G protein coupled receptors s siehler, g milligan (cambridge university, 2011)

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G Protein-Coupled Receptors
This text provides a comprehensive overview of recent discoveries
and current understandings of G protein-coupled receptors (GPCRs).
Recent advances include the first mammalian non-rhodopsin GPCR
structures and reconstitution of purified GPCRs into membrane discs
for defined studies, novel signaling features including ­oligomerization,
and advances in understanding the complex ligand pharma­cology and
physiology of GPCRs in new assay technologies and drug targeting.
The first chapters of this book illustrate the history of GPCRs based
on distinct species and genomic information. This is followed by
­discussion of the homo- and hetero-oligomerization features of GPCRs,
including receptors for glutamate, GABA B, dopamine, and chemokines.
Several chapters are devoted to the key signaling features of GPCRs.
The authors take time to detail the importance of the pathophysiological function and drug targeting of GPCRs, ­specifically β-adrenoceptors
in cardiovascular and respiratory diseases, ­metabotropic glutamate
­receptors in CNS disorders, S1P receptors in the immune system, and
Wnt/Frizzled receptors in osteoporosis.
This book will be invaluable to researchers and graduate students in
academia and industry who are interested in the GPCR field.
Dr. Sandra Siehler is a Research Investigator at the Novartis Institutes
for BioMedical Research in Basel, Switzerland. Dr. Siehler is a member
of the American Society for Pharmacology and Experimental Therapeutics and the British Pharmacological Society.
Dr. Graeme Milligan is Professor of Molecular Pharmacology at the
University of Glasgow. He is actively involved in numerous associations, such as the Biochemical Society and the British Pharmacological Society. Dr. Milligan was awarded the Ariens Award for Pharmacology from the Dutch Pharmacological Society in 2006.

G Protein-Coupled
Receptors
Structure, Signaling, and Physiology
Edited by
Sandra Siehler
Novartis Institutes for BioMedical Research

Graeme Milligan
University of Glasgow


CAMBRIDGE UNIVERSITY PRESS

Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore,
São Paulo, Delhi, Dubai, Tokyo
Cambridge University Press
The Edinburgh Building, Cambridge CB2 8RU, UK
Published in the United States of America by Cambridge University Press, New York
www.cambridge.org
Information on this title: www.cambridge.org/9780521112086
© Cambridge University Press 2011
This publication is in copyright. Subject to statutory exception and to the
provision of relevant collective licensing agreements, no reproduction of any part
may take place without the written permission of Cambridge University Press.
First published in print format 2010
ISBN-13

978-0-511-90991-7

eBook (NetLibrary)

ISBN-13

978-0-521-11208-6

Hardback

Cambridge University Press has no responsibility for the persistence or accuracy
of urls for external or third-party internet websites referred to in this publication,
and does not guarantee that any content on such websites is, or will remain,
accurate or appropriate.


Contents

List of Figures
List of Tables
List of Contributors
Introduction

page vii
xi
xiii
1

Part I: Advances in GPCR Protein Research
1

2

The evolution of the repertoire and structure
of G protein-coupled receptors
Torsten Schöneberg, Kristin Schröck, Claudia Stäubert,
and Andreas Russ
Functional studies of isolated GPCR-G protein complexes
in the membrane bilayer of lipoprotein particles
Adam J. Kuszak, Xiao Jie Yao, Sören G. F. Rasmussen,
Brian K. Kobilka, and Roger K. Sunahara

5

32

Part II: Oligomerization of GPCRs
3

GPCR-G protein fusions: Use in functional dimerization analysis
Graeme Milligan

53

4

Time-resolved FRET approaches to study GPCR complexes
Jean Phillipe Pin, Damien Maurel, Laetitia Comps-Agrar,
Carine Monnier, Marie-Laure Rives, Etienne Doumazane,
Philippe Rondard, Thierry Durroux, Laurent Prézeau, and Erin Trinquet

