription.
Chromatin remodeling complexes are attracted to the promoter site by
the activation domains of the transcription factors. Referring back to the
yeast modules listed in Table 16.1, The SWI/SNF module is recruited by the
acidic activation domain present in many transcription factors. The SAGA

16.6 Composition and Structure of the Basal Transcription Machinery

393

Table 16.2. Nuclear receptors and their ligands: Common abbreviations for the receptors are given within the
parentheses.
Steroid receptors
Androgens (AR)
Estrogens (ER)
Cortisol (GR)
Aldosterone (MR)
Progestins (PR)

Nonsteroid receptors
Ecdysone (EcR)
All-trans retinoids (RAR)
9-cis retinoids (RXR)
Thyroid hormone (TR)
Vitamin D3 (VDR)

histone acetyltransferase module can also be recruited in this manner. The

recruitment of the chromatin remodeling machinery is not dependent
on the presence of either the TBP (TATA binding protein) or RNA polymerase II at the promoter.

16.5 Nuclear Hormone Receptors Are

Transcription Factors
The genomes of complex metazoans contain large numbers of transcription
factors. An early estimate is that there are over 3000 transcription factors
encoded in the human genome. Nuclear hormone receptors, with over 150
members, form the largest family of transcription factors. These receptors
can be grouped into two large families, designated as Class I (steroid) and
Class II (nonsteroid). Representative members of these two families are
listed in Table 16.2.
Steroid hormones and other small lipophilic molecules such as vitamin
D3 are able to pass through the plasma membrane and enter the cell interior where they bind to nuclear receptors. Following ligand binding, the
steroid receptors dimerize, translocate into the nucleus from the cytoplasm,
and bind their cognate responsive elements. Class II receptors differ from
those of Class I in that they can bind to responsive elements in the absence
of ligand binding. Many Class II receptors readily form heterodimers and
bind to a variety of coactivators.

16.6 Composition and Structure of the Basal

Transcription Machinery
The basal transcription machinery consists of RNA polymerase II and a set
of general transcription factors (GTFs). These components, listed in Table
16.3, make up a minimal set of transcription initiation factors that support
recognition and low (basal) levels of transcription from most promoter

394

16. Gene Regulation in Eukaryotes

Table 16.3. Basal transcription machinery: Listed are the components, the number
of subunits in each component, their mass, and their role in initiating transcription
(discussed further in the main text).
Component
TFIID-TBP
TFIID-TAFIIs
TFIIB
TFIIF
RNA Poly II
TFIIE
TFIIH

Number of subunits

Molecular weight (kDa)

Function

1
12+
1
2
12
2
9

38
15250
35
30, 74
10220
34, 57
3589

TATA binding protein

Positive/negitive regulators
TFIIF/Poly II binding
Shuttles Poly II
Catalytic
Stim. TFIIH melting
Stim. melting/escape

sites. The minimal set of general transcription factors assembled at the promoter includes in order of recruitment TFIID, TFIIB, TFIIF, TFIIE, and
TFIIH. Another GTF, TFIIA, is sometimes recruited to the promoter, and
this (disposable) unit assists in TBP binding and stabilization. The assembly of the minimal set at the core promoter is depicted schematically in
Figure 16.1(b).
TFIID is believed to be the rst component of the transcription initiation complex to be recruited to the promoter site. It is the major sequencespecic GTF, and is a common target of TF activation domains. TFIID
contains the TATA box-binding protein (TBP), and a collection of TBPassociated factors (TAFs).The TBP subunit is a general transcription factor.
It recognizes the TATA sequence in its minor groove and is present in all
RNA polymerization operations. The TAFs are promoter-selective and
different mixes of these factors are present in different promoter sites.
Recruitement of TFIID triggers the recruitment of TFIIA and then TFIIB
followed by TFIIF bound to RNA polymerase II, and after that TFIIE, and
then TFIIH. These additional GTFs that follow establish protein-protein
contacts and link to the promoter site through the TBP. The TBP bends the
DNA and forms a saddle with its inner surface in contact with the DNA
molecule covering an 8-bp region of DNA.

16.7 RNAP II Is Core Module of the

Transcription Machinery
The RNAP II molecule is composed of 12 subunits designated as Rpb1
through Rpb12. The yeast RNAP II molecule contains 4565 amino acid
residues and has a mass of 514 kDa. The sizes of the subunits vary considerably. The largest two, Rpb1 (192 kDa) and Rpb2 (139 kDa), account for
the bulk of the mass of the polymerase. A top view of the entire assembly
is presented in Figure 16.6.

16.8 Regulation by Chromatin-Modifying Enzymes

395

Figure 16.6. Top view of RNAP II determined by means of X-ray diffraction measurements at 3.1 and 2.8 resolution: Ten of the subunits are shown in this gure.
Superimposed on this structure is a representation of a DNA molecule entering the
RNAP II, and an arrow indicating the direction of transcription. The gure was prepared using Protein Explorer with atomic coordinates deposited in the Protein Data
Bank under accession number 1I50.

As shown in Figure 16.6, the DNA enters the assembly through a cleft
formed by Rpb1 and Rpb2. The DNA is gripped by protein jaws but
cannot pass straight through the molecule because of a protein wall and
makes a right angle bend facilitating the addition of nucleotide triphosphates (NTPs). The NTPs enter through a funnel and pass through a pore
to reach the active site for polymerization. Exit of the nascent messenger
RNA chain is guided by several other structural elements, most notably by
rudder, lid, and clamp elements. Most of these functional elements belong
to the large Rpb1 and Rpb2 subunits. These subunits and the assignments
of functional roles to portions of each are depicted in Figure 16.7.

16.8 Regulation by Chromatin-Modifying Enzymes

Chromatin-modifying enzymes regulate the accessibility of the DNA to
transcription factors and the basal transcription machinery. Recall from
Chapter 2 that histone tails undergo several different kinds of posttranslational modications.The main targets of the modications are amino groups
situated at the end of lysine side chains (see Figure 2.13). Regulatory

396

16. Gene Regulation in Eukaryotes

Figure 16.7. Functional roles of the large subunits of RNA polymerase II: (a) Rpb1
subunit and (b) Rpb2 subunit. The gure was prepared using Protein Explorer with
atomic coordinates deposited in the Protein Data Bank under accession number
1I50.

enzymes functioning as histone acetyltransferases (HATs) catalyze the

addition of acetyl groups, while histone deacetylases (HDACs) do the opposite, catalyzing the removal of these groups. Acetyl groups are not the only
ones added and removed from histone tails. Phosphoryl, methyl, and
SUMO groups are added and removed as well, as was discussed in
Chapter 2.
Transcriptionally inactive heterochromatin is tightly compacted. Sites
where transcription initiation and regulation take place are inaccessible to
the proteins responsible for these actions. In contrast, transcriptionally
active euchromatin has a far more open shape, where transcription factors
and the basal transcription machinery bind are accessible to the responsible proteins. The covalent addition of acetyl groups to the side chains
reduces the net positive charge on the histone tails thereby weakening their
attraction to the DNA strands. The addition of these groups counteracts the
natural tendency for the chromatin bers to fold into compact nucleosomal
units making transcription difcult to impossible. Upon acetylation, the promoter sites become far more accessible to the transcription machinery.
The enzymes that mediate the modications on the H3 and H4 tails are
quite specic. This aspect, together with the presence of so many potential
modication sites, has led to the suggestion that there is a histone code that
determined whether a particular gene or cluster of genes is silenced or not.
An example of how this might work is supplied by data regarding the H3
lysine 9 (K9) site. As indicated in Figure 2.13 this is a site that can be either
acetylated or methylated. The selection process is a competitive one. Acetylation at K9 blocks methylation at that site, and vice versa. The silencing

16.9 Multiprotein Complex Use of Energy of ATP Hydrolysis

397

Figure 16.8. A peptide fragment of histone H3 doubly methylated on Lys9 (shown

in black) complexed with the chromodomain of HP1 (shown in gray): Residues
Gln5, Thr6, Ala7, and Arg8 of the H3 peptide form beta sheet interactions with
residues Glu23, Tyr24, Val26, Asn60 and Asp62 of the chromodomain. The peptide
residues are labeled in (a) while the chromodomain residues are labeled in (b). In
(a) the chromodomain backbone is highlighted, while in (b) a space-lled representation is presented. The atomic structures were determined using X-ray crystallography. The gure was prepared using Protein Explorer with atomic coordinates
deposited in the Protein Data Bank under accession number 1KNA.

process for that site works in the following way. An HDAC acts on K9 to
remove an acetyl group. Next, a histone methyltransferase-specic for that
site catalyzes the covalent addition of a methyl group. Then, heterochromatin (adapte) protein 1 (HP1) binds to the methylated K9 site and blocks
transcription. The binding of HP1 to the methylated histone tail is shown
in Figure 16.8.

16.9 Multiprotein Complex Use of Energy of

ATP Hydrolysis
There are two basic kinds of chromatin modiers. There are modiers of
chemical afnity between histones and DNA, and there are hydrolyzing
enzymes that break and reform bonds between histones and DNA. The rst
group consists of enzymes such as the HATs and HDACs that act on the
histone tails, covalently modifying them through the addition or subtraction of acetyl, methyl, or phosphoryl groups as just discussed. The second
group encompasses several large multisubunit complexes that use the
energy of ATP hydrolysis to disrupt interactions between histones and the
DNA. There are three classes of ATP-dependent multisubunit complexes.
Each contains at least one ATPase plus additional subunits. Some of the

398

16. Gene Regulation in Eukaryotes

Table 16.4. Three families of ATP-dependent SWI chromatin remodeling complexes: AbbreviationsATP-dependent chromatin assembly and remodeling factor
(ACF); chromatin accessibility complex (CHRAC); chromatin organization modier
(Chromo); Imitation SWI (ISWI); nucleosome remodeling factor (NURF); remodels
the structure of chromatin (RSC); sucrose nonfermenting (SNF); switch (SWI);
Yeast (y); Drosophila (d); human (h).
Family
SWI/SNF
ISWI
Mi-2

Complexes

Recognition domain

ySWI/SNF, hSWI/SNF, yRSC

dCHRAC, dNURF, dACF
Mi-2

Bromodomain
SANT domain
Chromodomain

complexes identied to date in various organisms are summarized in

Table 16.4.

16.10 Protein Complexes Act as Interfaces Between TFs

and RNAP II
Chromatin modifying enzymes do not interact with the basal transcription machinery, but instead make it possible for that machinery to nd
and attach to the promoters. Other complexes then take over and further
support transcription. These interfacing complexes link activators and
enhancers to the basal machinery attached to the core promoter. These
complexes function as interfaces integrating and conveying to RNA polymerase II and its suite of general transcription factors instructions embodied in the transcription factors.
The best studied of these interfacing complexes is the Mediator complex found in yeast. This complex consists of about 20 different proteins
arranged into three modules. Figure 16.9 illustrates how this complex can
serve as an interface between the core machinery and the transcription
factors bound to activator and enhancer sites. As can be seen in this gure
the mediator complex forms a bridge between the regulatory proteins and
basal transcription machinery at the core promoter. This bridging action is
especially important for proteins bound at distal enhancer sites brought into
close proximity to the core promoter through bending and looping of the
DNA.
The yeast mediator complex has a number of metazoan counterparts.
These complexes each contain homologs of a number of the yeast subunits
along with additional members specically needed to deal with the metazoan regulatory pathways. Some of the metazoan complexes are quite large
with more than a dozen members, while others are smaller composed of
subsets of the larger complexes. As is the case for the Mediator, the physical dimensions are large enough to act as bridges. For example, the planar
dimensions of the activation-recruited coactivator-large (ARC-L) complex

16.11 Alternative Splicing to Generate Multiple Proteins

399

Figure 16.9. Yeast Mediator complex: Shown are the three Mediator modules
The Srb4 head domain, the Med9/10 body domain, and the Gal11/Sin4 tail
domain, each named for prominent members of the module.

are 420 by 185 with distinct head, body, and tail regions formed by specic groups of subunits. The smaller coactivator required for Sp1 activation
(CRSP) complex, which consists of a subset of the ARC-L subunits, is
still 360 by 145 . Another mammalian Mediator complex, called
SMCC/TRAP (SRB- and MED-containing cofactor complex/thyroid
hormone receptor-associated protein), has about 25 members. These large
mediator complexes attach to the RNAP II CTD and form holoenzymes
with a total mass of about 1.5 MDa.

16.11 Alternative Splicing to Generate

Multiple Proteins
The remarkable efciency of genetic coding is exemplied by the relatively
modest increases in genome size in going from the yeast to the worm and
the y, and to vertebrates and man. A central feature in making this possible is the modular organization of the genome and the proteins so
encoded. In higher eukaryotes, genes consist of short coding sequences, or
exons, separated from one another by longer noncoding or intervening
sequences, or introns.The exons are typically 50 to 400 nucleotides in length,
while introns can be as long as 200,000 nucleotides. Whereas most yeast premRNAs do not contain introns, and those that do usually contain only a
single one, in higher eukaryotes a single gene may contain as many as 50
introns. The introns are included in pre-mRNAs produced by transcription
from the DNA templates. In splicing, introns situated in between pairs of
anking exons are removed. These intervening sequences are removed
from pre-messenger RNAs (pre-mRNAs) to form mature mRNAs containing a selected set of exons that is used for protein synthesis.

400

16. Gene Regulation in Eukaryotes

Figure 16.10. Alternative splicing of pre-messenger RNAs: Alternative splicing is

an efcient method of generating multiple proteins from a single primary transcript
in a controlled way. (a) The dotted lines indicate the intervening DNA sequences
that are spliced out to produce a mature messenger RNA molecule containing three
exons. (b) A different mix of exons is selected; the middle exon is skipped; that is,
it is spliced out too, resulting in a mature mRNA molecule consisting of exon1 and
exon3.

In alternative splicing, different sets of pre-mRNA sites are selected for

splicing. This process can generate several different isoforms, or splice variants, of a single gene. By varying the mix of splice sites a variety of messenger RNAs (mRNAs) can be generated differing from one another by
the presence of certain exons and the absence of others that have been
skipped. This selection process is illustrated schematically in Figure 16.10.
Alternative splicing generates a variety of tissue and compartment-specic
proteins from a single gene; it is commonplace in the expression of genes
that encode control layer proteins, especially those that function in the
nervous and immune systems.

16.12 Pre-Messenger RNA Molecules Contain

Splice Sites
The pre-mRNA molecule contains exons, introns, and regulatory sequences
that provide binding sites for the splicing machinery and regulatory proteins. Three sites are depicted in Figure 16.11. The rst is the 5 site characterized by the presence of a binding sequence containing a guanine-uracil
(GU) pair within a longer GURAGU-like sequence, where R is a purine.

16.13 Small Nuclear RNAs (snRNAs)

401

Figure 16.11. Organization of the pre-messenger RNA molecule: A representative

pre-messenger RNA molecule is depicted consisting of two exons, an intron, and
three accompanying regulatory sites. The introns are usually much longer than the
exons, but are shown shortened for illustrative purposes. Nucleotides characteristic
of the regulatory sequences are shown at the bottom of the diagram.

The second binding site is the branch site. This site is characterized by the
nucleotide sequence YNYURAY, where Y is a pyrimidine. The letter A
in Figure 16.11 denotes this site. The next portion of the RNA molecule is
the pyrimidine tract, which as its name suggests is a string of pyrimidine
nucleotides. The last regulatory unit is the 3 splice site characterized by
either a CAG sequence or a UAG sequence. Splicing is mediated by proteins that bind to particular stretches of DNA just as transcription is mediated by proteins that bind to promoter sites.

16.13 Small Nuclear RNAs (snRNAs)

A family of small nuclear RNA (snRNA) molecules found in the cell
nucleus forms the catalytic core of the spliceosome, the name given to the
machinery responsible for splicing out introns from the pre-mRNAs. These
snRNAs are enriched in uridine and are referred to as the U1 snRNA, U2
snRNA, and so on. The U4 and U6 snRNAs are bound to one another
through base pairing and operate as a single unit. The snRNAs function in
tight association with from 60 to 100 proteins to form ribonucleoprotein
particle (RNP) complexes. These RNP complexes are often referred to as
small nuclear ribonucleoprotein particles (snRNPs) or snurps. The premRNAs produced during transcription are also complexed with proteins.
The combined structures are referred to as heterogeneous nuclear RNP
(hnRNP) particles, and the proteins are called hnRNP proteins.
The spliceosome is assembled in several stages. Each stage is identied
by the presence of a particular set, or complex, of snRNPs as depicted in
Figure 16.12. The rst step in spliceosome assembly is the recruitment of
the U1 snRNP to the 5 splice site. Several auxiliary proteins assemble at
the branch point and polypyrimidine (Py) tract at this time. The rst stage
where the U1 snRNP binds to the 5 splice site sequence is known as the

402

16. Gene Regulation in Eukaryotes

Figure 16.12. Splicing of a pre-mRNA by the spliceosome: Splicing takes place

through the formation of splicing complexes. In the rst step, U1 binds to the 5
splice site forming the early (E) complex. In the next step, U2 binds the branch point
forming the A complex. This action is followed by recruitment of U4/U6 and U5 to
form the B complex. U4 and U1 leave and the others complete the splicing process.

commitment stage (complex E). The next stage, the assembly of the U2
snRNP at the branch point, assisted by the auxiliary proteins, is referred to
as the presplicing stage (complex A). In the third stage, a preassembled
U4/U6 U5 complex is recruited to the U2 snRNP and these components
are joined by the U1 snRNP. All components of the spliceosome are now
assembled into an early splicing machine (complex B) and, as shown in
Figure 16.12, the 5 and 3 ends of the intron have been brought together
to form a loop.
Once the rst phase of the splicing process is complete, the catalytic
phaseearly splicing and late splicing (complex C)begins. In the catalytic
phase the spliceosome removes the intron and joins the anking exons. The
catalytic process involves several steps and is accompanied by extensive
conformational changes and rearrangements of the spliceosome. The U2,
U5, and U6 snRNPs form the catalytic core of the spliceosome. These components remain attached to the removed intron, while U1 and U4 leave the
spliceosome earlier. At the end of the catalytic stage the introns and Py
tracts have been removed leaving the selected set of exons joined together
in a mature mRNA molecule.
Transcription and splicing are coupled processes. Splicing begins while
the pre-mRNA is still being formed, during the transcription elongation
process, rather than after completion. As noted above, long introns separate rather short exons from one another. The coupling of transcription with

16.14 How Exon Splices Are Determined

403

splicing helps bring together the exons in the correct manner over the large
distances and enables components of the transcription machinery to recruit
proteins required for splicing.

16.14 How Exon Splices Are Determined

In addition to the principal splice sites shown in Figure 16.11, there are a
number of auxiliary splice sites. These auxiliary sites may be located in
either exons or in introns and provide places where splicing regulators may
bind. Auxiliary sites located within exons are called exonic splice enhancer
(ESE) and exonic splice silencer (ESS) sites, depending on which regulatory outcome is supported. Similarly, intronic sites are termed intronic splice
enhancer (ISE) and intronic splice silencer (ISS) sites. Two kinds of proteinshnRNPs and SR proteinsbind to these auxiliary regulatory RNA
sequences.
SR proteins are a family of essential splicing factors ranging in size from
20 to 75 kDa. Members of the family are characterized by the presence of
an arginine-serine-rich (RS) domain of variable length at their carboxyl terminal and one or more RNA recognition motifs (RRMs) in their amino terminal.These proteins are sequence-specic RNA binding proteins that have
multiple roles in splicing. They play essential roles in the assembly of the
spliceosome at splice sites and in the selection of alternative splice sites.
Figure 16.13 depicts the assembly of a representative set of splicing
factors along a section of a pre-mRNA molecule. Two auxiliary splicing
factors (U2AF65 and U2AF35) associated with the U2 snRNP have been
recruited to the Py tract and 3 splice site by SR proteins bound at nearby
ESEs. These proteins, and other auxiliary factors, recruit or inhibit components of the spliceosome from assembling at the pre-mRNA and recognizing nearby splice sites. Proteins that bind enhancer or silencer sites can be
either SR proteins or hnRNPs.

Figure 16.13. SR (serine-arginine-rich) proteins recruit U1 and U2 snRNPs to

splice and branch sites: Auxiliary splicing sites and regulatory proteins that bind
them determine which exons are spliced together. AbbreviationsExonic splice
enhancer (ESE) site; exonic splice silencer (ESS) site; intronic splice enhancer (ISE)
site; intronic splice silencer (ISS) site.

404

16. Gene Regulation in Eukaryotes

Figure 16.14. Crystal structure of the N-terminal region (UP1) of the hnRNP A1:
Each subdomain contains an RRM and the overall structure of the module is that
of an extended RNA binding surface. The crucial amino acid sequences are located
on the two middle beta strands of each subdomain. The gure was prepared using
Protein Explorer with atomic coordinates deposited in the Brookhaven Protein
Data Bank under accession number 1UP1.

Like other regulatory proteins the hnRNP proteins are modular in

design. They contain one or more RNA recognition motifs (RRMs) that
facilitate their participation in the complexes. An example of an RNA
binding domain for an hnRNP called A1 is presented in Figure 16.14. The
RNA binding module consists of a pair of subdomains, I and II. Each subdomain consists of a four-stranded antiparallel beta-pleated sheet and two
alpha helices off to one side. The overall structure is that of an extended
RNA binding surface.
The relative concentration of splicing proteins, and the mix of regulatory
sites, jointly determine which combination of exons is assembled into the
mature messenger RNA molecule. Different cell types in a multicellular
organism will express a different mix of proteins that bind to the auxiliary
regulatory sites, and a given cell may alter the mix of splicing regulators
over time to produce proteins with slightly altered properties.

16.15 Translation Initiation Factors Regulate Start

of Translation
The machinery involved in translation initiation serves as an important end
point for stress signaling. Before discussing the mechanisms underlying the
control of translation it is worthwhile to examine the organization of
mRNAs and the machinery that carries out translation. Messenger RNA
molecules contain special structures at their 5 and 3 termini. A cap structure is added to its 5 end. The cap consists of a 7-methylguanylate (a CH3
group appended to the 7 position of the G base) plus a triphosphate group
that links the 7-methylguanylate to the sugars at the 5 end of the RNA
molecule. The cap is referred to as an m7GpppN structure, where N is any

16.15 Translation Initiation Factors Regulate Start of Translation

405

nucleotide. A string of perhaps as many as 200 adenylates is appended to

the 3 terminus of the mRNA. This addition is referred to as a polyadenylate tail or poly (A) tail. The cap and poly (A) additions are separated from
the mRNA coding region by anking noncoding regions and start and stop
codons (Figure 16.15).
A set of proteins called eukaryotic initiation factors, or eIFs, is responsible for assembling the ribosomal 40S and 60S subunits at the mRNA molecule. These proteins are listed in Table 16.5. Translation initiation begins
at the 5 end of the mRNA. An initiator transfer RNA (tRNA) is a tRNA
molecule loaded with a methionine (Met), the amino acid encoded by the
AUG start codon. For translation to begin, the Met-tRNA initiator and the
small and large ribosomal subunits must be recruited and positioned at the
AUG start site of the mRNA. This occurs in a staged manner as shown in
Figure 16.16. In the rst main step the loaded tRNA molecule and small
ribosomal subunit associate. The Met-tRNA molecule is guided to its
correct position by the initiation factor eIF2 (eukaryotic initiation factor 2)
that forms a complex with the Met-tRNA and a GTP molecule. This terti-

Figure 16.15. Organization of the mature mRNA molecule: The coding region is
anked by a pair of noncoding regions, a 5 end cap, and a 3 end polyadenylate tail.

Table 16.5. Vertebrate initiation factors.

Initiator
factor

Molecular mass
(kDa)

eIF1A
eIF2
eIF2B
eIF3
eIF4F

17
126
277
330
291

eIF4B
eIF5

69
45

Function
Promotes Met-tRNA, 40S, mRNA binding
GTP binding protein, guides Met-tRNA onto 40S
GEF for eIF2
Binds to 40S, prevents 60S binding
Complex of eIF4E, eIF4G, and eIF4A; eIF4E
binds to the cap; eIF4G interacts with eIF3 to
augment binding of the preinitiation complex;
eIF4A is an RNA-dependent ATPase and an
RNA helicase
Facilitates ribosome-mRNA binding
Stimulates hydrolysis of GTP associated with eIF2

406

16. Gene Regulation in Eukaryotes

Figure 16.16. Assembly of the ribosomal 40S and 60S subunits at the mRNA molecule: At the end of the process the 40S subunit, 60S subunit, and Met-tRNA are
positioned at the AUG start codon. This positioning is guided by the eIFs with the
eIF4E subunit gripping the cap and the eIF4G subunit serving as a scaffold.

ary complex associates with the small (40S) ribosomal subunit to form a
43S preinitiation complex.
Next, the preinitiation complex attaches to the mRNA near its 5 end. An
initiation factor complex, eIF4F, binds to the m7GpppN cap and prepares the
way for the attachment of the preinitiation complex to the mRNA. Upon
attachment the preinitiation complex scans in the 5 to 3 direction for the start
AUG codon and halts upon arrival.Another initiation factor,eIF5,stimulates
the hydrolysis by eIF2 of GTP to GDP, resulting in the dissociation of the initiating factors from the assemblage leading to the attachment of the large
(60S) ribosomal subunit, and translation elongation can begin.

16.16 eIF2 Interfaces Upstream Regulatory Signals and

the Ribosomal Machinery
The translation initiation factors have a crucial role in guiding the 40S and
60S ribosomal subunits to the start codon. As a result, if the translation initiation factors are prevented from carrying out their tasks, translation will
cease. Prevention can be accomplished by phosphorylating certain serine

16.17 Critical Control Points for Protein Synthesis

407

residues on eIF2 and eIF2B, thereby placing translation under the rm

control of upstream signals conveyed by protein kinases.
The eIF2 translation initiation factor is a GTPase. As noted in Table 16.5,
eIF2B functions as a GEF for eIF2, and eIF5 operates as its GAP. The eIF2
initiation factor contains several subunits. Its alpha subunit can be phosphorylated on Ser51 by a number of upstream kinases. One of the kinases
that phosphorylates the subunit is protein kinase R (PKR). As discussed
earlier in Chapter 9, interferon signaling acts in part through this kinase to
halt protein synthesis when a virus infection is detected (through the presence of dsRNAs). Among the other kinases that target eIF2 are GCN2, a
kinase that phosphorylates eIF2 in response to poor nutrient conditions,
and PERK, a kinase that phosphorylates eIF2 in response to ER stresses.
The eIF2 initiation factor is deactivated by phosphorylation and activated
by dephosphorylation. When eIF2 is phosphorylated it binds eIF2B tightly,
thereby sequestering both and preventing their translational activities. The
translation initiation factor eIF2B catalyzes the conversion of GDP back
to GTP, a step that is required for continued cycling and reformation of
Met-tRNA-GTP-eIF2 complexes. It, too, can be phosphorylated, by GSK3
among others, to prevent its activities. In response to growth and survival
signals, Akt is activated and when this kinase phosphorylates GSK3, eIF2B
is freed of the inhibitory activities of GSK3.

16.17 Critical Control Points for Protein Synthesis

The eIF2 and eIF2B proteins are not the only targets of upstream regulatory kinases and phosphatases. The cap-binding, translation initiation factor
eIF4E is phosphorylated at Ser209 in response to growth factor, hormonal
and mitogenic signaling, and it is dephosphorylated in response to heat
shock. Phosphorylation enhances the ability of eIF4E to bind to eIF4G
and/or the cap and thus promotes translation. The extent to which eIF4Es
are phosphorylated matches the overall translation level at different stages
of the cell cycle. These levels are low during G0 phase, increase through G1
and S phases, and then rapidly drop during M phase. Several MAP kinase
pathways are involved in phosphorylating eIF4E. These include the RasRaf-Mek-Erk that relays growth/mitogenic signals, and the p38 MAP kinase
that relays stress signals. Both of these pathways activate Mnk1, a kinase
that integrates MAP kinase signals, and in response phosphorylates eIF4E
on Ser209.
The cap-binding protein eIF4E is the target of negative regulation by a
family of small 10 to 12 kDa-eIF4E binding proteins (4E-BPs).These regulators compete with eIF4G for binding to eIF4E. The ability of the 4E-BPs
to compete for binding is dependent on their phosphorylation status. Phosphorylation decreases the binding afnity of the 4E-BPs for eIF4E and
leads to dissociation.The best characterized member of the family is 4E-BP1

408

16. Gene Regulation in Eukaryotes

Figure 16.17. Structure of the human 4E-BP1 protein: The cap-binding protein
eIF4E, along with its 4E-BPs, is a critical control point for protein synthesis. The
seven identied phosphorylation sites are indicated along with the N-terminal
RAIP (Arg-Ala-Ile-Pro) motif, the C-terminal TOS (Phe-Glu-Met-Asp-Ile) motif
and the eIF4E binding site. The RAIP and TOS motifs are essential for proper phosphorylation by upstream kinases.

(Figure 16.17).The regulation of eIF4E by 4E-BP1 was discussed in Chapter

6. Recall from that earlier discussion that, under good growth/nutrient conditions, target of rapamycin (TOR) proteins phosphorylate and thus activate a
protein called Tap42 that ensures that 4E-BP1 does not bind and inactivate
eIF4E (Figure 6.10). Under poor growth conditions Tap42 is not activated by
TOR and 4E-BP1 is free to inhibit the activity of the translation initiation
factor.The 4E-BP proteins are phosphorylated on a number of serine/threonine residues by several kinases in a hierarchical fashion. For example, they
are phosphorylated on Thr37 and Thr46 by Akt activated in the PI3K
pathway, and once primed by Akt they are further phosphorylated by hTOR
on Ser65,Thr70 and Ser83.These stratied phosphorylation events induce the
dissociation of 4E-BP1 from eIF4E.

References and Further Reading

Promoter Architecture
Bell AC, West AG, and Felsenfeld G [2001]. Insulators and boundaries: Versatile
regulatory elements in the eukaryotic genome. Science, 291: 447450.
Blackwood EM, and Kadonaga JT [1998]. Going the distance: A current view of
enhancer action. Science, 281: 6063.
Calhoun VC, Stathopoulos A, and Levine M [2002]. Promoter-proximal tethering
elements regulate enhancer-promoter specicity in the Drosophila antennapedia
complex. Proc. Natl. Acad. Sci. USA, 99: 92439247.

Structure of RNAP II
Cramer P, Bushnell DA, and Kornberg RD [2001]. Structural basis of transcription:
RNA polymerase II at 2.8 ngstrom resolution. Science, 292: 18631876.
Gnatt AL, et al. [2001]. Structural basis of transcription: An RNA Polymerase II
elongation complex at 3.3 resolution. Science, 292: 18761882.
Woychik NA, and Hampsey M [2002]. The RNA polymerase II machinery: Structure illuminates function. Cell, 108: 453463.

References and Further Reading

409

Chromatin-Modifying Enzymes
Boyer LA, et al. [2000]. Functional delineation of three groups of the ATP-dependent
family of chromatin remodeling enzymes. J. Biol. Chem., 275: 1886418870.
Eissenberg JC [2001]. Molecular biology of the chromo domain: An ancient chromatin module comes of age. Gene, 275: 1929.
Horn PJ, and Peterson CL [2002]. Chromatin higher order folding: Wrapping up
transcription. Science, 297: 18241827.
Narlikar GJ, Fan HY, and Kingston RE [2002]. Cooperation between complexes that
regulate chromatin structure and transcription. Cell, 108: 475487.
Vignali M [2000]. ATP-dependent chromatin-remodeling complexes. Mol. Cell Biol.,
20: 18991910.
Zeng L, and Zhou MM [2002]. Bromodomain: An acetyl-lysine binding domain.
FEBS Lett., 513: 124128.

Mediator Complexes
Asturias FJ, et al. [1999]. Conserved structures of Mediator and RNA polymerase
holoenzyme. Science, 283: 985987.
Boube M, et al. [2002]. Evidence for a mediator of RNA polymerase II transcriptional regulation conserved from yeast to man. Cell, 110: 143151.
Dotson MR, et al. [2000]. Structural organization of the yeast and mammalian mediator complexes. Proc. Natl. Acad. Sci. USA, 97: 1430714310.
Lewis BA, and Reinberg D [2003]. The Mediator coactivator complex: Functional
and physical roles in transcriptional regulation J. Cell Sci., 116: 36673675.
Malik S, and Roeder RG [2000]. Transcriptional regulation through Mediator-like
coactivators in yeast and metazoan cells. Trends Biochem. Sci., 25: 277283.
Taatjes DJ, et al. [2002]. Structure, function, and activator-induced conformations of
the CRSP coactivator. Science, 295: 10581062.

Theory
Levine M, and Tjian R [2003]. Transcriptional regulation and animal diversity.
Nature, 424: 147151.
Orphanides G, and Reinberg D [2002]. A unied theory of gene expression. Cell,
108: 439451.

Alternative Splicing
Cceres JF, and Kornblihtt AR [2002]. Alternative splicing: Multiple control mechanisms and involvement in human disease. Trends Genet., 18: 186193.
Faustino NA, and Cooper TA [2003]. Pre-mRNA splicing and human disease. Genes
Dev., 17: 419437.
Graveley BR [2000]. Sorting out the complexity of SR protein function. RNA, 6:
11971211.
Krecic AM, and Swanson MS [1999]. hnRNP complexes: Composition, structure,
and function. Curr. Opin. Cell Biology, 11: 363371.
Maniatis T, and Reed R [2002]. An extensive network of coupling among gene
expression machines. Nature, 416: 499506.
Smith CWJ, and Valcrcel J [2000]. Alternative pre-mRNA splicing: The logic of
combinatorial control. Trends Biochem Sci., 25: 381388.

410

16. Gene Regulation in Eukaryotes

Staley JP, and Guthrie C [1998]. Mechanical devices of the spliceosome: Motors,
clocks, springs, and things. Cell, 92: 315326.

Regulation of Translation Initiation

Dever TE [2002]. Gene-specic regulation by general translation factors. Cell, 108:
545586.
Gingras AC, Raught B, and Sonnenberg N [2001]. Regulation of translation initiation by FRAP/mTOR. Genes Dev., 15: 807826.
Jacinto E, and Hall MN [2003]. TOR signaling in bugs, brain and brawn. Nature Rev.
Mol. Cell Biol., 4: 117126.
Kosak M [1999]. Initiation of translation in prokaryotes and eukaryotes. Gene, 234:
187208.
Pain VM [1996]. Initiation of protein synthesis in eukaryotic cells. Eur. J. Biochem.,
236: 747771.
Proud CG [2002]. Regulation of mammalian translation factors by nutrients. Eur. J.
Biochem., 269: 53385349.
Wang XM, et al. [2003]. The C terminus of initiation factor 4E-binding protein 1
contains multiple regulatory features that inuence its function and phosphorylation. Mol. Cell. Biol., 23: 15461557.

Problems
16.1 Transcription factors and cofactors can promote or inhibit transcription in several ways. List some of these ways. For each way give the
location/site of action along the DNA, the specic interaction that
mediates the response, and the effect of the binding interaction on
transcription.
16.2 The translation initiation factor eIF2 is a GTPase. Several different
modes of action of GTPases were presented in Chapter 13. For
example, Ras acted one way as a switch while Cdc42 cycles continuously, and thus operates in a different manner from Ras. Which of the
several GTPase modes of operation does eIF2 follow?
16.3 One of the early events in apoptosis is the shutting down of the translation machinery. Give some examples of how the apoptosis regulatory machinery discussed in the last chapter might signal and regulate
protein synthesis through interactions of apoptosis signaling/regulatory proteins with the translation initiation factors.

17
Cell Regulation in Bacteria

Bacteria are not only highly efcient organisms, but they are also the unseen
majority wherever they exist. Bacteria are found in large numbers in the
open oceans (1.2 1029), in soils (2.6 1029), in oceanic subsurfaces (3.5
1030), and in terrestrial subsurfaces (~1 1030). Each human body hosts
some 1013 bacteria. One of the locales of high concentration is the gut. From
500 to 1000 different species of bacteria populate the human intestinal tract.
The greatest concentration of bacteria is in the colon where there are about
1011 bacteria per gram of bulk material. This number is far greater than the
amount of bacteria living on the surface of the skin. From 1000 to 10,000
bacteria live on each square centimeter of skin. These numbers yield a total
skin population of about 300,000,000 bacteria.
Most of the bacteria that reside in the gastrointestinal (GI) tract are
anaerobic, harmless, and even benecial. One of the species of resident bacteria is the Gram-negative bacterium Bacteroides thetaiontaomicron. This
bacterium acquires and hydrolyzes dietary polysaccharides that would
otherwise be indigestible. Bacteroides thetaiontaomicron possesses a large
number of genes that encode signaling proteins and gene regulators allowing the bacteria to communicate with and regulate their local environments.
A symbiotic relationship is established between bacterial ora and their
host environment. The bacteria stimulate vascularization and development
of intestinal villi and stimulate epithelial cells to secrete bacterial nutrients.
The bacteria generate simplied carbohydrates, amino acids, and vitamins.
Not all bacteria found in the GI tract are harmless. One of the occupants
is the Gram-positive bacterium Enterococcus faecalis. This is an opportunistic pathogen that occupies a variety of ecological niches including soil,
sewage, and water. Inside the GI tract, this bacterium causes uninary tract
infections and is highly resistant to vancomycin, the preferred antibiotic
for treating gram-positive bacterial infections. The propensity for this bacterium to harm its host is tied to genetic material acquired through horizontal gene transfer (HGT) from other bacteria. More than 25% of its
genome consists of DNA acquired from other species.

411

412

17. Cell Regulation in Bacteria

17.1 Cell Regulation in Bacteria Occurs Primarily at

Transcription Level
Messenger RNAs are rapidly turned over in bacteria. Their half-lives are
far shorter than eukaryotic mRNAs. Whereas eukaryotic mRNAs have
half-lives on the order of an hour, bacterial mRNA are degraded within a
few minutes. Since the steady state concentration of any molecule is directly
proportional to its half-life, the rapid degradation of selected molecules
ensures that the bacterium can respond rapidly to changing environmental
conditions. The rapid degradation of proteins is coupled to an equally rapid
capability for synthesizing new proteins.
The machinery for regulating gene expression in bacteria is highly
streamlined compared to that for eukaryotes. Bacterial DNA is not
wrapped in histones, and chromatin-modifying enzymes are not required.
Their genes do not possess introns, and a splicing stage is not needed. There
are few noncoding regions, and the fraction of DNA that codes for proteins
ranges from 87 to 95%. Transcription and translation are contiguous, and
the two processes take place at the same time in the same place. In many
species extra-chromosomal DNA is present. It is organized into one or
more small circular plasmids containing collections of genes that perform
specialized functions.
Bacteria such as Streptomyces coelicolor, Escherichia coli, Bacillus subtilis and Caulobacter crescentus possess genomes comparable in size to the
unicellular yeast genome, and some, for example, Calothrix, have genomes
approaching Drosophila in size. These bacterial species possess a sophisticated control layer that enables them to alter their metabolism, morphology, and lifestyle. Some can execute developmental programs that produce
differentiated progeny and make possible colonial behavior. The rst part
of this chapter is devoted to an examination of gene regulation in bacteria,
the counterpart to the discussions in the last chapter dealing with gene
regulation in eukaryotes. The second part of this chapter deals with the
more sophisticated forms of cell regulation in bacteria. Special attention is
given to how some of these modes help cause disease in humans while other
modes promote benecial symbiotic relationships.

17.2 Transcription Is Initiated by RNAP Holoenzymes

Transcription in bacteria is carried out by RNA polymerase (RNAP)
holoenzymes. Whereas eukaryotes possess three kinds of RNA polymerases, bacteria utilize a single kind. The bacterial RNAP molecule is
composed of a large and rather nonspecic core RNAP unit and a small
regulatory and targeting subunit, the sigma factor (Table 17.1).
The core unit is composed of four subunitsa pair of alpha subunits, and
two larger subunits, b and b, responsible for the enzymatic activities of the

17.2 Transcription Is Initiated by RNAP Holoenzymes

413

Table 17.1. Components of the E. coli RNA polymerase holoenzyme.

Subunit
a
b, b
s

Molecular weight (kDa)

Function

36.5
150.6, 155.2
20 to 70

Targeting and assembly

Catalytic activities
Promoter recognition

Figure 17.1. Organization of the bacterial promoter: (a) The primary DNA binding
regions are shown together with the transcription start site, InR. (b) Arrangement
of the subunits of RNA polymerase together with a catabolite activator protein
(CAP) transcriptional activator dimer.

polymerase. The alpha subunit contains two domains connected by a exible hinge. As shown in Figure 17.1b, each alpha subunit has an N-terminal
domain (aNTD) that interfaces the subunit with the beta subunits, and a
C-terminal domain (aCTD) that mediates interactions with the DNA promoter and with regulatory proteins. The core, written symbolically as a2bb,
has a total mass of about 380 kDa. There are several different kinds of sigma
factors, and their masses vary from 20 to 70 kDa.The sigma factors are DNA
sequence-specic binding proteins that recognize and bind the two specic
promoter sites listed in Table 17.2. The bacterial transcription-competent
RNAP holoenzyme is thus of the form a2bbs.
The association of a sigma factor and the core RNAP unit is transient.
Shortly after initiation of transcription, the sigma factor dissociates from
the core unit, which proceeds with transcript elongation. This dissociation
makes possible the attachment of a different sigma factor subsequent to
termination of the transcription event. If alternative sigma factors are
present in sufcient numbers, they will out-compete the initial sigma factor
for binding to the core unit. The core is thus reprogrammed to bind to an
alternative promoter site and transcribe a different set of genes.

414

17. Cell Regulation in Bacteria

Table 17.2. Bacterial s70 promoter architecture.
Regulatory region
-10 box
-35 box
UP element

Consensus sequence

Binding

5-TATAAT-3
5-TTGACA-3
AT-rich tracts

s
s, TFs
aCTD, TFs

17.3 Sigma Factors Bind to Regulatory Sequences

in Promoters
DNA promoters that bind bacterial RNAP subunits and transcription
factors contain a pair of core regulatory sequences separated by a spacer
region. The regulatory sequence nearest the transcription (+1) start site is
the -10 box, and it is centered 10 bp immediately upstream of that site. This
region is separated by a spacer from the -35 box, the second core regulatory region centered 35 bp upstream. The amount of agreement between
-10 and -35 box sequences and the consensus sequences has a regulatory
role. Genes are strongly expressed at sites where the agreement is good
and are weakly transcribed at sites where the agreement is not optimal. In
many promoters, there is a third regulatory site located further upstream.
Referred to as the upstream (UP) sequence its precise location varies from
promoter to promoter (Figure 17.1a).
Different sigma factors recognize different promoter sequences. The
consensus sequences listed in the second column of Table 17.2 are recognized
only by primary sigma factor (i.e., by s70). Each of the groups of alternative sigma factors listed in Table 17.3 has its own characteristic -10 and
-35 consensus sequences.There are two families of sigma factors, s70 and s54.
Members of the s54 differ from those in the s70 in several ways. One of the
ways they differ is that instead of recognizing sequences -10 and -35 bp
upstream of the start site they recognize sequences centered -12 and
-24 bp upstream.

17.4 Bacteria Utilize Sigma Factors to Make Major

Changes in Gene Expression
Bacteria such as E. coli and B. subtilis express several kinds of sigma factors.
The primary sigma factors, s70 in E. coli and sA in B. subtilis, regulate genes
active all the time that perform housekeeping functions and support normal
vegetative growth. This latter term denotes situations where nutrients
are plentiful. Rapid cell growth and cell division typically produces
exponential increases in the bacterial population. A second sigma factor
is utilized in situations where exponential growth is not supported. The

17.4 Bacteria Utilize Sigma Factors

415

Table 17.3. Sigma factors of E. coli and B. subtilis.

Sigma factor
Primary

Heat shock

Flagellar and chemotactic

Sporulation

Extracytoplasmic function (ECF)

s54 family

Bacteria and designation

70

E. coli s , B. subtilis s
E. coli sS (s38)
E. coli sH (s32)
E. coli sE (s24)
B. subtilis sB (s37)
E. coli sF (s28),
B. subtilis sD
B. subtilis sH

B. subtilis sE
B. subtilis sF
B. subtilis sG
B. subtilis sK
E. coli sFecI (s19),
B. subtilis sV, sZ

E. coli sN (s54),
B. subtilis sL

Control function
Housekeeping, vegetative
growth
Stationary phase
Stress response genes
Stress response genes
Stress response genes
Genes for motility and
chemotaxis
Postexponential,
competence and early
sporulation genes
Early mother cell genes
Early forespore genes
Late forespore genes
Late mother cell genes
Genes for iron uptake,
antibiotic production,
virulence factors, outer
membrane proteins
Nitrogen xation, genes for
degradative enzymes

bacteria modify many of their internal activities in these cases using the
stationary phase sigma factor sS. This sigma factor stimulates physiological changes needed in the presence of nutrient deprivation and toxic
chemicals.
Many stressful conditions have to be dealt with by bacteria. Some conditions, like temperature extremes, osmotic stresses, and pH effects, are
related to the chemical and physical environment. These stresses are dealt
with by stress response sigma factors. High temperature is a representative
example of a stressful condition. When high temperatures are encountered
bacteria such as E. coli and B. subtilis use sH and sB to trigger the synthesis of heat shock proteins. These stress response proteins refold proteins
that have become partially unfolded due to thermal effects, and if the proteins cannot be refolded tag them for destruction. Sporulation and extracytoplasmic function (ECF) sigma factors are two more families of sigma
factors involved in controlling specic stress responses. The sporulation
sigma factors control the development of spores in B. subtilis in response
to starvation conditions. The ECF sigma factors control a diverse set of
responses (for example, iron uptake, heavy metal resistance, antibiotic production, and induction of virulence factors) mostly involving the remodeling of the transport machinery located in bacterial membranes. Finally,
some members of the s54 family are involved in control of nitrogen
metabolism while others are involved in regulating stress responses.

416

17. Cell Regulation in Bacteria

17.5 Mechanism of Bacterial Transcription Factors

Most bacterial transcription factors contain a helix-turn-helix (HTH) motif
consisting of about 20 amino acid residues that binds DNA and is part of
a larger DNA binding domain. The DNA binding domain often has additional sites that contact DNA and aid in recognition. The transcription
factors bind to regulatory sequences in the promoters as dimers or as higher
order oligomers. Besides contacting the DNA, transcription factors also
contact one or more subunits of the RNAP holozenzme. Most often they
contact s and/or aCTD. However, some contact b, b, or aNTD. Promoters
often contain sites for attachment of multiple transcription factors, and by
this means integrate a variety of environmental signals into the transcription decision.
Bacterial TFs can be grouped into families according to characteristic
DNA sequences present in family members and related physiological functions. Members of more than a dozen families of transcription factors are
present in environmental bacteria such as Escherichia coli, Bacillus subtilis,
Vibrio cholerae, and Pseudomonas aeruginosa. A short list of the most
prevalent families present in these organisms is presented in Table 17.4. As
discussed in Chapter 1, the size of the control layer contracts rapidly when
the genome sizes are reduced. Minimalist bacteria such as Mycoplasma
genitalium have only a few transcription factors, or none at all. Social,
differentiating bacteria, which will be discussed later in this chapter, possess
genomes that are even larger than those of the environmental bacteria.
These organisms may well possess families of transcription factors needed
for differentiation that are absent in Table 17.4.
Bacterial transcription factors are HTH DNA binding proteins that integrate signals, and ne tune and regulate transcription in response to changes

Table 17.4. Transcription factors, their actions, and typical cellular roles.
Family
AraC/XylS
ArsR
AsnC
Crp
DeoR
GntR
LacI/GalR
LuxR
LysR
MarR
MerR/SoxR
TetR/AcrR

Actions

Cellular role

Activator
Repressor
Dual
Dual
Repressor
Repressor
Repressor
Activator
Dual
Repressor
Dual
Repressor

Virulence, sugar metabolism

Heavy metal resistance
Amino acid biosynthesis
Global responses
Sugar metabolism
Carbon metabolism
Carbon uptake
Biosynthesis, glycerol metabolism
Amino acid biosynthesis
Antibiotic resistance
Heavy metal resistance
Antibiotic resistance

17.6 Many TFs Function as Response Regulators

417

in their environment. First and foremost, the transcription factors regulate

metabolic activities. Sugar metabolism, carbon uptake and metabolism, and
amino acid biosynthesis are heavily represented in the list of cellular roles
in Table 17.4. The second main function of the transcription factors is to
regulate physiological responses to potentially harmful substances in the
environment. Noteworthy in the table are transcription factors for arsenic
resistance (ArsR), for mercury, copper, zinc, and cobalt resistance (MerR),
and for tetracycline and other antibiotics resistance (TetR).

17.6 Many TFs Function as Response Regulators

Many transcription factors function as response regulators in two-component
signal transduction systems. Two-component signal transduction systems
are the main devices used by bacteria to sense their environments. The
bacterial chemotactic system discussed in Chapter 6 is the prototype example
of a two-component system. It is used to sense nutrients and noxious
compounds in the local environment and relay these signals to a agellar
motor so that a bacterium can swim towards nutrients and away from harmful
substances. More commonly, two-component systems are used to relay
environmental information directly to sites on chromosomes and plasmids
that regulate transcription.
Recall that two-component systems consist of a sensor module and a
response regulator module. In two-component systems that regulate transcription the sensor module is a single transmembrane protein and the
response regulator is a single cytoplasmic protein. The sensor, a histidine
protein kinase, contains an N-terminal input unit and a C-terminal transmitter unit. The input unit responds to environmental stimuli by stimulating the autophosphorylation of the transmitter on a specic histidine
residue. The high energy phosphoryl group is then transferred to the second
component of the system, the response regulator. The response regulator
contains an N-terminal regulatory domain and a C-terminal DNA binding
domain. The N-terminal domain catalyzes the transfer of the phosphoryl
group to a conserved aspartate residue in its own domain. The C-terminal
output unit possesses an HTH DNA-binding domain that functions as a
transcription factor. The output unit is activated in response to phosphorylation state-dependent conformational changes in the regulatory domain.
The second column of the Table 17.4 describes the regulatory function
(action) of the transcription factors. Some TFs are activators: They are
positive regulators that stimulate transcription at higher frequencies and/
or for greater durations. Other transcription factors are repressors: They
are negative regulators that block or mechanically inhibit transcription.
Still other transcription factors can act either as activators or as repressors:
They are dual regulators and their specic actions will vary with cellular
context.

418

17. Cell Regulation in Bacteria

Transcription activators bind to sites upstream of the -35 box. Some bind
in the vicinity of the UP element while others bind to sites further upstream.
Many of these transcription activators exert their inuences by binding to
aCTD. Because of its exibility, aCTD can bind to activators over distances
from -35 to -100 bp.Transcription factors that bind to distant upstream sites
are often referred to as transcription enhancers. The DNA molecule itself
has a certain intrinsic exibility. Sequences such as A-T tracts are especially
bendable. This exibility plus bending brought on by protein binding
enables activators to come into physical contact with subunits of the
holoenzyme. Transcription repressors bind to sites close to the transcription
start site. These proteins prevent the holoenzyme from initiating transcription either by sterically inhibiting access or by binding the holoenzyme so
tightly that it cannot release and start transcription.

17.7 Organization of Protein-Encoding Regions and

Their Regulatory Sequences
Bacterial DNA is organized into operons. Each operon contains DNA
sequences that encode proteins and the upstream sequences for attachment
of the RNA holoenzyme and regulatory proteins (Figure 17.2). Regulatory
information is conveyed in several ways: It is imparted through the promoter recognition sequences that provide attachment sites for specic
sigma factors: it is conveyed by degree of agreement between the promoter
sequences and the consensus sequence, and by the length of the intervening spacer sequences, both inuencing the strength of the transcription; and
it is supplied by the presence of binding sites for transcription activators
and repressors.
In contrast to transcription in eukaryotes, several genes can be transcribed from each promoter into an mRNA molecule. The protein-coding
sequences follow one another and the protein-coding region terminates
with a characteristic stop sequence that mediates the release of the polymerase from the DNA. Multiple gene transcription makes it possible for a
promoter to regulate its own activity by implementing a simple negative
feedback loop. Two kinds of proteinsstructural and regulatorycan be

Figure 17.2. Bacterial operon: Shown are ve genes, labeled A through E, controlled by a single gene regulatory region partitioned into repressor (O), core promoter (P), and enhancer (E) sections.

17.8 The Lac Operon Helps Control Metabolism in E. coli

419

expressed at the same time. In negative feedback, regulatory proteins shut

down transcription from their own promoters, thus providing a mechanism
for automatically terminating transcription an appropriate time after the
onset of transcription as determined by the threshold concentration. In
many instances the regulatory proteins control the assembly of the cotranscribed structural proteins into membrane-bound structures.
Cassettes have the following properties. They are sets of operons that
encode proteins performing a set of related functions. They are usually
found on plasmids and enable the bacteria to respond to hostile conditions.
The operons are positioned in the vicinity of one another, and because of
their location on plasmids can be transferred from one bacterium to
another. Some cassettes encode proteins involved in either antibiotic
production or antibiotic resistance. Others, called virulence cassettes or
pathogenicity islands, encode virulence factors such as toxins and proteins
required for erection of morphological structures involved in the toxins
secretion and delivery to targets.
Regulons are sets of operons located at well-separated loci along the
chromosome; they encode proteins involved in a common physiological
response. Many regulons are organized by sigma factors. By binding to multiple sites along the chromosome they can either upregulate or downregulate 100 or more proteins. Examples of sigma factor associated regulons
include the E. coli sS stationary phase regulon, the E. coli s32 heat shock
regulon, and the B. subtilis sB stress response regulon.

17.8 The Lac Operon Helps Control Metabolism in

E. coli
Bacteria optimize their metabolism to take maximal advantage of their
environments. Bacteria such as E. coli possess a broad range of metabolic
strategies. They are able to metabolize a variety of sugars including glucose,
lactose, and maltose, and make best use of the nutrients available in their
environment. E. coli is the most studied bacteria and serves as a model
system for how these organisms rapidly adjust their metabolism to environmental conditions. The genes involved in regulating whether glucose or
lactose is metabolized form the lac operon. They are listed in Table 17.5,
and their arrangement in the lac operon is illustrated in Figure 17.3.
Table 17.5. Genes belonging to the E. coli lac operon.
Gene

Function

LacI

Repressor

LacZ
LacY
LacA

b-galactosidase
b-galactoside permease
b-galactoside transacetylase

Description
Binds to transcription start site and part of the
promoter, inhibiting RNAP
Hydrolyzes lactose to glucose plus galactose
Transports lactose into the cell
Nonessential enzyme

420

17. Cell Regulation in Bacteria

Figure 17.3. Organization of the lac operon: (a) Constitutive expression of the lac
repressor protein (LacI) blocks transcription from the lac promoter when it binds
to the operator. (b) Binding of LacI is blocked by allolactose allowing cAMP-bound
CAP proteins to bind and stimulate transcription by the NRA polymerase (RNAP).

Glucose is the favored sugar and if it is plentiful the expression of proteins needed to metabolize alternative nutrients is inhibited. The LacI
repressor is constitutively expressed and under plentiful glucose conditions
binds downstream of the -10 and -35 sites and blocks RNAP from initiating transcription. When lactose is present in the environment, some of it
will enter the cell and, in the form allolactose, will bind to the LacI repressor. The lactose binding induces structural changes in LacI and the repressor is no longer able to block RNAP.
The lac promoter is a weak one. The sequences at the -10 and -35 sites
are not optimal for RNAP binding and the polymerase needs the binding
activities of an activator, the catabolite activator protein (CAP), to efciently
transcribe the structural genes in the operon. CAP forms a complex with
cAMP. When glucose levels are high the cAMP levels are low and the
complex does not form. When glucose is absent, the cAMP levels are high
enough for the CAP/cAMP complexes to form. The complexes bind at a
site upstream of the RNAP binding site (CAP by itself does not), where
they help RNAP to bind and start transcribing the genes. In sum, under the
combined actions of the LacI (negative regulation) and CAP (positive
regulation), the lac genes are transcribed when lactose is present and
glucose is absent (Figure 17.3).
The structure of LacI is well suited for controlled interactions with DNA.
As shown in Figure 17.4, two of its domains, the so-called Headpiece and

17.9 Flagellar Motors Are Erected in Several Stages

421

Figure 17.4. Crystal structure of the Lac repressor bound to a DNA operator determined using X-ray crystallography: Several domains of the Lac repressor are
involved in the interactions. The headpiece domain binds to the DNA in the major
groove while the Hinge helix binds DNA in the minor groove. The core domain is
shown broken down into NH2 and CO2 subdomains that mediate interactions with
inducers and repressors. One other domain, the tetramerization domain that mediates interactions with other LacI proteins, is not included in the gure, but can be
seen in other crystal structures. The gure was generated using Protein Explorer
with atomic coordinates deposited in the Protein Data Bank under accession code
1EFA.

Hinge helix domains, bind to the DNA in its major and minor grooves,
respectively. The core domain enables the repressor to be regulated by allolactose, which induces allosteric transitions that prevent the protein from
binding DNA.
Regulatory proteins such as CAP are able to bend DNA. This ability can
be seen in Figure 17.5 where a CAP dimer is shown bound to its regulatory
DNA regions. The bending of the DNA is quite pronounced, and by this
means transcription factors and coactivators can make transcription either
easier or harder.

17.9 Flagellar Motors Are Erected in Several Stages

Bacteria can alter their morphology, either building up or disassembling
internal structures, such as gas vesicles, and membrane wall-attached structures, such as agellar motors, pili, and virulence cassettes. It takes more
than 50 genes to assemble a agellar motor system. In species such as

422

17. Cell Regulation in Bacteria

Figure 17.5. Crystal structure of DNA bending by a CAP dimer determined using
X-ray crystallography: The gure was generated using Protein Explorer with atomic
coordinates deposited in the Protein Data Bank under accession code 1RUN.

Escherichia coli and Salmonella enterica, these genes belong to a large agellar motor regulon. The genes in this regulon are organized temporally
into three classesearly, middle, and late (Figure 17.6).
A variety of cellular and environmental conditions are factored into the
decision to begin biosysnthesis of the agellar motor. The corresponding
signals impinge on a master control point, namely the promoter for the
hCD master operon, from which the early genes hC and hD are transcribed. The transcriptional activation complex then regulates the transcription of middle genes. These genes encode structural proteins and
assembly regulators for the basal body and hook of the agella. The timing
of events is carefully controlled during the assembly process. Two regulatory proteins are expressed during the middle phase. One of these, FlgM,
binds to and inhibits the second, FliA (s28). The inhibition on s28 by FlgM
is relieved after the middle stage components of the agellar motor have
been assembled. The s28 regulator then initiates transcription of the late
genes including those for the chemotactic receptors Tar, Tsr and Aer, and
the suite of Che proteins discussed in Chapter 6.

17.10 Under Starvation Conditions, B. subtilis

Undergoes Sporulation
Under starvation conditions, B. subtilis undergoes a program of asymmetric cell division resulting in two daughters, a large mother cell and a small
prespore that further develops into a spore that, in turn, aids in long term
survival. When nutrients are plentiful B. subtilis enters a vegetative state
where it multiplies exponentially. The vegetative growth sigma factor sA
governs this state. Under starvation conditions, B. subtilis alters its meta-

17.10 Under Starvation Conditions, B. subtilis Undergoes Sporulation

423

Figure 17.6. Assembly of the agella motor: Environmental signals arriving at the
master controller stimulate transcription of early (Class I) genes. FlhC and FlhD
jointly stimulate transcription of middle genes from middle (Class II) promoters.
FliA (s28) and FlgM (anti-s28) regulate transcription from late (Class III) promoters. Transcription of late genes is inhibited until completion of the basal body
and hook assembly signied in the gure by the sending of a checkpoint signal.

bolic and reproductive strategy. It leaves the vegetative state and converts
itself into an inactive spore that can survive for long periods of time. As
indicated in Table 17.3 ve sigma factors govern the transition from vegetative growth to spore. The rst to come into play is sH. This sigma factor
is followed in sequential fashion by sF, sE, sG, and nally sK. As the genes
selected by these sigma factors are expressed the bacterium ceases exponential growth. Its plasma membrane invaginates, partitioning the cell into
large and small compartments, each containing a chromosome generated
during the last round of DNA replication. Once the septum is completed
the large compartment, the mother cell, engulfs the small cell, the forespore, producing a cell within a cell. A series of coating stages follow where
the forespore and its chromosome are protected, and lastly the spore is
released through lysis of the mother cell; that is, the rupture of the plasma
membrane/cell wall and the dissolution of the outer cell.
A number of signals, integrated together, govern the decision whether
to undergo sporulation (Figure 17.7). These signals converge on the transcription factor Spo0A. This protein acts at the end of a phosphorelay
system involving kinases such as KinA/KinB, a phosphorelay protein Spo0B
and an upstream transmitter Spo0F. Signals sent through the phosphorelay,
and associated protein phosphatases such as RapA, RapB, RapE, and
Spo0E include metabolic information, DNA status, and cell density. These

424

17. Cell Regulation in Bacteria

Figure 17.7. Integration of sporulation signals: Environmental and intracellular

signals are integrated together into a phosphorelay consisting of at least two sensor
(input) kinases (KinA and KinB) a Spo0F transmitter, Spo0B intermediate, and
Spo0A response regulator, which functions as a transcription factor when phosphorylated. Negative regulatory signals are conveyed by several aspartyl phosphatasesRap family members dephosphorylate Spo0F and Spo0E dephosphorylates Spo0A.

signals inuence the phosphorylation status of Spo0A. The Spo0A transcription factor, along with KinA and Spo0F, is upregulated by sH. The
Spo0A operon contains the genes for sF and sE, and starts the expression
of spore and mother-specic genes.

17.11 Cell-Cycle Progression and Differentiation in

C. crescentus
C. crescentus is a gram-negative bacterium adept at survival in a variety of
aquatic environments. During every cell cycle it differentiates, producing as
one offspring a motile swarmer cell equipped with a single agellum at one
pole, and as the second offspring an immobile (sessile) stalked cell possessing a stalk rather than a agellum. While stalked cells are immediately
competent to undergo cell division, the swarmer is not. It must rst convert
itself into a stalked cell. The swarmer possesses a single agellum at one
pole, and this agellum is coupled to a chemotactic sensor system. During
G1 phase, the swarmer can move about seeking nutrients using its chemotactic system. At the beginning of S phase (Figure 17.8), the swarmer
degrades the agellum and chemotactic sensor system. A stalk develops at
the pole where the agellum was sited, and the cell elongates. The DNA is

17.11 Cell-Cycle Progression and Differentiation in C. crescentus

425

Figure 17.8. Asymmetric cell division in Caulobacter crescentus: A swarmer cell differentiates into a stalked cell, which then undergoes asymmetric cell division to
produce a swarmer cell and a stalked cell. CtrA gene expression suppresses DNA
replication. This master controller is shut down through regulated proteolysis at the
end of G1 phase and is started up again in the new swarmer cell near the end of S
phase.

replicated, the chromosomes move to opposite ends of the cell, and a new
agellum is formed at the other pole. In the next phase, G2, the cell divides
into a swarmer cell and a stalked cell.
The key element in the regulatory network that coordinates the morphological changes with the cell cycle progression is CtrA. This regulatory
protein is the counterpart to the Spo0A transcription factor discussed in
the last section. The CtrA master cell cycle controller controls a large
number of genes, in this case, about 25% of all the genes whose expression
varies in a cell cycle-dependent way in C. cresentus. Cellular localization,
proteolysis, and phosphorylation all have role in regulating the behavior of
CtrA. The controller is expressed in the swarmer, but not in the stalker. The
CtrA proteins that are present in the swarmer at the end of G1 phase
undergo proteolytic destruction and thus are absent in the S phase starker.
Pole proteins belonging to the agellum and chemotactic sensor are also
cleared by proteolysis. These and the subsequent regulatory processes are
facilitated by a number of two-component signal transduction systems that
localize to one or the other of the poles. One of these systems is DivJ/DivK.
This two-component system facilitates the localization of CtrA to the pole
that will produce a new agellum and upon cell division will belong to the
new swarmer. Another protein, the histidine kinase CckA phosphorylates
CtrA after DNA replication takes place, thereby activating CtrAs transcriptional activities. In response, CtrA regulates the transcription of genes
involved in agellar biogenesis, cell division, DNA methylation, and DNA

426

17. Cell Regulation in Bacteria

replication. In this way, localization, proteolysis, and phosphorylation

pattern the spatial and temporal actions of CtrA, enabling it to coordinate
morphogenic and cell cycle activities.

17.12 Antigenic Variation Counters Adaptive

Immune Responses
Bacteria can up- and downregulate expression of their genes fairly rapidly
and can move DNA about. One of the ways bacteria (and a number of singlecelled eukaryotes) exploit such capabilities is through antigenic variation,
the systematic alterations in antigens expressed on the cell surface.Antigenic
variation is used by a number of vector-borne bacterial and protozoal
pathogens to defeat the acquired immune response of vertebrates. The
vectors are typically arthropodstics,lice,ies,and mosquitoes.Well-studied
pathogens include several species of Borrelia responsible for relapsing fever,
African Trypanosomas, the protozoan causative agent of sleeping sickness,
and Plasmodium falciparum, the protozoan responsible for malaria.
As discussed in Chapter 9, the acquired (adaptive) immune response
takes several days to develop. It requires several steps: First there is antigen
recognition, then communication and convergence of leukocytes to the site
of infection, and nally clearance. In antigenic variation, the pathogen
utilizes the differential expression and switching between members of a
family of antigens among a population of clones. Multiple copies of the gene
encoding the antigen, each differing somewhat in its recognition epitope,
are maintained by the pathogen. By either differentially expressing different copies among different clones, or by switching from one copy to another
more rapidly than the response time of the immune system, the pathogens
evade recognition and simply wear out the immune system.
A variety of switching mechanisms are employed. Most are implemented
at the transcription level. One method is to silence a promoter at one site
and turn on expression at another. Alternatively, point mutations may be
used, or a gene from a silent site may replace another at an active site of
transcription through gene rearrangements. Still another method, one that
does not operate at the transcription level, is to differentially transport the
antigens to the surface. No matter what the mechanism, the result is the
same. The population of pathogens survives.

17.13 Bacteria Organize into Communities When

Nutrient Conditions Are Favorable
Bacteria talk to each other and share genetic material. Bacteria continually
secrete communication molecules that are sensed by other bacteria.The signaling informs the bacteria that others are present in their vicinity and

17.13 Bacteria Organize into Communities

427

Table 17.6. Bacteria and their collective behavior.

Bacterial species

Collective behavior

Gram-negative bacteria
Photobacterium scheri
Vibrio harveyi
Proteus mirabilis
Serratia liquefaciens
Myxococcus xanthus
Chondromyces crocatus
Pseudomonas aeruginosa
Escherichia coli

Luminescence
Luminescence
Swarming, migratory rafts
Swarming, migratory rafts
Fruiting body
Fruiting body
Antibiotic resistance, virulence factors
Virulence factors

Gram-positive bacteria
Staphylococcus aureus
Streptomyces coelicolor
Bacillus subtilis
Streptococcus pneumoniae

Virulence factors
Antibiotics, differentiation
Antibiotics, genetic competence
Genetic competence

enables them to sense their numbers. If the density of bacteria exceeds a

threshold for cooperative behavior then the bacteria express genes that
support that form of behavior. Any of a number of collective behaviors may
be exhibited, each depending on the species, its needs, and its environmental conditions. A sapling of these collective behaviors is presented in
Table 17.6.
When nutrient conditions are favorable so that migration is not necessary bacteria organize into highly structured communities. In forming these
associations the bacteria express sets of genes that differ from those utilized when free-living. The members of the bacterial communities differ in
morphology and physiology from their planktonic counterparts. In communities that are anchored to solid surfaces, the bacteria can develop elaborate three-dimensional structures. These colonies take on characteristics
usually associated with multicellular organisms. They exhibit simple forms
of differentiation and cellular specialization. The bacteria excrete molecules
that form a matrix, form channels that support the circulation of uids, and
share metabolic tasks. Some bacterial colonies are conical-shaped while
others are mushroom-shaped and grow to sizes that allow the naked eye to
observe them.
The rst bacteria discovered to form communities were the luminescent
marine organisms Photobacterium scheri and Vibrio harveyi. These bacteria were found to form symbiotic associations with several species of marine
sh and squids. The luminescent bacteria collect in the light organs of their
marine hosts. The concentrations of the bacteria are generally low in seawater, typically on the order of 100 cells per milliliter, well below any
threshold for luminescent behavior. However, the bacteria can reach high
concentrations, 1010 to 1011 cells/ml under favorable nutrient conditions in
the light organ. Once the threshold density is exceeded the bacteria collec-

428

17. Cell Regulation in Bacteria

tively express genes for luminencence. The relationship between bacteria

and animal host is a symbiotic one. The production of light by the bacteria
masks the host from prey residing below them by reducing the hosts
shadow against the sky, while the hosts provide nutrients for the bacteria.
Table 17.6 lists several prominent forms of communal behavior. One of
these is the production of antibiotics. These are natural byproducts of cellular metabolism. They are agents produced by microorganisms (fungi and
bacteria) that kill or inhibit other microorganisms. Fungi produce penicillin
and a closely related family of antibiotics, the cephalosporins. Streptomyces
produce tetracycline, streptomycin, and erythromycin. These natural
products interfere with protein synthesis and damage cellular membranes.
Bacillus subtilis produces bacitracin, an antibiotic often added to animal
feed.
Other widely utilized forms of coordinated behavior are the production
of virulence factors and genetic competence. Virulence factors (e.g., toxins)
are cellular products that enhance bacterial survival in hostile host
environments. Natural genetic competence is the ability of bacteria to
acquire large pieces of exogenous (extracellular) DNA from their local
microenvironment.

17.14 Quorum Sensing Plays a Key Role in Establishing

a Colony
Bacteria use cell-to-cell signaling to take a census of how many fellow bacteria are present in their local environment. The cell density-determining
process is known as quorum sensing, and it operates in the following
manner. The bacteria secrete signaling molecules called autoinducers. When
the local environment is restricted in some manner so that the molecules
do not simply diffuse away, the concentration of autoinducers will increase
in a manner that reects the number of bacteria secreting the molecules.
The quorum sensing system responsible for secreting autoinducers and
sensing their concentration is an integral part of the bacterial cell control
system. Once the concentration exceeds a threshold level, a program of
gene expression is initiated that leads to alterations in morphology and
physiology, and supports communal behavior.
Many different kinds of signaling molecules are utilized in cell-to-cell
communication. Some of these are species-specic while others enable the
bacteria to communicate between members of different species. Gram-negative bacteria utilize short chain acetyl homoserine lactases (AHLs) to
signal one another (Figure 17.9). These molecules are able to diffuse across
the cell membranes and bind to cytoplasmic receptors of the recipient bacterium. Gram-positive bacteria utilize oligopeptide autoinducers (AIPs).
Signaling by oligopeptides in gram-positive bacteria resembles signaling by
peptide hormones in eukaryotes. The autoinducers do not diffuse across the

17.14 Quorum Sensing Plays a Key Role in Establishing a Colony

429

Figure 17.9. Quorum sensing in Gram-negative bacteria: One component of the

system, the autoinducer synthase generates AHL signaling molecules that freely
leave and enter bacterial cells. These are bound by autoinducer receptors, which can
then stimulate transcription.

Figure 17.10. Quorum sensing in gram-positive bacteria: Signaling peptides are

shipped out of the cell by means of a transporter. The signals (autoinducer peptides
or AIPs) bind to bacterial two-component signals transduction systems, which transduce signals into the cell and stimulate gene transcription.

membranes. Instead, they are shipped out of the sender by ABC transporters, and diffuse over to and subsequently bind transmembrane receptors of the recipient. The latter receptors operate as the sensor components
of two-component signal transduction systems (Figure 17.10).
In some species (e.g., B. subtilis and S. pneumoniae) quorum sensing triggers a bacterial state called genetic competence that is geared for the uptake
of exogenous DNA. In others a threshold-exceeding density of cells induces

430

17. Cell Regulation in Bacteria

a virulence response (S. aureus) and the expression of virulence genes (P.
aeruginosa), or the formation of a biolm, a colony of bacteria collectively
exhibiting an increased resistance to antibacterial agents.

17.15 Bacteria Form Associations with Other Bacteria

on Exposed Surfaces
Whenever possible, bacteria establish surface-attached communities known
as biolms. These structures can be formed by members of one species or
by members of multiple species. Bacteria can freely switch between freeliving and communal forms of organization, and communities can form,
grow, collectively migrate, and disperse. When bacteria organize themselves
into biolms they become difcult to eradicate. The biolms contain not
only the bacterial cells but also a protective matrix of secreted molecules,
secreted by the bacteria, that protects the members of the colony from
harmful agents in their environment. Actinomyces, Lactobacillus, and
Streptococcus lms can appear as plaque on teeth; Staphylococcus and
Escherichia coli lms can emerge on medical instruments and surgical
implants, and Pseudomonas aeruginosa and Escherichia coli colonies can
form in the body.
Pseudomonas aeruginosa forms elaborate structures on surfaces. This
highly adaptive species is an opportunistic pathogen, and is often encountered in the treatment of cystic brosis patients and burn victims. The formation of a biolm protects the P. aeruginosa from antibiotic applications,
increasing their resistance to the antibiotics by more than a hundredfold.
These bacteria are highly resistant to antibiotic treatment due in part to the
presence of their self-generated polymeric matrix that encases the colony
and protects it from the actions of antibiotics.
Biolms are also encountered in urinary tract infections. Most urinary
tract infections are due to uropathogenic strains of Escherichia coli
(UPEC). These bacteria are able to form podlike bulges on the surface of
the bladder. Like the P. aeruginosa biolms formed in lungs, the UPEC cells
are encased in a protective shell that insulates them from antibiotics. They
are able to persist as pods for several months undetected, and are utterly
resistant to standard courses of antibiotics.

17.16 Horizontal Gene Transfer (HGT)

Bacterin share genetic information through horizontal gene transfer
(HGT). Horizontal gene transfer differs from vertical gene transfer that
takes place between parents and their offspring. In HGT genetic information is transferred between distantly related species. There are several different mechanisms for affecting the lateral transfer of genetic material. One

17.17 Pathogenic Species Possess Virulence Cassettes

431

of these is conjugation, in which two cells establish direct contact with one
another. Sex pili are formed and genetic material in the form of a plasmid
is sent from the donor bacterium to the recipient. This is perhaps the most
common way of carrying out horizontal gene transfer. A second way of
transferring genetic material is through transduction, where a bacteriophage picks up genetic material from one bacterium and delivers it to
another. A third way of acquitting genetic material is through transformation, in which a bacterium becomes competent to capture exogenous DNA
from its local environment as discussed in the previous section.
Resistance to antibiotics can be spread rapidly through horizontal gene
transfer. One consequence of horizontal genetic transfer is that groups of
genes that endow one bacterial species with antibiotic resistance can be transferred in minutes to hours to another species.The transfer of genetic material,
that is, the sharing of blueprints for making useful proteins among bacteria, is
a particularly potent adaptive response.It is far more efcient to simply transfer a complete set of genes than to have each species invent for itself the
genes one by one by means of random mutations. In examining the complete
genome sequences of these pathogens it becomes clear that entire regions of
DNA have been acquired from other species in the past.
Antibiotics are agents synthesized by fungi and bacteria that kill competing microbes. There are several different kinds of antibiotics. Some
antibiotics act on bacteria by causing damage to their cell walls. Other
antibiotics impede the ability of the bacteria to carry out protein synthesis
or to disrupt the replication of DNA or to interefere with metabolism. Penicillin-type antibiotics such as Ampicillin and Methicillin, cephalosporin
antibiotics, and Vancomycin attack bacterial cells walls. Protein synthesis
impeding antibiotics include tetracycline, aureomycin, streptomycin, and
Erythromycin. Quinolones disrupt DNA replication, and sulfur drugs interfere with bacterial metabolism.
There are several ways of conferring resistance to antibiotics. One way is
to synthesize biomolecules that destroys the antibiotics. Another approach
is to pump the antibiotics rapidly out of the cell thereby keeping its intracellular concentration so low that it is ineffective. Still another way of
dealing with the antibiotic is to synthesize cellular agents that bind antibiotics and neutralize them thereby acting as intracellular blockers.

17.17 Pathogenic Species Possess Virulence Cassettes

Most bacterial strains are harmless, but some can be deadly. In pathogenic
strains additional proteins such as toxins and mbriae components are typically encoded on plasmids and other mobile genetic elements that can be
readily transferred and exchanged between species. It is these additional
proteins that endow bacteria with their disease-causing capabilities. For
example, the standard laboratory strain of Escherichia coli K12 is harmless.

432

17. Cell Regulation in Bacteria

Its genome is 4.6 MBp long, while the pathogenic strain of E. coli O157 is
considerably larger5.4 MBp in length. In E. coli O157 there are O
islands and K islands, clusters of genes that encode a total of 1387 proteins not found in the K12 strain. The additional proteins encoded by the
E. coli O157 strain endow the bacterium with enterohaemorrhagic abilities.
One of the most striking ndings from sequencing complete genomes of
microbes is that bacterial genomes are riddled with phage genes. It appears
that bacteriophages are a major source of mobile genetic material moving
in and out of bacteria. In many species, pathogenicity and virulence of the
bacterial strain are tied to the presence of a prophage, the inactive (lysogenic) bacteriophage whose genes have been integrated into the bacterial
genome. An example of this coupling is provided by group A Streptococcus
(GAS) strains. These bacteria promote sore throats, impetigo, acute rheumatic fever (the major heart disease of children), necrotizing fasciitis (the
disease caused by esh-eating bacteria), and toxic shock syndrome. The
various strains (serotypes) of GAS are given M designations according
to differences in a family of streptococcus cell surface proteins called M
proteins, which impede the engulfment by phagocytes. The M18 strain,
responsible for acute rheumatic fever contains phage genes that encode the
disease-causing toxins. Another strain, M3, which produces toxic shock syndrome and necrotizing fasciitis, has a different set of phage genes from the
M18 strain, and the strain most often responsible for sore throats, M1, has
yet another set of phage genes.
Pathogenicity islands, or virulence cassettes, are clusters of functionally
related virulence genes that are absent in nonpathogenic organisms. The
term virulence denotes the ability to cause disease. A virulence factor is
the designation given to an individual disease-causing or disease-promoting agent of an organism. Some examples of virulence factors are toxins,
surface adhesion molecules, control molecules, and virulence regulatory
proteins (transcription factors). Virulence cassettes, consisting of ensembles
of these proteins, may be located on plasmids or on other mobile genetic
elements that can be transferred from one bacterium to another. They may
be found on the bacterial chromosome, as well. In all cases they encode a
complete suite of proteins needed to promote virulence.
The virulence cassettes of enteropathogenic E. coli (EPEC) strains allow
them to build their own signal transduction pathway in a host cell, which
they use to remodel the hosts cytoskeleton. The bacteria rst send soluble
proteins into the host cell membrane. The proteins are then modied to
form physiologically functional receptors for its bacterial ligands, thereby
establishing a rm contact between cells. Upon appropriating the communication machinery the bacterium uses the hosts resources to erect a
pedestal on the host cells surface. This pedestal supports the subsequent
assembly of a bacterial colony.
Virulence cassettes are used to transfer agents from bacterium to host.
The most common forms, known as Type III and IV secretion systems, are

17.18 Bacterial Death Modules

433

encountered in many pathogenic bacterial species. They are similar enough

to one another to be interchangeable and to have been acquired by horizontal transfer from species to species. The best characterized system of this
sort is the Yop virulon, a Type III system encoded on a plasmid. It contains
(i) a secretion apparatus, (ii) a delivery system that sends bacterial proteins
into the eukaryotic host, (iii) a control unit, and (iv) an agent effector
module that disarms the host signal transduction network. The objective of
Yersinia is to survive and replicate in the lymphoid tissue of its host. The
prime danger to survival is from phagocytes produced by the host organisms immune system. The virulons inactivate the phagocytes by activating
the phagocytes apoptosis (suicide) circuitry. Yersinia avoids attracting the
attention of the immune system. The secretion apparatus and delivery
system is activated by cell-to-cell contact and inserts agents into the target
in a highly directional way so that there is little or no leakage into the intercellular spaces.

17.18 Bacterial Death Modules

Bacteria employ death modules to kill competing bacteria and in some
instances to commit suicide. Bacteria are able to synthesize and release
toxins without themselves being harmed. To accomplish this feat the bacteria synthesize the antidote to the toxin at the same time that they make
the toxin. The genes for both toxin and antidote, or antitoxin, usually reside
on a plasmid and are transcribed from the same promoter (Figure 17.11).
The toxin is long-lived while the antidote is unstable and short-lived. As
long as the plasmid is present and active both toxin and antidote get made
and the bacterium is protected against the former.
Toxin-antidote pairings can serve as the core element in suicide modules.
As is the case for self-protection, a long-lived toxin is paired with a short-

Figure 17.11. Bacterial death modules: The genes for toxin and antitoxin (antidote)
reside side by side on a plasmid. When the genes are expressed the antidote binds
the toxin and prevent it from binding to its substrate. In response to suicide signals,
proteolytic enzymes degrade the antidote; the toxin then binds to its substrate
leading to cell death.

434

17. Cell Regulation in Bacteria

lived antidote that antagonizes the activity of the toxin. If the plasmid is
disabled the antidote will not be made, and because it has a short half-life
the antidote will decay away rapidly. When the antidote is removed either
by loss of the plasmid or by some other means such as proteolytic cleavage, the toxin is free to degrade its substrate, usually an enzyme critical for
cellular survival, and the host cell dies. It forming colonies, bacteria sometimes use death modules of this type to selectively remove a potion of their
population. This may be viewed as a bacterial form of programmed cell
death.

17.19 Myxobacteria Exhibit Two Distinct Forms of

Social Behavior
Myxobacteria are gram-negative, aerobic soil bacteria that feed on decomposing vegetation. When nutrient supplies are plentiful species such as M.
xanthus form feeding swarms that glide together over solid surfaces and
prey on other bacteria. The members of a swarm are free to come and go,
explore other feeding sites, and establish colonies. This type of social behavior changes when nutrients are no longer available. Under starvation conditions the bacteria organize into a differentiated hemispherical structure
called a fruiting body. Rod cells inside the fruiting body differentiate into
spores that are subsequently dispersed. When these spores encounter a
hospitable environment they reestablish a feeding colony of gliding
bacteria.
The transition from feeding swarm to fruiting body is initiated by
starvation signaling. When bacteria are deprived of essential nutrients
they reduce or halt protein synthesis, DNA replication, and cell division
and enter a stationary phase. Any such alteration in cellular physiology is
referred to as a stringent response. One of the main elements of the stringent response is the accumulation of the highly phosphorylated, guanosine
(penta)tetraphosphate in the cell. In bacteria, RelA (a synthase) and SpoT
(a hydrolase) synthesize pppGpp and ppGpp from GTP and GDP using
ATP as a donor. These nucleotides serve as intracellular second messengers
of starvation conditions. Under adequate nutrient conditions these molecules are maintained in the bacterial cell at low, steady state concentrations.
Starvation conditions, in the case of M. xanthus, a lack of amino acids or
carbon compounds supplied by prey, stimulate an increased ppGpp production by RelA. The ppGpp molecules bind and alter the activity of RNA
polymerase shifting the global pattern of gene expression from growth to
a stationary phase. In Streptomyces coelicolor, the stringent response triggers an increase in antibiotic production and morphological differentiation.
In M. xanthus, the buildup in ppGpp upregulates several asg genes, one
result of which is to release proteases that generate a mixture of amino acids
and peptides collectively referred to as A-factors.The A-factors are secreted

17.20 Structure Formation by Heterocystous Cyanobacteria

435

from the myxobacteria and act as quorum-sensing signals. If the concentration of A-factors is adequate, fruiting body formation proceeds.
A-signaling in M. xanthus is followed by C-signaling that determines
which cells in the nascent fruiting body are fated to become spores and
which cells will retain their original rod shape. Transmission of C-signals
occurs between cells that are in close, end-to-end contact. Transmission is
ineffective when neighboring cells lie side by side. The circuitry involved in
C-signaling closely resembles that used in Notch signaling in eukaryotes. Csignals transmitted between ends of cells activate FruA, a transcription
factor that stimulates the expression of the csgA gene that encodes the Csignals. This positive feedback loop increases FruA activity above thresholds for its activation of frz genes and then dev genes. The frz genes encode
proteins that mediate the aggregation of the cells into mounds, and the dev
genes encode proteins that promote the differentiation of rod cells into
spores. Because of the geometric constraint on C-signaling, cells in the
periphery never experience threshold-exceeding concentrations of FruA
while those in the nascent fruiting body reach critical signal densities.

17.20 Structure Formation by Heterocystous

Cyanobacteria
Heterocystous cyanobacteria form differentiated structures with properties
usually associated with multicellular eukaryotes. Cyanobacteria are phototrophs and, like plants, are capable of oxygenic photosynthesis in which
CO2 is removed and O2 is produced.These organisms have minimal requirements for survivalwater, light, CO2, and some salts that supply N2, iron,
phosphorus, and sulfur. For many years the cyanobacteria were referred to
as blue-green algae, but are now recognized as being Gram-negative bacteria. Ancestral cyanobacteria rst appeared some 3.5 billion years ago and
are believed to be responsible for the oxygenation of the Earths atmosphere. Photosynthesis in cyanobacteria is carried out in thylakoid membranes in the same way as it is done in plant cells. Similarities in size and
structure, the presence of chlorophyll A, photosystems I and II, and similarities in control elements provide evidence that present day chloroplasts
and cyanobacteria share a common ancestry.
Cyanobacteria are an essential part of the worlds ecology. Not only do
they release oxygen but they also x nitrogen (and carbon). Most organisms cannot make use of atmospheric nitrogen. Cyanobacteria are able to
x nitrogen, that is, they can convert atmospheric nitrogen into inorganic
nitrogen that can be used for biological purposes by other organisms. They
are a main component of marine plankton, the dense collection of (mostly)
small drifting organisms that cover the worlds oceans and produce 90% of
the planets oxygen. They are also found in large numbers in the surface
regions of lakes, are a major component of communities known as micro-

436

17. Cell Regulation in Bacteria

bial mats found in the hot springs of Yellowstone National Park, and populate nearly all ecosystems.
Members of genera such as Calothrix and Anabaena can form differentiated structures. The structures take the form of long laments in which
nitrogen-xing heterocysts are interspersed at regular intervals among photosynthetic vegetative cells. The heterocysts reduce N2 to ammonia but do
not carry out photosynthesis or carbon xation. The vegetative cells, on the
other hand, perform photosynthesis and x carbon. The technical problem
being solved through multicellularity is the need to separate photosynthesis from the nitrogen-xing due to the inactivation by oxygen of the nitrogenase complex responsible for nitrogen-xing. The heterocysts possess a
protective envelope and turn off their photosystem II. They export inorganic nitrogen to their neighbors and import nutrients. About 1 in 10 to 20
cells in a lament is a heterocyst. Cell-to-cell signaling is used to establish
and maintain the pattern of cell differentiation just as it does in multicellular eukaryotes. Most strikingly, the heterocysts are terminally differentiated cells, like those in metazoan tissues.

17.21 Rhizobia Communicate and Form Symbiotic

Associations with Legumes
Rhizobia is the name given collectively to several genera of rod-shaped,gramnegative bacteria that x nitrogen and make it available for use by legumes.
In poor soils they provide a major source of xed nitrogen to grain legumes
such as soybeans, peas, and beans, as well as to forage legumes such as alfalfa
and clover. Rhizobia are free-living soil bacteria that enter and colonize plant
root cells. They stimulate the formation of nodules, specialized root organs,
and in the nodules they convert atmospheric nitrogen into ammonia and
export it to their hosts, and receive carbon compounds in exchange.
The process whereby the bacteria and host plant establish their symbiotic association involves several stages of signaling between bacteria and
plant root, rounds of gene expression, and differentiation and cellular/tissue
remodeling. To initiate the process, the plant roots exude signaling compounds called avinoids that attract the bacteria to the root surface. The
rhizobia attach to young root hairs and signal the root cells by secreting
glycolipids called nod factors. In response to nod factors, the plant cells
starts to remodel their actin and microtubule cytoskeletons and form
nodules. First, the bacteria become entrapped between cell walls of stimulated (deformed) root hairs, and the plant cell walls undergo hydrolysis
resulting in lesions. New cell wall material is then deposited resulting in the
formation of a tubule called an infection thread that is lled with rhizobia.
The infection threads are the doorways to the root cells. The infection
thread network expands into the root cortex while the bacteria multiply.
Eventually, the bacteria exit the network of threads and enter the cytoplasm

References and Further Reading

437

of the root cells. A nodule is formed containing a plant cell-derived peribacteroid membrane. This membrane envelops the bacteria, which multiply
and differentiate into bacteroids. In some symbiotic associations the bacteroids differ greatly in morphology compared to their free-living forms,
becoming several times larger and Y-shaped in form. Within the nodules
the bacteroids and legumes exchange metabolites, which pass through pores
and channels in the peribacteroid and bacteroid membranes.
Initial plant bacteria-signaling events not only launch the nodule-forming
process but also confer specicity and lter out potentially harmful associations from helpful symbiotic ones. Flavinoids secreted by the plants bind to
NodD proteins situated in the cytoplasmic membrane of the bacteria. The
NodD proteins are members of the LysR family of transcription factors.They
serve as sensors of plant signals and as activators of genes whose promoters
contain a 49 Bp sequence called a nod box. Binding by favinoid-NodD complexes results in the activation of the NodA, NodB, and NodC genes common
to all rhizobia. They encode enzymes that catalyze the synthesis of the
lipooligosaccharide core, or backbone, of the nod factors. Structural nod
genes that are species-specic are expressed at the same time. These genes
encode enzymes that modify or decorate the nod factor backbones in a
manner that depends upon the structure of the avinoids.

References and Further Reading

Gilmore MS, and Ferretti JJ [2003]. The thin line between gut commensal and
pathogen. Science, 299: 19992002.
Whitman WB [1998]. Prokaryotes: The unseen majority. Proc. Natl. Acad. Sci. USA,
95: 65786583.
Xu J, and Gordon JI [2003]. Honor thy symbionts. Proc. Natl. Acad. Sci. USA, 100:
1045210459.

Gene Organization
Busby S, and Ebright RH [1994]. Promoter structure, promoter recognition and
transcription activation in prokaryotes. Cell, 79: 743746.
Ptashne M, and Gann A [1997]. Transcription activation by recruitment. Nature, 386:
569577.

Sigma Factors
Cannon WV, Gallegos MT, and Buck M [2000]. Isomerization of a binary sigma-promoter DNA complex by transcription activators. Nature Struct. Biol., 7: 594601.
Gralla JD [2000]. Signaling through sigma. Nature Struct. Biol., 7: 530532.
Sharp MM [1999]. The interface of s with core RNA polymerase is extensive, conserved, and functionally specialized. Genes Dev., 13: 30153026.

Flagellar Assembly
Aldridge P, and Hughes KT [2002]. Regulation of agellar assembly. Curr. Opin.
Microbiol., 5: 160165.

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17. Cell Regulation in Bacteria

Chilcott GS, and Hughes KT [2000]. Coupling of agellar gene expression to agellar assembly in Salmonella enterica, Servovar typhimurium. and Escherichia
coli. Micro. Mol. Biol. Rev., 64: 694708.
Kalir S, et al. [2001]. Ordering genes in a agella pathway by analysis of expression
kinetics from living bacteria. Science, 292: 20802083.

Sporulation
Burbulys D, Trach KA, and Hoch JA [1991]. Initiation of sporulation in B. subtilis
is controlled by a multicomponent phosphorelay. Cell, 64: 545552.
Fabret C, Feher VA, and Hoch JA [1999]. Two-component signal transduction in
Bacillus subtilis: How one organism sees its world. J. Bacterial., 181: 19751983.
Jiang M, et al. [2000]. Multiple histidine kinases regulate entry into stationary phase
and sporulation in Bacillus subtilis. Mol. Microbiol., 38: 535542.
Perego, M, Glaser P, and Hoch JA [1996]. Aspartyl-phosphate phosphatases deactivate the response regulator components of the sporulation signal transduction
system in Bacillus subtilis. Mol. Microbiol., 19: 11511157.

Cell Differentiation in Caulobacter

Domain IJ, Quon KC, and Shapiro L [1997]. Cell type-specic phosphorylation and
proteolysis of a transcriptional regulator controls the G1-to-S transition in a bacterial cell cycle. Cell, 90: 415424.
Jenal U [2000]. Signal transduction mechanisms in Caulobacter crescentus development and cell cycle control. FEMS Microbiol. Revs., 24: 177191.
Laub MT, et al. [2000]. Global analysis of the genetic network controlling a bacterial cell cycle. Science, 290: 21442148.
Quon KC, et al. [1998]. Negative control of bacterial DNA replication by a cell cycle
regulatory protein that binds at the chromosome origin. Proc. Natl. Acad. Sci.
USA, 95: 120125.
Shapiro L, McAdams HH, and Losick R [2002]. Generating and exploiting polarity
in bacteria. Science, 298: 19421946.

Antigenic Variation
Barbour AG, and Restrepo BL [2000]. Antigenic variation in vector-borne
pathogens. Emer. Infect. Dis., 6: 449457.

Quorum Sensing
Davies DG, et al. [1998]. The involvement of cell-to-cell signals in the development
of a bacterial biolm. Science, 280: 295298.
Fuqua WC, Winans SC, and Greenberg EP [1994]. Quorum sensing in bacteria:
The LuxR-LuxI family of cell density-responsive transcriptional regulators.
J. Bacteriol., 176: 269275.
Kleerbezem M, et al. [1997]. Quorum sensing by peptide pheromones and two-component signal transduction systems in Gram-positive bacteria. Mol. Miocrbiol., 24:
895904.
Schauder S, and Bassler BL [2001]. The language of bacteria. Genes Dev., 15:
14681480.
Van Delden C, and Iglewski BH [1998]. Cell-to-cell signaling and Pseudomonas
aeruginosa infections. Emer. Infect. Dis., 4: 551560.

Problems

439

Biolms and Disease

Anderson GG, et al. [2003]. Intracellular bacterial biolm-like pods in urinary tract
infections. Science, 301: 105107.
Costerton JW, Stewart PS, and Greenberg EP [1999]. Bacterial biolms: A common
cause of persistent infections. Science, 284: 13181322.
Singh PK, et al. [2000]. Quorum sensing signals indicate that cystic brosis lungs are
infected with bacterial biolms. Nature, 407: 762764.

Horizontal Gene Transfer, Antibiotic Resistance, and Bacteriophages

Casjens S [2003]. Prophages and bacterial genomics: what have we learned so far?
Mol. Microbiol., 49: 277300.
Frankel G, et al. [1998]. Enteropathogenic and enterohaemorrhagic Escherichia coli:
More subversive elements. Mol. Microbiol., 30: 911921.
Hacker J, Hentschel U, and Dobrindt U [2003]. Prokaryotic chromosomes and
disease. Science, 301: 790793.
Karaolis DKR, et al. [1999]. A bacteriophage encoding a pathogenicity island, a
type-IV pilus and a phage receptor in cholera bacteria. Nature, 399: 375379.
Koch AL [2003]. Bacterial walls as target for attack: Past, present and future
research. Clin. Microbiol. Rev., 16: 673687.
Lowy FD [2003]. Antimicrobial resistance: The example of Staphylococcus aureus.
J. Clin. Invest., 111: 12651273.
Ochman H, Lawrence JG, and Groisman EA [2000]. Lateral gene transfer and the
nature of bacterial innovation. Nature, 405: 299304.
Wagner PL, and Waldor MK [2002]. Bacteriophage control of bacterial virulence.
Infect. Immun., 70: 39853993.