67

5

Signaling of dopamine receptor homo- and heterooligomers
Ahmed Hasbi, Brian F. O’Dowd, and Susan R. George

90

6

Functional consequences of chemokine receptor dimerization
Mario Mellado, Carlos Martínez-A., and José Miguel Rodríguez-Frade

111

Part III:  GPCR Signaling Features
7

G protein functions identified using genetic mouse models
Stefan Offermanns

125

8

Kinetics of GPCR, G protein, and effector activation
Peter Hein

145

v

vi

Contents
9

RGS-RhoGEFs and other RGS multidomain proteins as effector
molecules in GPCR-dependent and GPCR-independent cell signaling
José Vázquez-Prado and J. Silvio Gutkind

159

10

Adenylyl cyclase isoform-specific signaling of GPCRs
Karin F. K. Ejendal, Julie A. Przybyla, and Val J. Watts

189

11

G protein-independent and β arrestin-dependent GPCR signaling
Zhongzhen Nie and Yehia Daaka

217

12

Assays to read GPCR modulation and signaling

Ralf Heilker and Michael Wolff

231

Part IV: Ligand Pharmacology of GPCRs
13

Assessing allosteric ligand-receptor interactions
Ivan Toma Vranesic and Daniel Hoyer

247

14

7TM receptor functional selectivity
Terry Kenakin

270

Part V:  Physiological Functions and
Drug Targeting of GPCRs
15

β-Adrenoceptors in cardiovascular and respiratory diseases
Michele Ciccarelli, J. Kurt Chuprun, and Walter J. Koch

287

16

Role of metabotropic glutamate receptors in CNS disorders
Richard M. O’Connor and John F. Cryan

321

17

S1P receptor agonists, a novel generation of immunosuppressants
Rosa López Almagro, Gema Tarrasón, and Nuria Godessart

380

18

Wnt/Frizzled receptor signaling in osteoporosis
Georges Rawadi

398

Index
Color plates follow page 32.

415


Figures

1.1.

Evolutionary occurrence of the different GPCR families

in eukaryotes.

page 7

Signatures of positive selection during human evolution
and examples for detection methods.

19

1.3.

Phylogenetic trees of primate TAAR3 and TAAR4 subtypes.

23

2.1.

Illustration of reconstituted HDL particles.

34

1.2.

2.2. Schematic overview of GPCR reconstitution into HDL particles.

36

2.3. Monomeric GPCRs are capable of functional G protein coupling.

38

2.4. Theoretical models of monomeric (left) and dimeric (right)
rhodopsin coupling to transducin (Gt ).

42

2.5. Molecular models of the conformational changes in β2AR.

46

2.6. Schematic model of the β2AR conformational changes induced
by agonist, inverse agonist, neutral antagonists and Gs heterotrimer
interactions.

47

3.1.

4.1.

Reconstitution of function by co-expression of a pair of
inactive dopamine D2-Gαo1 fusion proteins: unaltered
potency of dopamine.

59

Conditions for energy transfer to occur between two fluorophores.

70

4.2. Emission spectra for CFP-YFP FRET (A), luciferase-YFP BRET (B),
and Eu3+ cryptate d2 TR-FRET (C).

71

4.3. Biophysical properties of the TR-FRET fluorophore pairs.

73

4.4. Structure and properties of two different Eu3+ cryptates.

74

4.5. Comparison of the emission spectra of Eu cryptates TBP (black)
and Lumi4 Tb (gray).

75

4.6. Testing the proximity between cell surface proteins using anti-tag
antibodies conjugated with TR-FRET compatible fluorophores.

77

3+

4.7.

TR-FRET between antibody-labeled GABAB subunits measured
at various receptor expression level.

79

4.8. Using the snap-tag to label cell surface proteins.

80

4.9. Using the ACP-tag to label cell surface proteins.

82

4.10. GABAB dimers analyzed with snap-tag-TR-FRET.

83
vii


viii

Figures
4.11. GABAB oligomers revealed with snap-tag-TR-FRET.
8.1.