Virulence Cassettes and Factors

Aizenman E, Engelberg-Kulka H, and Glaser G [1996]. An Escherichia coli chromosomal addiction module regulated by 3,5-bispyrophosphate: A model for
programmed bacterial cell death. Proc. Natl. Acad. Sci. USA, 93: 60596063.
Cornelis GR, and Wolf-Watz H [1997]. The Yersinia Yop virulon: A bacterial system
for subverting eukaryotic cells. Mol. Microbiol., 23: 861867.
Groisman EA, and Ochman H [1996]. Pathogenicity islands: Bacterial evolution in
quantum leaps. Cell, 87: 791794.

Death Modules and Programmed Cell Death

Gonzlez-Pastor JE, Hobbs EC, and Losick R [2003]. Cannibalism by sporulating
bacteria. Science, 301: 510513.
Webb JS, et al. [2003]. Cell death in Pseudomonas aeruginosa biolm development.
J. Bacteriol., 185: 45854592.

Problems
17.1 Nonlamentous cyanobacteria solve the problem of separating photosynthesis from nitrogen-xing in a way different from heterocystous
cyanobacteria. The nonlamentous forms carry out photosynthesis
during the day and x nitrogen at night. The separation of metabolic

440

17. Cell Regulation in Bacteria

tasks is made possible by the presence of an internal clock that synchronizes the organisms metabolic processes with Earths 24-hour
rotation (circadian) period.
Circadian clocks are found in plants where they regulate the
opening and closing of plant leaves with respect to the daily cycle of
light and dark. They are found in protists, fungi, and animals, as well.
All share a common set of properties such as the use of feedback loops
and the introduction of time delays. How might a circadian clock be
set up in a bacterium? How might the mechanism(s) used to x the
period and phase differ from those employed by eukaryotes?
Suggested Reading
Dunlap JC [1999]. Molecular bases for circadian clocks. Cell, 96: 271290.
Ishiura M, et al. [1998]. Expression of a gene cluster KaiABC as a circadian feedback process in cyanobacteria. Science, 281: 15191523.
Xu Y, Mori T, and Johnson CH [2003]. Cyanobacterial circadian clockwork: Roles
of KaiA, KaiB and the KaiBC promoter in regulating KaiC. EMBO J., 22:
21172126.
Young MW, and Kay SA [2001]. Time zones: A comparative genetics of circadian
clocks. Nature Rev. Genet., 2: 702715.

17.2 Some bacteria swim from place to place using a rotating agellum at
the back end to provide a driving force in the uid medium. Swimming is not the only method exploited by bacteria for motility.Another
means of locomotion is gliding motility. Social bacteria use this type
of motion, sometimes called twitching motility, quite often. Describe
the components of a locomotion system that might allow a bacterium
to glide along a surface.
Suggested Reading
Wall D, and Kaiser D [1999]. Type IV pili and cell motility. Mol. Microbiol., 32: 110.
Wolgemuth C, et al. [2002]. How myxobacteria glide. Curr. Biol., 12: 369377.

18
Regulation by Viruses

Viruses consist of nucleic acid cores encased in protein sheaths. The protein
sheaths surrounding and protecting the nucleic acid cores are referred to
as capsids.Viral capsids enclose either DNA or RNA.As might be expected,
the viruses belonging to the former class are known as DNA viruses, while
members of the latter group are called RNA viruses. Some RNA viruses
covert their RNA to DNA, and from there pass through both a transcription and a translation stage to make new viruses. These viruses are called
retroviruses. Other RNA viruses do not convert their RNA to DNA.
Instead, their RNA functions as messenger RNA molecules that are used
directly in a translation stage to synthesize proteins.
Many viruses use lipids and carbohydrates acquired from their hosts to
make an envelope. They encase their caspsids in lipids and carbohydrates
picked up from plasma membrane or from organelles such as the nucleus,
ER, or Golgi apparatus. These viruses are referred to as enveloped viruses.
Glycoproteins are an important component of the viral envelopes. These
proteins mediate contact with and entry into the host cell through their
ability to bind to receptors on the hosts plasma membrane.
Viral genomes vary greatly in size. Some, like those of the picornaviruses,
are small, less than 10 kBp, and encode on the order of 10 proteins. Others,
such as poxvirus genomes, are large. They range in size from 130 to 300 kBp
and encode ~200 proteins, approaching those of the mycoplasmas in size of
their genomes. In all cases, the viruses require assistance and resources from
the host cell to replicate and propagate. For that reason their genomes
encode not only genes of a structural character that encode proteins for the
capsid and envelope, but also genes involved in replication and genes of a
signaling and regulatory character. The viral signaling and regulatory genes
encode proteins that regulate viral replication, disrupt and disable host
immune/protective responses, and interact with cellular systems in order to
use them for transport and replication.
In the rst part of the chapter the viral cycle of entry, replication, and exit
will be examined. This discussion will be followed by a general sketch of
some of the ways viruses deal with the immune system. The remainder of
441

442

18. Regulation by Viruses

the chapter is devoted to detailed discussion of how signaling and regulatory proteins make possible the survival of specic viruses. The hepatitis
C virus responsible for chronic hepatitis, cancer-causing viruses, human
immunodeciency virus (HIV)the causative agent of acquired immunodeciency syndrome (AIDS), and bacteriophages will be examined.

18.1 How Viruses Enter Their Host Cells

Viruses enter their host cells using receptors and the cellular machinery for
membrane fusion and endocytosis. Viruses cannot pass directly through the
plasma membranes of cells. Instead they possess proteins in their exposed
surfaces that bind to receptors on host cells triggering their ingestion into
the cell. Enveloped viruses, for example, contain proteins in their envelopes
that distinguish the correct host cells from non-host cells and initiate the
process whereby the virus gains entry to the cell interior. There are two
main ways of entering the host cell. In endocytic entry, the virus enters the
cell in vesicles associated with clathrin-coated pits and caveolae. In this
mode of entry, the viral nucleic acid and the capsid are enclosed in a transport vesicle that is pinched off from the plasma membrane. In the nonendocytic mode of entry, the viral envelope fuses with the plasma membrane
leading to release of the nucleic acid encased in its capsid into the
cytoplasm.
In order to replicate, viruses must rst gain entry into the cell, and then
disassemble, replicate DNA, synthesize components, reassemble, and exit.
Viruses that use eukaryotic hosts for their replication have special challenges. They must navigate in an environment containing a dense cytoskeleton and numerous organelles partitioned into many subcompartments. The
viruses, even small ones, are too big to traverse intracellular distances by
means of passive diffusion, but instead must rely on the cells machinery for
directed active transport involving the use of the cytoskeleton and transport vesicles. Viral capsids and viruses packaged in transport vesicles attach
to dynein (and kinesin) motors and move along microtubules to the nucleus.
The viruses rst move along actin bers and then along microtubules to
reach their sites of replication, and once replicated and assembled move
again along the cells active transport highway to reach the cell periphery.

18.2 Viruses Enter and Exit the Nucleus

in Several Ways
Viruses that replicate in the nucleus must pass through the nuclear pore
complex, rst to get into the nucleus and then again to leave. In some cases,
the viruses travel back and forth, in and out of the nucleus several times.
Viruses exploit several mechanisms for passage through the nuclear pore

18.3 Ways that Viruses Exit a Cell

443

complex, their choice depending on the virus type and size. Virus particles
of diameter less than 30 to 40 nm are small enough to pass through the NPC
intact, without dissolution of the capsid, but larger viruses cannot pass
through without some rearrangement and capsid disassembly. These larger
viruses must either wait for dissolution of the nuclear envelope during
mitosis, or they must at least partially disassemble to pass through the NPC
during interphase.
Proteins that are actively transported into the nucleus are referred to as
being karyophilic. These proteins rely on importins and Ran GTPases for
passage through the NPC in the manner discussed in Chapter 11. These proteins have nuclear localization signals that interact with importins a and b.
Many viruses enter and leave the nucleus in the same manner as cellular
proteins. In the case of small viruses that can pass through the NPC intact,
the capsids contain proteins bearing nuclear localization signals (NLSs).
Herpes simplex virus Type 1 (HSV-1) is an example of a virus that is too
large to pass into the nucleus without disassembly. HSV-1 is an enveloped
virus that fuses with the plasma membrane of the host cell. The intact capsid
is released into the cytoplasm and is transported to the nucleus where it
docks. In order to gain entry to the nucleoplasm of its nondividing host it
expels its DNA directly into the nucleoplasm in a spring-loaded-like
fashion, leaving its icosahedral 125-nm wide capsid outside the nucleus.
This is not the only mechanism utilized by large viruses to get their DNA
into the nucleus. In the case of adenoviruses such as Ad2, the capsids are
rst disassembled and then the DNA is transported through the NPC into
the nucleus. This is accomplished with the use of NLSs resident on proteins
that chaperone the DNA through the NPC. Viruses such as HIV-1 utilize
yet another mechanism. These viruses are able to bind directly to the
nuclear pore, and their DNA can enter the nucleus without requiring the
assistance of importins.

18.3 Ways that Viruses Exit a Cell

Viruses forms buds in endosomal vesicles that are transported to the plasma
membrane. Viruses are able to exit a cell in at least two different ways. They
may exit one at a time through membrane fusion, or they may egress in
mass through cell lysis. They also may leave by some other less common
means. Enveloped viruses acquire their envelopes from the host cells by
budding. They may bud off of an internal membrane or generate a bud from
the plasma membrane. These viruses typically exit the cell through membrane fusion where one virus at a time fuses with the plasma membrane
and releases the virus into the extracellular spaces. The second common
means for release, cell lysis, is typically associated with nonenveloped
viruses. These viruses are released in large numbers all at once in a process
that causes the rupture of the plasma membrane and death of the host cell.

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18. Regulation by Viruses

In order to exit the cell, virus particles interact with the cellular machinery responsible for multivesicular body (MVB) biogeneisis. MVBs are a
type of endosome having a multivesicular appearance; they play a role in
receptor internalization, protein sorting operations, and viral budding. In
their role as devices for internalization and protein sorting, the MVBs transport receptors from the plasma membrane to lysosomes for either degradation or recycling back to the plasma membrane. These transport and
sorting operations are referred to as the MVB sorting pathway.
The proteins that participate in the vascular protein-sorting in MVBs are
called class E Vpss. The reason for this name is that, in the absence of
vascular protein-sorting (Vps) proteins, abnormal endosomes are formed
called class E compartments. The class E Vps proteins form complexes
called endosomal-sorting complexes required for transport (ESCRTs). In
humans, there are four distinct ESCRT complexes. Members of these complexes interact with the exiting viruses through late (L) domains. Late
domains are found in virus gag proteins in some viruses and in matrix proteins in others (virus gag and matrix proteins will be discussed shortly).
These L domains mediate interactions with the ESCRTs. The motifs found
in L domains are protein-protein interaction domains. The three most
common binding motifs are PXXP, PPXY, and YXXL. The rst of these
binds to proteins with SH3 domains; the second binds proteins possessing
WW domains, and the third provides attachment for clathrin-associated
adapter proteins. The last named motif is often found in cytoplasmic tails
of proteins involved in sorting and shipping.

18.4 Viruses Produce a Variety of Disorders in Humans

Viruses are responsible for colds and infections, immunodeciency disorders, and cancers. Viruses that infect humans include both DNA and RNA
viruses. One of the main families of DNA viruses infecting humans is the
herpesvirus (Herpes simplex virus, or HHV) family. This family of viruses
is a large one and includes the Epstein-Barr virus (HHV-4) and Kaposis
sarcoma associated herpes (HHV-8). Another common DNA family is the
adenovirus family responsible for producing respiratory infections, and still
another is the poxvirus family (vaccinia) responsible for smallpox and
cowpox. Several families of RNA viruses cause diseases in humans, among
which are colds, hepatitis, inuenza, measles, mumps, polio, rabies, and
AIDS. Members of the most prominent families of viruses and the diseases
they cause are listed in Table 18.1. All of these viruses have been studied
extensively in the laboratory along with viruses that infect bacteria (bacteriophages), plants, and arthropods (e.g., baculoviruses).
Viruses differ from bacteria in the way they damage hosts: Bacteria
release toxins while viruses kill their host cells when they escape from them.
In spite of differences in damage-causing mechanisms, both kinds of

18.5 VirusHost Interactions Underlie Virus Survival and Proliferation

445

Table 18.1. Viruses and some of the diseases they cause: AbbreviationsReverse
transcribing (RT); single-strand negative sense (-ss); single-strand positive sense
(+ss); double strand (ds).
Virus family

Type of virus

Adenovirus
Herpesvirus

DNA
DNA

Papovavirus
Poxvirus
Calicivirus
Filovirus
Flavivirus
Orthomyxovirus
Paramyxovirus

DNA
DNA
+ssRNA
-ssRNA
+ssRNA
-ssRNA
-ssRNA

Picornovirus
Rhabdovirus
Rheovirus
Togavirus
Lentivirus
Oncornavirus

+ssRNA
-ssRNA
dsRNA
+ssRNA
RT RNA
RT RNA

Diseases
Respiratory infections
Chickenpox, cold sores, encephalitis, genital
sores, Kaposis sarcoma, mononucleosis,
roseola, shingles
Warts
Cowpox, smallpox
Norwalk
Ebola, Marburg
Yellow fever, hepatitis C
Inuenza A and B
Measles, mumps, respiratory, colds (children),
pneumonia (adults)
Polio, hepatitis A, common cold
Rabies
Intestinal infections, respiratory infections
Rubella
AIDS
Human T cell leukemia

pathogens utilize many of the same strategies to evade detection and

suborn host defenses. Like bacteria, viral genomes contain both structural
and regulatory genes. The genomes of viruses encode proteins comprising
their caspsids, envelopes, and other components. The genes encoding these
structural proteins are referred to as structural genes. In addition, viral
genomes contain a number of nonstructural genes. These genes encode proteins involved in replication and proteins of a regulatory nature.

18.5 VirusHost Interactions Underlie Virus Survival

and Proliferation
Viruses interact with their hosts in order to survive. They manipulate elements of the host control layer in order to create environments that support
replication and proliferation.They turn off antiviral activities, prevent apoptosis, and alter the cell cycle progression. Shown in Table 18.2 are some
examples of eukaryotic control proteins targeted by viruses. One of the
groups of signaling proteins targeted by viruses is the cytokine group that
coordinates the activities of leukocytes. The cytokine signaling events are
disrupted by the viruses, thereby reducing the effectiveness of the immune
system in responding to the viral invasion.
Another group of signaling elements targeted by the viruses are the
antigen-presenting elements that present peptides on their surface derived

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18. Regulation by Viruses

Table 18.2. Eukaryotic cell components targeted by some well-known viruses.

Control layer elements
Antigen-presenting elements
Apoptosis system

Cytokine signaling

Interferon signaling

Viruses (and signaling elements interfered with)

Adenovirus (MHC class I, II), Epstein-Barr virus (MHC,
class II)
Adenovirus (Fas, Bcl-2, caspases), baculovirus (caspases),
cowpox (Fas, caspases), Epstein-Barr virus (Bcl-2),
vaccinia (caspases)
Cowpox (TNF), human herpesvirus-8 (CC and CXC
chemokines and their receptors), vaccinia (IL-1b,
IFNa/b)
Adenovirus (Jak/STAT), baculovirus (PKR), inuenza virus
(PKR), poliovirus (PKR), reovirus (dsRNA)

from the viruses of the invaded cells. A third set of targets is the proteins
that regulate apoptosis. Cells infected by viruses are killed and induced to
undergo apoptosis. The viruses manipulate this protective response; in some
cases they turn off apoptosis and in others they use the process to hitch a
ride out of the cell. Finally, receptors bound in the plasma membrane are
targeted by viruses in order to gain entry into the cell.

18.6 Multilayered Defenses Are Balanced by

Multilayered Attacks
Pathogens such as viruses and bacteria enter a body, or host, establish themselves in a specic locale, or niche, multiply, cause damage, and exit. Host
defenses are mounted at multiple levels. The outermost layer is the erection of mechanical barriers such as the skin, and chemical barriers such as
antibacterial defensins. Next, the innate immune response is triggered and
then the adaptive immune response is.
The approach used by viruses to interfere with the hosts control system
is a multilayered one. Viruses, for example, overcome the acquired immunity response (antigen-presenting elements) by interdicting expression of
the MHC Class I and II products at the transcription level at the translation level, by forcing the retention of the surface molecules in the cytoplasm, by accelerating their removal from the surface, and by stepping up
their deactivation.
The survival of a pathogen in a host is critically dependent on its ability
to deal with an arsenal of host defenses.Viruses and interferon systems have
coevolved. One striking consequence of this coevolution is that practically
all viruses have developed some level of resistance to the interferon system.
The counterattacks take several forms and are multileveled, like the response to acquired immunity. Depending on the specic virus and target,
the virus may

18.8 Hepatitis C Virus Disables Host Cells Interferon System

447

block the production of interferons,

supply interferon decoys,
block interferon signaling, and
block the ability of the interferon signaling targets to properly act.

At the most fundamental level, what is happening is that both host and
pathogen (viral and bacterial) have coevolved. Both have developed control layers tuned to the other, promoting adaptation and evolution of each.
The overall result is a balance, an equilibrium situation of coexistence of
host and bacterial and viral pathogens.

18.7 Viruses Target TNF Family of Cytokines

The TNF family of cytokines and their receptors, and the apoptosis machinery, are targeted by viruses. The TNF family of cytokines and their receptors are central signaling elements of the adaptive immune response.
Members of this family regulate secondary lymphoid organ development,
lymph node formation, and apoptosis signaling. A variety of viruses including hepatitis C virus, Epstein-Barr virus, adenoviruses, and poxviruses elude
destruction by the adaptive immune response by expressing proteins that
alter the operation of the TNF machinery.
The hepatitis C virus core protein is a structural gene product that is able
to bind to and modulate TNF family receptors. In response to these binding
events cytokine signaling is impaired and cytotoxic T lymphocytes do not
adequately respond to the viral invasion. Adenoviruses target the cellular
apoptosis machinery triggered by the TNF family member Fas (Table 18.2).
The viruses are able to induce the internalization of this receptor, thereby
turning off the ability of the cells to kill the virus. Adenoviruses use other
mechanisms as well to elude the adaptive immune response. For instance,
they interfere with antigen presentation and T cell recognition. Each of the
adenoviral evasion tactics utilizes a different subset of viral gene products.
Cells infected with a virus are usually induced to undergo apoptosis in
order to prevent viral replication. Interestingly, some viruses have evolved
ways to use this mechanism to their own advantage. They induce cell death
at the end of their replication cycle with the result that they are encapsulated in a protective package that is taken up by neighboring cells. By this
means they spread and elude detection at the same time.

18.8 Hepatitis C Virus Disables Host Cells

Interferon System
The hepatitis C virus is believed to infect more than 2% of the worlds
population. The virus causes chronic hepatitis and can lead to cirrhosis
of the liver and/or liver cancer. The virus is a member of the Flaviviridae

448

18. Regulation by Viruses

Figure 18.1. Organization of the genome of the hepatitis C virus: Proteins shown
as dark-shaded boxes are structural (S) in character while those in light-shaded
boxes are nonstructural (NS). From left to right the proteins are: Cnucleocapsid
protein, sometimes called the core protein; E1 and E2glycoproteins belonging
to the viral envelope; p7linker protein of unknown function; NS2 through NS5B
NS enzymes. Arrows denote cleavage sites. Solid arrows indicate sites cleaved by
host cellular signalases; the dashed arrow indicates the site cleaved by NS2, and the
dotted arrows denote sites cleaved by NS3.

family of viruses, which includes the yellow fever virus and several forms
of encephalitis-inducing virus.
The hepatitis C virus (HCV) contains a single strand positive sense RNA
(+ssRNA) molecule approximately 9.6 kBp in length that functions as a
messenger RNA. The genome encodes a single open reading frame that is
preceded at the 5 end by a noncoding region and terminated at the 3 end
by another such region. The resulting polypeptide chain, or polyprotein,
is proteolytically processed during and after translation into 10 proteins
(Figure 18.1). The four amino-terminal-most are structural in character. The
rst protein, C, encodes the capsid protein, and the next two, E1 and E2,
are glycoproteins that are inserted in the viral envelope. It is customary to
refer to proteins forming the virus capsid and envelope as structural proteins and proteins involved in replication and signaling/regulation as nonstructural. The fourth structural protein, p7, is a linker that separates the
structural from nonstructural proteins.
The nonstructural genes encode several different kinds of enzymes. The
NS5B gene encodes an RNA-dependent RNA polymerase that is responsible for viral replication. The NS2 and NS3 genes encode proteases that,
along with cellular proteases, chop up the large polyprotein into the structural and nonstructural proteins at the sites indicated in Figure 18.1. The
NS4B and NS5A gene products are regulatory in nature. The regulatory
gene products along with the E2 glycoprotein target the interferon system
of antiviral defense, allowing the viruses to survive and proliferate even
when that system is activated.
Recall from Chapter 9 that the cells rst line of defense to a viral attack
is the interferon system. During viral replication dsRNA is produced and
the detection of these molecules alerts the cell that viruses are present. A
sensor of dsRNA called protein kinase R (PKR) activates the interferon
response. PKR shuts off translation and stimulates transcription of a host

18.9 Human T Lymphotropic Virus Type 1 Can Cause Cancer

449

of antiviral factors. Interferons are produced that signal to other cells

through interferon receptors and Jak/Stat proteins to generate a global
response to the viral invasion. The favored treatment for patients with hepatitis C is IFNa, or a-interferon. However, this form of treatment, the best
available at present, is ineffective in more than half of patients. In these
many cases, the virus is able to overcome the antiviral activities of the
interferons.
The interferon line of defense is breached by hepatitis C virus through
its NS5A, NS4B, and E2 proteins, which inhibit the activity of PKR. The
NS5A protein inhibits not only the PKR protein but also the IRF1 transcription factor, which acts downstream of PKR to trigger transcription of
antiviral genes from the ISRE promoter. The E2, NS5A, and NS4B proteins
enhance translation from what is termed an HCV internal ribosomal entry
site, or IRES. The net effect of E2, NS4B, and NS5A is to throttle back
expression of antiviral genes and maintain the translation capabilities of the
host cell so that the viruses can replicate.

18.9 Human T Lymphotropic Virus Type 1 Can

Cause Cancer
Certain retroviruses can induce the transformation of normal cells into cancerous ones. One group of retroviruses that can trigger cancers is the oncogenic (oncornaviruses) viruses, typied by the Rous sarcoma virus. This
virus has picked up an oncogene from a host some time in its past, then
deliveres it to its new hosts when it invades a cell. In the case of Rous
sarcoma virus, the oncoprotein is Src, a tyrosine kinase involved in the
growth pathway. This protein was discussed in several places in the text. To
distinguish cellular from viral forms, one is designated as c-Src and the
other as v-Src. The v-Src oncoprotein is of considerable historical importance, as it was the rst oncoprotein to be discovered.
A different mechanism is used to transform cells by viruses such as
bovine leukemia virus (BLV) and human T lymphotropic virus type 1
(HTLV-1). The latter is associated with T cell leukemia. These retroviruses
do not carry oncogenes. Instead regulatory proteins are encoded in their
genomes, and these proteins along with promiscuous, virally supplied promoters that accompany them fatally alter the operation of the cellular
control layer. Viruses belonging to this group cause a variety of cancers in
livestock, while HTLV-1 and -2 promote cancer (adult T cell leukemia) in
humans.
The organization and placement of control sites is an essential feature of
a genome. Retorviruses bring their own control sites with them. These
control sites are known as long terminal repeats, or LTRs. There is an LTR
at the 5 end and the other at the 3 end. The one at the 5 end is used for
transcription control. Each LTR is composed of three regions: a U5 region,
an R region, and a U5 region. The U3 region controls viral transcription

450

18. Regulation by Viruses

Figure 18.2. Retroviral control region: (a) A minimal retrovirus, containing gag,
pol, and env genes, is shown bounded at both ends by long terminal repeats (LTRs)
and a pX region near the 3 end. (b) An expanded view of the 5 LTR showing its
U3, R, and U5 regionsA further expanded view of the U3 region depicts a canonical set of DNA binding sites (responsive elements) for transcription factors. Abbreviations: cyclic AMP responsive element (Cre); glucocorticoid responsive element
(Gre); initiation, or start, site for RNA transcription (InR), located at the boundary
between U3 and R.

while the R and U5 regions are essential for viral replication. As illustrated
schematically in Figure 18.2, the U3 region is endowed with binding sites
for many of the key families of eukaryotic transcription factors. These sites
are capable of binding to both viral and cellular regulatory proteins.
A unique feature of HTLV-1 is the presence near the 3 end of its genome
of a region called pX that codes for several regulatory proteins. One of
these proteins, called Tax, binds to CREB proteins at the various Cre sites
not only in the viral genome but also in the cellular genome. Tax interacts
with a variety of cellular regulators of transcription including not only
CREB and cofactors but also NF-kB. Through its interactions with the cellular regulators, Tax changes the regulatory decisions of the T cells control
circuitry, immortalizing and transforming the cells.

18.10 DNA and RNA Viruses that Can Cause Cancer

Listed in Table 18.3 are a number of viruses that are known to transform
cells in humans. The list includes both DNA and RNA viruses. The HTLV1 virus appears in the table as a member of the retrovirus group that promotes T cell leukemias in adults. One of the recent additions to the list of
viruses known to promote cancer is Kaposis sarcoma-associated her-

18.10 DNA and RNA Viruses that Can Cause Cancer

451

Table 18.3. Tumor viruses.

Virus
DNA tumor viruses
Hepatitis B virus
Herpesviruses
Epstein-Barr virus
Kaposis sarcoma virus
Papillomaviruses
RNA tumor viruses
Hepatitis C virus
Retroviruses

Type of tumor
Liver cancer
Burkitts lymphoma,
nasopharylgeal carcinoma
Kaposis carcinoma
Cervical cancer
Hepatocellular carcinoma
Adult T cell leukemias

Figure 18.3. Approximate locations of regulatory genes encoded in the Kaposis

sarcoma-associated herpesvirus (KSHV) genome: The 141-kB genome is anked by
terminal repeat sequences (gray). The coding region (clear) contains two classes of
regulatory genes. The rst of these are the genes pirated from host genomes. The
members of this group can be recognized by the presence of a letter v in front of
familiar gene names. The second group consists of regulatory genes unique to the
virus and its close relatives, such as the Epstein-Barr virus. Three examples are
included in the gure: K1, latency-associated nuclear antigen type 1 (LANA-1), and
latency-associated membrane protein (LAMP). Other abbreviationsComplement
binding protein (CBP); macrophage inammatory protein (MIP); interferon regulatory factor (IRF).

pesvirus (KSHV). Viruses such KSHV have pirated a large number of regulatory genes from their hosts, and through expression of these genes alter
the cell cycle and growth responses of the host so that they serve the virus.
They use the pirated genes, as well as genes that are unique to the viruses
to degrade both the innate immune response and the adaptive immune
response, and can transform cells, producing a number of cancers in
humans.
Some of the most prominent regulatory genes in the KSHV genome are
shown in Figure 18.3. The rst group of genes depicted in the gure encodes

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18. Regulation by Viruses

two antiapoptotic proteins. The viral Bcl-2 protein acts at the PTPC to
inhibit apoptosis, and the viral FLIP acts at the DISC to throttle back death
receptor signaling by preventing activation of caspase 8. The next set of
gene products regulates the cell cycle so that it becomes more conducive
to viral replication. The viral cyclin protein (vCyc) is similar to the cellular
D cyclins and primarily binds to Cdk6. The cell cycle genes are followed in
the gure by a considerable number of genes that jointly regulate immune
responses and cell growth. The LAMP protein is a 12-pass transmembrane
protein that inhibits signaling from the B cell receptor. The other proteins
further shift the life-death signaling balance towards growth and away from
apoptosis, and suppress Th1 immune responses.

18.11 HIV Is a Retrovirus

The HIV-1 virus is a member of the retrovirus family. Like others in that
family the packaging of the virus is fairly elaborate. The virus capsid is
encapsulated in a matrix that, in turn, is enclosed inside an envelope. Two
copies of an ssRNA molecule are contained within the capsid. Each ssRNA
molecule encodes 9 polygenes and 15 genes overall. The structure of the
virus is depicted in Figure 18.4.
The organization of the HIV-1 genome is depicted in Figure 18.5. As was
the case for the other viruses, a combination of structural and nonstructural
genes is encoded. The structural genes are located on group-specic antigen
(gag) and envelope (env) genes. These are proteolytically processed to yield
seven protein-coding genes. The core, or CA, gene encodes the capsid
protein, and the MA gene encodes the matrix protein. The third gag gene
is the nucleocapsid gene, NC. It encodes a protein that associates with the
ssRNA molecule. The last gag segment is the p6 gene that contains a late
domain that mediates docking, budding, and release of the HIV virus at the
cell surface.
The envelope polygene encodes two glycoproteinsgp120 surface (SU)
and gp41 transmembrane (TM). These proteins form complexes in the

Figure 18.4. Structure of the human

immunodeciency virus (HIV): The
virus is shown with its surface-bound
gp120 and transmembrane gp41 proteins
protruding out from the envelope of the
virus.

18.12 Role of gp120 Envelope Protein in HIV

453

Figure 18.5. Organization of the HIV-1 genome: Abbreviations of regulatory

genesNegative effector (nef); regulator of viral expression (rev); transactivator of
viral transcription (tat); virulence infectivity factor (vif); virulence protein regulatory (vpr); virulence protein unknown (vpu).

plasma membrane. They facilitate binding and entry of the virus into the
host cell. All retroviruses contain gag, env, and pol genes. The pol gene
encodes enzymes required for replication of the virus. One of the enzymes
is a reverse transcriptase (RT). This enzyme catalyzes the polymerization
of a DNA molecule from the RNA molecule. This is a reverse, or retro,
information transfer operation, hence the name retrovirus. The pol gene
also encodes a protease (PR) that cleaves viral proteins following their
translation, and an integrase (IN) that helps integrate the viral DNA into
the host genome. There are six regulatory genes. These genesvif, vpr, vpu,
nef, tat, and revare unique to HIV and along with gp120 are responsible
for HIVs lethal behavior. These virulence-promoting genes are discussed
next in greater detail.

18.12 Role of gp120 Envelope Protein in HIV

The gp120 envelope protein mediates contact with and entry into the host
cell.The gp120 protein is extensively modied in the Golgi where it acquires
a set of complex sugars. The glycoproteins are then transported to the cell
surface where they fuse with the budding viruses and are incorporated into
their envelopes. The gp120 envelope protein interacts with receptors on the
cell surface, most notably with CD4 receptor on helper T cells, and uses the
chemokine CCR5 receptors as coreceptors, sometimes along with the
CXCR4. Binding is sequential. Binding to CD4 triggers conformational
changes leading to binding to the chemokine receptors. Membrane fusion
and entry into the cell follows these binding events.
Neutralizing antibodies bind to epitopes on viral surface molecules such
as gp120 and block the ability of those proteins to bind receptors on host
cells. The viruses are prevented from receptor-binding and entry into the
cell, and are thus neutralized. In the case of HIV, most neutralizing antibodies are unable to recognize the gp120 glycoprotein. This glycoprotein
avoids detection by occluding its antibody-binding sites. Antibodies bind
gp120 in two places. One of these overlaps the CD4 binding site and is

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18. Regulation by Viruses

Figure 18.6. Crystal structure of the HIV-1 gp120 envelope glycoprotein in a

complex with CD4 and a neutralizing antibody determined by X-ray crystallography: (a) The gp120 glycoprotein is shown in a bound to the CD4 receptor expressed
on helper T cells and to a Fab antibody fragment called 17b. (b) Expanded view of
the gp120 coreThe structure is shown rotated 90 degrees about a vertical axis from
the view shown in part (a). The gure was generated using Protein Explorer with
atomic coordinates deposited in the Brookhaven Protein Data Bark under accession code 1GC1.

occluded by the V1/V2 loop. The other overlaps the chemokine binding site
and is occluded by the V2 and V3 loops (Figure 18.6). This is not the only
technique used. A second way HIV avoids neutralizing antibodies is by conformational masking.This is a thermodynamic effect, tied to conformational
changes, that allows gp120 to bind to CD4 while simultaneously avoiding
neutralization by the antibodies. Yet another way used by gp120 to avoid
discovery is glycosylation. A large number of changing sugar groups are
added, about 20 glycans for each gp120 molecule. These act as a constantly
shifting shield against neutralizing antibodies.

18.13 Early-Acting tat, rev, and nef Regulatory Genes

Prior to entry into the nucleus the HIV-1 RNA is transcribed into double
stranded DNA by the reverse transcriptase (RT). A preinitiation complex
(PIC) is formed consisting of the viral DNA, integrase (IN), multiple matrix
(MA) protein copies, nucleocapsid protein (NC), p6, and Vpr. The PIC
binds to the pore and is then sent into the nucleus using NLSs embedded
in viral and associated cellular proteins.
The 9-kB HIV genome encodes 15 proteins, but nascent transcripts are
spliced in about 30 different ways to produce a variety of messenger RNA

18.13 Early-Acting tat, rev, and nef Regulatory Genes

455

Figure 18.7. Regulation of transcription elongation by Tat: A portion of the HIV

promoter is shown highlighting the upstream NF-kB and Sp1 binding sites, and
TATA box. The RNAP II is depicted with several of its cofactors transcribing the
DNA into an RNA chain. A portion of the RNA chain (shown in black), the TAR,
is bound by Tat which then recruits the Cdk9/Cyclin T1 complex. The Cdk9 kinase
hyperphosphorylates the C-terminal domain (CTD) of the RNAP II molecule.

molecules. In the early stages of HIV infection, short 2-kB mRNAs are produced and exported from the nucleus. These short transcripts encode Tat,
Rev, and Nef proteins, and the genes for these are referred to as early acting
genes. Later in the infection cycle longer 4-kB variants and full length
mRNAs are made, and the genes transcribed at that time are termed late
acting genes.
The HIV virus not only supplies its own promoter, as do other retroviruses, but also supplies its own transcription activator, Tat. The Tat protein
is an RNA binding protein. It binds to a segment of nascent HIV RNA
chain called the transactivating response (TAR) region located at the 5 end.
A number of cellular proteins recognize the TAR sequence and are
recruited to the site. As depicted in Figure 18.7, these include Cyclin T1 and
its cyclin-dependent kinase Cdk9, which hyperphosphorylates the Cterminal domain of RNAP II. In the absence of hyperphosphorylation, the
transcripts generated by RNAP II tend to be short. Hyperphosphorylation prevents premature elongation termination and enables the enzyme
to catalyze full length transcripts.
Rev is a sequence-specic RNA binding protein that binds to a region
called the Rev responsive region (RRE) located in env gene transcripts.
The Rev protein contains a nuclear export sequence (NES) and mediates
the export of all transcripts except those of tat, rev, and nef from the nucleus
into the cytoplasm, where they are translated into the virus components. In
the early stages of an HIV infection, before Rev levels are sufcient to
support its export function, transcripts longer than 2 kB that get made
collect in the nucleus. In the later stages of HIV infection, Rev facilitates
the export of longer and full length transcripts from the nucleus.
The Nef protein helps the virus to control signaling by its host and creates
an environment conducive to replication of the virus. One of the key targets

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18. Regulation by Viruses

of Nef is the MHC-I complex on the cell surface. Recall that MHC-I complexes are used to present antigens derived from, for example, viruses, on
the surface where they can be recognized by killer cells of the immune
system. Nef stimulates the internalization of these receptors and directs
them to the Golgi. Another target of the Nef protein is the CD4 receptor.
These are internalized and sent to digestive compartments. The MHC-I and
CD4 receptors are not the only immune system signaling elements targeted
by Nef. It also downmodulates the CD28 coreceptor. All of these actions
help disable signaling between cells of the immune system, enabling
infected cells to evade detection and destruction. In macrophages, which
along with CD4 T cells are the main targets of HIV, Nef induces the release
of chemokines that attract resting T cells and help to transmit the virus to
uninfected cells.

18.14 Late-Acting vpr, vif, vpu, and vpx

Regulatory Genes
Viral protein U (Vpu) is found exclusively in HIV-1, while viral protein X
(Vpx) is restricted to HIV-2 and simian immunodeciency virus (SIV). Vpu
assists in the degradation of CD4 in the endoplasmic reticulum and in the
exit of the virus from the cell. It binds CD4 in the ER and targets it for
degradation in the ubiquitin-proteosome pathway thereby preventing its
translocation to the plasma membrane.
Viral protein R (Vpr) encoded by the vpr gene, is a small protein, containing just 96 amino acids. It is soluble and is able to pass through membranes. Vpr acts in several ways to increase HIV survival in cells that it
infects. It assists in the nuclear localization of the PIC, and triggers arrest
of the cell cycle at G2/M transition by blocking Cdk1/Cyclin B complex formation that drives the cell into mitosis. The Vpr protein acts as a modulator of apoptosis. It migrates to the mitochondria where it works together
with proteins resident in the PTPC to shift the balance either towards or
away from apoptosis. It can work with the antiapoptotic Bcl-2 protein to
inhibit apoptosis and work at the ANT and VDAC to increase mitochondrial membrane permeability and promote apoptosis.
Viral infectivity factor (Vif) counters the antiviral activities of a cellular
agent with a long nameapolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, or APOBEC3G. While the retroviral RNA is
being reverse-transcribed into DNA this enzyme catalyzes the conversion
of C nucleotides to U nucleotides. These alterations prevent replication
of retroviruses in cells, referred to as being nonpermissive, that express the
APOBEC3G enzyme. The Vif protein renders this antiviral enzyme ineffective by binding to it and speeding up its degradation in the ubiquitinproteosome pathway.

18.15 Bacteriophages Two Lifestyles: Lytic and Lysogenic

457

18.15 Bacteriophages Two Lifestyles:

Lytic and Lysogenic
Bacteriophages, or phages, are bacteria-infecting viruses. Bacteriophage
lambda is perhaps the best studied of this group of viruses. Like many
others it has two alternative lifecycleslytic and lysogenic. In its lytic cycle,
the virus attaches to its host, and its nucleic acidsdsDNAenter the cell
while the head and tail sections of the capsid (Figure 18.8) remain outside.
The DNA is rapidly replicated and capsid proteins are synthesized. The
virus components are reassembled and the resulting virions are released
from the host cell through lysis, in which the cell membrane ruptures, and
the viruses escape leaving behind a dying cell.
In its lysogenic cycle, the phage DNA is integrated into the host cell DNA
rather than being immediately replicated. It becomes a prophage, replicating as part of the bacterial hosts chromosome. It continues to do so until
environmental conditions are such that a lytic cycle is preferable. When
those conditions arise it switches from the lysogenic cycle to the lytic cycle.
It rapidly replicates itself and lyses the cell.
The organization of the bacteriophage lambda genome is depicted in
Figure 18.9. Several sets of nonstructural genes are shown. These genes
encode enzymes required for recombination (integration of the viral
genome into the hosts chromosome), replication, and lysis. Six regulatory
genes are interspersed among the aforementioned nonstructural genes.

Figure 18.8. Bacteriophage lambda: The capsid

of the bacteriophage is organized into a head
section, which contains the DNA, and a tail, which
assists in attachment of virus to its host and injection of the viral DNA into the bacterial host.

Figure 18.9. Organization of the bacteriophage lambda genome and its regulatory
genes: The organization of the six regulatory genes and two noncoding operator
regions are depicted in the lower portion of the gure.

458

18. Regulation by Viruses

Their gene products control the decision between the lytic and lysogenic
life.

18.16 Deciding Between Lytic and Lysogenic Lifestyles

The regulatory network used by the bacteriophage lambda to switch
between lysis and lysogeny is one of the best characterized genetic circuits.
This genetic network consists of six regulatory genes (N, Q, cI, cro, cII, and
cIII), three promoters (PL, PR, and PcI), and two noncoding operator regions
(OL and OR). The cI gene is called the lambda repressor because when it is
expressed its gene product, the CI protein, represses transcription of the
genes needed for viral replication thereby favoring the lysogenic lifestyle.
For the same reason the Cro protein is called the lambda counterrepressor
because when it is expressed it facilitates transcription of the genes needed
for the lytic lifestyle. The decision process is represented schematically in
Figure 18.10.
PL and PR are left and right promoters. PL is responsible for transcribing
the left hand portion of the network that includes cIII and N. PR is responsible for transcribing the right hand portion that includes cro, cII and Q. OL
and OR are noncoding operator regions each consisting of three cooperative binding sites. cI is the lambda repressor gene, which, when bound to
the operator sites, shuts down transcription of all lambda genes except that
of the repressor itself. It has its own promoters, PRE and PRM. These have
been represented in Figure 18.10 as PcI. When cI is expressed it binds to

Figure 18.10. Operation of the phage lambda decision circuit: The central portion
of the regulatory circuit in shown in an expanded view in the lower portion of the
diagram. The lysogenic state is selected when CI binds to the operator sites and
represses transcription from the left and right promoters. The lytic state is chosen
when the Cro protein binds to the operator sites.

18.17 Encoding of Shiga Toxin in E. coli

459

sites nearest the left and right promoters, thereby blocking transcription of
the other genes, and the phage remains a prophage. The cro protein product
is an antirepressor that shuts off cI when it is bound to the operator sites.
As shown in Figure 18.10, it favors the innermost sites in the left and right
promoters. When it binds to these sites it shuts off cI transcription while
allowing transcription of the other genes from the left and right promoters.
This circuit exploits both positive and negative feedback.
One of the most interesting aspects of the phage decision circuit is the
occurrence of cooperativity in the two operator regions. Binding of a
protein to a site in one of the operator regions increases the probability
that a second protein will bind to a nearby site that would otherwise be
unoccupied due to weak binding. The initial binding events thus serve to
recruit additional proteins to the regulatory regions where they jointly
control transcription. This type of control is not unique to bacteria or to
phages; it was encountered earlier in the chapter on eukaryotic gene
regulation.
The decision circuit operates under environmental control. The decision
circuit is sensitive to good versus poor growth conditions and to exposure
to ultraviolet radiation. The outcome of the race between lysogeny and lysis
is determined by the concentration of CII in the cell. These proteins act as
enhancers of cI transcription. The concentration of CII in a healthy cell is
usually maintained at a low level by the activity of host proteases that are
sensitive to glucose levels. When nutrients are plentiful, CII levels are low
resulting in a stable lytic state of replication and release of new phages.
The CII concentration under poor bacterial growth conditions is elevated
leading to CI levels that are high enough to send the phage into a lysogenic
state.
The phage can transition from lysogeny to lysis in response to DNA
damage arising from UV or ionizing radiation. RecA proteins are synthesized by the host in response to ultraviolet damage to the cellular DNA.
These proteins stimulate the repair of damaged DNA by inactivating the
LexA repressor. The LexA repressor is similar to the lambda repressor CI.
When phage lambda is present, RecA targets CI as well. There is an
increased expression of Cro proteins leading to a lytic stage where the virus
escapes from the UV damaged cell.

18.17 Encoding of Shiga Toxin in E. coli

Shiga toxin genes are encoded by a lambda-like phage in enterohaemorrhegic Escherichia coli. In the last chapter, it was noted that harmless strains
of bacteria such as the harmless K12 variety of E. coli differ from their
pathogenic relatives in that the latter contain additional genes acquired
mostly through horizontal gene transfer. Bacteriophages are a common
means of transfer of DNA from one bacterial species to another. The Shiga

460

18. Regulation by Viruses

Figure 18.11. Phage genes incorporated into the genomes of several strains of
enterohaemorrhagic E. coli: The toxin genes StxA and StxB are situated in between
the replication and late antitermination (Q) genes on the left and lysis genes on the
right.

toxin genes that render strains of E. coli such as O157 : H7 so harmful

provide an excellent example of this mechanism. As shown in Figure 18.11
the Shiga toxin genes StxA and StxB genes are encoded in E. coli O157 :
H7 in a stretch of DNA almost identical to that of bacteriophage lambda.
The gene sequence depicted in the gure is found not only in the O157 : H7
strain but also in the several other E. coli and Shigella dysenteriae strains.
Shiga toxin genes are composed of catalytic A subunits and glycolipidbinding B subunits. They are regulated by factors intrinsic to the bacterial
genome and by the bacteriophage life cycle. There is a binding site in the
operator region upstream of the A gene subunit for the iron binding Fur
protein. When iron is plentiful the protein is able to bind to the operator
and block transcription. This repression is relived in low-iron environments.
Production of toxins is also increased when the bacteriophage replicates
and is released from the host cell through lysis. This places the genes for
the toxins under the control of environmental factors such as increased
H2O2 levels, which can damage DNA, and the presence of antibiotics.

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General Reading
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Viral Entry into the Cell

Greber UF [2002]. Signalling in viral entry. Cell. Mol. Life Sci., 59: 608626.
Overbaugh J, Miller AD, and Eiden MV [2001]. Receptors and entry cofactors for
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Sieczkarski SB, and Whittaker GR [2002]. Dissecting virus entry via endocytosis.
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Virus Cytoplasmic Transport

McDonald D, et al. [2002]. Visualization of the intracellular behavior of HIV in
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Seisenberger G, et al. [2001]. Real-time single-molecule imaging of the infection
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Sodeik B, Ebersold MW, and Helenius A [1997]. Microtubule-mediated transport
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Viral Movement In and Out of the Nucleus

Salman H, et al. [2001]. Kinetics and mechanism of DNA uptake into the cell
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Trotman LC, et al. [2001]. Import of adenovirus DNA involves the nuclear pore
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Viral Release and Budding

Freed EO [2002]. Viral late domains. J. Virol., 76: 46794687.
Katzmann DJ, Odorizzi G, and Emr SD [2002]. Receptor downregulation and
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McDonald D, et al. [2003]. Recruitment of HIV and its receptors to dendritic cellT cell junctions. Science, 300: 12951297.
Pelchen-Matthews A, Kramer B, and Marsh M [2003]. Infectious HIV-1 assembles
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Hepatitis C Virus
Bartenschlager R, and Lohmann V [2000]. Replication of hepatitis C virus. J. Gen.
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Goodbourn S, Didcock L, and Randall, RE [2000]. Interferons: Cell signalling,
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Katze MG, He YP, and Gale M, Jr. [2002]. Viruses and interferons: A ght for
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Large MK, Kittlesen DJ, and Hahn YS [1999]. Suppression of host immune response
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HIV-1
Boshoff C, and Weiss R [2002]. Aids-related malignancies. Nature Rev. Cancer, 2:
373382.
Emerman M, and Malim MH [1998]. HIV-1 regulatory/accessory genes: Keys to
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Karn J [1999]. Tackling Tat. J. Mol. Biol., 293: 235254.
Kwong, PD, et al. [2002]. HIV-1 evades antibody-mediated neutralization through
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Lum JJ [2003]. Vpr R77Q is associated with long-term nonprogressive HIV infection and impaired induction of apoptosis. J. Clin. Invest., 111: 15471554.

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Marin M, et al. [2003]. HIV-1 Vif protein binds the editing enzyme APOBEC3G and
induces its degradation. Nature Med., 9: 13981403.
Sheehy AM, Gaddis NC, and Malim MH [2003]. The antiretroviral enzyme
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Stevenson M [2003]. HIV-1 pathogenesis. Nature Med., 9: 853860.
Swigut T, Shohdy N, and Skowronski J [2001]. Mechanism for downregulation of
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Swingler S, et al. [1999]. HIV-Nef mediates lymphocyte chemotaxis and activation
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KSHV
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Problems
18.1 Engineered viruses are being devised for use in cancer therapy. The
goal of researchers is to nd natural viruses, or create genetically
modied ones, that can kill cancer cells while leaving normal cells
unharmed. This main idea behind creation of these oncolytic viruses
is to take advantage of the altered signaling pathways in the tumors
by devising viruses that selectively target those cells.

Problems

463

The rst viruses to be used in this way are the adenoviruses. These
viruses and other like them stimulate host cells to pass through the
cell cycle. If the host cell does not pass into S phase the viruses cannot
replicate their DNA. Adenoviruses encode 30 to 40 proteins that are
expressed sequentially. First immediate early genes, then early genes
and nally late genes are expressed. The rst protein to be made is
E1A. This control protein binds to the pRB protein of the host cell
and inactivates it (Figure 14.11). The E1B protein is made soon thereafter. It binds to p53 and inactivates it, too. These binding and inactivation events ensure entry of the host cells into S phase.
Recall from Chapter 14 that p53 and pRb central cell cycle controller circuitry and are mutated in most cancers. The inactivation of
these genes is an important step in transforming the host cell into one
that supports replication of the adenoviruses. How would you engineer an adenovirus to kill cancerous cells possessing mutated p53 proteins while leaving normal cells unharmed?
18.2 Some cells are permissive with respect to a virus while others are not.
In a permissive cell, the virus can gain entry by binding a receptor and
once inside the cell the viruses can replicate themselves. In nonpermissive cells, there arent any receptors that can be exploited for entry,
and if they do the signaling pathways do not support completion of
the viral replication cycle.
One of the signaling pathways often altered in cancer cells is the
Ras signaling pathway. One of the downstream targets in this growthpromoting pathway is the antiviral agent PKR. As mentioned earlier,
when activated PKR shuts down proteins synthesis thereby preventing viral replication. Phosphorylation by one or more kinases acting
downstream from Ras represses this activity. How would you engineer
an oncolytic virus to kill cancerous cells possessing mutated Ras
proteins while leaving normal cells unharmed? Note: dsRNA viruses
often encode an anti-PKR protein.

19
Ion Channels

The plasma membranes of nerve and muscle cells differ from those of other
cells in the body. Whereas the potential across the plasma membrane of any
cell will change when the ion permeability across the membrane is altered,
only in nerve and muscle cells will the converse occur. That is, in nerve and
muscle cells, the ion permeability will change as a result of a change in membrane potential. This property is a consequence of the presence in the
plasma membrane of large numbers of ion channels that open and close in
response to changes in voltage. When an ion channel opens, up to 10 million
ions can ow though its conducting pore per second, generating a few
picoamperes (10-12 amps) of current. The presence of a few thousand of
these channels endows the plasma membrane with the ability to actively
conduct action potentials. Ion channels are responsible for all electrical
signaling, and regulate a variety of processes including osmotic balance,
hormone release, heartbeat, motor movement, learning, and memory.
Ion channels render the plasma membrane permeable to the passage of
certain inorganic cations (Na+, K+, Ca2+) and anions (Cl-). The channels are
composed of several subunits arranged in a way that creates a hollow pore
that permits the one-way passive diffusion of ions from either outside the
cell in, or inside the cell out. Some channels permit the passage of sodium,
while others allow potassium ions or calcium ions or chloride ions to pass
through. Ion channels are not open all the time, but rather open and close
in response to extracellular ligand (neurotransmitter) binding, intracellular
ligand binding, or to changes in membrane voltage. They possess one or
more gates that mechanically open and close their pores to the passage
of ions in response to the regulatory signals.
The rst part of the chapter is devoted to an examination of how membrane potentials arise and how action potential can be generated from
voltage-dependent changes in the permeability of the plasma membrane. A
number of key biophysical concepts such as channel gating, and voltagedependent activation and deactivation will be introduced along with the
Nernst and HodgkinHuxley equations. The second part of the chapter is
dedicated to the mechanisms that make properties such as gating possible.
465

466

19. Ion Channels

These mechanisms are revealed by electron microscopy and X-ray crystallography studies of the atomic structure of the ion channel proteins.

19.1 How Membrane Potentials Arise

Membrane potentials arise from ion concentration differences and selective permeability. Nerve and muscle cells, unlike other cell types in the body,
possess appreciable membrane potentials or membrane potential differences. Their membrane potentials arise from differences in ionic concentrations between outside and inside environments and from the selective
permeability of the membrane to specic ion species due to the presence
of ion channels. To see how ion concentrations and selective permeability
give rise to membrane potentials four ion species will be considered
sodium (Na+), potassium (K+), chloride (Cl-), and A-. The A- ion species
represents the net negative charge of the large biomolecules residing within
the cell. The four ion species are distributed outside and inside the cell
subject to two conditions:
Osmotic balanceThe net concentration of ions outside the cell must
equal the net concentration of ions inside the cell.
Charge balanceThe net charge both outside the cell and inside the cell
must be zero.
The rst condition ensured that the cell will not be subject to osmotic
stresses and the second condition is a charge equalization requirement in
each compartment. The concentrations of these four ion species for a representative nerve cell are depicted in Figure 19.1(a). As can be seen by

Figure 19.1. Ion concentrations inside and outside the cell and membrane permeability: (a) Inside and outside are separated by an impermeable membrane
represented by the double dashed lines. (b) The membrane separating the two
compartments contains ion channels and is permeable to passage of potassium and
chloride ions into the cell and sodium ions out of the cell. Ion concentrations are
shown in units of millimoles per liter (mM/liter or mM).

19.1 How Membrane Potentials Arise

467

adding up the concentrations the osmotic and charge conditions are

satised.
The membrane separating inside and outside in part (a) of the gure is
impermeable to the passage of ions. Since there is no membrane permeability there is no membrane potential. Consider now what happens when
ion channels are added as in part (b) of Figure 19.1. Channels for K+, Na+,
and Cl- ions are now present, but not for A- ions, which are too large to
pass through an ion channel. The sodium and chloride channels will be neglected for the moment, with the main attention on the K+ and the A- ion
concentration.
Potassium channels allow potassium ions to passively diffuse out of the
cell. They will be driven to do so by the potassium concentration gradient
set up because the potassium concentrations outside and inside the cell
differ. As potassium ions diffuse out of the cell a net negative charge will
build up within the cell since the negative charge associated with the Aions exceeds the positive charge of the remaining potassium ions. The
buildup of negative charge will oppose the outward ow of positively
charged potassium ions, eventually reaching the point where it is strong
enough to halt the ow of ions. At this point an equilibrium situation is
reached. There is a potential difference across the membrane and this
potential just balances the concentration gradient. The potential difference
required to halt the ow of potassium ions through the potassium channels
is the potassium membrane potential, similar to the situation for other ion
species. These potentials are given by the Nernst equation:
EX =

RT [X]out
ln
.
zF
[X]in

(19.1)

In the Nernst equation, R is the universal gas constant, T is the absolute

temperature, F is Faradays constant, z is the valence of the ion (+1 for
sodium and potassium; +2 for calcium, and -1 for chloride), and X stands
for the ion species under consideration. At a temperature of 37 Celsius,
RT/F = 26.73 mV, and switching from natural logarithms to base 10
logarithms:
EX =

[X]out
61.54
log
.
z
[X]in

(19.2)

The potentials given by the Nernst equation are commonly referred to as

reversal potentials, or alternatively, as equilibrium potentials. They can be
either positive or negative, depending on the sign of z and on which concentration is greaterinside or outside. As noted in Table 19.1, the reversal potentials for sodium and calcium are positive while those for potassium
and chloride are negative.
The plasma membrane of cells is more permeable to some ions than to
others. It is far more permeable to potassium ions than to sodium ions, and

468

19. Ion Channels

Table 19.1. Values for typical concentrations of four main kinds of ions inside
and outside mammalian skeletal muscle cells are presented along with the
values for the equilibrium potential calculated from the Nernst equation.
Ion species

[X]out(mM)

Na+
K+
ClCa2+

145
4
123
1.5

[X]in(mM)
12
155
4
10-4

Ex(mV)
+67
-98
-92
+129

somewhat more permeable to potassium ions than to chloride ions. The

resting potential of a membrane (Vm) is given by combining the currents
from the various ion species, taking into account not only the concentrations outside and inside but also the relative permeabilities. A simple
expression, known as the GoldmanHodgkinKatz equation, incorporates
both of these factors:
Vm = 61.54 log

[K]out + ( pNa pk )[Na]out + ( pCl pk )[Cl]in

.
[K]in + ( pNa pk )[Na]in + ( pCl pk )[Cl]out

(19.3)

As is customary, the calcium contribution, which is small, has been neglected. In Eq. 19.3, pK, pNa and pCl are permeability constants. The appearance of their ratios in Eq. 19.3 takes into account the relative permeabilities
of the membrane to the different ion species. Typical resting potentials are
in the range -60 to -80 mV. These values are far closer to the potassium
equilibrium potential than to the sodium equilibrium potential. The closeness of the membrane resting potential to the potassium reversal potential
is a reection of a far greater permeability for potassium than for either
sodium or chloride. As their permeability ratios in the GHK equation
become negligible, the expression for the resting potential approaches that
of the Nernst equation for potassium.The reversal potential may be thought
of as supplying a driving force for changes in resting potential.

19.2 Membrane and Action Potentials Have

Regenerative Properties
Action potentials are generated by the joint action of fast-acting sodium
channels and delayed-acting potassium channels. The generation of an
action potential begins with a threshold-exceeding depolarization that
opens sodium channels. When this happens the permeability of the membrane to sodium increases; according to the GHK equation the membrane
depolarizes as its potential moves towards the equilibrium value for
sodium. The open sodium channels permit the entry of sodium cations into
the cell, which depolarize the membrane even further allowing for an

19.2 Membrane and Action Potentials Have Regenerative Properties

469

opening of still more sodium channels. This continues for only a short while.
The positive feedback of sodium channel openings stimulating the further
opening of sodium channels is terminated when the channels inactivate.
At this point, the potassium channels, which also open in response to
depolarization, start to dominate. The buildup of positive charge inside the
cell through the entry of sodium cations is now countered by a decrease in
positive charge through the exit of potassium cations. This slower activity
moves the membrane potential towards the reversal potential for potassium
ions. The opening of potassium channels continues for some time resulting
in the repolarization of the membrane, and contributing to the shut down
of the sodium channels. At the start of the cycle the resting potential is
about -60 mV. The membrane depolarizes to the extent that its potential
reaches positive values due to the sodium inux. It repolarizes to hyperpolarized values of more than -70 mV driven by the potassium efux and then
relaxes back to its resting potential. The hyperpolarization stage is important. It is needed to relieve the inactivation of the sodium channels, and
prepare the membrane for the next round of activation and action potential generation.
As shown in Figure 19.2, the time course of the ow of sodium ions
through their channels differs from that of potassium ions through their
channels. Sodium and potassium channels are both activated by depolarization, but the sodium channels open for a short time and then shut down

Figure 19.2. Time courses of sodium and potassium currents through a plasma
membrane: Plotted are relative permeability values for the two ion species, that is,
the number of open channels per unit surface area as a fraction of the peak number
open channels/unit area for each ion species. Time is measured from initiation of an
action potential by a threshold exceeding depolarization.

470

19. Ion Channels

even if the membrane potential has not changed from its depolarized value.
That is, the sodium channels inactivate after being activated for a short time.
Potassium channels activate more slowly than sodium channels, reaching
their peak conductivity values after the sodium channels shut down.
However, once they reach their peak conductivity values, the potassium
channels remain open for some time. That is, potassium channels are
noninactivating.
To summarize, sodium channels launch and generate the rising part of
the action potential; positive feedback sustains it, and potassium channels
generate the slowly declining following portion of the pulse. The positive
feedback loop of the sodium channels is the key to generation of action
potentials that propagate down axons without diminishing. Once a small
depolarization is started in a limited region of space, the spread of ions laterally to neighboring spaces triggers a growing depolarization in that space
and from there to the next neighboring space and so on. The pulse does not
die out since a small depolarization triggers a large one, that is, the signal
is amplied, as a result of the positive feedback. Thus, once started, a pulse
propagates along without losing strength, that is, the pulses are regenerative. The existence of a threshold all-or-nothing response is a consequence
of the positive feedback from the sodium channels and negative feedback
from the potassium channels. Negative feedback is present because the
depolarization-induced exit of potassium hyperpolarizes the membrane,
thereby acting negatively to shut down the potassium ion ow. As a result
of both forms of feedback there is an all-or-nothing response. It takes a
certain amount of depolarization to trigger the positive feedback dominated sodium inux, and once started the process runs to completion.

19.3 HodgkinHuxley Equations Describe How Action

Potentials Arise
In 1952, Alan Hodgkin and Andrew Huxley constructed a mathematical
model of the time course of sodium and potassium permeability changes in
the giant axon of the squid. There are two ways to preserve and speed up
the conduction of electrical pulses over long distances in an axon: either by
increasing the diameter or by adding an insulating fatty sheath (myelination). The squid giant axon is unmyelinated and makes use of the rst of
the two solutions to the conduction problem. It is enormous, with a diameter that can reach 1 mm. It can be easily manipulated in the laboratory,
and is thus is an ideal system for laboratory studies of how pulses are transmitted down nerves. The HodgkinHuxley model represents the culmination of decades of study by Hodgkin, Huxley, Katz, and many others on how
electrical pulses are produced, and how they propagate, in nerve tissue. The
development of the model was preceded by their detailed experimental
studies of the ow of electrical current through the surface membrane of

19.3 HodgkinHuxley Equations Describe How Action Potentials Arise

471

the squid giant axon. In these studies, a method, the voltage clamp, was
developed that allowed researchers to directly measure current ow across
the membrane of the axon.
The basic elements of the HodgkinHuxley model are a set of ionic currents separated into distinct contributions from sodium and potassium ions,
and from the leakage current, representing the fairly constant and voltageindependent, background leakage of ions through the plasma membrane.
These three currents are related to membrane permeabilities, which are
expressed in terms of ionic conductances gNa, gK and gL, and the displacements of the membrane potential from the reversal potentials for the ion
species. The relationship between current, conductance, membrane potential, and reversal potential is from Ohms law,
I x = gx (Vm - Ex ).

(19.4)

In the above, X denotes the ion species under consideration, Vm the membrane potential, Ix is its current, Ex is its reversal potential, and gx is its conductance, the inverse of its resistance Rx, that is,
gx = 1 Rx .

(19.5)

In elementary circuits, R is a constant, and Ohms law says that current and
voltage are linearly related. This is not the case for a biological membrane.
Although the linear form of Ohms law is retained in the HodgkinHuxley
equations, the sodium and potassium conductance are no longer constants,
but instead are treated as functions of the membrane voltage.
Because of the nonconducting nature of the lipid bilayer the plasma
membrane can be thought of as a capacitor that permits a buildup of charge
on its surfaces. If a current is applied part will contribute to charging up the
plasma membrane and part will ow through the membrane into the cell.
The net ionic current is the sum of the sodium, potassium, and leakage currents plus any external applied or synaptic currents. The capacitive current
IC is, from Faradays law,
IC = C

dVm
.
dt

(19.6)

In this expression, C is the capacitance, and dVm/dt is the time rate of change
in the voltage. The membrane voltage does not instantaneously adapt to the
ow of charge in and out of the cell. Instead, it takes a certain amount of
time for charge to build up on the membrane and the voltage to adjust to
the current ow. The complete circuit equation, expressing the conservation
of charge, is
C

dVm
= - g K (Vm - EK ) - g Na (Vm - ENa ) - g L (Vm - EL ) + I ,
dt

(19.7)

where I is any applied or synaptic current and EL is leakage current.

An equivalent circuit for the membrane and its ion channels is presented
in Figure 19.3. The HodgkinHuxley equation describes an electrical circuit

472

19. Ion Channels

Figure 19.3. Electrical circuit described by the HodgkinHuxley equation: As is

customary, the capacitor residing in the capacitive current branch is represented by
a pair of short parallel lines, and the batteries in the ionic current branches by pairs
of short-long lines denoting cathode (-) and anode (+). Arrows crossing through
the resistors symbolize their non-ohmic character. Arrows show the direction of the
current ow, sodium and chloride ions inward and potassium ions outward.

with branches for sodium current, potassium current, leakage current,

capacitive current, and any external or synaptic currents. If desired, any
other ionic currents such as chloride current can be added to the equation
in the same way. Ionic currents are small compared to the ionic concentrations outside and inside a neuron and thus do not change the concentrations appreciably. Therefore, the concentration gradients are represented in
the circuit diagram by simple batteries that supply currents, one type of
battery for each ion species. The voltage across the terminals in these batteries is the reversal potential for that ion species represented by the Nernst
equation: approximately +60 mV for sodium, -90 mV for potassium, and
-75 mV for chloride. Time and voltage-varying resistors are present in each
branch of the circuit.

19.4 Ion Channels Have Gates that Open and Close

The non-ohmic character of membrane resistance is of considerable signicance. In a non-ohmic, or rectifying, resistor, the ow of ions through a particular kind of ion channel is directional. That is, ions will pass through the
ion channels in one direction more easily than in the other direction. If the
ions pass more easily into the cell, the ion channel is inward rectifying, and
if they pass more readily out of the cell, the ion channel is outward rectifying. The rectifying character of the membrane permeability naturally leads
to the introduction of gates that open and close to permit the ow of ions
through the channels.

19.4 Ion Channels Have Gates that Open and Close

473

To model the opening and closing of sodium and potassium channels in

response to depolarization of the nerve membrane, Hodgkin and Huxley
introduced three gates. Recall that the sodium channels rapidly open as the
membrane is depolarized and then shut after a short time interval. Hodgkin
and Huxley describe this behavior in terms of two gates: an activation gate
and an inactivation gate. Initially, at the resting membrane potential, the
activation gate is shut and the inactivation gate is open. Once the threshold depolarization, for activation is exceeded, the activation gate opens
allowing sodium ions to enter the cell. Further increases in depolarization
allow the activation gates to open fully, but the inactivation gate, which
operates more slowly, closes in response to the increasing depolarization,
thereby shutting down the ow of sodium ions.
The third gate introduced by Hodgkin and Huxley is the activation gate
for potassium. This gate is also opened by depolarization, but much more
slowly than the activation gate for sodium. As the gate for the potassium
channel opens in response to depolarization, potassium ions leave the cell,
thus opposing the effect of the sodium entry, and eventually overcoming it
to repolarize the cell. The repolarization accelerates the further closing
of sodium channels and leads to the hyperpolarization of the membrane.
When this occurs the inactivation gate is opened; the activation gate is shut,
and the membrane relaxes back to the resting potential, ready for another
round of excitation.
Activation and inactivation gates have sigmoidal (S-shaped) dependencies on voltage. The differences between activation and inactivation gates
are illustrated in Figure 19.4. As shown in the gure, inactivation gates open
when the membrane is hyperpolarized and shut when the membrane is
depolarized. Activation gates work the opposite way. They open when the
membrane is depolarized and shut when the membrane is hyperpolarized.
Hodgkin and Huxley called the sodium and potassium gates m-, h-, and
n-gates. That is, a fast m-gate describes the activation process and a slow
h-gate describes the inactivation process for the sodium entry, while the
potassium channels are controlled by a single n-gate. The sigmoidal dependence of their opening and closing was approximated by algebraic terms
cubic for the m-gate, linear for the h-gate, and quadratic for the n-gate. In
mathematical terms, dimensionless variables m, h, and n were introduced,
each a function of Vm. These variables assume values between 0 and 1 corresponding to completely closed and completely open. The sodium conductance was expressed as
g Na = g Na m3 h.

(19.8)

Similarly, the potassium conductance was written as

g K = g K n4 .
3

(19.9)

The quantities m h and n represent the fractions of open sodium and potassium channels, respectively, and the g-bars are constants representing the

474

19. Ion Channels

Figure 19.4. Activation and inactivation functions and resulting membrane

currents: The activation (diamonds) and inactivation (squares) functions represent
the amount of current owing through the ion channels relative to the maximum
possible currents. These quantities are plotted as functions of the membrane
voltage.

maximum conductances occurring when the channels are completely open.

These core equations are supplemented by a set of rate constants and rate
equations for the m-, h-, and n-gate variables.

19.5 Families of Ion Channels Expressed in Plasma

Membrane of Neurons
The human genome contains hundreds of genes that encode ion channel
subunits. The repertoire of ion channels is further increased through extensive use of alternative splicing. Still more ion channel forms are generated
by mixing together different combinations of subunits. The resulting ion
channels differ from one another in their biophysical properties in several
ways. The differ in their

ion selectivity and direction of ion movement,

gating mechanism,
topology and assembly plan,
time course of activation and inactivation, and
reversal potentials for activation and inactivation.

19.5 Families of Ion Channels Expressed in Plasma Membrane

475

Table 19.2. The four families of ion channels found in the plasma membrane of
neurons: Except for the outward directed potassium channels, ion channels allow
for the inward selective diffusion of the ion species listed in the second column.
AbbreviationsHyperpolarization-activated cyclic nucleotide gated (HCN);
chloride channel of the CLC family (ClC); nicotinic acetylcholine receptor
(nAChR); g-aminobutyric acid Type A, Type C (GABAA,C); 5-hydroxytryptamine
Type 3 (5-HT3); a-amino-3-hydroxyl-5-methyl-4-isoxazole propionate acid
(AMPA); N-methyl-d-aspartate (NMDA).
Ion channel

Selectivity

6TM cation
Calcium
HCN
Potassium
Sodium

Subunit topology

Channel assembly

6TM, loop
24TM, 4 loops

Tetrameric
Monomeric

18IM

Dimeric

4TM

Pentameric

3TM, loop

Tetrameric

Ca2+
Na+, K+
K+
Na+

Voltage-gated anion
ClC

Cl-

Cys-loop receptor
nAChR
GABAA,C
Glycine
5-HT3

Cations
Anions (Cl-)
Anions (Cl-)
Cations

Glutamate receptor
AMPA
kainate
NMDA

Na+, K+
Na+, K+
Ca2+

Ion channels have several central properties. One of these is selectivity

the ability to pass some ion species through the membrane rapidly while
preventing passage of other ion species. A closely related property is direction of ion movement. Ion channels let ions pass through in one way only.
Some let ions into the cell while others let ions leave the cell, but none allow
two-way passage. Another essential property of an ion channel is gating
the ability to open and close the pore through which the selected ions pass
in response to voltage across the membrane and to ligands, intracellular and
extracellular.
The different kinds of ion channels can be grouped into four large
families. As can be seen in Table 19.2, two of the families open and close
in response to changes in membrane voltage, and are thus said to voltagegated. One of these families permits the selective passage of cations and the
other family allows the passage of anions. The other two families are gated
by ligands. For that reason they are designated as receptor ion channels. The
voltage-gated ion channels, of which there are many different kinds, can be
further distinguished by their time courses for activation and deactivation,
and by their reversal potential. That is, by how fast they open and close, and
at which membrane voltages, as was discussed earlier in the chapter. Finally,

476

19. Ion Channels

the different families of ion channels are characterized by their subunit

composition (assembly plan) and by the manner in which the polypeptide
chain for each subunit threads back and forth through the membrane
(topology).

19.6 Assembly of Ion Channels

Ion channels are assembled from multiple subunits arranged in regular
ways to form pores through which the ions diffuse. The threading patterns
characteristic of the subunits belonging to each of the four families of ion
channels are presented in Figure 19.5. In the case of the voltage-gated
cation channels, the core unit, or basic module, consists of a polypeptide
segment that passes back and forth through the plasma membrane six times
so that the N- and C-terminals are both on the intracellular (cytoplasmic)
side. As illustrated in Figure 19.5(a), there is a loop (helix) between the fth
the sixth transmembrane segments (TM5, TM6). The loop regions from
each subunit jointly form the conducting pore and selectivity lter (the nar-

Figure 19.5. Ion channel subunit threading patterns: (a) Voltage-gated potassium
channelTransmembrane helices 5 and 6 along with the indicated loop form a
conducting pore. The positively charged residues in transmembrane segment 4 plus
portions of transmembrane helices 13 form the voltage sensor. (b) Bacterial ClC
showing the threading of 18 alpha helices of varying lengths. (c) Acetylcholine
receptor ion channel indicating the presence in the amino terminal portion of a
conserved Cys loop near the membrane surface. (d) AMPA receptor ion channel
showing the locations of S1 and S2 segments, which, along with the Flop segment,
form the ligand-binding domain.

19.6 Assembly of Ion Channels

477

Figure 19.6. Single chain voltage-gated cation channel topology: Shown in the
gure is a single 6 4-pass polypeptide chain topology characteristic of bacterial
KscA potassium channel.

rowest part of the pore) that allows certain ion species to ow through. In
channels that are voltage-gated, transmembrane segment 4 (TM4) contains
a number of positive changes (basic arginine or lysine amino acid residues),
and along with elements of TM13 functions as the voltage sensor. In channels whose gates are controlled by intracellular ligands, binding sites for the
regulators are location on the C-terminal segment that follows TM6.
Voltage-gated cation channels are assembled in one of two ways. In the
Shaker family of voltage-gated potassium channels, four of six TM core
units are arranged into the ring. In many voltage-gated sodium channels
and voltage-gated calcium channels, the amino acid residues replicated
among the four subunits form domains belonging to a single, long (2000
amino acid residues), 6 4 = 24-pass, polypeptide chain. In either assembly
mode, the result is a fourfold symmetric ring structure in the plasma membrane with a pore in the center. A typical threading pattern for a 24-pass
transmembrane protein, the bacterial KscA potassium channel, is presented
in Figure 19.6.
The threading pattern and assembly plan for voltage-gated anion channels are radically different from those of the cation channels. As shown in
Figure 19.5(b), the polypeptide chain of a ClC chloride channel subunit consists of 18 distinct alpha helices of varying lengths. Some of the helices pass
through the membrane, but others do not and one helix resides entirely in
the cytoplasm. As will be discussed shortly, the anion channel is formed by
two of these subunits, and in place of a single pore in the center there are
two off-center pores.
The two families of ligand-gated ion channels can be further divided into
several smaller families according to the types of ligands, or neurotransmitters, being bound. Some ligand-gated ion channels permit the passage
into the cell of cations, while others allow anions such as chloride to enter
the cell.When an anion enters the cell the net charge inside the cell becomes
more negative and the membrane voltage shifts to more hyperpolarized
values. Since action potentials are generated by threshold-exceeding depolarizations, the entry of negative ions such as chlorine suppresses ring.
Neurotransmitters such as GABAA,C and glycine that act through anionic
receptors are referred to as inhibitory neurotransmitters. Conversely,

478

19. Ion Channels

neurotransmitters such as glutamate and acetylcholine that act though

cationic receptors are referred to as excitatory neurotranmitters.

19.7 Design and Function of Ion Channels

Ion channels are modular in design and function as small molecular
machines. Voltage-gated ion channels are modular in design. Each ion
channel is a small molecular machine with core (alpha) subunits that form
its pore, voltage sensor, and selectivity lter. Voltage-gated sodium, potassium, and calcium channels possess one or two regulatory b subunits.
Calcium channels contain not only a 6TM a1 core subunit and a cytoplasmic b subunit, but also an extracellular a2 subunit linked to a single-pass
transmembrane d subunit by disulde bridges (the a2-d subunit), and, in
skeletal muscle and some neurons, a 4TM g subunit. These subunits all have
regulatory roles in the operation of the channels.
The gates of the ion channels are electro- and chemo-mechanical devices
that open and close by means of conformational changes. The voltage
sensor (or ligand-binding domain) is connected to the helices forming the
pore. In response to voltage changes (or ligand binding), the sensor module
does mechanical work on the helices lining the pore to alter its conformation from a closed to open one. A variety of regulatory mechanisms are
encountered in the various ion channels. These include direction-sensitive
blocking elements and regulation by second messengers, G protein bg subunits, and protein kinases and phosphatases.
Receptor ion channels alter conformation in response to ligand binding.
As was the case for other signaling receptors, the underlying mechanism is
usually an allosteric one. The receptors contain multiple ligand-binding
sites. Binding of a ligand at a regulatory site in the protein changes the electrostatic environment resulting in the stabilization of the protein in a different subset of conformations. As a result the binding properties at active
sites are changed in a reversible way. The mechanism is an indirect one
in that binding at the regulatory site inuences binding at a physically
removed primary, or active, site through conformational changes rather
than by direct physical contact.

19.8 Gates and Filters in Potassium Channels

Potassium channels have spatially separated voltage gates and selectivity
lters. A crucial feature of a voltage sensitive ion channel is its ability to
sense changes in voltage and use these changes in voltage as an energy
source to drive the opening and closing of the channel gate. The threedimensional atomic structures of several potassium channels have been
determined in a series of landmark studies. The resulting data reveal how

19.9 Voltage-Gated Chloride Channels Form a Double-Barreled Pore

479

Figure 19.7. Potassium channel voltage sensor: In the absence of depolarizing currents, the membrane is hyperpolarized. The positive charges in helix S4, along with
the other components of the S1 to S4 voltage sensor, maintain the channel in a
closed conguration. When depolarizing currents are present the S1 to S4 helices
move towards the outer leaet, and as a result the pore helices S5 and S6 shift their
orientation, thereby opening the pore.

the channels open and close in response to changes in membrane voltage

and how these machines selectively permit potassium ions to ow through.
The representation of the subunit as being composed of equal-sized
helices each passing through the membrane is a simplication that emphasizes the sequential connections between the helices. In the threedimensional structure of the subunit some of the helices are not only not
perpendicular to the plane of the membrane but actually lie within the
membrane parallel to its surfaces. The voltage-operate gate works in the following way. Charged groups in S4 together with a portion of S3 and the
loop connecting it to S4 form a charged paddle. This paddle lies entirely
within the lipid bilayer, and changes its orientation in response to changes
in membrane voltage. As illustrated in Figure 19.7, when the membrane is
depolarized the paddle tugs on other helices pulling open the pore to let
the ions through.
The selectivity lter is separated in position from the gate. The subunit
6-pass chain contains an intramembrane loop between segments 5 and 6.
These last two segments and the loop from each of the four subunits of the
protein form the ion selectivity lter and pore. As can be seen in Figure
19.8, the overall shape of the channel is that of an inverted tepee with
the broad portion including the intramembrane loops near the top and the
narrowest portion at the bottom.

19.9 Voltage-Gated Chloride Channels Form a

Double-Barreled Pore
The second set of voltage-gated ion channels is the anion, or chloride,
channel family. These ion channels are often referred to as the ClC family
to distinguish them from the cystic brosis transmembrane conductance

480

19. Ion Channels

Figure 19.8. Potassium channel pore:
Shown is the tetramer formed by membranespanning S5 and S6 segments and connecting
intracellular loop from each domain from a
bacterial (KscA) potassium channel. The
data determined by means of X-ray crystallography were rendered as a ribbon diagram
showing the a-helices and connecting loops.
The top of the tetramer lies in the extracellular space and the bottom is in the intracellular region. The gure was generated using
Protein Explorer with atomic coordinates
deposited in the Brookhaven Protein Data
Bank under accession code 1BL8.

regulator (CFTR) chloride channels implicated in cystic brosis, and the

GABA and glycine receptors, which are ligand-gated and not voltage-gated.
The ClC ion channels are found in the plasma membrane and in the membranes of internal organelles. These anion channels are not responsible for
generating action potentials, but instead regulate the excitability of muscle
and nerve cells, the acidication of internal organelles, and cell volume.
The X-ray crystal structure of two bacterial ClC family members reveal
a radically different architecture and pore structure from the voltage-gated
cation channels. In place of the four (or ve) subunits used to form a centrally positioned conducting pore, only two subunits are utilized. Each
subunit is quite large, and consists of 18 a-helices, some short and others
long enough to pass through the membrane. Each subunit is large enough
to form its own pore, and two of these subunits assemble into the chloride
ion channel.
A side view of the ion channel formed by a pair of identical subunits is
presented in Figure 19.9. As can be seen, the channel possesses a pair of
off-center pores. As was the case for the cation channels the helices are oriented in a variety of ways. These varied orientations are not arbitrary but
instead bring together the charged residues in the right way, from different
positions in the polypeptide chain, to form the gate and selectivity lter.

19.10 Nicotinic Acetylcholine Receptors Are

Ligand-Gated Ion Channels
Ligand binding controls the gates in the other two superfamilies of ion
channels. One of these families is the cys-loop, or acetylcholine, family. In
this family, the polypeptide chains comprising the subunits pass through the
plasma membrane four times. The name for this family is derived from one

19.10 Nicotinic Acetylcholine Receptors Are Ligand-Gated Ion Channels

481

Figure 19.9. The ClC chloride ion channel determined by X-ray crystallography: A
side view of the dimeric chloride-selective ion channel is presented. The approximate locations of the selectivity lter in the two subunits are denoted by the circled
Xs. The gure was generated using Protein Explorer with atomic coordinates
deposited in the Brookhaven Protein Data Bank under accession code 1KPK.

of the structural properties of the subunits. Their extracellular amino terminal contains a cys-loopa pair of disulde-bridged cysteines separated
by a stretch of 13 amino acid residues.
Nicotinic acetylcholine receptor ion channels are responsible for excitatory signaling at the connections between nerve and muscle cells, or neuromusclular junctions, located throughout the body. There are two types of
acetylcholine receptor ion channelsnicotinic and muscarinic. The former
bind to the nicotine whereas the latter binds to the muscarine, like nicotine
an alkaloid. Those nicotinic receptors, located at neuromuscular junctions,
are termed muscle-type nAChRs, while those found at neuron-to-neuron
contacts in the spinal cord ganglia and in the brain are referred to as neuronal nAChRs. As their name indicates, the neuronal nicotinic receptors
mediate nicotine addiction in smokers, whereas muscle-relaxing, paralyzing, and spasm-promoting toxins target neuromuscular nAChRs. Examples
of toxins that target nicotinic acetylcholine receptors are atropine, curare,
and scopolamine, all from plants, and cone snail and cobra toxins.
The nAChRs are pentamers, assembled in a symmetric or nearly symmetric ring with a pore in the center. There are 17 known vertebrate subunits. These subunits belong to one of ve groupsa, b, g, d, and e. Neuronal
receptors are typically homopentamers of a particular a subunit or heteropentamers of a and b subunits. The muscle-type of nAChR is less varied.
These receptors consist of two a1 subunits, plus one each of a b1, d, and g
or e subunit.
Perhaps more has been learned about the acetylcholine receptor than
any other neurotransmitter-gated ion channel. It has been studied with

482

19. Ion Channels

increasing resolution by electron microscopy for 30 years. Receptor ion

channel subunits consist of several segments connected by exible disordered loops. As a result of this architecture it is difcult to impossible to
make crystals from entire subunit chains. However, crystals can be fabricated from portions of the subunits. Receptor ion channels, like so many
other proteins, are highly modular, and this modularity can be exploited to
arrive at a consistent picture of how the ion channels work by studying the
channels part-by-part.
Each acetylcholine receptor subunit possesses an N-terminal ligandbinding domain, a transmembrane segment, and an intracellular region. As
depicted in Figure 19.5(c), the extracellular, ligand-binding domain is quite
large. There are four transmembrane segments and there is a substantial
cytoplasmic region. The ligand binding domain is capable of binding not
only to agonists, but also to antagonists. When an agonist binds the ligand
binding domain the channel formed by the transmembrane segments
rapidly opens to permit the passive diffusion of cations leading to a depolarization of the muscle cells postsynaptic membrane and contraction of
the muscle.
The acetylcholine-binding protein (AChBP) from the snail L. stagnalis is
almost identical in size and in residues crucial for ligand binding to the Nterminal ligand binding domains of nAchRs. It lacks the transmembrane
and cytoplasmic segments present in mammalian AChRs. It can be crystallized and serves as an archetypal example of a ligand binding domain.
Shown in Figure 19.10 are two views of the pentamer formed by ve iden-

Figure 19.10. Structure of the ligand-binding domain of the acetylcholine-binding

protein: (a) Top view showing the ringlike arrangement of the ve subunits to form
a conducting pore. Each subunit is shown in a different shade of gray. (b) Side view
of the ring highlighting the two units located in the foreground. The N-terminus is
at the top and the C-terminus is at the bottom of each subunit. The gure was generated using Protein Explorer with atomic coordinates deposited in the Brookhaven
Protein Data Bank under accession code 1I9B.

19.11 Operation of Glutamate Receptor Ion Channels

483

tical AChBPs. The pore formed by the ve subunits is clearly visible in this
gure. As might be expected the residues lining the inner wall of the pore
are highly hydrophilic, and many are charged. A ligand-binding site is
present in a cavity formed at the interface between each adjacent subunit.
The cavity, positioned towards the outside of the ring, is formed by a set of
loops from one subunit and a series of beta strands from the other unit.
Since there are ve interfaces, the ligand binding domain can bind up to
ve ligands.

19.11 Operation of Glutamate Receptor

Ion Channels
The second family of ligand gated ion channels is the glutamate family. Glutamate receptor ion channels are prominent components of sensory processing systems and contribute greatly to learning and memory processes.
They are found on the postsynaptic side of sensory/information-processing
neurons. Referring back to the HodgkinHuxley model, the cys-loop and
glutamate receptor ion channels give rise to synaptic currents in the postsynaptic cells. The ow of ions through these channels leads to changes in
membrane voltage. By this means, the ligand-gated ion channels communicate and work together with the voltage-gated channels in these cells to
control membrane excitability and convey signals. In addition to mediating
changes in membrane voltage, calcium is itself a second messenger and thus
functions as a key signaling intermediary.
The core chains of glutamate receptors pass through the plasma membrane three times. The N-terminal is positioned in the extracellular spaces
and the C-terminal is situated in the cytoplasm as illustrated in Figure
19.5(d). A pore-forming reentrant (M2) loop is situated between the rst
two transmembrane portions of the chain. Two extracellular sequences (S1
and S2) form the ligand-binding domain. One of these is located just after
the amino terminus; the other occurs between M3 and M4. The conducting
channel is assembled from four subunits. The subunits may be identical or
they may be mixtures of different subunit types. Because of their modular
structure, one kind of subunit may be swapped for another in response
to regulatory signals thereby altering the biophysical properties of the
channel.
Several kinds of receptors bind the neurotransmitter glutamate. Some
ionotropic glutamate receptors (iGluRs) bind AMPA with high afnity
while others bind either kainite or NMDA. The subunits belonging to each
of the three groups of iGluRs are listed in the table. Like the acetylcholine
receptors, the glutamate receptors are highly modular. They contain an Nterminal extracellular domain followed by an extracellular ligand binding
domain, three transmembrane segments plus a reentrant loop, and a Cterminal cytoplasmic region.

484

19. Ion Channels

Figure 19.11. The S1S2 ligand-binding
domain of the AMPA receptor: The S1,
S2, and Flop regions of the polypeptide
chain used in the S1S2 construct are identied in Figure 19.5(d). The gure was
generated using Protein Explorer with
atomic coordinates deposited in the
Brookhaven Protein Data Bank under
accession code 1FW0.

Glutamate receptor ion channels are responsible for most of the excitatory signaling in the brain. These receptors are most likely homo- and
heterotetrameric assemblages of subunits. They assemble in several stages.
Individual subunits rst associate to form dimers, and these dimers then
associate to form dimers of dimers. Like the voltage-gated ion channels, the
receptor ion channels have open and closed conformations. Ligand binding
results in stabilization of the open conguration through an allosteric mechanism. The receptors do not remain open indenitely, but rather desensitize and go back to a closed conformation soon (within milliseconds) after
ligand binding.
The S1S2 ligand-binding domain of an AMPA receptor is depicted in
Figure 19.11. The S1S2 domain depicted in this gure is a laboratory
construct made by cutting the polypeptide chain and then joining together
the S1 and S2 + Flop segments using a 13-residue-long linker. The result
is a ligand-binding domain that replicates the actions of the natural extracellular unit without the encumbrances of the other loops and transmembrane segments. As can be seen in Figure 19.11 the S1 and S2 semidomains
form a pair of jaws that grip the ligand. In the absence of ligand binding,
the jaws are open, but they close upon binding the ligand. The amount of
jaw closing is variable, and depends on which of several different kinds of
ligands are being bound. Binding by antagonists stabilizes the S1S2 domain
in an open conguration; the jaws partially close in response to binding
by partial agonists such as kainite, and close more fully in the presence of
glutamate.
The jaws are mechanically coupled of the gate. A linker that connects the
jaws to the membrane-spanning helices provides the mechanical coupling.
In the dimeric form, there are two linkers, one for each subunit. The linkers
move apart from one another as the jaws close and move nearer one
another when the jaws open. This movement opens and closes the gate.
When the jaws are open the gate is closed and when the jaws close through
agonist binding the gate opens.

References and Further Reading

485

Ions ow into the cell when the gate opens. The gate does not remain
open for very long, just a few milliseconds. The gate then closes even though
the ligand remains bound. The closing of the gate is brought on by
rearrangements of the dimer interface. The interface between subunits in
the dimer is placed under strain by the gate opening. The rearrangements
of the dimer interface relieve this strain by disengaging the conformation
changes brought on by agonist binding and shutting the gate, resulting in
receptor desensitization (to the ligand binding).

References and Further Reading

General References
Hille B [1992]. Ionic Channels of Excitable Membranes (2nd edition). Sunderland
MA: Sinauer Associates, Inc.
Kaczmarek LK, and Levitan IB [1987]. Neuromodulation. New York: Oxford
University Press, Inc.
Nicholls JG, Wallace BG, Fuchs PA, and Martin AR [2001]. From Neuron to Brain:
A Cellular Approach to the Function of the Nervous System (4th edition).
Sunderland MA: Sinauer Associates, Inc.

Voltage-Gated Potassium Channel

Doyle DA, et al. [1998]. The structure of the potassium channel: Molecular basis of
K+ conduction and selectivity. Science, 280: 6977.
Jiang YX, et al. [2003]. X-ray structure of a voltage-dependent K+ channel. Nature,
423: 3341.
Jiang YX, et al. [2003]. The principle of gating charge movement in a voltagedependent K+ channel. Nature, 423: 4248.
Perozo E, Cortes DM, and Cuello LG [1999]. Structural rearrangements underlying K+-channel activation gating. Science, 285: 7378.
Roux B, and MacKinnon R [1999]. The cavity and pore helices in the KcsA K+
channel: Electrostatic stabilization of monovalent cations. Science, 285: 100102.
Schrempf H, et al. [1995]. A prokaryotic potassium ion channel with two predicted
transmembrane segments from Streptomyces lividans. EMBO J., 14: 51705178.
Yellen G [2002]. The voltage-gated potassium channels and their relatives. Nature,
419: 3542.

Voltage-Gated Sodium Channels

Caterall WA [2000]. From ionic currents to molecular mechanisms: The structure
and function of voltage-gated sodium channels. Neuron, 26: 1325.

Voltage-Gated Anion (Chloride) Channels

Dutzler R, Campbell EB, and MacKinnon R [2003]. Gating the selectivity lter in
ClC chloride channels. Science, 300: 108112.
Dutzler R, et al. [2002]. X-ray structure of a ClC chloride channel at 3.0 reveals
the molecular basis of anion selectivity. Nature, 415: 287294.

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19. Ion Channels

Acetylcholine Receptor Ion Channels

Brejc K, et al. [2001]. Crystal structure of an Ach-binding protein reveals the ligand
binding domain of nicotinic receptors. Nature, 411: 269276.
Cordero-Erausquin M, et al. [2000]. Nicotinic receptor function: New perspectives
from knockout mice. Trends Pharmacol. Sci., 21: 211217.
Karlin A [2002]. Emerging structure of the nicotinic acetylcholine receptors. Nature
Rev. Neurosci., 3: 102114.
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Glutamate Receptor Ion Channels

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receptors is mediated by several discrete protein-protein interactions. Neuron, 13:
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Sun Y, et al. [2002]. Mechanism of glutamate receptor desensitization. Nature, 417:
2452253.

Problems
19.1 Using the Nernst equation with R = 8.31 Joule/deg (K) mol and F =
9.65 104 C/mol, calculate the equilibrium potential for potassium,
sodium, and chloride ions at 20 degree Celsius for the inside and
outside concentrations presented in Figure 19.1.
19.2 Calculate the reversal potential using the GoldmanHodgkinKatz
equation for the concentrations presented in Table 19.1 with the following ratios of permeability constants: pNa/pK = 0.05 and pCl /pK = 0.2.
19.3 Sketch the time course of the sodium and potassium permeabilities
using Figure 19.2 as the template. Sketch the behavior of the action
potential produced by the sodium and potassium currents over that
time period. Assume a maximum positive value of +20 mV and a
maximum negative value of -70 mV, and indicate in the plot where the
action potential will reach these maximum and minimum values.

20
Neural Rhythms

The focus in the last chapter was on the potassium and sodium ion channels responsible for generating action potentials in neurons. This subject is
developed further in the present chapter where the emphasis shifts towards
how ion channels generate rhythmic discharges in neurons. Voltage-gated
ion channels, currents owing through ligand-gated ion channels located in
chemical synapses (synaptic currents), and electrical synapses (gap junctions)all these contribute to the generation of neural rhythms. A variety
of patterns can be produced, some in small populations of cells and others
in large ones. Rhythmic patterns among large populations of neurons are
associated with different sleep states and with states of arousal and attention. Small circuits of neurons known as central pattern generators generate
rhythmic behavior in motor systems. These circuits control heartbeat,
regulate respiration, and control locomotion, chewing, and digestion.
This subject of the nervous system concludes in the last chapter, where
the focus will be on the chemical synapseon how it is organized and how
it promotes learning and memory formation. The organization of the
sensory cortices will be looked at along with how they process sensory
information that is relayed in from sensory organs. Special attention will be
given to the glutamate receptor ion channels and their central role in
sensory information processing, learning, and memory formation.
This chapter is divided into three parts. In the rst part, pacemaker cells
will be introduced in a discussion of heartbeat. In the middle part of the
chapter, the low frequency synchronous ring of large populations of cells
will be examined. The main focus will be on sleep oscillations and how these
forms of rhythmicity may change into abnormal epileptic seizures. The third
part of the chapter will deal with how central pattern generators drive
muscle contractions, drawing on several invertebrate systems for examples.

20.1 Heartbeat Is Generated by Pacemaker Cells

Heartbeat is generated by rhythmic discharges from nerve cells situated in
a small region of the heart called the sinoatrial node (SAN) located in the
wall of the right atrium. Impulses from the SAN are distributed rapidly
487

488

20. Neural Rhythms

throughout the heart by a specialized conduction system and cause the

rhythmic contractions of the heart muscle. Impulses from the SAN are sent
to the atrioventricular node (AVN) and left and right bundles of Purkinje
bers, and then to locations throughout the heart. The cells in the SAN that
generate the rhythmic impulses do not require rhythmic input, but instead
generate the rhythmic patterns by themselves. Other neurons in the conduction system respond to the pacemaker cells by ring in synchrony with
them. In the absence of input from the SAN, nerve cells in the AVN and
the Purkinje bers can act as pacemakers, but they re at a lower frequency.
These cells, along with other nonpacemaking cells, can be driven by the
SAN neurons and made to re in synchrony with them.
The ability to generate rhythmic discharges is due to the mix of ion channels expressed by the neurons in the SAN and other regions of the heart.
Since the function of the neurons in each of the regions is slightly different, the neurons in these regions express slightly different mixes of ion
channels. The main ion channels contributing to the action potentials in the
heart are listed in Table 20.1. The ion channels each have a specic function, as listed in column 3. The meanings of the terms appearing in that
column are illustrated in Figures 20.1 and 20.2.
The rst entry in Table 20.1, the sodium channel, generates fast upstrokes
that are pronounced in non-SAN neurons. Funny (f) channels in the heart
are responsible for diastolic depolarization in SAN neurons. These same ion
channels are called Ih channels in the brain and are referred to as HCN
channels in the next section. The term diastolic depolarization refers to the
rising (depolarization) portion of the action potential occurring at the very
end of the SAN action potential. This depolarization allows the SAN
neurons to immediately re another action potential after completion of a
previous one, and thus enables the SAN neurons to function as the pacemakers for the entire heart.The ring appears as the rising part of the action
potential for the SAN neuron that occurs after the late repolarization stage,
as shown in Figure 20.2.
Neurons express several different kinds of calcium channels. Some
calcium channels are called L-type while others are referred to as being
N- or P/Q- or R- or T-type. Most of the calcium channels are actiTable 20.1. Ion currents contributing to the ring properties of neurons
in the heart.
Cardiac current
INa
If
ICa,L; iCa,T
IKr,Ks
It0,sus

Description
+

TTX sensitive, inward Na current

Hyperpolarization-activated inward
nonselective cation (Na+, K+) current
L- and T-type inward Ca2+ currents
Rapid (r) and slow (s) delayed rectifying
outward K+ currents
4-AP sensitive, transient (t0) and sustained
(sus) outward K+ currents

Function
Fast upstroke
Diastolic depolarization
Plateau
Late repolarization
Early repolarization

20.2 HCN Channels Role in Pacemaker Activities

489

Figure 20.1. Stereotypic cardiac action potential: A stereotypic action potential of

the form found in the heart is depicted. The rapid depolarization of the membrane,
or upstroke, leads to a peak membrane potential of +10 to +20 mV. Once the peak
depolarization is achieved there is an early phase of repolarization followed by a
slow and sometimes at regime called a plateau, and then a late, or nal, repolarization phase a few hundred milliseconds after the start of the upstroke.

vated by large depolarization while a few, most notably the T-type channels,
require only mild depolarization of the membrane. The calcium channels
requiring large depolarization are referred to as high voltage-activated
(HVA) calcium channels whereas the T-type channels are termed low
voltage-activated (LVA). The calcium currents found in the heart help maintain action potential plateaus, the fairly at and slowly declining portions
of the action potentials. Lastly, the potassium currents related in the last
two sets of entries in Table 20.1 are responsible for the early and late phases
of action potential repolarization.