Sample FRET traces for early signaling processes.

8.2. Kinetics of different steps of signal transduction.
9.1.

Structure of multidomain RGS proteins including Gα12/13-regulated
Rho guanine exchange factors (p115-RhoGEF, PDZ-RhoGEF and LARG),
G protein coupled receptor kinase 2 (GRK2), RGS12 and 14, and Axin.

85
151
154

161

9.2. In migrating fibroblasts, Rho activation is important to promote
the removal of focal adhesions at the trailing edge in response to
lysophosphatidic acid.

163

9.3. GRK2 is an effector of Gβγ that phosphorylates agonist stimulated
G protein-coupled receptors initiating the process of desensitization.

165

9.4. Additional mechanisms of regulation of PDZ-RhoGEF and LARG
include activation in response to interaction of Plexin B with
Semaphorin, oligomerization and phosphorylation.

180

9.5. RGS12 and RGS14 regulate G protein signaling and Growth
Factor Receptor signaling.

182

10.1. Adenylyl cyclase is a membrane-bound enzyme that contains

an intracellular N-terminus, followed by a membrane-bound
region (M1).

191

10.2. The nine membrane-bound isoforms of adenylyl cyclase are classified
into four categories/groups based on their regulatory properties.

193

10.3. Visualization of adenylyl cyclase-GPCR interactions using
Bimolecular Fluorescence Complementation, BiFC.

206

11.1. β-Arrestin-mediated signaling.

220

12.1. HCS to monitor GPCR ligand binding, internalization, and
arrestin redistribution.

238

12.2. HCS to monitor GPCR-modulated second messenger responses
and ERK signaling.

240

13.1. Effects of the intrinsic efficacy β of the modulator B on the binding

properties of an orthosteric ligand A expressed in terms of receptor
occupancy and of the modulator B (β = 100, B varying from 0.01
to 3 x M) on the saturation curves of an orthosteric ligand A
represented in a logarithmic scale.

253

13.2. Effects of allosteric agonist or inverse agonist modulator on
concentration effect curves of an orthosteric agonist.

254

13.3. Effects of γ on the binding properties of a neutral antagonist.

254

13.4. Effects of γ on the activation curves of an orthosteric agonist.

255

13.5. Effects of δ of the modulator B on binding curves of an
orthosteric agonist (left) and neutral antagonist (right).

255

13.6. Effects of δ on activation curves of an orthosteric agonist.

256

13.7. Effects of CPCCOEt on glutamate-induced IP1 production

via mGluR1 receptors and binding.

259

14.1. Two opposing views of receptor activation.

271


Figures

ix

14.2. Vectorial flow of allosteric energy.

276

14.3. Biased antagonism.

279

14.4. Different views of receptor/agonist efficacy.

281

15.1. Sympathetic activation in heart failure.

299

15.2. Localization of the two most frequent polymorphisms within

the human β1-adrenoceptor.