20.2 HCN Channels Role in Pacemaker Activities

Hyperopolarization-activated cyclic nucleotide-gated channels contribute
to pacemaker activities in the sinoatrial node. The HCN (hyperpolarizationactivated cyclic nucleotide-gated) channel is regulated both by cyclic
nucleotides such as cAMP and by voltage. It is a member of the 6 TM family
of ion channels and possesses a cAMP-binding domain in its cytoplasmic

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20. Neural Rhythms

Figure 20.2. Action potentials in different parts of the heart: Shown are action
potentials in the sinoatrial node (SAN) and in the ventricles. The latter are delayed
by the amount of time required for the pulse from the SAN to reach the ventricular bers. The minimum, hyperpolarized values of the membrane potentials are
listed just below the dashed lines.
Table 20.2. Collective ring patterns observed in electroencephalograph recordings in the brain.
Type of activity

Frequency

Association

Delta waves
Theta waves
Alpha waves
Spindling oscillations
Beta waves
Gamma band

0.5 to 4 Hz
4 to 8 Hz
8 to 12 Hz
7 to 14 Hz
12 to 30 Hz
30 to 60 Hz

Deep sleep, abnormal states

Sleep onset, abnormal states
Drowsiness and relaxed activity
Quiescent sleep
Arousal, alert states, and information processing
Arousal, alert states, and information processing

C-terminal region. Its voltage regulation is unusual. Most ion channels are
activated by depolarization. As discussed in the last chapter, their activation gates are closed at resting membrane potentials but open when the
membrane is depolarized beyond some threshold level. The HCN channels
work the opposite way. They are activated when the membrane potential is
hyperpolarized beyond values in the range -50 to -70 mV. Whenever a
cell res an action potential the membrane hyperpolarizes and the HCN

20.2 HCN Channels Role in Pacemaker Activities

491

Figure 20.3. Activation functions for the hyperpolarization-activated cation

current: The HCN channels are activated by hyperpolarization. b-adrenergic
(adrenaline) stimulation shifts the activation function towards more depolarized
values, while cholinergic (acetylcholine) stimulation does the opposite, shifting the
activation function to more hyperpolarized values.

channel opens. Whereas the potassium channels responsible for the

descending phase of the action potential has a reversal potential that is
more negative than the resting membrane potential, the HCN channels
exhibits the opposite voltage relationship. Its reversal potential is in the
range from -20 to -30 mV, values that are less negative than that of the
resting potential. When the HCN channel opens both Na+ and K+ ions ow
into the cell to depolarize the membrane. The presence of HCN channels
provides a means of moving the membrane potential in the direction
needed to trigger action potentials and thus renders the cell more excitable.
The biophysical properties of ion channels can be altered by neuromodulators acting in a variety of ways. Two examples of neurmodulation
are included in Figure 20.3. Cardiac sinoatrial node neurons express badrenergic and cholinergic G protein-coupled receptors (GPCR). When
adrenaline binds to a GPCR, Gs alpha subunits are activated. As discussed
in Chapter 12, these subunits stimulate adenylyl cyclase to increase its the
production of cAMP. The cAMP molecules diffuse to and bind the cytoplasmic portion of the HCN ion channels. This binding shifts the activation
curve towards less negative membrane potentials. As a result the neurons
are able to re action potentials more rapidly and the heart rate goes up.
Acetylcholine has the opposite effect. When it binds its GPCR, Gi alpha
subunits are activated. These molecules bind to adenylyl cyclase and throt-

492

20. Neural Rhythms

tle back its production of cAMP. As a result the activation function shifts
to more negative membrane potentials and the heart rate slows down.

20.3 Synchronous Activity in the Central

Nervous System
Electroencephalographic (EEG) recordings of wave patterns in the brain
are a well-established tool for identifying abnormal conditions and various
stages of sleep and arousal. Several different kinds of wave patterns can be
produced in the brain, each characterized by a specic brain wave frequency
range (Table 20.2). These patterns fall into two groupslow frequency and
high frequency. The low frequency group includes delta waves, theta waves,
spindling oscillations, and alpha waves. These patterns are of large amplitude when observed in the EEG, and as such are produced by large populations of neurons undergoing synchronized ring. The high frequency
group includes the beta and gamma bands. The amplitudes of these rhythms
are far lower that those of the low frequency waves, indicative of participation by smaller populations of neurons. These patterns are not as sharply
delineated in the EEG recordings.
As indicated in Table 20.2, delta waves are associated with deep sleep and
with abnormal brain states such as coma and anesthesia. Theta oscillations
are most often associated with drowsiness and the onset of sleep. The amplitude of theta waves is elevated in a number of attention-related disorders.
Alpha waves are observed during drowsiness and relaxed activity, and
spindle activity associated with early stages of quiescent sleep. In contrast
to the low frequency regime, the high frequency activity is associated with
arousal and alert, attentive states, with complex motor and sensory processing tasks, and with rapid eye movement (REM) sleep.

20.4 Role of Low Voltage-Activated

Calcium Channels
Neurons can re action potentials in a number of ways. Neurons can be
silent and not re at all. Alternatively, they can act as spiking neurons and
generate a small number, just one or two, of action potentials at isolated
time intervals. Tonic ring is yet another mode. In tonic ring, a train of
action potentials is generated. The action potentials may be generated at
regular time intervals or they may be irregularly spaced in time. This type
of ring pattern is illustrated in Figure 20.4(a) for the case of regular
spacing. Yet another kind of ring pattern is rhythmic bursting. In this kind
of ring, illustrated in Figure 20.4(b), multiple sequences of action potentials are produced. Each sequence, or burst, consists of a train of closely
spaced action potentials. The intervals between each spike in the burst are
small compared to the time intervals between successive bursts, which are

20.4 Role of Low Voltage-Activated Calcium Channels

493

Figure 20.4. Rhythmic ring patterns, plots of

membrane potential versus time: Stereotypic
plots of current versus time are shown for
(a) tonic ring and (b) rhythmic bursting.

somewhat variable. The inverse of the average burst-to-burst time interval

is the burst frequency. These frequencies can vary from less than one per
second to 150 or 200 per second.
The creation of trains and bursts of action potentials rather than single
spikes is an important component of the rhythmicity. One of the most
important ion channels contributing to the rhythmicity is the low voltageactivated (T-type) calcium channel. Whereas high voltage-activated calcium
channels require depolarization of about 40 mV away from resting membrane potentials in order to open, low voltage-activated calcium channels
only need about 10 mV of depolarization to open. When they do open and
let in calcium, the membrane is further depolarized, resulting in activation
of sodium channels and the generation of bursts of action potentials.
The T-type calcium channels operate in a way closely resembling the
sodium channels responsible for the upstroke of the action potential. That
is, they possess activating and deactivating functions as do the sodium channels and like the sodium channels the T-type calcium channels are activated
by depolarization. There are two main differences between the two kinds
of channels. The rst difference is that the calcium channel activation and
deactivation functions are centered at larger hyperpolarizations than those
for sodium. At the resting potential, the calcium channels are activating
whereas the sodium channels are deactivating. This means that the calcium
channels can open at and near the resting potential. The entry of calcium
through these channels depolarizes the membrane and triggers the opening
of the sodium channels leading to the ring of action potentials.
The second difference between the T-type calcium channel and the
sodium channel is in how fast they respond to changes in membrane potential. The sodium channels respond quickly to changes in potential. The
calcium channels activate more slowly, and deactivate considerably more
slowly. As the membrane voltage moves up and down rapidly due to the
sodium/potassium depolarization and repolarizations the calcium current

494

20. Neural Rhythms

increases and then decreases back to zero, terminating the burst of action
potentials. The calcium channels that open to start the burst then require a
large hyperpolarization in order to de-inactivate and start the next burst.
Tonic ring and rhythmic bursting are widely encountered in the brain,
and neurons can switch from one form to the other. Large populations of
neurons in the brain undergo slow rhythmic bursting during natural, slow
wave sleep, but switch their ring patterns from bursting to tonic ring in
waking states and rapid eye movement (REM) sleep. In order to transition
from a slow wave sleep state to either awake or REM sleep states, the delta,
alpha, and spindling patterns have to be abolished and replaced by the high
frequency tonic ring in the beta band (Table 20.1).
The waking up and REM sleep tasks are carried out by collections of
neurons that the wake-up system comprises, the reticular formation, located
in the brain stem. Neurons in the reticular formation release a number of
neuromodulators, the most prominent of which are acetylcholine (ACh),
norepinephrine (NE), and serotonin (5-HT). These neuromodulators inuence the activities of the neurons generating brain rhythms and also inuence muscle states. In the brain, high frequency activity replaces the low
frequency waves. The membrane potential is depolarized in response to the
neuromodulators, and the T-type calcium channels responsible for bursting
are shut down.

20.5 Neuromodulators Modify the Activities of

Voltage-Gated Ion Channels
Up to this point, the discussion has been limited to voltage-gated ion channels and to how individual neurons re. Except for two brief discussions of
the effect of neuromodulators such as adrenaline and acetylcholine, no
mention has been made of the role networks play in generating rhythms.
Network properties such as the manner in which neurons are connected to
one another and what signals are sent and exchanged, together with the
voltage-gated ion channels intrinsic to the individual neurons, jointly
determine the nature of the neural rhythms. Three forms of cell-to-cell
communication contribute to rhythmicity. These are communication by
neuromodulators, by neurotransmitters at chemical synapses, and by small
signaling molecules at electrical synapses (gap junctions).
Neuromodulators act on ion channels to produce slow, long-lasting
changes in the excitability of the neurons. Neuromodulators are secreted
by many different neurons and bind to G protein-coupled receptors on
target cells, in contrast to neurotransmitters, which bind to ion channels.
Diffusible signaling molecules such as acetylcholine can act either as a
neurotransmitter or as a neuromodulator. Acetylcholine acts as a neurotransmitter when binding to nicotinic (ion channel) receptors and as a
neuromodulator when binding to muscarinic (G protein-coupled) recep-

20.6 Connexins Mediate Rapid Signaling Between Cells

495

tors. Similarly, glutamate and GABA each can function either as a neurotransmitter or as a neuromodulator, depending on whether it acts through
ionotropic or metabotropic (G protein-coupled) receptors. The functional
characterization of a molecule as a hormone, as a neurotransmitter, or as a
neuromodulator is determined by the nature of its receptor and what kind
of cellular response is elicited upon binding.
Recall from Chapter 12 that G protein-coupled receptors activate heterotrimeric G proteins. Neuromodulators can exert their inuences on ion
channels in two ways. In direct regulation, G protein subunits, particularly
the Gbg subunits, diffuse along the inner face of the plasma membrane and
bind to the cytoplasmic portions of ion channel subunits, thereby modifying channel conductances. Alternatively, the neuromodulators can exert
their inuences indirectly by activating second messenger systems. The
second messengers either bind the ion channels, thereby modifying their
conductances, or they activate protein kinases and protein phosphatases,
which, in turn, phosphorylate or dephosphorylate ion channel subunits,
thereby modifying their conductances. The modied states of the ion
channel subunits have lifetimes that are long compared to the time that it
takes to generate and propagate action potentials, and thus the modulatory
inuences are long-lasting ones.

20.6 Gap Junctions Formed by Connexins Mediate

Rapid Signaling Between Cells
Gap junctions are specialized communication channels that permit the rapid
exchange of metabolites, ions, and second messengers such as Ca2+ and
cAMP between pairs of neurons. The channels are nonselective and when
open allow ions of mass less than about 1 kDa to pass directly from the cytoplasm of one cell to another.These communication channels are often found
in opposing dendritic processes and are widely distributed among neurons.
Because of their direct connectivity, communication through gap junctions,
or electrical synapses, as these pores are often called, is more rapid than is
possible through chemical synapses. This rapid means of exchanging cytoplasmic signaling elements enables the communication partners to coordinate their ring activities. This synchronizing capability is utilized heavily
during the embryonic development of sensory circuits such as those found
in the retina.Although there is no sensory input at that time, the neurons are
spontaneously active. They continually communicate with one another in
order to rene the circuits formed during the initial connecting stages. Communication through gap junctions plays a similar role during development
of motor systems, and contributes to adult motor behavior as well.
Gap junctions are formed by connexons, hemipores the span the plasma
membrane of a cell.A gap junction between two adjacent cells is created when
a connexon hemipore embedded in the plasma membrane of a cell aligns and

496

20. Neural Rhythms

docks with a connexon situated in the plasma membrane of an opposing cell.

Once joined the two connexons form a communication channel permitting
one- and two-way ow of small charged and uncharged molecules. The size
restriction prevents the passage of larger molecules such as nucleic acids and
proteins. Gap junctions tend to cluster together into local regions of the
plasma membranes of adjacent cells. The two cells remain separated by a
gap of from 2 to 4 nm, hence the name gap junction.
Each connexon is erected from six connexins arranged in a symmetric
fashion to form a hollow pore through which small molecules can diffuse.
Some connexons contain a single type of connexin and the connexon mates
with an identical connexon. In these cases two homomeric connexons form
a homotypic gap junction. Alternatively, a connexon can be assembled from
more than one kind of connexin to form a heteromeric connexon. Such a
connexon can dock with either the same kind of connexon or with a different kind of connexon on the opposing cell membrane. Thus, there are a
variety of homomeric and heteromeric connexons, and these can combine
in different homotypic and heterotypic ways to form gap junctions.

Figure 20.5. Connexin topology and assembly into gap junctions: (a) Connexins
pass back and forth through the plasma membrane four times. Starting from the Nterminal end the transmembrane segments are designated as M1 through M4. The
N- and C-terminal portions of the polypeptide chains are situated in the cytoplasm
of the host cell. The C-terminal end is longer than the N-terminal end and may form
a loop. The two extracellular loops, E1 and E2, mediate connexin to connexin
contacts while the three cytoplasmic portionsthe N- and C-terminal ends and the
cytoplasmic loop (CL) connecting M2 to M3are sites for regulation of the pore.
(b) Six connexins arrange themselves in a symmetric fashion to form a hemipore
called a connexon. Two connexons, one from each cell, form the conduction pore,
or gap junction. These remain separated from one another by a 2- to 4-mm gap,
denoted in the gure by the pair of parallel lines.

20.7 Synchronization of Neural Firing

497

Like ion channels, gap junctions are voltage-gated. Gap junctions will
open and close, and change their permeabilities, in response to alterations in
potential difference. The state of the pore is responsive to differences in
membrane potential of the two cells (transjunctional voltage differences)
and to differences in potential across plasma membranes, i.e., between the
cytoplasm and extracellular spaces (inside-outside voltage differences). Gap
junctions are also responsive to biochemical changes in the intracellular environmentfor instance, the pores will close when exposed to low pH levels.
In addition, gap junctions are subject to regulation by phosphorylation.

20.7 Synchronization of Neural Firing

Gamma-aminobutyric acid (GABA) is the preeminent inhibitory neurotransmitter in the brain. As indicated in Table 19.2, GABA binds to receptors belonging to the cys-loop family of ion channels. When these ion
channels open, Cl- anions enter the cell and drive the membrane potential
towards more negative, hyperpolarizing values. There are three kinds of
GABA receptors: GABAA and GABAC receptors are chloride ion channels, while GABAB receptors are 7TM, G protein-coupled receptors.
GABA-releasing neurons are found throughout the CNS interspersed
among a larger population of excitatory, glutamate-releasing neurons. Glutamate acts through members of the glutamate family of receptor ion channels to allow Na+, K+, and Ca2+ cations to enter the cell. Most cells in the
central nervous system (CNS), inhibitory and excitatory, express GABA
receptors. These along with glutamate-binding NMDA and AMPA receptor ion channels are the predominant receptor ion channels.
GABA-releasing inhibitory neurons, referred to as interneurons, make
contacts with other interneurons to form interneuron circuits. They also
make contacts with excitatory cells, especially those expressing N-methyld-aspartate (NMDA) receptors and capable of repetitive bursting behavior. They excitatory cells make contacts with each other, as well, and
together the excitatory and inhibitory cells form large networks of interconnected cells that exhibit the population oscillations seen in the EEGs.
Inhibition mediated by GABAergic neurons and by gap junctions synchronizes the ring of neurons. The GABAergic interneurons synchronize
their ring through release of GABA and the subsequent activation of the
chloride channels. Their synchrony is greatly improved, and in some situations predominantly mediated, by gap junctions. There are a number of different kinds of interneurons. Each population of interneurons expresses a
particular kind of gap junction that enables it to communicate with other
interneurons of the same kind. For example, one kind of interneuron, the
fast spiking (FS) interneuron, will communicate through gap junctions with
other FS interneurons. The networks of interneurons collectively act as a
pacemaker circuit for the larger circuits involving the excitatory cells. In
these situations, the gap junctions operate in combination with NMDA

498

20. Neural Rhythms

receptors that endow the neuron with intrinsic rhythmic bursting properties. In the absence of the gap junctions and the inhibitory connections, each
excitatory neuron would carry out its program of rhythmic bursting in a distinct and uncoordinated manner. When the inhibitory circuitry is present,
the cells synchronize their ring patterns. They can undergo rhythmic discharges at low frequencies and also at the higher frequencies characteristic of the beta and gamma bands of the EEG.

20.8 How Spindling Patterns Are Generated

Spindle waves (Figure 20.6) are generated during the early stages of quiescent sleep. Large populations of neurons in the thalamus and cortex undergo
rhythmic discharges in the 7 to 14 Hz range. These oscillations wax and wane
with a 1- to 3-second period. That is, the oscillations steadily increase in magnitude up to a maximum and then decrease until they vanish some 1 to 3
seconds after they started. After a silent period of 3 to 20 seconds they start
up again, and the waxing and waning pattern repeats itself.
Spindle rhythms are generated in thalamic neurons. Two populations of
cells are involvedinhibitory neurons (GABAergic) in the thalamic reticular nucleus that make reciprocal connections with excitatory neurons in the
thalamic relay nucleus. When pulses of GABA are released at the presynaptic terminal of thalamic reticular neurons, inhibitory postsynaptic potentials
(IPSPs) are produced in the relay nuclei. These hyperpolarizing events activate the Ih channels, which permit the generation of action potentials in the
relay neurons, which through their reciprocal synapses with the reticular
neurons trigger membrane depolarization and re-excite the reticular cells.

20.9 Epileptic Seizures and Abnormal Brain Rhythms

Excitation mediated through glutamergic cationic channels must be in
balance with inhibition mediated by GABAergic anionic channels. One of
the consequences of an imbalance between excitation and inhibition is the

Figure 20.6. Plot of membrane potential

versus time for 7- to 14-Hz spindle oscillations: The train of action potentials rst
waxes and then wanes in amplitude.

20.10 Swimming and Digestive Rhythms in Lower Vertebrates

499

occurrence of epileptic seizures. These abnormal events are generated when

there is too much excitation (hyperexcitabilty) leading to aberrant rhythms
and epileptic seizures.
Because of their ability to generate rhythmic discharges, neurons in the
neocortex, thalamus, and hippocampus are ideal sites for aberrant rhythms
leading to epileptic events and seizures. These regions of the brain contain
large numbers of excitatory, intrinsically bursting pyramidal cells that are
massively interconnected with one another, and smaller populations of
several different kinds of inhibitory interneurons that, as just discussed, not
only make connections with one another but also make contact with large
numbers of excitatory pyramidal cells.
One of the best-studied forms of epilepsy is the absence seizure,or petit mal
epilepsy.This type of seizure appears in EEGs as an aberrant 3-Hz spike-andwave pattern.Absence seizures occur most often in children and adolescents,
and are characterized behaviorally as brief (~10 s), nonconvulsive interruptions in consciousness. The spike-and-wave patterns are generated by the
general thalamic/thalamocortical circuitry that produces the spindling patterns. One of the likely causes of this type of aberrant brain wave pattern is
malfunctioning GABAA receptors. These receptors are erected from mixtures of different GABA subunits. The different kinds of GABAA receptor
subunits are designated as a, b, g, d, e, p, or r. Several different isoforms exist
for many of the subunit types. Ion channels built from r subunits are called
GABAC ion channels.The GABA channels are constructed from ve subunits
and, like the other ion channels, its subunits are arranged to form a hollow
conducting pore. A stoichiometric combination that is widely distributed
throughout the brain is one asubunit,two bsubunits and two gsubunits.Mutations in the g2 subunit have been implicated as a possible cause of absence
seizures in several experimental studies, while theoretical modeling efforts
have demonstrated how malfunctions in GABAA and GABAB receptors can
generate aberrant 3-Hz spike-and-wave rhythms.
That mutations in an ion channel subunit can cause aberrant rhythms and
have behavioral consequences is not unique to the GABA and absence
seizures. Rather, mutations in other ion channel subunit exits have similar
behavioral consequences. These include mutations in the channel-forming
a subunit of high voltage-activated, P/Q-type calcium channels, in other
GABA subunits, and in potassium and sodium channel subunits. Thus, there
is an entire ensemble of ion channel disorders, or channelpathies.

20.10 Swimming and Digestive Rhythms in

Lower Vertebrates
Central pattern generators located in the spinal cord of lower vertebrates
generate swimming and digestive rhythms. The rhythmic movements of
muscles are controlled by neurons located in the brain stem and spinal cord.

500

20. Neural Rhythms

These neurons are organized into small neural circuits called central pattern
generators (CPGs). The CPGs may be thought of as elementary circuits,
autonomous modules built from small numbers of neurons that are used to
drive locomotion activities such as walking and swimming, breathing, and
chewing and digestion. Many of the principles governing their operation
are shared by other rhythm-producing systems such as those responsible
for sleep and alert behavior. Processes that generate rhythms, processes
such as reciprocal connections involving inhibition, and burst-promoting
voltage-gated ion channels, are common to all rhythm-producing circuits.
Motor neurons supply input to muscles that drives their contractions.
They are the last stage of neural connectivity before the muscles. Inputs
from different drivers such as the CPGs converge upon these cells. Some
of the regulatory inputs come directly to the motor neurons while others
converge upon the CPGs to regulate their ring patterns.
Invertebrates are widely used as model systems for studying central
pattern generators.The invertebrate CPGs exhibit all of the main properties
of vertebrate CPGs, yet are small enough to be well characterized at the
network and ion channel levels.The invertebrates selected as model systems
include the nudibranch Tritonia diomedea, the sea snail Aplysia californica,
the decapod crustaceans Panulirus interruptus (spiny lobster) and Cancer
borealis (rock crab), and the medicinal leech Hirudo medicinalis.
The stomatogastric ganglion (STG) of the lobster and crab is the site of
a pair of central pattern generators that supply digestive rhythms. These
crustaceans swallow their food whole. The material ingested is broken down
in the stomach through rhythmic contractions of gastric teeth and the
stomach. Several different kinds of rhythms are produced during digestion.
One of these is the gastric rhythm that controls the gastric teeth, and
another is the pyloric rhythm that regulates the food sorting and sifting
machinery. The rhythms are supplied by two CPGs. The CPG responsible
for the pyloric rhythm contains 14 neurons. Of these 13 are motor neurons
whose output drives the muscle contractions, and one is a pacemaker
neuron. The CPG responsible for gastric rhythms contains just 11 neurons.
However, its wiring and operation is more complex than that of the pyloric
circuit, and the discussion will be limited to the latter.
The pyloric circuit is presented in Figure 20.7. There are six different
kinds of neurons in the circuit. One each of these neuron types and its connectivity is shown in the gure. Connection leading to cells outside the CPG
are omitted to maintain clarity. As can be seen in the diagram the neurons
in the pyloric central pattern generator are connected to one another by
electrical synapses and by chemical synapses. The AB neuron functions as
the pacemaker neuron for the circuit. It produces bursts of action potentials at regular intervals. Electrical synapses link the AB and PD neurons
to one another. As a result the PD neuron res in synchrony with the AB
neuron, and these two neurons may be regarded jointly as the network
pacemaker. As is shown in Figure 20.7, these two neurons establish contact
with all the downstream motor neurons.

20.10 Swimming and Digestive Rhythms in Lower Vertebrates

501

Figure 20.7. Crustacean pyloric circuit: The central pattern generator responsible
for pyloric rhythms consists of anterior burster (AB), pyloric dilator (PD), ventricular dilator (VD), inferior cardiac (IC), lateral pyloric (LP), and pyloric (PY)
neurons. Resistor symbols denote electrical synapses, and lines terminating in lled
circles represent inhibitory chemical synaptic connections.

Figure 20.8. Mutual inhibition: Two cells, A and B, are coupled to one another by
means of inhibitory synaptic connections. Because of this coupling the two cells
alternatively re synchronized sequences of action potentials. The dashed lines
denote the gradual depolarization of the membrane potential towards the plateau
potential and threshold for ring action potentials.

The second main feature of the network is wide usage of mutual inhibition in which pairs of cells are reciprocally connected to each other and
inhibit one another. Mutual inhibition produces alternative ring pattern
that can drive the phased contractions of opposing muscles. A stereotypic
pattern of alternating ring by a pair of cells mutually inhibiting one
another is depicted in Figure 20.8. In the pyloric circuit, the AB and PD
neurons re rst, inhibiting the ring of the downstream neurons until the
bursting is completed. The downstream neurons then re in a sequence

502

20. Neural Rhythms

determined by the synaptic connections and by the mix of ion channels

expressed by each neuron.
Two kinds of neurotransmitters are released at the presynaptic terminalsglutamate and acetylcholine. These neurotransmitters diffuse across
the synaptic cleft and bind to receptors expressed on the postsynaptic
cell. In crustaceans, glutamate and acetylcholine both act as inhibitory, not
excitatory, neurotransmitters. The glutamate receptors are members of an
inhibitory glutamate receptor ion channel (IGluR) family. They resemble
the GABA and glycine receptors, and open to permit the entry of Cl- ions
into the cell, as do the crustacean acetylcholine receptor ion channels.

20.11 CPGs Have a Number of Common Features

Many of the key design elements found in the stomatogastric ganglion
(STG) pyloric circuit are encountered in other central pattern generators.
Some of these properties, such as the occurrence of certain sets of ion channels, operate at the cellular level. Other properties, such as mutual inhibition, are related to how the circuits are built, and operate at the network
level. A summary of design properties common to many CPGs is presented
in Table 20.3.
One of the most common properties is spike frequency adaptation. Small
conductance, calcium activated potassium channels are quite sensitive to
small changes in calcium concentration. Although the effect of sodium and
potassium is small compared to their concentrations within the cell, the
same is not true for calcium. Its concentration in the cell in quite low, and
small changes in calcium concentration can be sensed and responded to.
The small conductance, calcium-activated potassium channels open in
response to changes in calcium concentration. The effect of these openings
is to hyperpolarize the membrane and decrease its excitability in response
to sustained ring. The sequence of pulses exhibits an increasing time lag
between successive pulses, and eventually reaches a steady state ring
frequency.
Table 20.3. Amine and peptide neuromodulators expressed in humans: Abbreviations for specic neuromodulators (excepting the general opioid and tachykinin
categories) are shown in parentheses.
Amines
Acetylcholine (ACh)
Dopamine (DA)
Epinephrine (E)
Histamine (HA)
Norepinephrine (NE)
Octopamine (OCT)
Serotonin (5-HT)

Peptides
Corticotropin-releasing factor (CRF)
Cortistatin (CST)
Opioids (dynorphins, endorphins)
Oxytocin (OT)
Somatostatin (SST)
Tachykinins (substance P, neurokinins)
Vasopressin (AVP)

20.11 CPGs Have a Number of Common Features

503

Table 20.4. Properties of central pattern generators.

Property
Cellular
Rhythmic bursting

Plateau potentials

Spike frequency adaptation

Post-inhibitory rebound

Network
Input signals that initiate rhythms
Feedback signals that regulate
rhythms
Highly interconnected circuits that
generate rhythms
Pacemaker cells that generate
rhythms
Regulatory
Extrinsic
Intrinsic

Description
Pacemaker and other rhythm-producing
neurons express HCN ion channels that
support rhythmic bursting
Increase excitability resulting from a stable
state lying close to thresholds for action
potential generation
Small conductance, calcium-activated
potassium channels allow for adaptation of
the ring rate
Low voltage-activated (T-type) calcium
channels permit the temporary increase in
excitability
Input from sensory neurons, and from neurons
in brain stem control areas
Mechanical and chemical signals that are used
to ne tune the rhythms
Mutual inhibition and gap junctions ensure the
proper ring sequences and produce synchrony
Cells that entrain other cells in the CPG

Neuromodulatory signals sent from neurons

outside the pattern generation circuit
Neuromodulatory signals sent and received
within the rhythm-producing circuit

Reciprocal (mutual) inhibition is another widely encountered network

property. The underlying phenomenon is a simple one. Suppose that two
cells, A and B, expressing the ion channels mentioned in Table 20.4, mutually inhibit one another. Further suppose that A res rst. It will inhibit B
from ring while it is bursting, but then will exhibit spike frequency adaptation or simply cease to re. This cessation will release B from its inhibition. B will re and inhibit A from ring until it too has completed its ring
sequence. A will then be released from inhibition and the cycle will repeat.
Alternatively, if the B cell being inhibited exhibits a gradual depolarization
of its membrane, as illustrated in Figure 20.8, it will escape from the inhibition when its membrane potential depolarizes sufciently. When it starts
ring it will inhibit the A cell from ring and shut down its burst. This will
continue until the membrane of the A cell depolarizes and so on.
Another property commonly encountered in rhythmically ring neurons
is the presence of plateau potentials. Many motor neurons exhibit bistability. Recall that stable states are long-lived states. If a system is in a stable
state it will return to that state whenever it is subjected to small perturba-

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20. Neural Rhythms

tions and disturbances. Plateau potentials are membrane potentials that,

like the resting membrane potentials, are stable states. Plateau states occur
at depolarizations greater than that of the resting membrane potential.
When a membrane is at a plateau potential, it is far more able to repeatedly re action potentials, even in the absence of sustained excitatory input
from synapses. As a result neurons possessing plateau potentials exhibit two
types of behavior. When at their resting membrane potential, the neurons
do not re action potentials in the presence of modest sustained depolarizations, and thus are silent. In contrast, when they at their more depolarized plateau potential, even modest sustained depolarizations will trigger
the repeated ring of action potentials. Compare the ring pattern shown
in Figure 20.4(b) to the one presented in Figure 20.8. The cells producing
the bursts in Figure 20.8 possess plateau potentials; their membrane potentials remain elevated after each action potential. This situation differs from
the one depicted in Figure 20.4 in which the potential returns to values close
to the resting potential after each action potential.
Last, referring again to Table 20.4 post inhibitory rebound refers to the
paradoxical situation that inhibition can actually promote excitability. Some
ion channels require hyperpolarization to de-inactivate and others need
hyperpolarization in order to activate. If a neuron expressing these ion
channels is subjected to a strong hyperpolarization that is quickly terminated, its membrane can rapidly rebound, depolarizing and ring an action
potential.

20.12 Neural Circuits Are Connected to Other Circuits

and Form Systems
Central pattern generators do not work in isolation. Rather, they receive
inputs from sensory and higher information-processing areas and send
outputs to motor neurons controlling muscles. In situations where multiple
circuits must work together to generate motor rhythms, the CPGs communicate laterally to other CPGs. Properties of a typical mammalian network
such as the convergence of multiple input signals and feedback control are
listed in Table 20.4. The human respiratory system provides an excellent
example of both of these properties.
In humans, neurons organized into a CPG in a section of the medulla
called the Prebtzinger complex generate respiratory rhythms. This CPG
receives input from a variety of sources denoting emotional, sleep-related,
environmental, and motor activity states. The output from the CPGs drives
pools of motoneurons controlling the respiratory musclesthe diaphragm,
and the intercostal and abdominal musclesthat pump air in and out of
the lungs. The degree of resistance to airow is regulated by smooth muscle
in the bronchial walls, and by muscles controlling the diameter of the upper
airways, namely, the laryngeal, pharyngeal, and hyperglossal muscles.

20.13 A Variety of Neuromodulators Regulate Operation

505

The respiratory system is a feedback control system. Respiratory neurons

send output to the diaphram, intercostal muscles, abdominal muscles, and
accessory muscles of respiration. Mechanoreceptors supply feedback information on the effect of the ring patterns on the muscles. Information is
supplied by stretch receptors, located in the bronchial smooth muscle, by a
variety of irritant receptors located in the airway epithelium, and by C-ber
receptors located within the walls of the alveoli. Information on dissolved
gases is provided by peripheral (arterial) oxygen and carbon dioxide receptors, and by central carbon dioxide (acidication) medullary receptors that
monitor the cerebrospinal uid (CSF).
Several different patterns can be generated by the respiratory CPG.
Under normal breathing conditions the CPG generates a fairly regular,
eupneic ring pattern. However, under conditions of oxygen starvation/
hypoxia, the patterns generated within the CPG will change to either a
sighing rhythm or to a gasping rhythm. One of the features that characterize neurons lying within the CPG is the presence of neurokinin-1 receptors,
the receptors for the peptide neuromodulator, substance P. The neurons
expressing these receptors not only generate respiratory rhythms, but also
alter their ring frequency in response to the presence or absence of substance P and other neuromodulators that, in turn, reect oxygen status and
other types of regulatory information.

20.13 A Variety of Neuromodulators Regulate

Operation of the Crustacean STG
The neurons that make up the crustacean stomatogastric ganglion express
receptors for a number of amine and crustacean-specic peptide (CSP) neuromodulators. Receptors are present for (amines) serotonin, dopamine, and
octopamine, and for (CSPs) crustacean cardioactive peptide (CCAP), proctolin, and cholecystokinin (CCK). These neuromodulators not only alter the
ring patterns of the central pattern generators, but also can trigger aggressive behavior. Most of the CSPs are released by neurons that send their
axons to the STG. The usual effect of the peptides is to initiate one or more
of the rhythmsgastric, pyloric, and cardiac sac. In some instances, a neuropeptide such as red pigment-concentrating hormone (RPCH) triggers the
functional reconguration of the circuits. The reconguration may take
the form of circuit fusion or appear as alterations in circuit membership. In
the former case, two or more circuits may fuse together so that they work
together rather than independently. In the latter instance, a neuron, previously ring as part of one circuit may switch its ring properties and become
part of another circuit.
The amine neuromodulators act on the ion channels, enhancing some currents and inhibiting others. They exert their inuences by either shifting the
activation and or deactivation curves or by increasing the maximal con-

506

20. Neural Rhythms

ductances for the ion channels. These changes lead to alterations in phase
of one neuron relative to others within the CPG and change the number of
action potentials generated in a given time interval.
As indicated in Table 20.4, neuromodulatory signals can originate either
within a CPG or externally to it. There are many sources of external inputs
in the central nervous system. In vertebrates these sources are organized
into centers that project out to, and make contact with, neurons in most
areas of the brain. These centers are able to inuence the response properties of many circuits simultaneously. Intrinsic modulation is far more specic in its actions. In the stomatogastric ganglion, modulatory inputs are
supplied by higher brain centers and by feedback from sensory neurons.
These modulatory inputs are sensitive to the outputs of the circuit being
modied and are local to that circuit. These inuence the rhythms being
produced and the attendant behaviors.

20.14 Motor Systems Adapt to Their Environment

and Learn
The nudibranch gastropod Tritonia diomedea is preyed upon by starsh.
When Tritonia comes into contact with a foot of a starsh, it changes its
behavior from a slow-moving feeding mode to a fast-moving escape swimming mode. The circuitry that makes this escape response possible includes,
besides the sensory neurons, a command interneuron that receives sensory
information, and a central pattern generator that receives information from
the command dorsal ramp interneuron (DRI) to start the escape swim
rhythm. The CPG contains three kinds of interneuronsan interneuron
called C2, a pair of ventral swim interneurons (VSIs), and three dorsal swim
interneurons (DSIs). The output from the CPG is sent to an array of left
and right exion motor neurons that drive the muscles responsible for
escape swimming (Figure 20.9).
In Tritonia, the swim CPG produces either a swimming rhythm or a
nonswimming withdrawal response. The transformation of nonswimming
into swimming rhythms involves modulatory signaling by serotonin. The
command interneuron sends swim signals to the DSIs. In response to these
initiating signals, the DSIs release serotonin, which binds to receptors on the
C2 neuron. In response to serotonin binding, the C2 interneuron increases
the amount of neurotransmitter released from its presynaptic terminals and
exhibits an increased excitability. The effect of the increased neurotransmitter release is to strengthen the synaptic connection from the C2 interneuron
back to the DSIs thereby increasing the DSIs excitability through this positive feedback loop. (In the absence of the increased neurotransmitter
release, the DSIs are repressed by inhibitory signaling between themselves).
The increased excitability of the C2 neuron enables it to trigger and maintain the DSIs swim rhythm. If the ring rate of the C2 neuron falls below
the swim threshold, escape swimming will not occur. In the terminology of

20.14 Motor Systems Adapt to Their Environment and Learn

507

Figure 20.9. Tritonia escape circuit: Upstream signals conveyed by a set of about
80 S cells are relayed to the command neuron, the dorsal ramp interneuron (DRI),
which sends its synaptic output to a set of three dorsal swim interneurons (DSIs)
belonging to the central pattern generator. The CPG contains, besides the DSIs, a
pair of ventral swim interneurons (VSIs) and a single cerebral cell 2 (C2) neuron.
The three kinds of CPG neurons make synaptic contacts with a set of about 55
dorsal and ventral exion motor neurons. In the diagram, sharp arrows denote
excitatory connections; lled circles represent inhibitory connections, and the
hybrid arrow/circle symbol denotes a multiple component (excitatory and
inhibitory) monosynaptic contact.

Table 20.4, the alteration in the ring pattern of C2 by serotonin released by

the DSIs is an example of intrinsic neuromodulation.
The serotonin-mediated changes in rhythmicity are long-lasting in that
they outlast the stimuli that produced them. When the serotonin molecules
bind to GPCRs on the postsynaptic cell, they activate Gi alpha subunits of
the heterotrimeric G proteins docked nearby. These subunits stimulate
adenylyl cyclase to increase its synthesis of cAMP second messenger molecules leading to alterations in the way the ion channels operate and the
cell responds to further stimuli. The organism will exhibit an increased
responsiveness to further noxious stimuli and a reduced responsiveness to
harmless ones. The process of more rapidly responding to the dangerous
stimuli is called sensitization, and the process of responding less strongly to
benign stimuli is known as habituation. Both types of adaptive change
are examples of learning and have been extensively studied in the snails,
crustaceans, and leech.

508

20. Neural Rhythms

References and Further Reading

General References
Hille B [1992]. Ionic Channels of Excitable Membranes (2nd edition). Sunderland
MA: Sinauer Associates, Inc.
Kaczmarek LK, and Levitan IB [1987]. Neuromodulation. New York: Oxford
University Press, Inc.
Llins RR [1988]. The intrinsic electrophysiological properties of mammalian
neurons: Insights into central nervous system function. Science, 242: 16541664.
Nicholls JG, Wallace BG, Fuchs PA, and Martin AR [2001]. From Neuron to Brain:
A Cellular Approach to the Function of the Nervous System (4th edition).
Sunderland MA: Sinauer Associates, Inc.

Heartbeat
Boyett MR, Honjo H, and Kodama I [2000]. The sinoatrial node, a heterogeneous
pacemaker structure. Cardiovasc. Res., 47: 658687.
Schram G, et al. [2002]. Differential distribution of cardiac ion channel expression
as a basis for regional specialization in electrical function. Circ. Res., 90: 939950.

HCN Channels
DiFrancesco D [1999]. Dual allosteric modulation of pacemaker (f) channels by
cAMP and voltage in rabbit SA node. J. Physiol., 515: 367376.
Ludwig A, et al. [1998]. A family of hyperpolarization-activated mammalian cation
channels. Nature, 393: 587591.
Zagotta WN, et al. [2003]. Structural basis for modulation and agonist specicity of
HCN pacemaker channels. Nature, 425: 200205.

Thalamocortical Rhythms
Huguenard JR, and Prince DA [1992]. A novel T-type current underlies prolonged
Ca2+ dependent burst ring in GABAergic neurons of rat thalamic reticular
nucleus. J. Neurosci., 12: 38043817.
McCormick DA, and Huguenard JR [1992]. A model of the electrophysiological
properties of thalamocortical relay neurons. J. Neurophysiol., 68: 13841400.
Munk MHJ, et al. [1996]. Role of reticular activation in the modulation of
intracortical synchronization. Science, 272: 271274.
Steriade M, Amzica F, and Contreras D [1996]. Sunchronization of fast (3040 Hz)
spontaneous cortical rhythms during brain activation. J. Neurosci., 16: 392417.
Steriade M, McCormick DA, and Sejnowski TJ [1993]. Thalamocortical oscillations
in the sleeping and awake brain. Science, 262: 679685.
von Krosigk M, Bal T, and McCormick DA [1993]. Cellular mechanisms of a
synchronized oscillation in the thalamus. Science, 261: 361364.

Epilepsy
Crunelli V, and Leresche N [2002]. Childhood absence epilepsy: genes, channels,
neurons and networks. Nature Rev. Neurosci., 3: 371382.
Velazquez JPL, and Carlen PL [2000]. Gap junctions, synchrony and seizures. Trends
Neurosci., 23: 6874.

References and Further Reading

509

Gap Junctions
Bruzzone R, White TW, and Paul DL [1996]. Connections with connexins: The
molecular basis of direct intercellular signaling. Eur. J. Biochem., 238: 127.
Evans WH, and Martin PEM [2002]. Gap junctions: Structure and function. Mol.
Mem. Biol., 19: 121136.
Kumar NM, and Gilula NB [1996]. The gap junction communication channel. Cell,
84: 381388.
Simon AM, and Goodenough DA [1998]. Diverse functions of vertebrate gap
junctions. Trends Cell Biol., 8: 477483.
Unger VM, et al. [1999]. Three-dimensional structure of a recombinant gap junction
membrane channel. Science, 283: 11761180.

GABAergic Neurons and Gap Junction-Mediated Synchrony

Galarreta M, and Hestrin S [2001]. Electrical synapses between GABA-releasing
interneurons. Nature Rev. Neurosci., 2: 425433.
Galarreta M, and Hestrin S [1999]. A network of fast-spiking cells in the neocortex
connected by electrical synapses. Nature, 402: 7275.
Gibson JR, Belerlein M, and Connors BW [1999]. Two networks of electrically
coupled inhibitory neurons in neocortex. Nature, 402: 7579.
Tams G, et al. [2000]. Proximally targeted GABAergic synapses and gap junctions
synchronize cortical interneurons. Nature Neurosci., 3: 366371.

Central Pattern Generators

Grillner S, et al. [1998]. Intrinsic function of a neuronal networkA vertebrate
central pattern generator. Brain Res. Revs., 26: 184197.
Grillner S, et al. [1995]. Neural networks that co-ordinate locomotion and body
orientation in lamphrey. Trends. Neurosci., 18: 270279.
Marder E, and Bucher D [2001]. Central pattern generators and the control of
rhythmic movement. Curr. Biol., 11: R986R996.
Marder E, and Calabrese RL [1996]. Principles of rhythmic motor pattern
generation. Physiol. Rev., 76: 687717.
Nusbaum MP, and Beenhakker MP [2002]. A small-systems approach to motor
pattern generation. Nature, 417: 343350.

Respiration
Gray PA, et al. [1999]. Modulation of the respiratory frequency by peptidergic input
to the rhythmogenic neurons in the Prebtzinger complex. Science, 286: 1566
1568.
Lieske SP, et al. [2000]. Reconguration of the neural network controlling multiple
breathing patterns: Eupnea, sighs and gasps. Nature Neurosci., 3: 600607.
Ramirez JM, and Richter DW [1996]. The neuronal mechanisms of respiratory
rhythm generation. Curr. Opin. Neurobiol., 6: 817825.

Motor Learning and Memory

Kandel ER [2000]. The molecular biology of memory storage: A dialogue between
genes and synapses. Science, 294: 10301038.

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20. Neural Rhythms

Problems
20.1 A small number of ion channels have particularly prominent roles in
generating rhythmic patterns of action potentials. One of these, the
HCN channel, was discussed in Section 20.2 while another, the T-type
calcium channel, was discussed in Section 20.4. Make a sketch of the
activation and deactivation functions for the T-type calcium channel
and the sodium channels responsible for action potential upstrokes.
Recall from the discussion in Section 20.4 that at the resting potential
the T-type calcium channel is activating while the sodium channel is
deactivating. In the plots of current versus membrane potential, indicate the location of a resting potential and position the four curves so
they satisfy the relationship just mentioned.
20.2 The utility of a gradually depolarizing membrane potential was discussed in Section 20.11 and illustrated in Figure 20.1. As noted in that
discussion, this behavior greatly enhances excitability in cells possessing plateau potentials. When two cells of this type are coupled through
mutual inhibition they can synchronize their ring acting as oscillatory
units that produce motor rhythms. List some types of currents that
promote gradual declining membrane potentials of the sort depicted
in the gure.

21
Learning and Memory

The learning and memory formation processes carried out by the snails and
other invertebrates is certainly more modest than that of humans, yet the
two are similar. Each involve alterations in behavioral responses to stimuli
brought on by experience. Learning is the adaptive process whereby
changes in behavior are induced in response to experience. Memory is the
record, or trace, underlying the changes in behavior. The records in all
organisms, snails and mice, ies and humans, are stored in the patterns of
connection between neurons. During learning and memory formation some
connections are strengthened while others are weakened. For instance, in
the case of sensitization discussed at the end of the last chapter, connections between neurons were strengthened. This led to behavioral changes.
The snails were able to respond with increased rapidity to noxious stimuli.
The term efciency of synaptic transmission refers to the magnitude of
the response generated in a postsynaptic neuron when an action potential
is generated in the presynaptic neuron. In many situations, the efciency of
the transmission is not xed but instead varies in ways that depend on use.
The changes in efciency can be transient, lasting for a few milliseconds, or
can be permanent, lasting for a lifetime. Connections may be strengthened
or be weakened or disappear altogether. Synaptic plasticity is the term used
to describe the use-dependent, or adaptive, changes in the efciency of
synaptic transmission between pre- and postsynaptic cells.
The ability to vary connection strengths is an important one. It allows the
nervous system to form its precise connections and neural circuits during
development; it gives rise to a unique personality for each individual; and
it makes possible learning throughout life. Cortical circuits develop over
time in several stages. The rst stage of circuit development involves neurite
outgrowth, growth cone pathnding and then synaptogenesis. As the circuits become functional and can respond to synaptic input, ineffective and
improper connections are pruned, and neurons that are no longer needed
die off through programmed cell death. The process of varying the strengths
of synaptic connections continues throughout life. It underlies the development of simple motor skills as well as highly coordinated eye and hand
511

512

21. Learning and Memory

movements. It allows associations to be made that are needed for survival

(for example, of sights, sounds, touches and odors indicative of danger), and
makes possible learning and remembering and forgetting.
The pre- and postsynaptic terminals of nerve cells are highly enriched in
signaling molecules. Most major classes of signaling molecules discussed in
the preceding chapters are present in the synapses. Thee include ion channels, enzymes such as protein kinases, protein phosphatases and GTPases,
G protein-coupled receptors, cell adhesion molecules, and nonenzymatic
scaffold, adapter, and anchor proteins. The molecular composition of the
synapses will be examined following a brief overview of how neurons are
organized in the brain. The remainder of the chapter is devoted to explorations of learning and memory formation. The preeminent experimental
model of how this is accomplished is called long-term potentiation. It is
studied most often in vitro using slice preparations from the hippocampus
of rodents. Findings from these experiments as well as from experiments of
fear conditioning and drug dependence, which can be thought of as an aberrant form of learning and memory, will be explored, as well.

21.1 Architecture of Brain Neurons by Function

Neurons in the brain are organized into architecturally distinct functional
areas. The large-scale division of the brain into brain stem, limbic system,
cerebellum, and cerebral cortex is depicted in Figure 21.1. The rst of the
aforementioned large-scale regions, the brain stem, contains a number of
structures whose activities were discussed in the previous chapter. Neurons
in the medulla oblongata and pons regulate rhythmic movements associated with breathing and heartbeat, while cells in the reticular activating
complex control alertness and the sleep/wake cycle. The limbic system is
situated just above the brain stem. It includes the hippocampus, and
the amygdala, which mediates emotional responses such as fear and
aggression.

Figure 21.1. Architecture of the brain

showing its major divisions: Shown is a side
view with the front of the head on the left
and the back of the head on the right. The
partitioning of the cerebral cortex is
depicted along with the locations of the
brain stem, cerebellum, and limbic system.

21.1 Architecture of Brain Neurons by Function

513

Figure 21.2. Architecture of the brain showing the sequestering of brain functions
within specic areas of the brain: The locations of the major sensory areas are shown
along with motor areas and sites where forms of learning and memory, and higher
processing functions, as most strongly associated.

The cerebellum is a large region that sits behind the brain stem and below
the occipital lobe.Neurons in this region of the brain integrate and coordinate
motor actions and mediate motor learning. Cerebellar neurons control
balance and posture, and ensure that ne motor actions are accurate. The
large section of cortex sitting above and about these structures is partitioned
into frontal, parietal, occipital, and temporal lobes. The temporal lobe sits
above and to the outside of the brain stem at about the level of the ears. As
indicated in Figure 21.2, neurons in this region are involved in the senses of
sound,taste,and smell,and in memories thereof.Information of a tactile character, that is, the sense of touch, is processed by neurons located in the
somatosensory cortex that lies above in the parietal lobe. This region is situated next to regions where associations and interpretations are made from
information sent from the primary visual cortex and other primary sensor cortices.Behind this region,at the back of the head in the occipital lobe,(primary)
visual information is received and processed. Lastly, the nonmotor portions
of the frontal cortex are used for making judgments and intelligent decisions.
The premotor cortex controls head and eye movements, while speech is centered in Brocas area and smell in the olfactory center.
The presence of a large region devoted to motor and sensory information
processing is unique to mammals.This region is highly differentiated radially
into layers and laterally into regions. As rst noted by Brodmann in 1909,
regions of cortex involved in processing sensory information are organized in
the radial direction into six layers and in the lateral direction into at least 50
distinct areas. The numbers of sublayers, and their thicknesses and arrangements, vary from area to area in the cortex.Within each area there are cells of
a particular size and arborization, with a distinct set of inputs, outputs, and

514

21. Learning and Memory

intercortical connections.The overall organization of the neocortex is that of

a patchwork of areas each with its own cellular organization, or cytoarchitecture. Brodmann based his partitioning of the cortex upon differences in
appearance that he was able to observe using a light microscope. In more
modern approaches, functional MRI and other biophysical methods are used
to assign functions to anatomical regions. The anatomical regions, called
Brodmanns areas,correspond approximately to functional areas,so that each
anatomically distinct area carries out some unique function.

21.2 Protein Complexes Structural and Signaling

Bridges Across Synaptic Cleft
Communication occurs when neurotransmitters released from the presynaptic terminal diffuse across the synaptic cleft and bind to receptors on the
postsynaptic side. Before this communication can occur, a minimal set of
molecular components must be assembled into a functional unit, the synapse.
Active zones on the presynaptic side that contain the machinery for neurotransmitter release must align with the postsynaptic density (PSD) on the
postsynaptic side containing the receptors for the neurotransmitters. These
activities must take place as the two parts of the signaling unit are formed.
These pre- and postsynaptic portions of the synapse are aligned and stabilized by proteins that form bridges from one side of the cleft to the other.
Several of the most prominent bridging pairsNeurexins-to-Neuroligins,
SynCAMs-to-SynCAMs and EphB2s-to-EphrinBsare depicted in Figure
21.3. Neurexins are brain-specic cell surface proteins containing one or
more LNS (laminin, neurexin, sex hormone-binding globulin) domains. The
LNS domains, approximately 190 amino acid residues in length, act as cell
surface recognition modules. They are found in a variety of extracellular
matrix proteins and in cell surface receptors, typically in combination with
multiple EGF repeats. Neuroligins act as ligands for Neurexins. They are
brain-specic, single-pass, transmembrane proteins found on the opposing
surface to the neurexins. Neurexin-Neuroligin cell-cell adhesion complexes
form a bridge between the pre- and postsynaptic terminals. Neurexin interacts with CASK on the presynaptic side, and neuroligin binds to the PSD95 proteins on the postsynaptic side. The interactions with each another and
with the cytoplasmic CASK and PSD-95 proteins stabilize and maintain the
alignment between active zones on the presynaptic side and the postsynaptic density at the postsynaptic terminal.
More than one kind of signaling complex ties together the active zone
on the presynaptic side and the PSD at the postsynaptic terminal. Members
of the immunoglobulin superfamity of cell adhesion molecules called
synCAMs establish homophilic contacts between proteins expressed on
the two opposing surfaces. As is the case for other proteins that perform a
structural role mediating surface adhesion, the synCAMs also have a

21.3 The Presynaptic Terminal and the Secretion of Signaling Molecules

515

Figure 21.3. Transsynaptic proteinprotein interactions: Protein pairs form bridges

that physically link pre- and postsynaptic terminals. The protein pairs are tied to
scaffolding proteins on the two sides of the synapse. Many, if not most, of the scaffolding proteins have PDZ domains. Kalirin, which binds EphB2, is a Rho GEF.

signaling role. They appear to supply signals telling nascent presynaptic

terminals to differentiate into a mature signaling form. The neurexinneuroligin complexes serve a similar signaling role. These too instruct the
cells to form differentiated presynaptic terminals. Several families of cell
adhesion molecules participate in bridging actions. Neuron-specic cadherins form bridging complexes, too. These proteins are anchored at both
sides of the synaptic cleft to b-catenins.
Another family of proteins having an important role in synapse formation is the EphB receptor tyrosine kinases and their Ephrin ligands. The
EphB2 receptor signals through the Rho GEF kirilin to the actin cytoskeleton, thus helping in dendritic spine morphogenesis. In synapses that signal
using glutamate, N-methyl-D-aspartate (NMDA) receptors are an early
occupant of the postsynaptic density. Kirilin helps establish NMDA receptor aggregates at newly created synapses.

21.3 The Presynaptic Terminal and the Secretion of

Signaling Molecules
When action potentials arrive at the presynaptic terminal, voltage-gated
calcium channels open and calcium ions ow into the terminal. The calcium
ions bind to calcium sensors attached to the neurotransmitter-lled vesicles.

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21. Learning and Memory

Figure 21.4. Neurotransmitter release at the presynaptic terminal: In response to

the arrival of an action potential, calcium enters the cell through voltage-gated Ca2+
channels (VGCCs) and triggers fusion and release of neurotransmitters from neurotransmitter-lled synaptic vesicles. The neurotransmitter molecules diffuse across
the synaptic cleft and bind to receptors embedded in the terminal membrane of the
postsynaptic cell.

Binding of the calcium to the sensor triggers conformational changes

leading to vesicle fusion and release of the contents. Once released, the
neurotransmitters diffuse across the synaptic cleft and bind to receptors
embedded in the postsynaptic membrane. This set of steps is depicted
schematically in Figure 21.4.
The amount of neurotransmitter released at a presynaptic terminal
depends on several factors. Synapses in the central nervous systems are, in
general, small. Their amount of neurotransmitter released at one time is not
only small but also can be quite variable. The amount of neurotransmitter
released can,for example,depend on timing.If a large number of pulses arrive
at the presynaptic terminal in a short period of time,the pool of available neurotransmitter vesicles can be depleted. The release of neurotransmitter will
therefore monotonically decline with each later-arriving action potential.
Synapses with large pools will release more neurotransmitter than those with
small pools. The term active zone refers to the exact site along the presynaptic terminal where the vesicles fuse and release neurotransmitter. The
amount of neurotransmitter release will depend on the number of active sites
per terminal and on the number and size of the vesicles.
Several protein complexes regulate vesicle movements and neurotransmitter release in the presynaptic terminal.These have been organized under

21.3 The Presynaptic Terminal and the Secretion of Signaling Molecules

517

three headings in Table 21.1. The rst set of signaling elements in the table
is the voltage-gated calcium channel grouping that converts membrane
depolarization into a calcium signal. Included under this heading are a
number of signaling proteins that allow neuromodulators to inuence the
strength of the calcium signals and thus the amount of neurotransmitter
released into the cleft.
The second grouping contains vesicle regulators such as the soluble NSF
attachment protein receptor (SNARE) complex. SNARE proteins regulate
the nal stages of neurotransmitter releasedocking and fusion of the
vesicle with the plasma membrane resulting in release of the vesicle contents into the synaptic cleft. There are two classes of SNARE proteins
those that bind to the vesicle surface (v-SNAREs) and those that attach to
the plasma membrane (t-SNAREs). Interactions between the two kinds of
SNARES regulate the release of neurotransmitter. One of the v-SNAREs,
synaptotagmin, contains a calcium-binding C2 domain and functions as the
calcium sensor. Conformational changes brought on by calcium binding are
conveyed to the t-SNAREs with which it interacts promoting fusion and
vesicle content release.
The other signaling complexes in the vesicle regulator grouping help
shepherd vesicles into the presynaptic terminal and to the plasma membrane, and then regulate membrane fusion. Neurotransmitter-lled vesicles
Table 21.1. Main components of the presynaptic terminal at CNS synapses:
AbbreviationsN-ethylmaleimide-sensitive fusion protein (NSF); soluble NSFattachment protein (SNAP); soluble NSF attachment protein receptor (SNARE);
vesicle-associated membrane protein (VAMP); CaMK/SH3/guanylate kinase
domain protein (CASK).
Presynaptic terminal component

Function

Channels and their modulators

Voltage-gated calcium channels N- and P/Q-type calcium channels; enable calcium to
enter the cell in response to membrane depolarization
GPCRs
Gbg subunits negatively regulate N-type channels
Protein kinase C
Positively regulates N-type channels
Vesicle regulators
v-SNAREs

t-SNAREs
Sec6/8 complex
Sec1/Munc18
NSF, a-SNAP
Scaffolds and adapters
CASK
Mint1
Veli

Synaptobrevin (VAMP) and synaptotagmin mediate

vesicle docking and fusion. Synaptotagmin functions
as a calcium sensor
Syntaxin and SNAP-25 mediate vesicle docking and
fusion
Targeting of vesicles to the plasma membrane
Regulators of Syntaxin
Dissociation of the SNARE complex
Attaches to N- and P/Q-type channels and Mint1; aligns
vesicles
Attaches to CASK and N- and P/Q-type channels
Member of the CASK, Mint1, Veli adapter complex

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21. Learning and Memory

are transported along the cytoskeleton into the presynaptic terminal. Upon
arrival at the terminal the vesicles are targeted to the plasma membrane by
a multiple subunit protein complex called Sec6/8. Another set of proteins,
Sec1/Munc18, act both as positive and as negative regulators of SNAREmediated fusion through interactions with Syntaxin. Yet another set of molecules, NSF and a-SNAP, direct the dissociation of the SNAREs permitting
their reuse.
The third grouping presented in Table 21.1 encompasses the scaffold and
anchor proteins. These proteins help organize the complexes in the presynaptic terminal. As is the case in any cell, these proteins provide attachment
sites where proteins that must work together can bind in close proximity to
one another.

21.4 PSD Region Is Highly Enriched in

Signaling Molecules
The region just below the postsynaptic membrane is the postsynaptic
density (PSD). The PSD is a disclike structure that sits opposite the active
zone in the presynaptic terminal (Figure 21.5). It is highly enriched in struc-

Figure 21.5. The postsynaptic density: Shown as some of the types of signaling proteins that are found in the postsynaptic density at excitatory synapses in the central
nervous system. Glutamate released at the presynaptic terminal diffuses across the
synaptic cleft and binds to NMDA and AMPA receptors leading to membrane depolarization, calcium inux, alterations in AMPA receptor population, and gene
expression. Short-term modulatory effects are mediated by GPCRs such as
mGlu5R, leading to G protein subunit- and second messenger-binding to ion channels, and phosphorylation of ion channels by protein kinases.

21.4 PSD Region Is Highly Enriched in Signaling Molecules

519

Table 21.2. Main components of the PSD at excitatory glutamergic synapses:

Abbreviationsa-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA); Nmethyl-d-aspartate (NMDA); calcium/calmodulin-dependent protein kinases II
and IV (CaMKII and CaMKIV); glutamate receptor interacting protein (GRIP);
guanylate kinase-associated protein (GKAP).
PSD component
Receptors
AMPA receptor
NMDA receptor
Metabotropic receptors
Kinases and phosphatases
aCaMKII and CaMKIV
Protein kinase A
Protein kinase C b, g isoforms
ERK2 type MAPKs
Protein phosphatase 1
Anchors, scaffolds, and adapters
AKAP79
GRIP
PSD-95
SynGAP
GKAP
Shank
Homer

Function
GluRs: Key component of active synapses
NRs: Dual voltage and ligand-gated allowing entry of
calcium into the cell
mGluRs: Signal to IP3 regulated intracellular calcium
stores
Central signaling element, involved in both short and
long term forms of synaptic plasticity
Central signaling element, involved in both short and
long term forms of synaptic plasticity
Signaling element, involved in short term forms of
synaptic plasticity
Signaling element, involved in both short and long term
forms of synaptic plasticity
Regulator of homeostasis and learning
Sequesters protein kinase A, protein kinase C, and
calcineurin near receptors and ion channels
Attaches to GluR2 and GluR3
Attaches to NR2s
Activates MAP kinase signaling
Link between PSD-95 and GKAP
Link between Homer and GKAP
Attaches to mGluRs and to Shank

tural (cytoskeleton) and regulatory (receptor, ion channel, scaffolding, signaling) proteins. The several different kinds of regulatory proteins have
been placed into three groupings in Table 21.2. The rst group contains the
receptors for neurotransmitters. Glutamate is the predominant neurotransmitter at excitatory synapses in the central nervous system. Several different kinds of glutamergic receptors are found in the PSD of excitatory
synapses in the CNS. Some glutamergic receptors are ion channels
(ionotropic) while others are G protein coupled receptors (metabotropic).
Two kinds of ionotropic glutamate receptorsthe a-amino-3-hydroxy-5methyl-4-isoxazolepropionate (AMPA) receptors and the N-methyl-daspartate (NMDA) receptorsare intimately connected with synaptic
plasticity. Rather than listing a large number of receptors in the table, only
the three main classes of glutamergic receptors have been included in the
table. These are the principal receptors involved in learning and memory
formation.

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21. Learning and Memory

The second group in the table consists of several different kinds of serine/
threonine kinases and phosphatases that are central signaling elements
in the postsynaptic density. The third grouping contains connecting
and supporting agents such as GTPases functioning as molecular adapters,
and anchoring and scaffolding proteins that help organize the signaling
complexes. These signaling elements form a matrix within the PSD that
allows ion channels to cluster together in close proximity to second messenger and downstream signaling elements. The postsynaptic density is
enriched in cytoskeleton proteins, especially in actin and actin-related
cytoskeleton components.

21.5 The Several Different Forms of Learning

and Memory
There are several different kinds of learning. The forms of learning
described earlier with regard to Tritonia and Aplysiasensitization and
habituationare nonassociative in character. Repeated stimulation of
noxious stimuli produce sensitization while repeated exposures to a harmless stimulus lead to habituation, a reduced responsiveness. These forms of
learning do not require a second pairing stimulus to elicit behavioral
changes.
Associative learning, in contrast, involves a pairing of stimuli. The earliest
and still most famous example of associative learning is Pavlovs dog experiments.In these and similar experiments,an initial nondangerous event,called
the conditioned stimulus (CS), is paired repeatedly with a biologically significant (potentially dangerous) event called the unconditioned stimulus (US).
Over time, the relationship between the CS and US is learned; that is, the
behavioral responses adapt in response to the experiences. In a typical laboratory experiment, a rat is given a tone, a neutral nondangerous CS, which is
paired with an electrical shock, the dangerous US.After as few as one or two
exposures, the tone will elicit a number of physiological and behavioral adaptations. The rat will freeze in place and exhibit reex actions. Heart rate and
blood pressure will change, and stress hormones will be released. In Aplysia,
the associative learning counterpart is called long-term facilitation. In this
process, an electric shock to the tail serves as the US, and a weak touch to the
siphon functions as the CS. The snail learns to associate the two stimuli and,
because of the association with the electrical shock, alters its behavioral
response to the weak touch over time.
The circuitry responsible for the Aplysia gill withdrawal response is
depicted in Figure 21.6. The axon of the sensory neuron that conveys touch
signals contacts a dendrite on a motor neuron. The sensory neuron that
conveys the electric shock signals also makes contact with the motor
neuron. As shown in the gure the neuron has a synaptic connection with
an interneuron that releases serotonin from its axon terminal.The serotonin

21.6 Signal Integration in Learning and Memory Formation

521

Figure 21.6. Circuitry underlying the associative Aplysia gill withdrawal response:
Three different kinds of neurons form the circuitsensory neurons that detect and
respond to electrical shock (SN1) and touch (SN2), a serotonin-releasing interneuron (INT), and a motor neuron (MN).

diffuses across the synaptic cleft to the axon terminal of the touch sensory
neuron where the two sets of signals converge.
Just as there are several types of learning, there are several kinds of
memory. Short-term memory refers to memories that are labile (responsive
to changes in conditions under which they are created) and are only transiently formed. They are lost if not converted into stable memory traces.
Short-term labile memories involve celluar changes that utilize cellular
resources that are already available and are local to the synapse or synapses
undergoing modication. In contrast, long-term stable memory traces typically involve gene expression and protein synthesis leading to architectural
modications that produce the lasting changes in synaptic transmission.
This events stimulated by the release of serotonin are depicted in Figure
21.7. These include stimulation of adenylyl cyclase leading to the production of cAMP and its subsequent activation of the catalytic subunits of
protein kinase A. Protein kinase A phosphorylates the nearby potassium
channels and, as a result, currents through potassium channels are reduced.
The depolarization accompanying arrival of the action potential in the axon
terminal lasts longer. More calcium is able to enter the terminal resulting
in a greater release of neurotransmitter. Two routes are illustrated in the
gure. One of the routes leads to ion channel modication, a short-term
memory trace. The other route leads to the nucleus and results in changes
in the pattern of gene expression, producing a longer lasting memory trace.

21.6 Signal Integration in Learning and

Memory Formation
Synapses were rst described by Ramn y Cajal in the 1890s. It took several
years for the idea to be accepted that nerve cells were not hard-wired to
one another, but instead communicated using chemical messengers that
diffuse across a narrow gap between pre- and postsynaptic terminals. The

522

21. Learning and Memory

Figure 21.7. Signaling in the learning pathway in Aplysia leading to short- and longterm facilitation: Serotonin stimulates an increased production of cAMP by adenylyl
cyclase through actions of GPCRs and Ga subunits.A transient facilitation of the signal
response is produced by phosphorylation of ion channels by the catalytic subunit
(PKAC) of protein kinase A.This modication alters the biophysical properties of the
ion channel. Long-term responses are elicited by sustained serotonin signaling resulting in gene transcription and morphological changes at the synaptic terminal.

efciency of the transmission process depends on both presynaptic and

postsynaptic factors. First, it depends on the amount of neurotransmitter
release from the presynaptic terminal for a given depolarization or action
potential. Second, it depends on how strong a response that release elicits
at the postsynaptic side. The strength of the presynaptic signal and the postsynaptic response depends on the mix of ion channels and signal receptors
and on their biophysical states.
In the case of short-term facilitation in Aplysia, the increased efciency
in synaptic transmission is implemented at the presynaptic side of the cleft.
Calcium inux resulting from membrane depolarization and serotonin
induced G protein signals converge on adenylyl cyclase, which responds by
increasing cAMP production leading to changes in the amount of neurotransmitter being released from the terminal. Adenylyl cyclase acts as a
signal integrator, serving as a moleculer device for detecting the temporal
coincidence of electric shock and weak touch stimuli.
NMDA receptors perform a similar function, but, instead of acting on the
presynaptic side of the cleft, the NMDA receptors act at the postsynaptic

21.7 Hippocampal LTP Is An Experimental Model of Learning & Memory

523

terminal. The key biophysical property of these receptors is that they are
both voltage-dependent and glutamate-dependent thereby allowing them to
integrate signals and to evaluate associativity. These receptors work in the
following way:At the membranes resting potential, the NMDA receptor ion
channel is closedit is physically blocked by an Mg2+ ion that sits inside the
opening and blocks the pore.When the membrane is depolarized sufciently
the Mg2+ block is relieved, and once glutamate is bound, calcium ions can
pass through the channel and enter the cell. The voltage dependence is the
source of the receptors integrative property. No single synapse by itself can
depolarize the postsynaptic membrane sufciently to relive the magnesium
block. Rather, many synaptic inputs acting in close spatial and temporal
proximity to one another are needed to adequately depolarize the membrane. When depolarization and ligand binding occurs within an appropriate time window, the NMDA channel will open. Thus, the NMDA receptor
provides synapses with a way of determining whether the associativity conditions for synaptic modication have been satised or not.
One of the ways that both the efciency of synaptic transmission and
the ability to vary this property can be controlled is through the balance
between AMPA and NMDA receptors. At many postsynaptic terminals
only NMDA receptors are active during development. These synapses
remain silent at resting potentials because of the Mg2+ block, but are
capable of transmitting signals when the membrane is depolarized by one
means or another. In response to entry of calcium into the terminal through
the NMDA receptors, AMPA receptors become active. The presence of
active AMPA receptors allows the synapses to respond rapidly to glutamate
and generate stronger responses at the postsynaptic terminal to the release
of glutamate. AMPA receptors and AMPA receptor trafcking have a
prominent role in learning and memory formation and for that reason are
highlighted in Figure 21.5.

21.7 Hippocampal LTP Is an Experimental Model of

Learning and Memory
The hippocampus is a horseshoe-shaped structure belonging to the limbic
system. The hippocampus receives input from a number of sensory information processing areas. It, along with its surrounding structures, the
entorhinal cortex, perirhinal cortex, and parahippocampal region, are
believed to be the place where short term memories are stored and then
shipped out to long term, more permanent memory storage locations
through a process called memory consolidation. The hippocampal system is
thought to be responsible for recalling spatial relationships between objects
in the environment and for spatial navigation.
Hippocampal long-term potentiation (LTP) is a widely studied mammalian form of synaptic plasticity. This process is elicited in the laboratory

524

21. Learning and Memory

Figure 21.8. Hippocampal circuit in the rodent used in studies of LTP: The core
circuit includes a set of one-way connections from the entorhinal cortex (EC) to the
hippocampal dentate gyrus (DG), cornu ammonis 3 (CA3) region, CA1 region, subculum (Sub), and back to the EC and to other cortical regions. LTP is studied in the
CA3 region, where there is a dense network of recurrent connections, and in the
connections between the CA3 and CA1 neurons. Two sets of connections converge
on the CA1 neuronsthe Schaffer collaterals from the (ipsilateral) CA3 and the
commissural bers from the contralateral (contra) CA3.The names commonly given
to the various axons are shown.

in brain slices prepared from portions of the rodent hippocampus called the
CA1 and CA3 regions. The general architecture of the hippocampus
showing the locations of the CA1 and CA3 regions is presented in Figure
21.8. Two sets of connections are noted in the gurerecurrent connections
between cells in CA3, and connections from CA3 to CA1. In either circuit,
when a tetanic stimulus (i.e., a train of high frequency pulses) is delivered
to the presynaptic cell, or alternatively a series of pulses at low frequency
is delivered and the event paired with a laboratory-supplied depolarization
of the postsynaptic membrane, the efciency of synaptic transmission is
increased. The increase, or potentiation, of the synaptic efciency can be
generated in milliseconds, yet it lasts for minutes or even hours.

21.8 Initiation and Consolidation Phases of LTP

The initiation phase of LTP is followed by a consolidation phase involving
gene expression. The transition from a short lived form of memory formation to a more permanent one lasting for days, weeks, and even years is
through a process of memory consolidation. Gene expression is required
during this phase of memory formation. The cyclic AMP (cAMP) signaling
pathway is activated in which protein kinase A and downstream MAP
kinases interact with cAMP response element-binding protein (CREB)
transcription factors that bind to cAMP response elements (CREs) in promoters. This pathway can be activated in several ways. It can be activated
through calcium regulation of adenylyl cyclases, and by neuromodulators

21.9 CREB Is the Control Point at the Terminus of the Learning Pathway

525

Figure 21.9. Signaling in the learning pathway in hippocampal neurons leading to

consolidation of LTP: Calcium entry into the cell through NMDA receptors and Ltype calcium channels leads to activation of downstream acting serine/threonine
kinases. Key signaling intermediates include the GTPases Ras, and Rap1. These are
activated in a Ca2+ and cAMP-dependent manner, respectively, thereby integrating
and routing signals from these second messengers to the nucleus via a Raf-MEKERK MAP kinase complex.

such as serotonin and dopamine that bind to GPCRs, which act through
heterotrimeric G proteins to stimulate ACs to produce cAMP.
The MAP kinase and PKA routes are highlighted in Figure 21.9. As
shown in this gure, calcium entry either through NMDA receptors or
through L-type calcium channels leads to the activation of a number of
serine/threonine kinasesprotein kinase A, protein kinase C, MAP kinases,
and calcium/calmodulin-dependent protein kinases II and IV (CaMKII and
CaMKIV), not all of which are shown the gure. Several different actions
can occur depending on the duration of the synaptic signaling. Short-term
actions can take place in the neighborhood of the receptors using resources
already available. If the synaptic signaling is sustained over time, long-term
changes can occur involving changes in gene expression.

21.9 CREB Is the Control Point at the Terminus of the

Learning Pathway
The learning pathway terminates when the serine/threonine kinases arrive
in the nucleus and phosphorylate the cAMP response element-binding
protein (CREB) on Ser133. In more detail, a number of kinases translocate

526

21. Learning and Memory

Figure 21.10. Downstream signaling events leading to consolidation of memories:

AbbreviationsImmediate early gene (IEG); late gene (LG); cAMP response
element-binding protein (CREB); p300/CREB-binding protein (CBP).

to promoter sites in the nucleus containing cAMP responsive elements

(CREs). The kinases so involved include protein kinase A, protein kinase
C, MAP kinases, and CaM kinase IV. Members of the CREB family of transcription factors bind to these sites. The CREB proteins remain inactive if
not phosphorylated on Ser133. Phosphorylation on Ser133 by kinases such
as PKA results in binding by p300/CREB-binding protein (CBP). As noted
earlier, these are histone acetylases (HATs) that work together with the
basal transcription machinery to initiate a program of transcription leading to formation of new synapses and morphological changes to old ones
(Figure 21.10). As is the case for other transcriptional control points, a
variety of factors acting positively and negatively regulate CREB-mediated
transcription.
Transcription takes place in several stages, starting with immediate early
genes and ending with late genes. For example, among the rst genes to be
transcribed in serotonin-mediated facilitation in Aplysia are those whose
products extend to duration of the PKA signaling. This stage is followed by
the transcription and synthesis of transcription factors such as C/EBP, which
leads to the creation of new synaptic connections. This same overall set of
signaling activities is observed in other forms of learning and memory, and
in other test animals, so that there is a common family of mechanisms that
extends across phyla from Aplysia to ies to mice.

21.10 Synapses Respond to Use by Strengthening

and Weakening
In 1947, Donald Hebb formulated an associative rule governing usedependent changes in synaptic transmission. Hebbs rule is couched in terms
of cellular ring by a pair of cells (Figure 21.11). It says:When a when an axon
of cell A is near enough to excite cell B and repeatedly and persistently takes

21.10 Synapses Respond to Use by Strengthening and Weakening

527

Figure 21.11. Hebbs rule: (a) In response to the ring of an action potential by
cell A, cell B res an action potential. In this situation, the depolarization of the
postsynaptic membrane contributes to the ring of an action potential by cell B. The
strength of that synapse is increased. (b) Cell B does not re an action potential
when cell A does. The depolarization at the postsynaptic membrane is not effective
in eliciting action potentials. In situations of this sort the strength of the synapse is
reduced.

part in ring it, some growth process or metabolic change takes place in one
or both cells such that As efciency, as one of the cells ring B, is increased.
The rule in this form is incomplete. It does not state what happens when the
postsynaptic cell fails to re.To complete the rule one adds a subrule that says:
When a presynaptic axon of cell A repeatedly and persistently fails to excite
the postsynaptic cell B,while cell B is ring under the inuence of other presynaptic axons, metabolic changes takes place in one or both cells such that As
efciency, as one of the cells ring B, is decreased. In other words, synapses
whose activation is strongly correlated with the ring of the postsynaptic cell
are strengthened, while those synapses whose activation is poorly correlated
with the ring of the postsynaptic cell, for example, by being silent while the
cell res, are reduced in efciency.
According to the expanded form of Hebbs rule two types of changes in
the efciency of synaptic transmission can occur: the synapse can be
strengthened or it can be weakened. The efciency of synaptic transmission
increases when a synaptic connection is strengthened and decreases when
the connection is weakened. Modications in synaptic efciency depend
upon the timing of the action potentials in the pre- and postsynaptic cells.
Repetitive pre- and postsynaptic rings occurring within 10 to 50 msec of
one another are able to inuence the efciency of synaptic transmission. If
cell A res its action potential before cell B res its action potential, then
the synaptic connections between the two cells will be increased. If, on the
other hand, cell B repeatedly res before cell A, then the strength of the
synapse will decrease. The situation is intrinsically asymmetric so that there
is a preferred direction of information ow.

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21. Learning and Memory

21.11 Neurons Must Maintain Synaptic Homeostasis

If the efciency of transmission increases in too many synapses of a neuron,
excitation-induced toxic effects, such as excessive calcium levels, may set in.
As a result the neuron may be harmed or even killed. In a typical Hebbian or
associative process, each synapse acts in a fairly independent manner. There
has to be some additional mechanism or constraint operating cell-wide that
can throttle back runaway positive or negative changes in synaptic efciency.
There are two, not necessarily distinct, ideas of how this may occur, both
supported by a body of experimental data. One of these is the notion of a
sliding modication threshold; the other is the idea of synaptic scaling.
In the sliding threshold model (Figure 21.12), a cell B possesses a threshold for synaptic modication. If the postsynaptic cells ring rate exceeds
that threshold, paired pre- and postsynaptic ring will increase the efciency of the synapse. If the postsynaptic cells ring rate falls below the
threshold value, the same correlated activity will diminish the efciency of
that synapse. The effect is a dynamic one. Cell B will continually adjust its
threshold according to the changes in the cells ring rate suitably averaged
over time. The threshold will slide up or down as the ring rate increases
or decreases. By moving up it becomes more difcult to produce further
increases, and becomes easier to elicit decreases. Similarly, if the threshold
goes down because of weak ring activity, it becomes easier to strengthen
synapses and harder to weaken them.
In synaptic scaling, like the sliding threshold model, the total activity in
the postsynaptic cell determines how that cell responds to neurotransmitter release. The ring rate reects the sum of all synaptic activities in the
postsynaptic cell. If the ring rate in the postsynaptic cell is already high all
synaptic strengths will be scaled back, and conversely if the ring rate is too
low the strength of each synapse will be scaled up. In mathematical terms,
the effect is a multiplicative one. The strength at a particular synapse is proportional to the product of the ring rate and the synaptic strength at that

Figure 21.12. Sliding threshold model: Shown is a plot of the modication predicted
by the sliding threshold model as a function of the total activity in the postsynaptic cell. Activity levels below that of the threshold produce long-term depression, or
LTD (negative modication), while those that exceed the threshold lead to longterm potentiation, or LTP (positive modication). The threshold adapts to the
overall activity in the postsynaptic cell averaged over a suitable time interval.

21.13 Areas of the Brain Relating to Drug Addiction

529

synapse. Since the scaling affects all synapses in the same way it may be
best thought of as a form of cellular homeostasis operating in addition to
Hebbian mechanisms.

21.12 Fear Circuits Detect and Respond to Danger

The forms of synaptic plasticity such as LTP that are observed in the hippocampus are not limited to that region. They are encountered in regions
of the brain ranging from the visual cortex to the amygdala to regions of
the brain involved in drug addiction. The amygdala is the central player in
fear conditioning. In fear conditioning experiments using rats, tone signals
are relayed through the thalamus to the auditory cortex and from the thalamus and auditory cortex to the amygdala. Foot shock signals are similarly
relayed into the somatosensory cortex and from there to the amygdala.
The amygdala is partitioned into several regions. Some signals rst enter
the lateral nucleus (LN) of the amygdala and then pass to the basal nucleus
(BN), while other signals enter the basal nucleus directly. These areas along
with the accessory basal nucleus function as the central input unit of the
amygdala that receive auditory, visual, somatosensory, and other forms of
sensory information from the thalamic relay nuclei and higher sensory cortical areas. These nuclei also receive input information from the hippocampus of a contextual character, that is, environmental cues about, for
example, place and space, associated with the experience. As shown in
Figure 21.13 connections from the input unit are made to several regions.
These include connections between the input unit and central nucleus,
which sends projections to a number of brain stem regions and by that
means controls the behavioral and physiological fear responses.
Fear conditioning produces synaptic modications quite similar to hippocampal LTP. As is the case for LTP in hippocampal slices, LTP in the
lateral and basal nuclei of the amygdala is dependent on postsynaptic depolarization. Calcium entry through NMDA receptors plays an important role,
as does activation of and signaling by protein kinase A and MAP kinases.
As was the case for LTP, there are several different forms ranging from
short-term transitory changes to long lasting ones requiring CREB transcriptional activity and protein synthesis.

21.13 Areas of the Brain Relating to Drug Addiction

Drug addiction involves areas of the brain that shape mood and generate
emotional responses. There are many different kinds of addictive drugs.
Prominent examples of addictive drugs are alcohol, amphetamines, cocaine,
morphine, and nicotine. Although the chemical makeup of each of these
substances is different, all drugs target populations of neurons situated in

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21. Learning and Memory

Figure 21.13. Circuitry involved in responses to fearful and dangerous sensory and
contextual stimuli: Abbreviations: LNLateral nucleus of the amygdala; BN
Basal nucleus; ABNAccessory basal nucleus; CNCentral nucleus. The hippocampal system responsible for relaying contextual information into the amygdala
from sensory regions consists of the perirhinal cortex, entorhinal cortex, and
hippocampus.

areas involved in mood and emotional responses and exhibit the following
set of characteristics:
the compulsion to take drugs,
loss of control in limiting intake,
entry into a negative emotional state when access to drugs is prevented,
and
relapses into addiction after periods of abstinence.
The changes induced by drug use are long-lived and involve changes at both
the cellular and molecular levels. The cells and circuits that underlie drug
addiction are located in several regions of the brain. They include cells
that express acteylcholine receptors and communicate using dopamine
(dopaminergic) and serotonin (serotonergic), and also include cells that
release glutamate (glutamergic) or GABA (GABAergic). The corresponding brain regions are associated with reward, mood, arousal, and cognition. Long-lasting changes in synaptic transmission similar to those
associated with learning and memory, but leading to addiction, take place
in these brain regions.
The sites of action of addictive drugs such as cocaine, amphetamines, and
nicotine lie deep in the brain, in regions associated with mood, emotions,
and learning. As shown in Figure 21.14, drugs act on neurons in the upper
part of the brain stem (ventral tegmental area and substantia nigra), the
limbic system (amygdala and hippocampus), and the mesolimbic system

21.14 Drug-Reward Circuits Mediate Addictive Responses

531

Figure 21.14. Side view of the brain showing the main sites of drug action: The
regions of drug actionthe ventral tegmental area, substantia nigra, nucleus accumbens, striatum, and prefrontal cortexare shown in lighter and darker gray shades.
Areas that form the nigrostriatal system are shown in light gray and those that the
mesocorticolimbic system comprises are presented in dark gray.

(nucleus accumbens). The amygdala and hippocampus are ancient

structures. The amygdala, which means almond-shaped, connects to hippocampus and prefrontal areas. Activity in this area of the brain is associated with strong emotions such as fear, aggression, and the ght or ight
response. The hippocampus, meaning seahorse-shaped, is located just
behind the amygdala. As discussed earlier in this chapter, this structure is
associated with learning and memory, and especially with the transfer of
information into memory.

21.14 Drug-Reward Circuits Mediate

Addictive Responses
Under the inuence of drugs, neurons involved in mood, arousal, and cognition organize into drug-reward circuits.The core circuitry involved in drug
addiction contains two systemsthe nigrostriatal system and the mesocorticolimbic system. The substantia nigra (SN) is located in the upper part of
the brain stem. It contains two groups of cell. One group, the dorsally
located cells (the SN pars compacta, or SNc), uses dopamine to communicate with the corpus striatum. The other group, the ventrally positioned
neurons (the SN pars reticulata, or SNr), utilizes GABA to communicate
with the thalamus. The nigrostriatal system consists of neurons in the substantia nigra pars compacta and the corpus striatum, or striatum. The mesocorticolimbic system consists of cells in the ventral tegmental area (VTA)
located near the SN, the nucleus accumbens (NAc) situated just above the

532

21. Learning and Memory

amygdala, and the prefrontal cortex that encompasses the nonmotor portions of the frontal lobe.
The most striking feature of cells in the two systems is the prominence
of dopamine-releasing neurons. Neurons in the ventral tegmental area
and in the substantia nigra contain large numbers of dopamine-releasing
neurons. When stimulated by drugs these neurons elevate the extracellular
dopamine levels in the nucleus accumbens and striatum. Dopamine promotes reinforcement, in which behavioral responses linked to rewards
increase in frequency over time. In the drug-reward circuits, dopamine functions as the reward signal in response to the taking of drugs such as cocaine,
and also as a stimulant of reward-seeking behavior.

21.15 Drug Addiction May Be an Aberrant Form of

Synaptic Plasticity
Drug addiction appears to be an aberrant form of synaptic plasticity, in
which circuits involved in LTP and LTD are altered. Glutamate and
dopamine work together in the VTA to promote addiction (Figure 21.15).
Addictive drugs such as nicotine act in at least three different ways in
the VTA. They bind to nicotinic acetylcholine receptors (nAChRs) on
dopamine-releasing cells triggering the release of dopamine. They also bind
to nAChRs on presynaptic terminals of glutamate-releasing cells resulting
in an increased release of glutamate. The glutamate, in turn, triggers an
increased production of dopamine by dopamine-releasing cells receiving

Figure 21.15. Schematic representation of a portion of the VTA-NAc drug-reward

circuitry: Shown is a representative VTA dopamergic neuron whose axon projects
into the nucleus accumbens and releases dopamine there. As explained in the text,
the dopamine release is regulated and, under the inuence of nicotine, potentiated
by a GABAergic neuron and by a glutamergic neuron, here depicted as located in
the VTA. Also included in the gure is the action of amphetamines and cocaine on
the dopamine transporter (DAT).

21.16 In Reward-Seeking Behavior, the Organism Predicts Future Events

533

the glutamate signals. The effect of the glutamate is to prolong the release
of dopamine results in an LTP-like potentiation of synaptic transmission
and increase in duration of the reward signal. (When nAChRs found on
dopamergic neurons in the VTA bind nicotine they depolarize the membrane. The nAChRs located on presynaptic terminals enhance glutamate
release, thus stimulating further dopamine release. When NMDA receptors
are present, long-term potentiation of synaptic transmission is produced.)
Thirdly, nicotine binds to GABA-releasing cells, which for a while throttle
back the actions of dopamine-releasing cells, but desensitization turns this
off over time, further promoting addictive effects of the drug.
A denition of synaptic plasticity useful in discussions of drug addiction
is one describing it as the ability of circuits and systems to modify their
responses to a stimulus brought on by prior stimulation, either of the same
type or of an associated form. The connections between drug addiction and
synaptic plasticity have been studied most intensively in the ventral tegmental area and the nucleus accumbens. Opiates and nicotine exert their inuences through interactions with neurons of the VTA, while cocaine and
amphetamines act on neurons of the NAc. As indicated in Figure 21.15,
cocaine and amphetamines impede the ability of the dopamine transporter
(DAT) to take up dopamine from the extracellular spaces. Homeostatic regulation is lost and the dopamine levels become excessive.
Neurons in the prefrontal cortex send and receive signals from VTA and
NAc neurons, and these regions are the locus of feelings of pleasure associated with drug addiction. The networks in these areas serve as the core of
the brains reward circuitry. Again, a key element in this reward circuitry,
that is, a feature that is common to all forms of drug abuse, is the elevation
of dopamine levels leading to pleasurable sensations.

21.16 In Reward-Seeking Behavior, the Organism

Predicts Future Events
To survive an organism must be able to decide between alternative
responses whenever it is searching for food or water, or avoiding danger,
or seeking a mate. In reward-driven behavior a positive reinforcement is
supplied to behavioral acts in order to strengthen future response to the
associated stimuli. Reward-driven behavior is a type of learning, quite like
the Hebbian form, in which a stimulus is paired with a reward or a punishment. This kind of learning is called predictive learning, the classical
example of which is Pavlovs experiments with dogs.
Pairing a reward/punishment with a stimulus does not always result in
learning. There is a second requirement: Some element of uncertainty or
novelty must be present. More formally, there must be some discrepancy,
or error, between the reinforcement predicted by the stimulus and the
actual reinforcement. If this reinforcement error is zero there is no learn-

534

21. Learning and Memory

ing since everything turns out as expected. The dopamine release can be
regarded as a teaching signal that triggers an increased alertness whenever
something interesting or unexpected has occurred. In doing so, it strengthens connections between neurons leading to LTP and drug addiction.

References and Further Reading

General References
Bear MF, Connors BW, and Paradiso MA [2001]. Neuroscience: Exploring the Brain
(2nd ed.) Baltimore, MD: Lippincott, Williams and Wilkins.
Kandel ER [2001]. The molecular biology of memory storage: A dialogue between
genes and synapses. Science 294: 10301038.
Kandel ER, Schwartz JH, and Jessel TM [2000]. Principles of Neural Science (4th
edition). Norwalk CT: Appleton and Lange.
Lamprecht R, and LeDoux J [2004]. Structural plasticity and memory. Nature Rev.
Neurosci., 5: 4554.

Synaptic Assembly
Biederer T, et al. [2002]. SynCAM, a synaptic adhesive molecule that drives synapse
assembly. Science, 297: 15251531.
Dalva MB, et al. [2000]. EphB receptors interact with NMDA receptors and regulate excitatory synapse formation. Cell, 103: 945956.
Dean C, et al. [2003]. Neurexin mediates the assembly of presynaptic terminals.
Nature Neurosci., 7: 708716.
Penzes P, et al. [2003]. Rapid induction of dendritic spine morphogenesis by transsynaptic EphrinB-EphB receptor activation of the Rho GEF Kalirin. Neuron, 37:
263274.
Takasu MA, et al. [2002]. Modulation of NMDA receptor-dependent calcium inux
and gene expression through EphB receptors. Science, 295: 491495.

The Presynaptic Active Zone

Atwood HL, and Karunanithi S [2002]. Diversication of synaptic strength: Presynaptic elements. Nature Rev. Neurosci., 3: 497516.
Chen YA, and Scheller RH [2001]. SNARE-mediated membrane fusion. Nature Rev.
Mol. Cell Biol., 2: 98106.
Dobrunz LE, and Stevens CF [1997]. Heterogeneity of release probability, facilitation and depletion at central synapses. Neuron, 18: 9951008.
Jahn R, Lang T, and Sdhof TC [2003]. Membrane fusion. Cell, 112: 519533.
Rizo J, and Sdhof TC [2002]. SNAREs and Munc18 in synaptic vesicle fusion.
Nature Rev. Neurosci., 3: 641653.
Yoshihara M, and Littleton JT [2002]. Synaptotagmin I functions as a calcium sensor
to synchronize neurotransmitter release. Neuron, 36: 897908.

The Postsynaptic Density

Bredt DS, and Nicoll RA [2003]. AMPA receptor trafcking at excitatory synapses.
Neuron, 40: 361379.

References and Further Reading

535

Impey S, Obrietan K, and Storm DR [1999]. Making new connections: Role of ERK
MAP kinase signaling in neuronal plasticity. Neuron, 23: 1114.
Kennedy MB [2000]. Signal-processing machines at the postsynaptic density.
Science, 290: 750754.
Kim JH [2003]. Presynaptic activation of silent synapses and growth of new synapses
contribute to intermediate and long-term facilitation in Aplysia.Neuron,40:151165.
Kim JH, and Huganir RL [1999]. Organization and regulation of proteins at
synapses. Curr. Opin. Cell Biol., 11: 248254.
Malinow R [2003]. AMPA receptor trafcking and long-term potentiation. Phil.
Trans. R. Soc. Lond. B, 358: 77714.
Sheng M, and Kim MJ [2002]. Postsynaptic signaling and plasticity mechanisms.
Science, 298: 776780.
Sweatt JD [2001]. The neuronal MAP kinase cascade: A biochemical signal integration system subserving synaptic plasticity and memory. J. Neurochem., 76: 110.
Thomas GM, and Huganir RL [2004]. MAPK cascade signalling and synaptic plasticity. Nature Rev. Neurosci., 5: 173182.
Ziff EB [1997]. Enlightening the postsynaptic density. Neuron, 19: 11631174.

Long-Term Potentiation
Bliss TVP, and Collingridge GL [1993]. A synaptic model of memory: Long-term
potentiation in the hippocampus. Nature, 361: 3139.
Lisman J, Schulman H, and Cline H [2002]. The molecular basis of CaMKII function in synaptic and behavioral memory. Nature Rev. Neurosci., 3: 175190.
Malenka RC, and Nicoll RA [1999]. Long-term potentiationA decade of
progress? Science, 285: 18701874.
McGaugh JL [2000]. MemoryA century of consolidation. Science, 287: 248251.

Bidirectional Synaptic Plasticity

Abraham WC, and Bear MF [1996]. Metaplasticity: The plasticity of synaptic plasticity. Trends Neurosci., 19: 126130.
Shouval HZ, Bear MF, and Cooper LN [2002]. A unied model of NMDA receptor-dependent bidirectional synaptic plasticity. Proc. Natl. Acad. Sci. USA, 99:
1083110836.