301

15.3. Inotropic support in patients with the Arg389Gly-;β1AR polymorphism.

302

16.1. Schematic representation of the general structure of metabotropic
glutamate receptors.

323

16.2. Schematic representation of the metabotropic glutamate receptor
classification and signaling pathway.

324

16.3. General synaptic localization of the different metabotropic
glutamate receptor subtypes.

325

17.1. Metabolic pathways of S1P.

381

17.2. G proteins coupled to the S1P receptors and downstream
signaling pathways.

384

18.1. Current general view of Frizzled receptor dependent Wnt signaling.

399

18.2. Schematic representation of frizzled and secreted frizzled
(sFRP) proteins.

400

18.3. Canonical Wnt signaling.

401

18.4. Canonical Wnt signaling effect on bone metabolism.

405



Tables

   1.1. Potentially Selected GPCR Genes in the Evolution of Modern
Human Populations Identified in Genome-wide Studies

page 20

   6.1. Summary of Chemokine Receptor Dimers

117

   7.1. Phenotypical Changes in Mice Lacking α-subunits of
Heterotrimeric G proteins

127

  10.1. Regulatory Properties of Adenylyl Cyclase Isoforms

192

  10.2. Subunits of the Heterotrimeric G proteins

194

  13.1. Examples of Allosteric Modulators for GPCRs

257

  14.1. Biased Agonists for AA Release and IP3 Production
via 5-HT2C Receptors

279

  15.1 βARs Subtypes

289

  16.1. Pharmacological Evidence Implicating Group I mGLuRs in Anxiety

328

  16.2. Pharmacological Evidence Implicating Group I mGLuRs
in Cognitive Disorders

330

  16.3. Pharmacological Evidence Implicating Group I mGLuRs
in Schizophrenia

332

  16.4. Pharmacological Evidence Implicating Group I mGLuRs
in Depression

334

  16.5. Pharmacological Evidence Implicating Group I mGLuRs
in Parkinson’s Disease

336

  16.6. Pharmacological Evidence Implicating Group I mGLuRs
in Epilepsy

339

  16.7. Pharmacological Evidence Implicating Group I mGLuRs
in Pain Disorders

342

  16.8. Pharmacological Evidence Implicating Group II mGLuRs in Anxiety

345

  16.9. Pharmacological Evidence Implicating Group II mGLuRs
in Cognitive Disorders

347

16.10. Pharmacological Evidence Implicating Group II mGLuRs
in Schizophrenia

350

16.11. Pharmacological Evidence Implicating Group II mGLuRs
in Parkinson’s Disease

352

xi


xii

Tables
16.12. Pharmacological Evidence Implicating Group II mGLuRs in Epilepsy

354

16.13. Pharmacological Evidence Implicating Group II mGLuRs
in Pain Disorders

355

16.14. Pharmacological Evidence Implicating Group III mGLuRs in Anxiety

358

16.15. Pharmacological Evidence Implicating Group III mGLuRs in Epilepsy

362

  17.1. S1P Receptors: Knockout Phenotypes and Biological Functions

385

  17.2. Diseases Described in Laboratory Animals in Which
Different Strategies Targeting S1P Have Reported Efficacy

392


Contributors

Rosa López Almagro, Ph.D
Research and Development Center
Almirall
Barcelona, Spain

J. Kurt Chuprun, Ph.D.
Center for Translational Medicine
Thomas Jefferson University
Philadelphia, PA
Michele Ciccarelli, MD
Center for Translational Medicine
Thomas Jefferson University
Philadelphia, PA
Laetitia Comps-Agrar, Ph.D.
Department of Molecular
Pharmacology
Institut de Génomique Fonctionnelle
Montpellier, France
John F. Cryan, Ph.D
Senior Lecturer
School of Pharmacy
Department of Pharmacology
and Therapeutics
University College Cork
Cork, Ireland
Yehia Daaka, Ph.D.
Department of Urology
UF Prostate Disease Center
University of Florida
College of Medicine
Gainesville, FL

Etienne Doumazane, Ph.D.
Department of Molecular
Pharmacology

Institut de Génomique
Fonctionnelle
Montpellier, France
Thierry Durroux, Ph.D.
Department of Molecular
Pharmacology
Institut de Génomique
Fonctionnelle
Montpellier, France
Karin F. K. Ejendal, Ph.D.
Postdoctoral Research Associate
Department of Medicinal Chemistry
and Molecular Pharmacology
School of Pharmacy and
Pharmaceutical Sciences
Purdue University
West Lafayette, IN
Susan R. George
Professor
Department of Pharmacology
and Toxicology
University of Toronto
Toronto, Ontario, Canada
Nuria Godessart, Ph.D.
Head of Autoimmunity Department
Almirall Laboratories
Llobregat, Spain