Fear Conditioning
Blair HT, et al. [2001]. Synaptic plasticity in the lateral amygdala: A cellular hypothesis for fear conditioning. Learn. Mem., 8: 229242.
McKernan MG, and Shinnick-Gallagher P [1997]. Fear conditioning incuces a lasting
potentiation of synaptic currents in vitro. Nature, 390: 607611.
Rogan MT, and LeDoux JE [1996]. Emotion: Systems, cells and synaptic plasticity.
Cell, 85: 469475.
Rogan MT, Stubli UV, and LeDoux JE [1997]. Fear conditioning induces associative long-term potentiation in amygdala. Nature, 390: 604607.
Schafe GE, et al. [2001]. Memory consolidation of Pavlovian fear conditioning: A
cellular and molecular perspective. Trends Neurosci., 24: 540546.
Shumyatsky GP, et al. [2002]. Identication of a signaling network in lateral nucleus
of the amygdala important for inhibiting memory specically related to learned
fear. Cell, 111: 905918.

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21. Learning and Memory

Drug Addiction
Berke JD, and Hyman SE [2000]. Addiction, dopamine, and ther molecular mechansism of memory. Neuron, 25: 515532.
Fiorillo CD, Tobler PN, and Schultz W [2003]. Discrete coding of reward probability and uncertainty by dopamine neurons. Science, 299: 18981902.
Hyman SE, and Malenka RC [2001]. Addiction and the brain: The neurobiology of
compulsion and its persistence. Nature Rev. Neurosci., 2: 6695703.
Koob GF, Sanna PP, and Bloom FE [1998]. Neuroscience of addiction. Neuron, 21:
467476.
Mansvelder HD, and McGehee [2000]. Long-term potentiation of excitatory inputs
to brain reward areas by nicotine. Neuron, 27: 349357.
Nestler EJ [2001]. Molecular basis of long-term plasticity underlying addiction.
Nature Rev. Neurosci., 2: 119128.
Saal D, et al. [2003]. Drugs of abuse and stress trigger a common synaptic adaptation in dopamergic neurons. Neuron, 37: 577582.
Schultz W, Dayan P, and Montague PR [1997]. A neural substrate of prediction and
reward. Science, 275: 15931599.
Waelti P, Dickinson A, and Schultz W [2001]. Dopamine responses comply with basic
assumptions of formal learning theory, Nature, 412: 4348.

Problems
21.1 How does the neuron know which synapses to strengthen and which
synapses to weaken when making long-term changes involving gene
expression and protein synthesis? In a Hebbian type of rule for changing the strength of synapses, only those synapses exhibiting correlated
ring are strengthened. Referring back to Figure 21.11(a), only the
middle synapse onto cell B is strengthened. The middle synapse
must be tagged in some fashion or other, so that the results of gene
expression can be applied to that synapse alone. How might that be
done?
21.2 In a Hebbian type of rule the postsynaptic cell res an action potential. This is a cell-wide signal that a set of associations has been made.
This may be viewed as a strong form of Hebbs rule. The weak form
of the rule would only require postsynaptic membrane depolarization
by some amount. In a weaker form of Hebbian rule, an action potential is not required, but rather the convergent signals produce a depolarization. How might action potential information get conveyed to
the dendrites to elicit an immediate strengthening of the synapse?
21.3 Several different experimental protocols are utilized in the hippocampal slice preparations. All of these involve pairing of spikes in
the pre- and postsynaptic cells. One of the most effective protocols is
that of tetanic stimulations. In the last chapter, at least four different
kinds of ring modes were discussed. Which of the different modes of

Problems

537

ring would be most effective in eliciting LTP in vivo and in an adult

where strong signaling is needed?
Suggested Reading
Martin KC, and Kosik KS [2002]. Synaptic tagging: Whos it? Nature Rev. Neurosci.,
3: 813820.
Paulsen O, and Sejnowski TJ [2000]. Neural patterns of activity and long-term synaptic plasticity. Curr. Opin. Neurobiol., 10: 172179.

Glossary

Acetylation The addition, catalyzed by acetyltransferases, of acetyl groups

to amino acid side chain nitrogens on target proteins.
Action potential Self-regenerating pulse of electrical activity that propagates down axons and is generated by the opening and closing of sodium
and potassium channels.
Active zone Region of the presynaptic axon terminal containing the
machinery for neurotransmitter release.
Adaptive immune response Immune response unique to vertebrates
involving antigen recognition and the production of antibodies.
Adapter proteins Nonenzymatic proteins that contain protein-protein
interaction domains and serve as intermediaries that allow signaling proteins that would otherwise not be able to communicate to do so.
Agonist A molecule that binds a receptor and induces the same response
in the receptor as the one triggered by the natural ligand.
Allosteric modication Shifts in equilibria between two preexisting populations of conformational states, brought on either by ligand binding or by
covalent attachment of groups. In an allosteric modication, binding at one
location in the molecule is able to alter how other portions of the molecule
respond to their binding partners because of the conformational changes
that accompany the shifts in equilibrium.
Anchor proteins Nonenzymatic proteins that attach to the plasma membrane and to membranes of organelles, and provide platforms for signaling
proteins to dock in close proximity to receptors and ion channels.
Angiogenesis The late stages of vasculogenesis in which an initial set
of tubules is rened through further differentiation, sprouting, and branching to form a mature vascular system containing arterial and venous
structures.
539

540

Glossary

Antagonist A molecule that binds a receptor, but the receptor does not
transmit a signal in response to the binding event. Drugs that bind in an
antagonistic fashion are known as blockers.
Antibiotic Biomolecules synthesized by fungi and bacteria that kill competing microbes.
Antibodies Receptors synthesized by B cells that recognize and bind
antigens.
Antigen (antibody generator) Foreign substance, derived from a pathogen
and expressed either on the outer surface of the pathogen or on the surface
of an antigen-presenting cell, that triggers the production of antibodies.
Antigenic variation Systematic alterations in the antigens expressed on
the outer surface of a pathogen.
Apoptosis Programmed cell death, in which there is an orderly disassembly of the cell that avoids harming neighboring cells. Also called cell suicide.
Apoptosome The major control point for converting internal stress
signals into apoptotic responses. It is located just outside the mitochondria
and is activated by the release of cytochrome c.
Associative learning Changes in the behavioral response to the weak stimulus that has been paired with a strong (positive or negative) stimulus.
Autocrine A signaling mode in which hormones secreted from a cell act
back on the cell releasing them.
Auxiliary spice sites Sites where splicing regulators bind. Auxiliary sites
located within exons are called exonic splice enhancer (ESE) and exonic
splice inhibitory (ESI) sites, depending on which regulatory outcome is supported. Similarly, intronic sites are termed intronic splice enhancer (ISE)
and intronic splice inhibitory (ISI) sites.
Bacteriophage Virus that infects bacteria; also called a phage.
Biolm A bacterial colony formed on exposed surfaces and exhibiting
cooperative behavior between members.
Branching morphogenesis Growth, invasion, and proliferation of cells
that form branched tubular structures that carry uids in the vasculature,
lungs, kidneys, and mammary glands.
Caspases Proteolytic enzymes that catalyze the cleavage of specic molecules in response to apoptosis signals.
Catch bond A bond that is strengthened by the external forces. The forcedriven enhancements in the lifetime of these bonds allow leukoctytes to be
captured by the walls of the blood vessels and begin rolling.

Glossary

541

Caveolae (little caves) Tiny ask-shaped invaginations in the outer leaet

of the plasma membrane that are detergent-insoluble, enriched in glycosphingolipids, cholesterol, and lipid-anchored proteins, and in caveolins,
a coatlike material.
Cell adhesion Cell-to-cell and cell-to-ECM attachment mediated by long
modular and exible glycoproteins expressed on opposing surfaces acting
as receptors and counterreceptors or ligands.
Cell fate The determination of which tissue or organ a particular cell
becomes a member of during embryonic development.
Cell polarity The asymmetric distributions of cellular components that
arise during development. In the case of nerve cells it produces striking differences in morphologyaxons at one end, dendrites at the other; in epithelial cells it gives rise to apical and basolateral plasma membrane domains.
Central pattern generators Circuits built from small numbers of neurons
that are used to drive the rhythmic ring of muscles responsible for
activities such as walking and swimming, breathing, and chewing and
digesting.
Chaperones Proteins that help other proteins to fold into their native
state, shuttle proteins to their correct locations in the cell, prevent unwanted
aggregation, and assist in recovering and refolding proteins that have
become misfolded due to cellular stresses.
Checkpoints Signaling pathways that ensure that a cell cycle or assembly
process does not begin before a prior necessary process is completed.
Chemotaxis The process whereby a unicellular organism senses nutrients
and noxious substances in its local environment, and, in response, moves
towards the nutrients and away from the harmful chemicals.
Chromatin In eukaryotes, material from which chromosomes are made,
consisting of DNA wrapped around proteins called histories.
Chromophore Groups of atoms or molecules that act as pigments, imparting color to the materials in which they reside by absorbing light at some
wavelengths and scattering it at others.
Competence The ability of a bacterium to take up exogenous DNA from
its environment.
Conjugation A form of horizontal gene transfer in which bacteria establish direct contact with one another; sex pili are formed, and genetic material in the form of plasmid are sent from donor to recipient.
Control point Locations in the cell where environmental and regulatory
signals converge, integrate, and convent to cellular responses.

542

Glossary

Cytokines Small signaling proteins synthesized and secreted by leukocytes, most commonly, macrophages and T cells. They convey a variety of
instructions to leukocytes and to other cells such as neurons.
Death-inducing signaling complex (DISC) The name given to the control
point responsible for converting external death signals into apoptotic
responses. It is organized by death receptors at and just below the plasma
membrane.
Denatured state The ensemble of states that a newly synthesized protein,
or an unfolded protein, populates.
Dephosphorylation The removal catalyzed by protein phosphatases of
phosphoryl groups previously added to amino acid side chain hydroxyls on
protein substrates by protein kinases.
Desensitization The process whereby a G protein-coupled receptor, or
any other receptor, loses its responsiveness to binding by its ligand.
Diffraction Scattering of light by atoms, molecules, and larger objects
resulting in departures from rectilinear motion other than reection or
refraction.
Diffusion Thermally driven movement of particles in a uid from one
locale to another produced by random collisions of the particles with the
molecules of the uid.
Distal sites DNA regulatory regions where long-range interactions
between regulatory proteins and the basal transcription machinery take
place.They may be located upstream of the core promoter,downstream of the
core promoter,in between coding regions,and inside introns.Positively acting
transcription factors that bind at these sites are called enhancers, while negatively acting transcription factors are referred to as silencers.
Domain fold Stable arrangements of multiple secondary structure elements and of two or more structural motifs into independent folding units.
Efciency of synaptic transmission Magnitude of the response generated
in a postsynaptic neuron when an action potential is generated in the presynaptic neuron.
Electrostatic complementarity The matching of hydrophobic patches, the
complementary pairing of hydrogen bond donors and acceptors, and the
matching of positive and negative charges of basic and acidic polar residues
from one surface to the other of the interface.
Endocrine A signaling mode in which hormones are secreted into the
bloodstream and other bodily uids by specialized cells and travel large distances to reach multiple target cells.

Glossary

543

Endocytosis The process whereby plasma membrane proteins and materials captured at the cell surface are packaged into vesicles and shipped to
digestive compartments for processing and recycling.
Energy landscape A graphical depiction of how the number of states
available to a protein at each value of the potential energy varies as a function of a few signicant degrees of freedom.
Envelope Lipid/carbohydrate membrane derived from the host cell that
surrounds a viral capsid.
Euchromatin Transcriptionally active chromatin with an open shape that
permits transcription factors and the basal transcription machinery to
access promoters.
Exocytosis The packaging and shipping of newly synthesized proteins destined for export and use in the plasma membrane in vacuoles that move
over the rail system and fuse with membranes at their destination.
Exons Short coding sequences separated from one another by introns in
pre-messenger RNAs.
Focal adhesions Points of contact and adhesion between the cell and the
supporting extracellular matrix. They serve as control points where growth
and adhesion signals are integrated together to govern the overall growth
and movement of the cell.
Folding funnel The shape of the potential energy landscape that arises
because there are many high energy states and few low energy ones.
Gate The part of the ion channel that opens and closes the pore through
which ions pass.
GDP dissociation inhibitors (GDIs) Enzymes bind and maintain pools of
inactive GTPases by inhibiting the dissociation of GDP from the GDPase.
Glycosylation A common posttranslational modication to proteins destined for insertion in the plasma membrane in which covalently linked
oligosaccharides that extend out from their extracellular side are added.
The modied proteins are referred to as glycoproteins.
Glycosyl phosphatidylinositol (GPI) anchors Posttranslational modications to proteins that allow them to attach to the outer, or exoplasmic, leaet
of the plasma membrane. GPI anchors are made from a complex sugar plus
a phosphatidylinositol grouping.
Growth cones Sensory structures located at the tip of advancing axonal
and dendritic processes sent out from neurites. They explore, interact
with, interpret, and respond to signaling molecules in their local microenvironment.

544

Glossary

Growth factors Molecules that stimulate growth and development.

GTPase-activating proteins (GAPs) Enzymes that catalyze the hydrolysis of the GTPase-bound GTP to GDP.
GTPases Small enzymes that hydrolyze GTP and function as molecular
switches and timers. They are activated when bound to GTP and deactivated when bound to GDP.
Guanine nucleotide exchange factors (GEFs) Enzymes that catalyze the
dissociation of GDP from the GTPase.
Habituation Weakening of a behavioral response to a harmless stimulus
through repeated exposures to that stimulus.
Heterochromatin Transcriptionally inactive chromatin with a tightly compacted shape that prevents transcription factors and the basal transcription
machinery from accessing promoters.
Holoenzyme Inactive enzyme (apoenzyme) plus additional noncovalently
bonded biomolecules, either loosely bound cofactors or tightly bound prosthetic groups, required for full catalytic activity.
Homeostasis Physiological process by which a cell, tissue, or organism
balances and stabilizes internal conditions such as temperature, pH,
excitability, and cell type in the presence of external perturbations.
Horizontal gene transfer Lateral transfer of genetic information between
distantly related species through conjugation, transduction, and
transformation.
Hydrophilic A water-loving amino acid; a water molecule would rather
bind to this amino acid than to another water molecule.
Hydrophobic A water-hating amino acid; a water molecule would rather
bind to another water molecule than to this amino acid.
Inammatory response Set of physiological responses including fever and
pain, redness and swelling. A local environment is formed that promotes
migration of leukocytes to the infection site, the destruction of the invasive
agents, and the repair of damaged tissues.
Innate immune response Immune response involving recognition by
leukocytes of molecules situated on the outer surface of pathogens that are
characteristic of the pathogen.
Insulators DNA sequences that mark boundaries between independent
sections of DNA.
Interface Region of surface contact between two macromolecules
through which binding and communication take place.

Glossary

545

Internalization The removal of a receptor from the plasma membrane

through endocytic mechanisms.
Introns Long noncoding, or intervening, sequences situated in between
exons in pre-messenger RNAs.
Ion channels Membrane-spanning proteins forming narrow pores that
enable specic inorganic ions, typically Na+, K+, Ca2+ or Cl-, to passively
diffuse in a directional manner through cell membranes.
Ionotropic Receptor ion channel that opens and closes in response to
neurotransmitter binding.
Juxtacrine A signaling model in which messages are conveyed by direct
contact between a receptor on one cell and a cell surface-bound ligand or
counterreceptor on an adjacent cell.
Kinetic proofreading A cellular mechanism for improving the delity of
a process by tying it to a series of intermediate time- and energyconsuming steps. In receptor-ligand binding, differences in afnity are
converted to differences in signaling because of the intermediate timeand energy-consuming steps.
Kinetic trap A set of states forming a local minimum in the energy landscape and enclosed by energy barriers large compared to the thermal
energy.
Labile Readily undergoes change or breakdown.
Lateral inhibition Process whereby a cell adopting a particular cell fate
inhibits its neighbors from adopting the same fate.
Learning The adaptive process whereby changes in behavior are induced
in response to experience.
Leukocytes White blood cells; highly motile and short-lived cells that
move through the cardiovascular and lymphatic systems into damaged
tissues where they kill bacterial, protozoan, fungal and multicellular
pathogens, destroy cells infected with viruses and bacteria, and eliminate
tumor cells.
Lipid rafts Plasma membrane microdomains that are detergent insoluble
and enriched in cholesterol and sphingolipids. Unlike caveolae, they do not
contain caveolins and are not cavelike in shape but instead are at.
Long-term facilitation Strengthening of the behavioral response to a mild
touch to the tail that has been paired with an electric shock to the siphon,
an experimental model of associative learning in the Aplysia siphon withdrawal circuit.
Long-term potentiation Strengthening of the postsynaptic response to a
presynaptic action potential brought on by pairing a series of presynaptic

546

Glossary

action potentials with a postsynaptic depolarization or action potential, an

experimental model of associative learning in the rodent hippocampal
CA1CA3 regions.
Lysogenic Life cycle in which phage DNA is integrated into the host cell
DNA and the bacteriophage becomes a prophage, replicating as part of the
bacterial hosts chromosome.
Lytic Life cycle in which phage DNA is replicated and multiple virus particles are formed and escape from the cell by rupturing the cells plasma
membrane.
Matrix protein Associates with the inner layer of the viral envelope and
is situated in between the inner layer and the capsid.
Memory The record underlying the changes in behavior brought on by
learning.
Metabotropic receptors G protein-coupled receptors (GPCRs) that
activate their cognate heterotrimeric G proteins in response to neuromodulator binding.
Metastasis The process whereby cancer cells break away from their point
of origin, the primary tumor, enter the circulatory system, and invade other
organs, where they form secondary tumors.
Methylation The addition catalyzed by methyltransferases of methyl
groups to amino acid side chain nitrogens on target proteins.
Morphogens Signaling proteins expressed either on cell surfaces or
secreted into the extracellular spaces in the form of concentration gradients that are read by other cells to determine their developmental fate.
Motor neurons Supply input to muscles that drives their contractions, and
receive input from upstream sensory and control neurons.
Mutual inhibition Pairs of neurons that are reciprocally connected and
sequentially inhibit each others ring activities.
Native state A stable state of the folded protein. It is a state of minimum
Gibbs free energy at physiological temperatures and conditions.
Neuromodulators Signaling molecules secreted in a broader manner than
neurotransmitters; they modify the excitability of large numbers of target
cells by regulating the activities of their ion channels.
Neurotransmitters Signaling molecules released from the presynaptic terminal of a neuron and diffuse in a directional manner across the synaptic
cleft to the postsynaptic terminal of a neighboring neuron, where they bind
receptor ion channels.
Nucleocapsid The viral capsid plus its nucleic acid core.

Glossary

547

Nucleosome The fundamental repeating unit of chromatin. Nucleosomes,

146 base pairs of DNA wrapped about a histone octamer, are strung
together like beads on the string by means of linker segments.
Oncoproteins Proteins that operate in the signal transduction, integration, and regulatory pathways involved in cellular growth, multiplication, differentiation, and death, that when mutated stimulate
unregulated cell growth and proliferation thus promoting the development of cancer.
Operon DNA sequences that encode one or more proteins and the
upstream sequences for attachment of the RNA holoenzyme and regulatory proteins in bacteria.
Organizing centers Localized groupings of cells that secrete morphogens
that impart patterns of cell fates to elds of progenitor cells.
Pacemaker neuron Neurons that generate the rhythmic ring patterns
without requiring any rhythmic input.
Paracrine A signaling mode in which molecules are secreted into the
extracellular spaces by an originating cell and travel no more than a few
cell diameters to reach their target cell.
Pathogen (bacterial) Organisms possessing genes that encode virulence
factors and are situated on plasmids and other mobile genetic elements that
can be readily transferred and exchanged between species.
Permeability The propensity of a membrane to allow passage of certain
ions and molecules.
Permeability transition pore complex (PTPC) Also known as the permeability transition pore (PTP), this control point is formed at points of
contact between the inner and outer mitochondrial membranes. The PTPC
is a conduit for the passage of agents such as cytochrome c and
Smac/DIABLO that trigger apoptosome assembly and activation of
caspase 9, and is the major site for regulation by Bcl-2 proteins.
Phosphorelay A signal transduction system consisting of a hybrid sensor
unit, a histidine phosphotransfer protein, and a response regulator. Compared to the two-component system, the hybrid sensor unit contains an
extra module, an aspartate-bearing receiver, and a histidine phosphotransfer protein is situated in between the sensor unit and the response regulator.
Phosphorylation The reversible addition, catalyzed by protein kinases, of
phosphoryl groups to amino acid side chain hydroxyls on target proteins.
Plasmid Extrachromosomal DNA found in bacteria and encoding sets of
functionally related genes.

548

Glossary

Plateau potentials Stable membrane potentials occurring at depolarizations greater than that of the resting membrane potential. When a membrane is at a plateau potential it is far more excitable and can repeatedly
re action potentials even in the absence of sustained excitatory input from
synapses.
Platelets Cytoplasmic fragments of bone marrow cells called megakaryocytes that form clots that block blood ow at sites of injury.
Pleiotropic Multifunctional.
Polypeptide hormones Small compact polypeptide growth factors that
bind to receptor tyrosine kinases.
Pores Membrane-spanning proteins found in the outer membrane of
Gram-negative bacteria, mitochondria, and chloroplasts forming channels
that enable hydrophilic molecules smaller than about 600 Da to pass
through.
Post-inhibitory rebound A strong hyperpolarization that is quickly terminated and followed by a rapid depolarization leading to the ring of an
action potential.
Postsynaptic density Region of the postsynaptic dendrite terminal containing the machinery for neurotransmitter signal transduction.
Pre-initiation complex (PIC) Also known as the basal transcription
machinery the PIC consists of RNA polymerase II and a set of general transcription factors.
Pre-messenger RNA (pre-mRNA) Eukaryotic RNA molecule produced
by transcription from DNA. It contains exons, introns, and regulatory
sequences that provide binding sites for the splicing machinery and
regulatory proteins.
Primary splice sites Consist of (i) the 5 splice site characterized by the
presence of a binding sequence containing a guanine-uracil (GU) pair
within a longer GURAGU-like sequence, where R is a purine; (ii) the
branch site characterized by the nucleotide sequence YNYURAY, where Y
is a pyrimidine; (iii) the pyrimidine tract, a string of pyrimidine nucleotides;
and (iv) the 3 splice site characterized by either a CAG sequence or a UAG
sequence.
Primary structure The proteins covalent structure, the linear sequence of
amino acids linked to one another by peptide bond plus all disulde bonds
formed during folding.
Promoter Transcriptional regulatory region of DNA containing binding
and start sites for RNA polymerase II and binding sites for the transcription control elements.

Glossary

549

Protease See proteolysis.

Protein backbone The main chain, the set of repeating NCaC units covalently linked to one another by peptide bonds.
Protein folding The process whereby newly synthesized linear polypeptide chains spontaneously fold into functional three-dimensional forms.
Protein kinases Enzymes that catalyze the transfer of phosphoryl groups
to amino acid side chain hydroxyls on protein substrates using ATP as the
donor.
Protein phosphatases Enzymes that catalyze the removal of phosphoryl
groups previously added to selected amino acid side chain hydroxyls on
protein substrates by protein kinases.
Proteolyis The process of chopping up proteins by proteolytic enzymes,
or proteases, which cleave peptide bonds at specic residues.
Proteosomes Multisubunit complexes situated in the cytosol that degrade
ubiquitin-tagged proteins.
Proximal sites DNA regulatory regions situated upstream of the core
promoter. Proteins that stimulate transcription when they bind at these sites
are called activators while those that impede transcription are called repressors. DNA sequences that provide sites for attachment of coactivators and
corepressors and mediate long-range enhancer-promoter interactions are
called tethering elements.
Pumps Membrane-spanning proteins that actively transport ions and
molecules across cellular and intracellular membranes.
Quaternary structure In multisubunit (chain) proteins, the ensemble of
subunits and how they are arranged.
Quorum sensing Cell-to-cell signaling used by bacteria to determine the
density of fellow bacteria in their local environment.
Reactive oxygen species (ROS) Molecules possessing unpaired electrons
(free radicals) involving oxygen. These molecules have a tendency to take
electrons from other molecules, in many cases breaking bonds to acquire
them.
Receptors Transmembrane proteins that function as sensors of environmental stresses and as receivers of chemical messages. They transmit and,
in the process, convert the signals from an external outside-the-cell form to
an internal inside-the-cell one that can be understood and further
processed.
Rectifying Ion channels that allow the passage of ions in one direction
only.

550

Glossary

Regulon Sets of operons located at well-separated loci along the chromosome-encoding proteins involved in a common physiological response.
Response regulator In a two-component system, this unit functions as
the receiver of the transferred phosphoryl group and as an output unit for
the signal. Most output response regulators function as transcription
factors.
Responsive elements Transcriptional control points, consisting of
short DNA sequences located in promoters, where transcription factors
come together and bind in a sequence-specic manner to regulate
transcription.
Reversal potential The value of the membrane potential for a particular
ion species that exactly cancels out the ow of those ions through the membrane arising from concentration differences in that ion.
Rhythmic bursting Multiple sequences of action potentials in which each
sequence, or burst, consists of a train of closely spaced action potentials separated by large interbust intervals.
Robustness A property of a system with respect to one or more of its
parameters in which feedback damps out the effect of variations in the
value of the parameter(s) on the performance of the system.
Scaffold proteins Nonenzymatic proteins that enable signaling proteins
that must work together to attach in close proximity to one another.
Secondary structure The ensemble of short segments of the polypeptide
chain that fold into a geometrically regular, repeating structure, such
as alpha helices and beta sheets, that are stabilized by networks of
hydrogen bonds.
Second messengers Signaling intermediaries that tie together events
taking place at and just below the plasma membrane subsequent to ligand
binding. Acting as coactivators and allosteric regulators they help to recruit
and organize the proteins that function as receptors and intracellular signal
transducers.
Selectivity The ability of an ion channel to pass some ions through while
preventing passage of other ions.
Selectivity lter The part of the ion channel that selects which ions are
able to pass through the pore.
Senescence A nondividing stage of cellular life entered into when a cells
telomeres become critically shortened.
Sensitization Strengthening of a behavioral response to a noxious stimulus through repeated exposures to that stimulus.

Glossary

551

Sensor unit In a two-component system, this unit functions as a receptor

and plasma membrane signal transducer. It possesses a histidine kinase
activity that promotes autophosphorylation on a histidine residue, followed
immediately by a phosphotransfer operation.
Shape complementarity The propensity of the surfaces of two molecules
to geometrically t together so that multiple contacts can be established at
their interface.
Signal transduction The process of relaying messages and, in the process,
converting them from one form to another that can be understood by the
downstream signaling targets.
Slip bond A bond that is weakened by external applied forces. The forcedriven reductions in lifetime of these bonds allow rolling leukocytes to
detach from surface tethers at the right time while maintaining good adhesion to that place on the surface at earlier times.
Spike frequency adaptation Hyperpolarization of the postsynaptic
membrane brought on by calcium entry into the cell decreases its excitability. The sequence of pulses exhibits an increasing time lag between successive pulses and eventually reaches a steady-state ring frequency.
Spindle oscillations 7- to 14-Hz oscillations that wax and wane with a 1to 3-second period and are associated with early stages of quiescent sleep.
Spliceosome Machinery consisting of a family of small nuclear RNA
molecules responsible for removing introns and selected exons from
pre-mRNAs.
Stable (equilibrium) state Long-lived states of a system. In these states,
small perturbations and thermal uctuations are rapidly damped out so that
the behavior of the system is not appreciably altered.
Steroid hormones Small lipophilic growth factors that are synthesized
from cholesterol and bind to nuclear receptors.
Structural motif Stable arrangement of secondary structure elements into
small compact structures.
Synaptic A signaling mode in which neurotransmitters and neuromodulators diffuse across the synaptic cleft between the membranes of a pair of
pre- and postsynaptic cells.
Synaptic plasticity The use-dependent, or adaptive, changes in the efciency of synaptic transmission between pre- and postsynaptic cells.
Telomere A capping structure consisting of a series of TTAGGG repeats
and associated proteins that screen the ends of DNA strands from doublestrand DNA repair machinery.

552

Glossary

Tertiary structure The ensemble of structural motifs and domains in the

protein and how they are arranged.
Tonic ring Trains of unitary action potentials generated either at regular
time intervals or irregularly spaced in time.
Transcription factors Proteins that bind to DNA in a sequence-specic
manner and regulate the transcription of protein-coding and associated
sequences into RNA molecules.
Transduction A form of horizontal gene transfer in which a bacteriophage
picks up genetic material from one bacterium and delivers it to another.
Transformation A form of horizontal gene transfer in which bacteria
become competent to capture exogenous DNA from their local
environment.
Trophic factors Molecules that stimulate growth and development.
Tumor suppressors Similar to oncoproteins. They, too, are proteins that
operate in the signal transduction, integration, and regulatory pathways
involved in cellular growth, multiplication, differentiation, and death,
except, unlike oncoproteins, they normally act as brakes on growth. Whey
they suffer critical mutations these brakes on growth are removed.
Two-component system A signal transduction system consisting of a
sensor unit and a response regulator. The sensor unit possesses a histidine
kinase activity and catalyzes the transfer of the phosphate group to the
response regulator, which contains an aspartate residue that receives the
transferred phosphate group.
Ubiquitination The process of tagging and preparing proteins for proteolytic destruction by the proteosome. It involves sequential operations by
E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3
ubiquitin-ligase enzyme.
Vasculogenesis Creation of blood vessels to supply oxygen and nutrients
to newly formed tissues and remove waste products from them.
Virulence factors Bacterial products such as toxins and surface adhesion
molecules that enhance bacterial survival in hostile host environments and
cause disease.
Zymogen Enzyme synthesized as an inactive precursor that is made into
an active form by proteolytic cleavage and removal of a prodomain.

Index

14-3-3 anchor proteins, 255, 257t

in protein kinase B signaling,
176177, 176f
in TOR signaling, 134
3 splice site, see Pre-mRNAs
3.10 helix, 27t
5 splice site, see Pre-mRNAs
5-HT, see Serotonin
5-HT3 cys-loop receptors, 475t
5 TOP, see Terminal oligopyrimidine
tract
9-cis retinoids, 393t
A- currents, 466468, 466f
Abl, 258t
mutated in cancer, 333t, 340
A/B pocket proteins, 349, 350f
Absorption, of photons, see
Electromagnetic radiation
Acetylation, 33, 3839, 38f, 539
Acetylcholine, 288t, 494495
Acetylcholine receptors, 288, 289, 475t
chain topology, 480
in drug addiction, 532533, 532f
ligand binding, 482483, 482f
subunit types and assembly, 481
Acetyl coenzyme A, 39
Acetyl homoserine lactases, 428, 429f
Acetyltransferases, 39
Achaete-scute complex, see Notch
pathway
Acquired immunodeciency syndrome,
444, 445t; see also Human
immunodeciency virus
Actin laments, 2

Action potential, 468470, 539; see also

HodgkinHuxley equation
and neurotransmitter release,
515516, 516f
Activation function, 473474, 474f
Active site, 143
Active zone, 514, 515518, 516f, 517t,
539
Activin, 311313, 313t
Acute lymphocytic leukemia, 340
Acute rheumatic fever, 432
Acyl anchor, 34, 34t, 35f
ADAM metalloproteases, 310311
Adapter proteins, 113114, 114f, 529
Adaptive immune response, 188, 529
and antigenic variation, 426
Adenomatous poliposis coli protein,
315316, 316f
mutated in cancer, 333t, 338339
Adenosine, 290t, 293, 293t, 295
Adenosine diphosphate; see also
Protein kinases, Protein
phosphatases
as a local hormone, 293, 293t, 295
as a neuromodulator, 290t
Adenosine nucleotide translocator, 370,
370t
Adenosine triphosphate, 5; see also
Protein kinases, Protein
phosphatases
as a local hormone, 293, 293t, 295
as a neuromodulator, 290t
Adenovirus family, 445t, 446t, 447
Adenylyl (adenylate) cyclase, 172173,
173f, 280, 280t, 281, 281t, 282f
553

554

Index

Adenylyl (adenylate) cyclase (cont.)

in yeast general stress response, 130,
130f
stimulation by heterotrimeric G
proteins, 180182, 181f
Adherens junction, 227
Adrenal gland, 247, 285, 286287, 287t
Adrenaline, 286287, 287t, 290t
Adrenergic GPCR, 277t, 302303
Adrenocorticotropic hormone, 286,
287, 287t
Adult T cell leukemia, 449
Aequorea victoria, 57, 58
African Trypanosomas, 426
Agonist, 275, 529
A-kinase anchoring proteins, 181182,
181f
Aldosterone, 393t
Allosteric modication, 146, 529
All-trans retinoids, 393t
Alpha carbon, 26, 26f
a helix, 2730, 27t, 28f
a-interferon, 449
Alpha-secretase, see Secretases
Alpha waves, 490t, 492
ALS, see Lou Gehrigs disease
Alternative splicing, 399400
Alzheimers disease, 99, 100101, 100t,
310, 360
Amino acids, 2627, 27f, 27t, 71
abbreviations for, codes, 72t
in aqueous environments, 7274, 73t
size and shape, 7172, 72t
AMPA receptors, 519, 519t
and NMDA receptors, 523
biophysical properties, see Glutamate
receptors
in hippocampal LTP, 525f
in synaptic maturation, 523
receptor trafcking, 518f, 523
AMP-dependent protein kinase, 120t
Amphetamines, 532533, 532f
Amygdala, 512, 512f
areas and circuitry, 530531, 531f
Amyloid buildup
amyloid b protein, 100101, 100t
Lewy bodies, 100
neurobrillary tangles and tau, 99
plaques, 100

in neurological disorders, 100101,

100t
Amyloid precursor protein, 309310
Anabaena, 436
Anchor proteins, 113114, 114f, 124,
164f, 178179, 529
Androgen, 393t
Anergy, 212
Aneuploidy, 332
Annsen, 101, 103f
Angiogenesis, 249250, 529
Angiopoietins, 249
Angiotensin-converting enzyme, 291
Angiotensin GPCR, 277t
Angiotensins, 290291, 290t
Anion (chloride) channels, see
Chloride channels
Ankyrin repeat, 32t
Annealing, 102103, 103f
Antagonist, 275, 540
Anterior pituitary, 285, 286, 287t
Antibiotics, 419, 427t, 428, 431, 540
Antibodies, 188, 540
Anti-cancer drugs, and apoptosis,
379380
Antigenic variation, 426, 540
Antigen-presenting cells, 189, 189t
Antigens, 188, 540
Antihistamines, 276
Anti-oxidants, 371
AP-1 responsive genes, see Mitogenactivated protein kinase
modules
Apaf-1, 373, 373f
Aplysia californica, 500, 520
gill withdrawal response, 520521,
521f
learning pathway, 522, 522f, 526
Apoptosis, 12, 1718, 540
in the nervous system, 251
origin of term, 359
role of feedback, 375376, 376f,
380
through TNF family signaling,
199200, 200f
Apoptosis inducing factor, 372, 372t
Apoptosome, 373, 373f, 375376, 376f,
540
Apoptotic bodies, 359360

Index
Apoptotic proteins, mutated in cancer,
333
Arabidopsis response regulators,
152154, 153f
Arabidopsis thaliana
hormonal signaling, 152154, 152t,
153f
light sensing, 154156, 155f
Arachidonic acid, 294, 294f
ARF, 351, 351f, 354
Arf GTPase, 263t, 269270
Arginine-serine-rich domain, 403
Arrhenius formula, 108109
Aspirin, 294
Associative learning, 520, 540
Ataxia telangiectasia mutated, 120t,
333t, 342, 342t, 343f, 344,
345346, 352
ATM and Rad3-related, 120t, 342, 342t,
345346, 352
Atomic force microscopy, 232233
Atomic radii, 7374
Atrioventricular node, 488
Attention-decit hyperactivity
disorder, 291
Auditory brain area, 513, 513f
Autocrine signaling, 248, 540
Autoinducers, 428, 429f
Auxiliary splice sites, 540
Auxiliary splicing factors, 403
Auxiliary splicing sites, 403404, 403f, 540
Bacillus subtilis, 412, 416, 427t; see also
Sporulation
Bacteria, in the ecosystem, 411
Bacterial chemotaxis, see Chemotaxis
Bacterial communities, 426428, 427t
Bacterial receptors
chemotactic pathway, 150152, 150f,
150t
clustering, 148149
structure, 147, 147f
Bacteriophage lambda
decision circuit, 458459, 458f
genome, 457459, 457f, 458f
lambda counterrepressor, Cro, 458
lambda repressor, CI, 458
lifecycle, 457, 546
structure, 457, 457f

555

Bacteriophages, 432, 457, 540

Bacteriorhodopsin, 53
Bacteroides thetaiontaomicron, 411
Baculoviral IAP repeat, 374, 374f
Baculovirus, 446t
Bad, 176, 176f, 364, 364t
Barrier, see Energy barrier
Basal transcription machinery, 385386,
386t
Base excision repair, 340, 341t
Basement membrane, see Extracellular
matrix
Basic broblast growth factor, 249
Basic region-leucine zipper domain,
389391, 391f
Basophils, 189t
Bax, 333t, 364, 364t
B cell receptors, 207208
B cells, 189t
Bcl-2, 333t, 339340, 364, 364t
Bcl-2 homology domains, 363365, 365f
Bcl-2 proteins, 363364, 364t, 369371,
378
BCR-homology GTPase activating
domain, 168f
Bells equation, 243244
b-arrestins, 282285, 283f, 285f, 302
Beta-blockers, 275276
b-catenin, mutated in cancer, 333t,
338339
b-catenin pathway, in Wnt signaling,
315316, 316f
b-secretase, see Secretases
b sheet, 2730, 27t, 28f
Beta waves, 490t, 492
Bid, tBid, 364365, 364t, 367368, 368f,
376
Binding, and energy landscapes,
104105
Biolm, 430, 540
Biological membranes
diffusion, 185186
lipid composition, 162, 162t
microdomains, 163164
Bitter tastes, 300
Blastomeres, 320
Blood vessel physiology, 231
Bloom syndrome, 342
Boltzmann factor, 219

556

Index

Bond action during leukocyte rolling,

231232, 232f
experimental studies, 232233
slip and catch bonds, 233234
unbinding, 243245, 244f
Bond angles, 82f
Bond bending, 80f, 82f
Bond dihedrals, see Bond torsions
Bond lengths, 7576, 76t, 81f
Bond lifetimes, see Bond action during
leukocyte rolling
Bond stretching, 80f, 81f
Bond torsions, 8081, 80f, 82f
Bone morphogenetic proteins, 311313,
313t
Borrelia, 426
Bovine leukemia virus, 449
Bradykinin receptors, 295
Bradykinins, 293t, 295
Braggs law, 51, 51f, 67, 67f
Brain, functional architecture, 512514,
512f, 513f
Brainstem, 512, 512f
Brain waves, 490t, 492, 497498
Branching morphogenesis, 239, 540
Branch site, see Pre-mRNAs
Breast cancer proteins, 333t, 342345,
342t, 343f, 345f, 346
Bremsstrahlung, 50
Brodmanns areas, 513514
Bromodomain, 398t
Brownian motion, 135
Budding, viral, 443444
Burkitts lymphoma, 340
Burst ring, see Rhythmic bursting
C1 domain, 169170, 170t, 174,
177178, 177f
C2 domain, 169170, 170t, 174,
177178, 177f
in phospholipase C, 167f
in PI3 kinase, 168, 168f
C2H2 zinc nger domain, 32t
Ca2+/calmodulin-dependent kinase
classication of, 120t
in hippocampal LTP, 525526, 525f
Cadherin domain, 32t
Cadherins, 222f, 226228, 227f
Calcitonin GPCR, 277t

Calcium channels, voltage-gated, 475t,

479480, 481f
classication, 488489
in heartbeat, 488489, 488t
in hippocampal LTP, 525, 525f
in neurotransmitter release, 516517,
516f, 517t
low-voltage activated, 492494
single-chain topology, 477, 477f
Calcium currents, 468, 468t
Calcium GPCR, 277t, 278
Calcium signaling
and calmodulin, see Calmodulin
as a second messenger, 170172
cycling between ER and
mitochondria, 377378
in apoptosis, 375376, 376f
in cardiac cells, 179180, 180f
release from intracellular stores, 122,
165166, 167f
sequestration in intracellular stores,
170
types of signals, 376377
Calicivirus family, 445t
Calmodulin, 171172,172f
in FRET, 59, 60f
Calothrix, 412, 436
Cameleons, 59, 60f
cAMP response element-binding
protein, 391, 391f
Cancer borealis, 500
Cancer, 331332
and viruses, 450, 451t
hepatitis C, 447
human T lymphotropic virus,
449450, 450f
Kaposis sarcoma-associated virus,
450452, 451f
mutated proteins, 332334, 333t
Caretaker proteins, 333
Cargo, transport of, see Arf GTPase,
Rab GTPase, Ran GTPase
Casein kinase-2, classication of, 120t
CASK proteins, 514, 515f
Caspase 3, 367368, 368f, 371, 374,
375376, 376f
Caspase 6, 368, 376
Caspase 7, 362f, 374, 375
Caspase 8, 367368, 368f

Index
Caspase 9, 371, 373, 373f, 374, 375, 376,
376f
Caspase-activated deoxyribonuclease
protein, 367, 368f
Caspase recruitment domain, 257,
362363, 363t, 373, 373f
Caspases, 360363, 361f, 362f, 363t,
365366, 540
Cassettes, 419
Catabolite activator protein, see Lac
operon
Catch bonds, 540; see also Bond action
during leukocyte rolling
Catecholamines, 286
Catenins, 227
Caulobacter crescentus, 412; see also
Differentiation, in C. crescentus
Caveolae, 164, 541
Caveolins, 164
CD4, 8 receptors, see T cells
Cell adhesion, 541
Cell adhesion molecules, see Cell
adhesion receptors
Cell adhesion receptors, see Cadherins,
IgCAMs, Integrins, Selectins
and growth factor receptors in
cancer, 337338
as long glycoproteins, 221224
in immunological synapses,
212213
Cell cycle
checkpoints, 333, 346347
control system, 347, 348f, 348t
phases of, 2324
progression, 346347
Cell fate, 17, 541
Cell lysis, see Lysis
Cell polarity, 541
Cell regulation, 12
Cell wall, 2
Central pattern generators, 499500,
541
and motor learning, 506507
common features, 502504
in human respiratory system,
504505
in invertebrate motor systems,
500502
neuromodulation of, 505506

557

Central supramolecular activation

cluster, 213, 214
Cerebellum, 512f, 513
cGMP, 297, 298f
Channelpathies, 499
Chaperones, Chaperonins, see
Molecular chaperones
Charge balance, inside and outside the
cell, 466, 466f
CheA histidine kinase, 141144, 142f,
142t, 143f, 158159
Checkpoint kinases, 349350, 351f
Checkpoints, 541; see also Cell
cycle
Chemical shift, see Nuclear magnetic
resonance
Chemoafnity hypothesis, 236
Chemokine receptors, see
Chemokines
Chemokines, 191t, 193, 206207
Chemotaxis, 147152, 541
Chemotaxis (Che) proteins, 145146,
148149, 150t; see also CheA
histidine kinase
feedback, 150152, 150f
Chirality, 6061
Chloride channels, voltage-gated, 477,
480481
chain topology, 475t, 476f
conducting pore, 480, 481f
selectivity lter, 480, 481f
subunit assembly, 475t
Chloride currents, 466468, 466f,
468t
Chloroplasts, 3t, 45
Cholesterol, see Membrane lipids
Chromafn cells, 286
Chromatin, 5, 6, 22, 2426, 541
Chromatin remodeling, 386, 386t,
392393, 397398, 398t
Chromatin territories, 2426
Chromodomain, 397f, 398t
Chromophore, 49, 154t, 541
Chromosomes, 21
Chromosome translocations, see
Translocations
Ciliary neurotrophic factor, 251
Circadian rhythms, 155f, 156157
Circular dichroism, 6061

558

Index

c-Jun NH2-terminal kinase pathway, see

Mitogen-activated protein
kinase modules
CKI, in Wnt pathway, 315316, 316f
Class E compartments, 444
Class E Vpss, 444
Class I cytokine receptors, see
Hematopoietin cytokines
Class II cytokine receptors, see
Interferons
ClC family, see Chloride channels,
voltage-gated
Cluster of differentiation, 209213,
228t
Coat proteins, 269270
Cocaine, 532533, 532f
Coiled (Cajal) bodies, 25
Collagens, see Extracellular matrix
Colony-forming bacteria, 10; see also
Bacterial communities
Combinatorial control, 118119,
388389
Compartmentalization, 124
Competence, 541
Concentration gradients, 236238
Conditioned stimulus, 520
Conformational masking, by HIV, 454
Conjugation, 431, 541
Connexins, 495497, 496f
Connexons, 495497, 496f
Continuum electrostatics, 7980
Control layer, 1213, 13t, 15, 16
Controller proteins, mutated in cancer,
333t
Control point, 12, 117118, 387389,
388f, 541
Convergent extension, 317
Core transcription machinery, see Basal
transcription machinery
Corticotropin releasing hormone, 287t
Cortisol, 393t
Co-Smad, see Smad proteins
Costal-2, 318, 319f
Costimulation, 211213, 211t
Covalent bonds, energies of, 47
Covalent catalysis, 143144, 158
Covalent modication, see
Posttranslational modications
CREB, 522, 522f, 525526, 525f, 526f

CreutzfeldtJakob disease, 100, 100t

Crowding
and protein folding, 101
effect on diffusion, 135
Cryoelectron crystallography, 53
Cryptochromes, 49, 154, 154t, 155f,
156157
Csk, 258t, 259, 260
Cubitus interruptus, 318320, 319ff
Cyanobacteria, see Heterocystous
cyanobacteria
Cyclic adenosine monophosphate,
121
formation from ATP, 172173
signaling pathway, 180182
structure, 172173, 173f
Cyclic guanosine monophosphate, 280t,
281282
Cyclic nucleotide-gated ion channels,
298, 299f
Cyclin-dependent kinases,120t,
347349, 348f, 348t
Cyclins, 347349, 348f, 348t
Cyclooxygenases, 294, 294f
Cys loop receptors, types, 475t
Cystic brosis, 9899
Cytochrome c, 369, 370f, 371372, 373,
373f, 375376, 376f
Cytokines, 190191, 191tt, 192, 192f,
193, 201t, 542
Cytokinin signaling, see Arabidopsis
thaliana
Cytoskeleton, 2, 3
Cytotoxic T cells, 189t
Death domains, 196197, 200, 200f, 257,
257t, 367
Death effector domains, 257, 362363,
363t, 367
Death-inducing signaling complex,
365368, 368f, 542
Death ligands, 365
Death modules, bacterial, 433434, 433f
Death receptors, see Tumor necrosis
factor family
Deleted in colorectal cancer protein,
222, 229, 236t, 237238, 238f
Delta ligand, 306, 306t, 308, 309f
Delta waves, 490t, 492

Index
Denatured state, 95, 9698, 96t, 97f, 98f,
104105, 104f, 542
Dendritic cells, 188189, 189t
Deoxyadenosine triphosphate, 372,
372t, 373
Deoxyribonucleic acid, 5, 11, 14
Dephosphorylation, 542; see also
Protein phosphatases
Desensitization, 542
Detailed balance, see Principle of
detailed balance
Developmental pathways, 305306
Diacylglycerol, 122, 165166, 166f
Diastolic depolarization, 488, 488t, 490f
Differentiation, in C. crescentus,
424426, 425f
Diffraction, 542; see also Braggs law,
X-ray crystallography
Diffusion, 134136, 138, 185186, 542
Diffusion coefcient, 135136, 138
Dipole forces, see Macromolecular
forces
Discosoma striata, 57
Dishevelled adapter, 315, 315f, 316f
Disorder, see Entropy
Distal control region, 387389, 388f,
542
Disulde bridges, 73t, 7475
DNA-binding domains, see
Transcription factors
DNA damage, 331, 340342, 341t
DNA fragmentation factor, 367
DNA microarrays, 45, 46t, 6263
DNA-PK, 120t, 342t, 345346, 352, 354
Domain fold, 542; see also Domains
Domains, 29, 3032, 31f, 32t
Dopamine, 286, 290t
Dopamine GPCR, 277t, 291
Dopaminergic neurons, 532533, 532f
Dopaminergic system, 291292
Dopamine transporter, 532f, 533
Double-strand break repair, 341, 341t,
342346
Downstream promoter element,
387389, 388f
Drosophila, 305306, 321327
Drug addiction, 291, 529530
aberrant synaptic plasticity,
532533

559

characteristics, 530
reward circuitry, 531532, 532f
sites of action, 530531
Drugs, and G protein-coupled
receptors, 16
Dual specicity kinases, see Mitogenactivated protein kinase
modules
Dynorphins, 290, 290t
E1A, 351352, 351f
E2Fs, 348, 350f, 351352, 351ff, 354
Early repolarization, 488489, 488t,
489f
Ecdysone, 393t
Ectodomain shedding, 311
Efciency of synaptic transmission,
511, 526527, 527f, 542
EF hand domain, 32t
EF-Tu elongation factor, 263, 266
EGF-like domain, 32t
eIF4E binding proteins, see Eukaryotic
initiation factors
Electrical synapses, see Gap junctions
Electroencephalographic recordings,
492
Electromagnetic radiation, interactions
with matter, 4649, 48f; see also
Braggs law, X-ray
crystallography
Electromagnetic spectrum, 47, 47f
Electron crystallography, 53
Electrostatic complementarity, 78, 542
Emission, of photons, see
Electromagnetic radiation
Endocrine glands, 247
Endocrine pancreas, 285, 287288, 287t
Endocrine signaling, 248, 542
Endocytosis, 9, 268269, 543
Endonuclease G, 372, 372t
Endoperoxide H synthases, 294
Endoplasmic reticulum, 3t, 67, 8f
Endorphins, 290, 290t
Endosomes, 3t, 9
Endpoints, of signaling pathways, see
Control point
Energy barrier
kinetic shift, 105
thermodynamic shift, 105

560

Index

Energy landscapes, 9698, 96t, 97f, 98f,

104105, 543
for bond dissociation, 233, 233f,
243245, 244f, 245f
Energy-level diagrams, 48, 48f, 50f
Engrailed, 325326
Enhancer of split cluster, see Notch
pathway
Enkephalins, 290, 290t
Enterococcus faecalis, 411
Enthalpy, 9394, 9698, 97f, 98f
Entorhinal cortex, 524f
Entrainment and circadian rhythms,
154
Entropy, 89, 107
and Gibbs free energy, 9394
and protein folding, 9698, 97f, 98f
second law of thermodynamics,
9293
Envelope, 543
Enveloped viruses, 441, 442
Eosinophils, 189t
Eph receptors, 249250, 250f
Ephrins, 236t, 239240, 240f, 249250
in synapse stabilization, 515, 515f
Ephs, 236t, 239240, 240f
Epidermal growth factor, 247, 250251,
254
Epilepsy, 498499
Epstein-Barr virus, 444, 446t, 447
Equilibration, 9293, 92f
Equilibrium potential, see Reversal
potential
Equilibrium states, 95
Escherichia coli, 10, 412, 416, 427t, 430,
431432
enterohaemorrhagic, 432, 459460,
460f
enteropathogenic, 432
agellar motor assembly, 421422,
423f
lipopolysaccharides, 188
motility, 139, 149150
uropathogenic, 430
ESCRTs, 444
Estrogen, 393t
Ethylene-signaling pathway, see
Arabidopsis thaliana
Euchromatin, 40, 543

Eukaryotes, 23, 1012, 21

Eukaryotic initiation factors, 404406,
405t, 406f
in insulin signaling, 175, 175f
regulation by eIF2 and eIF2B, 205f,
406407
regulation by eIF4E and 4E-BPs,
132f, 407408, 408f
EVH1 domain, 256, 257t
Exact adaptation, 151
Excitatory neurotransmitters, 288289,
288t
Exocytosis, 9, 268269, 543
Exonic splice enhancer sites, see
Auxiliary splice sites
Exonic splice inhibitory sites, see
Auxiliary splice sites
Exons, 399400, 400f, 401f, 543
Exportins, 267
Extracellular matrix, 2, 3, 225226
Extracellular signal-regulated kinase
pathway, see Mitogen-activated
protein kinase modules
Extrachromosomal DNA, 2
Extremophiles, 1
FADD adapter, 367, 368f
Fanconi anemia, 342
Fanconi anemia complex, 343f
Faradays law, 471
Farnesyl anchor, 34, 34t, 35f
Fast folding, 96t, 97
Fast upstroke, 488, 488t, 489f
Fear conditioning, 529
circuitry, 529, 530f
Fibronectins, see Extracellular matrix
Fibronectin Type III domain, 32t
Filopodia, 235, 235f
Filovirus family, 445t
Finite-difference method, 78
First law of thermodynamics, see
Thermodynamics
Fixed infrastructure, 1213, 13t
Flagellar motor, 149150; see also
Chemotaxis
assembly, 421422, 423f
Flanking residues, 122123
Flavinoids, 436
Flavivirus family, 445t, 447449, 448t

Index
Fluorescence, 49, 5759, 60f
Fluorescence recovery following
photobleaching, 138
Fluorescence resonance energy
transfer, 46, 46t, 5859, 60f, 67
Fluorescent proteins, 5758, 58t, 138
Fluorophores, 49
Focal adhesion kinase, 258t, 261262,
262f
Focal adhesions, 260262, 543
Focal adhesion targeting sequence, 262,
262f
Folding cage, 102103, 103f
Folding diseases, see Misfolded
proteins and diseases
Folding funnel, 543; see also Protein
folding
Follicle-stimulating hormone, 286, 287t
Forkhead-associated domain, 255256,
257t
Forkhead transcription factors,
176177, 176f
Four-helix bundle, 29f
Free radicals, 371
Friction coefcient, 135136
Frizzled, 315, 315f
Frontal lobe, 512f, 513, 513f
Fruiting body, 434
Frustration, 107108
Functional epitopes, in binding,
202203
Funny (f) channels, see HCN channels,
voltage-gated
Fused genes, 340
Fused protein, 318, 319f
FYVE domain, 169170, 170t
GABA, 495
GABAA and GABAC receptors, 497
subunits and assembly, 499
GABAB receptors, 277t, 288, 288t, 497
GABA-releasing neurons, 497
in epilepsy, 498499
in spindling, 498
synchronization by, 497
Gamma rays, 47f
Gamma-secretase, see Secretases
Gamma waves, 490t, 492
Gap genes, 321322, 321t, 323324, 324f

561

Gap junctions, 495497, 496f

in synchronization, 497498
in the STG, 500, 501f
Gastric mill pattern, 500
Gastrulation, 320
Gates, in ion channels, 543; see
also HodgkinHuxley
equation
GDP dissociation inhibitors, in Rab
cycling, 269f, 543
Gel electrophoresis, 45, 46t, 63
General stress response pathway, in
yeast, 129131, 130f
General transcription factors, 385, 387f,
393394, 394t
Gene regulation, dened, 12, 1315
Genetic competence, 427t, 428, 429430
Genomes, sizes of, 910
Geranylgeranyl anchor, 34, 34t, 35f
Gibbs free energy, 9395
Glia, 234
Glial-derived neurotrophic factor, 251
Gli proteins, 318320, 319ff
Glucagon, 247
Glucagon GPCR, 277t, 287288, 287t
Glutamate, 288, 288t, 495
Glutamate GPCR, 277t, 278
Glutamate receptors, 475t
chain topology, 483
ligand binding, 483485, 484f
subunit composition and assembly,
483
Glycine, 288t
Glycine cys-loop receptors, 475t
Glycogen synthase kinase-3, 120t, 175,
175f
in Hh pathway, 318, 319f
in Wnt pathway, 315316, 316f
Glycolipids, see Membrane lipids
Glycoproteins, 36
Glycosylation, 36, 543
Glycosyl phosphatidyl inositol anchor,
34, 34t, 36, 164165, 543
GoldmanHodgkinKatz equation,
468
Golgi apparatus, 3t, 78, 8f
Gonadotropin-releasing hormone, 287t
G protein-coupled-receptor kinases,
120t, 282285, 283f, 285f

562

Index

G protein-coupled receptors, 112113,

112f, 275276, 276f, 276278,
277t
desensitization, 282284
in protein kinase C signaling,
179180, 180f
internalization, 284285
in the cAMP signaling pathway,
180182, 181f
in yeast general stress response, 130,
130f
in yeast pheromone response
pathway, 125128, 126f
G protein-linked inward rectied K+
channels, 289
G proteins, see Heterotrimeric G
proteins
Granulocytes, 189, 189t
Grb2, 252, 253f, 260f, 264
Greek key motif, 29, 29f
Green uorescent protein, see
Fluorescent proteins
GRIP, 515f, 519t
Groucho, 306
Group A Streptococcus, 432
Growth cones, 234235, 235f, 543
navigation, 236237, 236t
outgrowth, 235236, 236t
Growth factor receptors, in cancer,
336338
Growth factors, 544
Growth factor signaling, 167169, 169f
Growth hormone, 287t
Growth hormone-inhibiting hormone,
287t
Growth hormone-releasing hormone,
286, 287t
Growth regulators, mutated in cancers,
333, 333t
GTPase-activating proteins, 115, 116f,
544
Cdc42 family, 266267
Ras family, 264266
GTPases, 248, 262270, 544
function in the cell, 125
in yeast general stress response
pathway, 129131, 130f
in yeast pheromone response
pathway, 125128, 126f

Guanine nucleotide exchange factors,

115, 116f, 544
Cdc42 family, 266267
Ras family, 264266
Gustducin, 280, 300
GYF domain, 256, 257t
Habituation, 520, 544
in Tritonia, 506507
Hairpin, 30
Halobacteria, 53
HCN channels, voltage-gated
chain topology, 475t
neuromodulation, 491492, 491f
pacemaker activities, 489492
subunit assembly, 475t
Heartbeat, 487489, 488t, 489f, 490f
Heat, 90
rst law of thermodynamics, 9091
second law of thermodynamics, 9193
Heat shock proteins, see Molecular
chaperones
Hebbs rule, 526527, 527f
Hedgehog, 306, 317318, 318f
Hedgehog pathway, 317320, 319f
in cancer, 338339
in Drosophila development, 321t,
325326
Helix-loop-helix motif, 389390
Helix-turn-helix motif, 389390, 390f
Helper T cells, 191t, 192
Hematopoietin cytokines, 193, 200202,
201t
Hematopoietin receptors, 200202, 201t
Hemoglobin, 49, 99
Hepatitis C virus, 447449, 448f
Hepatocyte growth factor, see Scatter
factors
Herpes simplex virus type 1, entry into
the nucleus, 443
Herpesvirus family, 444, 445t, 446t
Heterochromatin, 40, 544
Heterocystous cyanobacteria, 435436,
439440
Heterogeneous nuclear RNP particles,
401403, 402f, 404f
Heterotrimeric G proteins, 113,
279280
activation by GPCRs, 279

Index
G a subunits, 279281, 280t, 281t,
282f
G bg subunits, 279, 281t, 283, 284,
284f, 285f, 289
in protein kinase C signaling,
179180, 180f
in the cAMP signaling pathway,
180182, 181f
in yeast general stress response, 130,
130f
in yeast pheromone response
pathway, 125128, 126f
regulation of ion channels, 297
High osmolarity glycerol pathway,
128129, 128f
Hippocampus, 512, 512f, 513f, 523,
530531, 531f; see also
Long-term potentiation
architecture and circuitry, 524, 524f
Hirudo medicinalis, 500
His-Asp phosphotransfer, 140141,
141f
Histamine, 290, 290t, 293, 293t
Histamine GPCR, 277t
Histidine kinases, 141145; see also
CheA histidine kinase
Histidine phosphotransfer protein, see
Phosphorelay
Histone acetyltransferases, 41, 395396
Histone deacetylases, 41, 395396
Histone modication, 14, 386, 386t,
395397
Histones, 6, 2223
posttranslational modications of,
4041, 41f
hMLH1, mutated in cancer, 333t, 341
hMSH2, mutated in cancer, 333t, 341
HodgkinHuxley equation, 470471
equivalent circuit, 471472, 472f
h, m, n gates, 472474
Holoenzyme, 544
Homeobox domain, 32t
Homeostasis, 11, 544
Homologous recombination, 341, 341t,
342344, 342t, 343f
Homology boxes, in CheA, see CheA
histidine kinase
Hookes law, see Macromolecular
forces

563

Horizontal gene transfer, 411, 430431,

544
Hormonal signaling, 247, 292293, 293t
Hot spots, 7879, 202203
Hox genes, 321322, 321t, 326327
Hsp70 chaperones, in yeast general
stress response, 129130, 130f;
see also Molecular chaperones
Hsp90 chaperones, see Molecular
chaperones
Human growth hormone, 202203,
202f, 254
Human immunodeciency virus,
452453, 453f
entry via CD4, chemokine receptors,
453
gag, pol, env genes, 452453
gp120 envelope protein, 453454,
454f
tat, rev, nef regulatory genes,
454456, 455f
viral structure, 452453, 452f
vpr, vif, vpu, vpx regulatory genes,
456
Human T lymphotropic virus type 1,
449450
Huntingtons disease, 100, 100t, 360
Hydrogen bonds, 2728, 47, 73t, 74
Hydrophilic, 73, 544
Hydrophobic, 73, 544
Hypothalamus, 247, 285, 286, 287t
Ibuprofen, 294
IgCAMs, 222, 222f, 225, 225f, 228229,
228t
Ih channels, see HCN channels, voltagegated
IkB proteins, see NF-kB signaling
module
IKK proteins, see NF-kB signaling
module
Immortalization, 354355
Immunoglobulin domain, 32t
Immunoglobulin superfamily, 207208
Immunological synapse, 212213
Importins, 267
Inactivation function, 473474, 474f
InaD scaffold, 297, 298f
Induced t, 105

564

Index

Inammatory response, 187, 230, 231,

544
Infrared radiation, 47f, 61
Infrared spectroscopy, 61
Inhibitor of apoptosis proteins, 374,
374f
Inhibitory glutamate receptors, 502
Inhibitory neurotransmitters, 288289,
288t
Initiator
eukaryotic (Inr), 386387, 387f
prokaryotic (InR), 413f
INK4a protein, 351, 351f, 354
Innate immune response, 187188, 198,
544
Inner mitochondrial transmembrane
potential, 375, 376f
Inositol 1,4,5-triphosphate, 165166,
166f
Insulators, 544
Insulin signaling, 247, 287, 287t; see also
Protein kinase B
Integrins, 223226, 224f, 225f, 261, 262f
Intercellular cell adhesion molecules,
see IgCAMs
Inter-chromatin granule clusters, 2526
Interface, 544
Interferon receptors, see Interferons
Interferons, 191t, 193, 201t, 205206,
206f
Interleukin-1b converting enzyme,
363t, 364
Interleukins, 190191, 191tt, 192f, 193,
200202, 201t
Internal conversion, 49
Internal energy, 9091
Internalization, 545
Interphase, 346347, 346f; see also
Nucleus, Cell cycle
Intracellular calcium, see Calcium
signaling
Intracellular stores, see Calcium
signaling
Intronic splice enhancer sites, see
Auxiliary splice sites
Intronic splice inhibitory sites, see
Auxiliary splice sites
Introns, 399400, 400f, 401f, 545
Invasive growth, 239

Ion channels, 4, 465466, 474478, 475t,

476f, 477f, 545
regulation by protein kinase A,
180182, 181f
Ion concentrations, inside and outside
the cell, 466468, 466f, 468t
Ionic conductances, 471
Ionizing radiation, and DNA damage,
331, 341, 346
Ionotropic receptors, 288289, 545
Ischemic preconditioning, 179
Islets of Langerhans, 247, 287
I-Smad, see Smad proteins
Isoelectric points, 63
Janus kinases, 203204, 204f, 205206,
206f, 258t
Jellyroll, 30
Juxtracrine signaling, 248, 545
Kainate receptors, biophysical
properties, see Gluatamate
receptors
Kaposis sacoma associated herpes
virus, 444, 451452, 451f
Karyophilic particles, 443
Keratinocytes, 251
Kinetic proofreading, 213214, 545
Kinetic shift, see Energy barrier
Kinetic trap, 96t, 97, 98t, 102, 105, 545
KIR receptors, 209
Krueppel-associated box domain, 32t
Ku module, see DNA-PK
Labile, 545
Lac operon, organization, 419421,
419t, 420f, 421f, 422f
Lambda repressor, 390, 390f
Lamellipodia, 235, 235f
Laminins, see Extracellular matrix
Late domains, 444
Lateral inhibition, 307308, 309f, 545
Late repolarization, 488489, 488t,
489f
Latrotoxin GPCR, 277t
Law of mass action, 217218
Leak current, 471472, 472f
Learning, 520, 545
Learning pathway, 122, 182

Index
LennardJones, 612 potential, 8183,
83f
Lentivirus family, 445t
Leucine-rich repeat, 32t
Leucine zippers, 390391, 391f
Leukocytes, 187, 188189, 189t,
190192, 230234, 243245,
545
Ligands, 16, 113
Light sensing, 154157, 154t, 155f
Lipid-binding domains, 169170,
170t
Lipid kinases, 165; see also
Phosphoinositide-3-OH kinase
Lipid phosphatases, see PTEN lipid
phosphatase
Lipid raft, 164, 545
Lipid second messengers, 122
Lipopolysaccharides, 187188, 198
Localization, see Compartmentalization
London forces, see Macromolecular
forces
Long-term facilitation, 520, 545
Long terminal repeats, 449450, 450f
Long-term memory, 521
Long-term potentiation, 512, 523524,
524f, 525526, 525f, 526f, 545546
Lou Gehrigs disease, 100, 100t, 360
Low-voltage-activated calcium
channels, see Calcium channels
LRP co-receptor, 315, 315f
Luminescent bacteria, 427428, 427t
Lutenizing hormone, 286, 287, 287t
Lymphocytes, 188189, 189t
Lysis, 443
Lysogenic cycle, see Bacteriophage
lambda
Lysosomes, 3t, 8, 8f
Lytic cycle, see Bacteriophage lambda
Macromolecular forces, 71, 7476, 76t,
7983, 81f, 82ff, 83f
Macrophages, 189t
Mad cow disease, 100
MAD homology domains, 313314
Magnetic dipole moments, 56
Magnetic resonance imaging, 57
Major histocompatability complexes,
207209, 213

565

Mammalian pheremones, 299

Mass action, see Law of mass action
Mass equilibration, see Equilibration
Mass spectrograph, 45, 46t, 6465, 69
Mast cells, 188189, 189t
Maternal effect genes, 321323, 321t,
323f
Matrix metalloproteinases, 332, 332t
Matrix protein, 546; see also Human
immunodeciency virus
Mdm2 protein, 351f, 352, 354
Mediator, 385386, 386t, 398399, 399f
Medulla oblongata, 512, 512f
Melanocortin GPCR, 277t
Melanocyte-stimulating hormone, 286,
287t
Melatonin GPCR, 277t
Memapsin proteases, 310
Membrane blebbing, 359360
Membrane lipids, 162, 162t, 163165,
163f
Membrane potentials, 466468, 467f,
468t, 468470
Membranes, see Biological membranes
Memory, 546
different forms, 521
Memory consolidation, 523, 524525
Mesolimbic system, 530531, 531f
Messenger RNAs
bacterial, 412
eukaryotic, 399400, 404408, 405f,
406f
Met, see Scatter factors
Metabolism, 45
Metabotropic glutamate receptors,
288289, 519t, 525f, 546
Metastasis, 331332, 546
Methylation, 33, 3839, 38f, 546
Methyl-accepting chemotactic proteins,
151
Methyltransferases, 39
Metropolis algorithm, 219220
Met-tRNA initiator, 405406, 406f
MichaelisMenton equation, 158159
Microarrays, 62
Microdomains, see Caveolae, Lipid raft
Microtubules, 2
Microvilli, 230
Microwaves, 47f, 61

566

Index

Misfolded proteins and diseases,

98101
Mismatch repair system, 341, 341t
Mitochondria, 3t, 45
Mitogen-activated protein kinase
modules, 120t, 196, 197f
in differentiation and growth
signaling, 253
in DISC signaling, 367, 368369, 368f
in high osmolarity glycerol pathway,
128129, 128f
in hippocampal LTP, 525, 525f
in pheromone response pathways,
125128, 126f
in T cell receptor signaling, 210f
in the postsynaptic density, 519t
in the TNF pathway, 200
in the Toll pathway, 199f
in Wnt pathway, 316f, 317
Mitosis phase, 346347, 346f
Molecular chaperones, 90, 101,
102104, 102t, 103f
Molecular dynamics, method of, 7980,
8384
Molecular mechanics, 7983
Molecular rotations, 61
Molecular vibrations, 61
Monoamine oxidase inhibitors, 292
Monte Carlo method, 219220
Morphogens, 305, 320321, 546
Mosaic proteins, 30
Motifs, 3032
Motor brain area, 513, 513f
Motor circuits and learning
in Aplysia gill withdrawal, 520521,
521f
in crustacean pyloric rhythms,
500502, 501f
in Tritonia escape swimming,
506507, 507f
Motor neuron, 546
Mre11 complex, 342344, 342t, 343f
m7GpppN cap structure, 404405, 405f
Multivesicular body, 444
Mutual inhibition, 501, 501f, 503, 503f,
546
Myc, 333t, 340, 351, 351f
Mycoplasma genitalium, 416
Mycoplasmas, 9

Myristoyl anchor, 34, 34t, 35f

Myxococcus xanthus, 427t, 434435
Naproxen, 294
Native state, 95, 9698, 96t, 97f, 98f,
104105, 104f, 546
Necrosis, 359
Necrotizing fasciitis, 432
Nernst equation, 467
Nerve bers, Ad and C, 293
Nerve growth factor, 247
Netrins, 236t, 237238, 238f
Neural cell adhesion molecules, see
IgCAMs
Neurexins, 514, 515f
Neurite, 234
Neurobrillary tangles, see Alzheimers
disease
Neurokinin GPCR, 277t
Neurokinins, 290, 290t
Neuroligins, 514, 515f
Neuromodulation, 289292, 290t, 502t,
546
extrinsic and intrinsic, 503t
in the reticular formation, 494
of central pattern generators,
505506
of HCN channels, 491492, 491f
of ion channels, 494495
Neuropilins, 236t, 239
Neurotransmitter release, 515518,
516f, 517t
Neurotransmitters, 288289, 288t, 546
Neurotrophin receptors, 251253, 252f,
253f
Neurotrophins, 251252
Neurulation, 320
Neutrophils, 189t
NF-kB responsive genes, see NF-kB
signaling module
NF-kB signaling module, 194195, 194f,
195f
antiviral activities, 205, 205f
in DISC signaling, 367, 368369, 368f
in immune responses, 194195
in neurotrophin signaling, 253f
in T cell receptor signaling, 210f
in the TNF pathway, 200f
in the Toll pathway, 199f

Index
Nicotinic acetylcholine receptors, see
Acetylcholine receptors
Nijmegen breakage syndrome, 342
Nijmegen breakage syndrome proteins,
333t, 342, 342t, 346, 354
Nitrogen xation
by cyanobacteria, 435436
by rhizobia, 436437
NK cells, 189t
NMDA receptors, 515, 519, 519t
and AMPA receptors, 523
biophysical properties, see Glutamate
receptors
dual voltage-ligand gated, 522523
in hippocampal LTP, 525, 525f
Nociception, 292293
Nod factors, 436437
Noncovalent bonds, 7576
Nonhomologous end-joining, 341, 341t,
342t, 344
Nonpolar side chains, 7374, 73t
Nonreceptor tyrosine kinases, 247,
258262, 258t
Nonsteriodal anti-inammatory drugs,
293295
Nonsteroid lipophilic hormones, 393,
393t
Noradrenaline, 286287, 287t, 290t
Norepinephrine, 494
Notch pathway, 3637, 306311, 306t,
307f, 308f, 310f
intracellular domain, 307, 308f
lateral inhibition, 307308, 309f, 545
Nuclear hormone receptors, 393, 393t
Nuclear import and export, 14
Nuclear magnetic resonance, 45, 46t,
5357, 54f, 55f, 68, 68f
Nuclear magneton, 56, 6768
Nuclear pore complex, 6, 267
passage by viruses, 442443
Nuclear spin, 54
Nucleocapsid, 546
Nucleocytoplasmic shuttling, 125128
Nucleoid region, 2
Nucleoli, 25
Nucleophile, 144, 158
Nucleosome, 22, 23, 23f, 24f, 547
Nucleotide excision repair, 340341,
341t

567

Nucleotide phosphodiesterases, see

Phosphodiesterases
Nucleus, 3t, 56, 2326
Nucleus accumbens, 530531, 531f,
531533, 532f
Occipital lobe, 512f, 513
Odorant receptors, 297299, 299f
Odorant signal transduction, 298299
Ohms law, 471
Olfactory receptors, 297, 298299
Olfactory signal transduction, 297299,
299f
Oligonucleotide arrays, 6263
Oligopeptide autoinducers, 428429,
429f
Omi/HtrA2, 372, 372t, 375
Oncolytic viruses, 462463
Oncoproteins, 332334, 333t, 547
Oncornavirus family, 445t
Oogenesis, in Drosophila, 322323
Open system, 89
Operon, 547
organization, 418419, 418f; see also
Bacteriophage lambda, Lac
operon
Opioid GPCR, 277t
Opsin GPCR, in rhodopsin, 295296
Opsins, 49
Order, 8990, 9698
Organelles, 3, 3f, 4
Organizing centers, 320321, 547
Orthomyxovirus family, 445t
Osmolytes, 76
Osmophobic forces, 7677
Osmotic balance, inside and outside
the cell, 466, 466f
Oxidative damage, see Reactive oxygen
species
Oxidative stresses, 371
Oxytocin, 290t
p21, 349350, 354
p300/CREB-binding protein, 526, 526f
p38 MAP kinase pathway, see Mitogenactivated protein kinase
modules
p53, 333t, 346, 347, 349354, 351ff,
353ff, 378379, 379f

568

Index

Pacemaker neurons, 488, 547

Pair rule genes, 321322, 321t, 324325,
324f
Palmitoyl anchor, 34, 34t, 35f
Pancreatic islets, see Islets of
Langerhans
Panulirus interruptus, 500
Papovirus family, 445t
Paracrine signaling, 248, 547
Paramyxovirus family, 445t
Parathyroid, 247
Parathyroid hormone GPCR, 277,
277t
Parathyroid hormone related protein
GPCR, 277, 277t
Parietal lobe, 512f, 513, 513f
Parkinsons disease, 100, 100t, 291,
360
Partition function, 219220
Patched receptor, 318, 318f
Pathogen, 547
Pathogenic bacteria, 431433
Pathogenicity islands, 419, 432
Pavlovs dog experiments, 520, 533
Paxillin scaffold, 260261, 262f
PDZ domain, 32t, 257, 257t
Peptide bond, 2627, 26f, 27f
Peripheral benzodiazepine receptor,
370, 370f
Peripheral supramolecular activation
cluster, 212213, 214
Periplasmic binding proteins, 278
Permeability transition pore, see
Permeability transition pore
complex
Permeability transition pore complex,
368, 368f, 369372, 370f, 372t,
375376, 547
Peroxisomes, 3t, 9
PEST motif, 306
Phagocytes, 189, 189t
Pheromone response pathway, 125128,
126f
Pheromones, 11
Philadelphia chromosome, 340
Phosopholipase C, 280t
Phosphadidylserine, 162t
Phosphatidylcholine, 162t, 163f
Phosphatidylethanolamine, 162t

Phosphatidylinositol, 163f, 165, 165t,

166f
Phosphatidylinositol 3,4,5 triphosphate,
165t, 169, 174175
Phosphatidylinositol 4,5 biphosphate,
165166, 166f
Phosphodiesterases, 172173, 280t,
281282
Phosphoinositide-3-OH kinase, 165,
167168, 168f
in activating protein kinase B, 169f
in DNA sensing and repair, 345346
Phosphoinositide-dependent kinase-1,
120t, 169f, 174, 177
in neurotrophin signaling, 253f
Phosphoinositides, 165, 165t, 166f
Phospholipase A2, 280t, 294f
Phospholipase C, 165166, 167ff
Phosphoprotein recognition modules,
254256, 257t
Phosphorelay, 139141, 141f, 547
in high osmolarity glycerol response
pathway, 128129, 128f
in plant hormonal signaling, 152154,
153f
Phosphorescence, 49
Phosphorylation, 33, 37, 38f, 146, 547;
see also Protein kinases
Phosphotyrosine binding domain,
169170, 170t, 255, 257t
Photobacterium scheri, see
Luminescent bacteria
Photoisomerization, 296, 296f
Photoreceptors, 49
Phototransduction, 295297, 298f
Phytochromes, 49, 154156, 154t, 155f
Picornovirus family, 445t, 446t
Pi helix, 27t
Pineal gland, 247
Pituitary gland, 247
Planar cell polarity pathway, 315, 316f,
317
Plancks constant, 47
Plancks formula, 4748
Plasmids, 2, 419, 547
Plasmodium falciparum, 426
Plateau potentials, 488489, 488t, 489f,
503504, 503t, 548
Platelet-derived growth factor, 249

Index
Platelets, 230, 548
Pleckstrin homology domains, 32t,
169170, 170t, 174
in phosphoinositide-dependent
kinase-1, 177f
in phospholipase C, 166, 167f
in protein kinase B, 174175, 177f
Pleiotropic, 13, 190, 548
Plexins, 236t, 239
Polar side chains, 7374, 73t
Polyacrylamide gel electrophoresis, see
Gel electrophoresis
Polyadenylate tail or poly (A) tail,
404405
Polymerase chain reaction, 62
Polypeptide hormones, 247248, 548
Polyproline (PP II) helix, 256
Polypyrimidine tract, see Pre-mRNAs
Pons, 512, 512f
Pores, 4, 548
Porins, see Pores
Post inhibitory rebound, 503t, 504,
548
Postsynaptic density, 514, 518520, 518f,
519t, 548
Postsynaptic terminal, see Postsynaptic
density
Posttranslational modications, 13, 21
anchors, 33, 34, 34t, 35ff
glycosylation, 33, 36
proteolysis, 33, 3637
to histones, 4041, 41f
to side chains, 33, 3640, 38ff
Potassium channels, voltage-gated, 475t
chain topology, 476f
conducting pore, 476477, 479, 480f
selectivity lter, 476477, 478479
subunit assembly, 476477
voltage sensor, 478479, 479f
Potassium currents, 466468, 466f, 468t,
468470, 469f; see also
HodgkinHuxley equations
Potential energy, of a macromolecule,
7983
Poxvirus family, 445t, 446t, 447
pRb, see Retinoblastoma protein
Prebtzinger complex, 504
Predictive learning, 533
Prefrontal cortex, 531f, 533

569

Preinitiation complex, 385, 386387,

386t, 387f, 548
Pre-messenger RNA, 548
Premotor cortex, 512f, 513, 513f
Pre-mRNAs, 399403, 401f, 402f
Prenyl anchor, 34, 34t, 35f
Presenilins, 309310, 310f
and Alzheimers disease, 99
Presynaptic terminal, see Active zone
Prickle, 317
Primary splice sites, 400401, 401f, 548
Primary structure, 548; see also Protein
structure
Principle of detailed balance, 217,
218219
Prions, 100, 100t
Progestins, 393t
Programmed cell death, see Apoptosis
Prokaryotes, 13, 910
Prolactin, 286, 287t
Prolactin-inhibiting hormone, 287t
Proline-recognition modules, 256, 257t
Promoter, 118
bacterial, 413f, 414, 414t; see also
Control point
eukaryotic, 386387, 387f, 548
Pro-opiomelanocortin, 286
Prostanoid GPCR, 277t, 294f
Prostanoids, 293t, 294
Proteases, 7
Protein backbone, 549; see also Protein
structure, Primary structure
Protein Data Bank, 5253
Protein dynamics, 7986, 104105
Protein Explorer, 43
Protein folding, 90, 9498, 96t, 97f, 98f,
549; see also Misfolded proteins
and diseases, Molecular
chaperones
Protein interfaces, 7778, 77t
Protein kinase A, 120t, 173174, 174t,
180182, 181f, 519t
catalysis, 182183, 183f
in Aplysia facilitation, 522, 522f
in hippocampal LTP, 525526, 525f,
526f
in the Hh pathway, 318320, 319ff
in yeast general stress response, 130,
130f

570

Index

Protein kinase A (cont.)

localization by anchoring proteins,
178179
Protein kinase B, 120t, 121, 173174,
174t, 174175, 175f, 177f
inhibition of Forkhead transcription
factors, 176177, 176f
in insulin signaling, 175, 175f
in neurotrophin signaling, 253f
in TOR signaling, 132f, 133f
regulation of Bad proteins, 176, 176f
Protein kinase C, 120t, 173174, 174t,
177178, 177f, 519t
localization by anchoring proteins,
178179
regulation of cardiac ion channels,
179180, 180f
Protein kinase R, 205, 205f, 448449,
448f
Protein kinases, 115116, 115f, 119123,
120t, 121f, 549
Protein phosphatase 1/2A, 519t
in ion channel regulation, 180182,
181f
in TOR signaling, 131132, 132f,
133134, 134f
Protein phosphatases, 115, 115f,
123124, 549
Proteinprotein interaction domains,
256258, 257t
Protein regulation, 1315
Protein structure, 2633, 26f, 27f, 27t,
28f, 31f, 32t
Proteolytic processing, 3637, 116117,
117f, 549
in Notch, 307f, 308311
Proteosome, 549
Proximal control region, 387389, 388f,
549
Proximal sites, 549
PSD-95 proteins, 514, 515f
Pseudomonas aeruginosa, 10, 416, 427t,
430
PTEN lipid phosphatase, 168169
Pumps, 4, 549
Purine GPCR, 277t, 295
Purkinje bers, 488
Pyloric circuit and pattern, 500, 501
Pyrin domains, 257

Quantum mechanics, 84
Quaternary structure, 549; see also
Protein structure
Quorum sensing, 428430, 429ff, 549
Rab GTPase, 263t, 268269, 269f
Radio waves, 47f
Rad proteins, 342344, 342t, 343f
Raf, 253f
Ramachandran plot, 27, 28f
Raman spectroscopy, 61
Random coils, 28
Ran GTPase, 263t, 267268, 268f
Raphe nuclei, 292
Rapid eye movement (REM) sleep,
492, 494
Ras-binding domain, in PI3 kinase,
168f
Ras GTPase, 253f, 263266, 263t, 265f
as activators of MAP kinase
modules, 196, 197f
in cell-cycle control, 351, 351f
in T cell receptor signaling, 210f
in yeast general stress response, 130,
130f
mutated in cancer, 333t, 334336,
335ff
Reactive oxygen species, 331, 341,
371372, 375376, 376f, 378, 549
Receiver domain, 140141, 141f
Receptor clustering, 148149
Receptor for activated C-kinase,
179180, 180f
Receptor interacting proteins, 368, 368f
Receptors, 12, 112113, 112f, 549
Receptor tyrosine kinases, 247,
252254, 253f
Reciprocal inhibition, see Mutual
inhibition
Recruitment, 114, 161
Rectifying, 549
Regulator-of-G-protein-signaling
domain, 256, 257t
Regulators of G protein signaling, 279
Regulons, 419, 550
Repair proteins, mutated in cancer,
333, 333t
Reporter protein, 59, 60f
Respiration, 504505

Index
Response regulators, 140141, 141f,
145146, 145f, 145t, 550
Responsive elements, 118, 387389,
388f, 550
Resting potential, 468
Restriction point, 346f, 347
Reticular formation, 494, 512, 512f
Retinal photoisomerization, 295296,
296f
Retinitis pigmentosa, 99
Retinoblastoma protein, 333t, 347349,
350352, 350f, 351ff, 354
Retrograde signals, 273
Retroviruses, 336, 441, 449450, 450f;
see also Human
immunodeciency virus
Reversal potential, 467, 550
Reward-driven behavior, 533534
Rhabdovirus family, 445t
Rheovirus family, 445t, 446t
Rhizobia, 436437
Rhodopsin GPCR, 154t, 277, 277t,
278279, 278f, 295296, 296f
Rho GTPase, 253f, 263t, 265f, 266267
as activators of MAP kinase
modules, 196, 197f
in the Wnt pathway, 316f, 317
Rhythmic bursting, 492494, 493f, 550
Ribonucleoprotein complexes, 401403,
402f
Ribosomal S6 kinase, 120t, 133, 133f
Ribosomes, 7, 131
RING nger, 32t, 374375, 374f
RNAP holoenzyme, bacterial, 412413,
413t, 413f, 414t, 418; see also
Sigma factors
RNA polymerase II, 385, 386387,
387f, 393, 394396, 394t, 395f,
396f
RNA recognition motifs, 404, 404f
Robustness, in the chemotactic
pathway, 150152, 150f,
550
Rossman fold, 29
Rouleaux, 231
Roundabout, 238, 238f
Roux sarcoma virus, 336
R-Smad, see Smad proteins
Ryanodine receptor, 377

571

Saccharomyces cerivisiae, 111

S-adenosyl-l-methionine, 39
SAGA complex, 386, 386t, 392393
Salmonella enterica, see Flagellar
motor, assembly
Salmonella typhimurium, 149150
Salt bridges, 73t, 7475
SANT domain, 398t
SARA anchor protein, 312f, 313
Sarco-endoplasmic reticulum calcium
ATPase, 377
Scaffold proteins, 113114, 114f, 124, 550
Scatter factors, 236t, 239
Scattering of X-rays, see X-ray
crystallography
Schizophrenia, 291
Secondary structure, 550; see also
Protein structure
Second messengers, 111112, 121122,
550
Secretases, 307f, 310, 310f
Secretin GPCR, 277278, 277t
Segment polarity genes, 321322, 321t,
325326
Selectins, 222, 222f, 229230, 230t,
233234
Selective permeability, 466468
Selectivity, 475, 550
Selectivity lter, 475, 479, 480, 550
Selector genes, 326
Semaphorins, 236t
Senescence, 354355, 550
Sensitization, 506507, 520, 550
Sensor unit, 140141, 141f, 551
Sensory neurons, in Aplysia, 520521,
521f
Serial triggering, 213214
Serine/threonine kinases, 119121
Serine/threonine phosphatases, 124
Serotonergic system, 292
Serotonin, 290, 290t, 292, 293t, 494
in Aplysia short- and long-term
facilitation, 522, 522f
Serotonin GPCR, 277t, 292
Serotonin reuptake inhibitor, 292
Serrate, 306, 306t
Severe combined immunodeciency, 346
Shank protein, 258f
Shape complementarity, 78, 551

572

Index

Shc, 252, 253f

Shiga toxin, 459460, 460f
Short-term memory, 521
Sickle cell anemia, 99
Sigma factors, 414, 415, 415t
ECF, 415, 415t
agellar/chemotactic, 415t, 421422,
423f
heat shock/stress, 415, 415t, 419
primary, 414, 415t
sporulation, 415, 415t, 422424
stationary phase, 414415, 415t, 419
Signaling complexes, 113114, 114f
Signaling pathway, 111
Signal receptors, see Receptors
Signals, 1011
Signal transduction, 12, 112, 551
Silent synapses, 523
Sinoatrial node, 487488, 488t, 489f,
490f
Skin cuts, 251
Slip bonds, 551; see also Bond action
during leukocyte rolling
Slits, 236t, 237238, 238f
Sloan Kettering Institute protein, 312f,
314
Smac/DIABLO, 372, 372t, 375
Smad proteins, 311, 312f, 313314, 313t,
314
Small nuclear RNA molecules, 401403
Small nuclear RNPs, 401
Small ubiquitin-related modier, 39, 40,
40f
Smoothened receptor, 318, 318f
Smurf E3 ligases, 314
SNARE proteins, 269, 517518, 517t
Sodium channels, voltage-gated, 475t
single-chain topology, 477, 477f
Sodium currents, 466468, 466f, 468t,
468470, 469f; see also
HodgkinHuxley equation
Somatosensory cortex, 512f, 513, 513f
Somatosensory system, 292293
Somatostatin, 247
Somatostatin GPCR, 277t, 287, 287t
Sore throats, 432
Sos Ras GEF, 253f, 265
Speckles, see Inter-chromatin granule
clusters

Speech brain area, 513, 513f

Sphingomyelin, 162t
Spike frequency adaptation, 502, 503t,
551
Spin, see Magnetic dipole moments,
Nuclear magnetic resonance
Spindling waves, 490t, 492, 498, 498f
Spinspin interactions, see Nuclear
magnetic resonance
Spliceosome, 401403, 551
Splice sites, see Pre-mRNAs
Splicing, 6, 2626; see also Pre-mRNAs
Splicing regulation, 14
Spongiform encephalopathies, 100
Sporulation, in B. subtilis, 422424, 424f
Src, 258t, 259260, 259f, 260f, 333t, 336,
449
Src homology 2, 3 domain, 32t,
166167, 167f, 168f, 169170,
170t, 255, 256, 257t
SR proteins, 403404, 403f
Stalked cell, C. crescentus, 424426,
425f
Staphylococcus aureus, 427t, 429430
START point, 346f, 347
STAT proteins, structure, 203204, 204f
in interferon signaling, 205206, 206f
Sterile a motif domain, 257258, 257t
Sterile (Ste) proteins, 126f, 127128,
128f
Steroid hormones, 247, 287t, 393, 393t,
551
StokesEinstein relationship, 136, 138
Stomatogastric ganglion, 500502, 501f
Strabismus co-receptor, 315, 315f
Streptococcus pneumoniae, 427t
Streptomyces coelicolor, 10, 412, 427t,
428, 434
Stress responsive element, in yeast, see
General stress response
pathway, in yeast
Striatum, 530531, 531f
Stringent response, 434
Structural motif, 29, 551
Substance P, 290, 290t, 293t
Substantia nigra, 291, 530531, 531f
Suicide modules, see Death modules,
bacterial
Sulfur bridges, see Disulde bridges

Index
Supersecondary structures, 29
Suppressor of Fused, 318, 319f
Suppressor of Hairless, 306t, 307, 308f
SWI/SNF complex, 386, 386t, 392393,
398t
Synapse, 213, 234235, 514515, 515f;
see also Postsynaptic density,
Presynaptic terminal
Synaptic cleft, 514515, 515f
Synaptic current, in HodgkinHuxley
equation, 471
Synaptic plasticity, 511, 526527, 527f,
528529, 528f, 536, 551
in drug addiction, 533
Synaptic signaling, 248249, 551
SynCAMs, 514515, 515f
T1, T2 cells, 191t, 192
TACE metalloprotease, 311
Tachykinins, 290
Tap42 protein, see Protein phosphatase
1/2A
Targets of rapamycin proteins, 120t,
131
Targets of rapamycin signaling,
131134, 132f, 133f, 134f
Tar receptor, see Bacterial receptors
TATA-binding protein, 386387, 387f,
394, 394t
TATA box, 386387, 387f
TBP-associated factors, 394, 394t
T cells, 189, 189t
CD4, CD8 co-receptors, 209210,
210f
cytokines, see Interleukins
differentiation, 192, 192f
receptors, 207212, 210f, 211t
Telomerase production, 354355
Telomere, 354355, 551
Temporal lobe, 512f, 513
Terminal oligopyrimidine tract, 133,
133f
Tertiary structure, 552; see also Protein
structure
TFIIA structure, 391, 392f
TFIIB-recognition element, 386,
387f
TGF-b pathway, 311314, 312f
in cancer, 338339

573

Therapeutic drugs, see Control layer,

Drugs
Thermal energies, 47
Thermal equilibration, see
Equilibration
Thermodynamics
rst law, 9091
Gibbs free energy, 9394
second law, 9193
Thermodynamic shift, see Energy
barrier
Theta waves, 490t, 492
Thymus, 247
Thyroid gland, 247
Thyroid hormone, 393t
Thyroid-stimulating hormone, 286, 287t
Thyrotropin-releasing hormone, 286,
287t
Tie receptors, 249250, 250f
Time-of-ight spectrometer, 6465,
6869
TNF-related apoptosis-inducing ligand
receptors, 366367, 367t, 369
Togavirus family, 445t
Toll/IL-1/Dorsal signaling, 191t, 193,
198199, 199f, 323
Tonic ring, 492494, 493f, 552
Tourette syndrome, 291
Toxins, 428, 433434, 433f
Toxic shock syndrome, 432
TRADD adapter, 367, 368f
TRAF adapters, 197, 198f, 252, 253f
in Toll signaling, 199f
in Tumor necrosis factor signaling,
196197, 200, 200f
TRAF2 adapter, 367, 368f
Transcription, 5, 5f, 2223
Transcription factors, 117119, 552
bacterial, 416418, 416t
eukaryotic, 387393, 388f, 390f, 391f,
392f
Transducin, 280t, 297
Transduction, bacterial, 431, 552
Transformation, bacterial, 431, 552
Transforming growth factor-b, 311312,
312f, 313t
Transition rate, 108109
Transition state, 104105, 158159, 159f
Translation, 5, 5f

574

Index

Translation initiation and regulation,

14; see also Eukaryotic initiation
factors, Messenger RNAs
Translocations, 340
Transport vesicles, 268270
Tricyclic antidepressants, 292
Tritonia diomedea, 500, 506507, 520
Trophic factor, 251, 552
Trp ion channels, 297, 298f
t-Snares, see SNARE proteins
Tumor necrosis factor family, 191t, 193,
199200, 200f, 366367, 367t,
368f
Tumor suppressors, 332334, 333t, 552
Two-component signaling, 139141,
140f, 140t, 152157, 552
bacterial transcription factors,
417418
Tyrosine kinases, 119120
Tyrosine phosphatases, 124
Ubiquitination, 3940, 40f, 552
Ultraviolet radiation, 47f, 61, 331, 341
Umami tastants, 299, 300
Unconditioned stimulus, 520
Unfolded state, see Denatured state
Van der Waals forces, see
Macromolecular forces
Vascular endothelial growth factor,
249250, 250f
Vasculogenesis, 249250, 552
Vasopressin, 290t
Vasopressin GPCR, 277t
Ventral tegmental area, 291, 530531,
531f, 531533, 532f
Venules, 231
Verlet algorithm, 87
Vibrio cholerae, 416
Vibrio harveyi, see Luminescent
bacteria
VIP/PACAP GPCR, 277t, 278
Viral diseases, 444445, 445t
Virulence cassettes, 419, 432433
Virulence factors, 427t, 428, 432, 552

Viral genomes, 441, 445

bacteriophage lambda, 457458, 457f
hepatitis C virus, 448, 448f
HIV, 452453, 453f
human T lymphotropic virus,
449450, 450f
Kaposis sarcoma virus, 451f
Viral host interactions, 445447, 446t,
447449, 448t
Viral movement, 442444
Viral oncogenes, 336
Viral structure, 441
bacteriophage lambda, 457, 457f
HIV, 452453, 452f
Viscosity, 135, 138
Visible light, 47f
Vision brain area, 513, 513f
Vitamin D3, 393t
Voltage-dependent anion channels, 370,
370f
Voltage-gated calcium channels, see
Calcium channels, voltage-gated
Vomeronasal organ, 299
v-Snares, see SNARE proteins
Water molecules, 74, 8485
WD-40 repeat, 32t
White blood cells, see Leukocytes
Wingless and Wnt, 314315, 315f, 316f
Wnt pathway, 314317, 316f, 321t,
325326
in cancer, 338339
Wound healing, 250251
WW domain, 256, 257t
X-ray crystallography, 22, 45, 46t, 47f,
4953, 51f, 52f
Yeast two-hybrid, 4546, 46t, 6162
Yersinia pestis, 433
Yop virulon, see Yersinia pestis
Zinc nger domain, 391, 392f
Zonula adherens junction, 227
Zymogens, 361, 552