xiii

xiv

Contributors
J. Silvio Gutkind, Ph.D.
Oral & Pharyngeal Cancer Branch
National Institute of Dental and
Craniofacial Research
National Institutes of Health
Bethesda, MD
Ahmed Hasbi, Ph.D.
Postdoctoral Fellow
Department of Pharmacology
and Toxicology
University of Toronto
Toronto, Ontario, Canada
Ralf Heilker, Ph.D.
Boehringer Ingelheim Pharma
GmbH & Co. KG
Department of Lead Discovery
Biberach, Germany
Peter Hein, MD, Ph.D.
Postdoctoral Researcher
Department of Molecular and Cellular
Pharmacology and Psychiatry
University of California at San
Francisco
San Francisco, CA
Daniel Hoyer, Ph.D.
Neuropsychiatry

Neuroscience Research
Novartis Institutes for BioMedical
Research
Basel, Switzerland
Terry Kenakin, Ph.D.
Department of Biological Reagents
and Assay Development
Molecular Discovery
GlaxoSmithKline Research and
Development
Research Triangle Park, NC
Brian K. Kobilka, MD
Professor
Depatment of Molecular and Cellular
Physiology
Stanford University
Stanford, CA

Walter J. Koch, Ph.D.
Center for Translational Medicine
Thomas Jefferson University
Philadelphia, PA
Adam J. Kuszak
Department of Pharmacology
University of Michigan
Ann Arbor, MI
Carlos Martínez-A., Ph.D.
Professor
Department of Immunology and
Oncology

Centro Nacional de Biotecnologia
Madrid, Spain
Damien Maurel, Ph.D.
Scientist
Ecole Polytechnique Fédérale de
Lausane
Lausane, Switzerland
Mario Mellado, Ph.D.
Research Scientist
Department of Immunology
and Oncology
Centro Nacional de
Biotecnologia
Madrid, Spain
Graeme Milligan, Ph.D.
Professor
Neuroscience and Molecular
Pharmacology
University of Glasgow
Scotland
Carine Monnier, Ph.D.
Department of Molecular
Pharmacology
Institut de Génomique Fonctionnelle
Montpellier, France
Zhongzhen Nie, Ph.D.
Department of Urology
UF Prostate Disease Center
University of Florida
College of Medicine

Gainesville, FL


Contributors
Richard M. O’Connor
School of Pharmacy
Department of Pharmacology
and Therapeutics
University College Cork
Cork, Ireland
Brian F. O’Dowd
Professor
Department of Pharmacology and
Toxicology
University of Toronto
Toronto, Ontario, Canada
Stefan Offermanns, MD
Director
Department of Pharmacology
Max-Planck Institute for Heart and
Lung Research
Hessen, Germany
Jean Phillipe Pin, Ph.D.
Director
Department of Molecular
Pharmacology
Institut de Génomique Fonctionnelle
Montpellier, France
Laurent Prézeau, Ph.D.
Department of Molecular

Pharmacology
Institut de Génomique Fonctionnelle
Montpellier, France
Julie A. Przybyla
Department of Medicinal Chemistry
and Molecular Pharmacology
School of Pharmacy and
Pharmaceutical Sciences
Purdue University
West Lafayette, IN
Sören G. F. Rasmussen, Ph.D.
Postdoctoral Scholar
Department of Molecular and
Cellular Physiology
Stanford University
Stanford, CA

xv
Georges Rawadi, Ph.D.
Business Development & Alliance
Manager
Galapagos
Romainville, France
Marie-Laure Rives, Ph.D.
Postdoctoral Research Fellow
Columbia University
New York, NY
José Miguel Rodríguez-Frade, Ph.D.
Research Scientist
Department of Immunology and

Oncology
Centro Nacional de
Biotecnologia
Madrid, Spain
Philippe Rondard, Ph.D.
Department of Molecular
Pharmacology
Institut de Génomique
Fonctionnelle
Montpellier, France
Andreas Russ, Ph.D.
Department of Biochemistry
University of Oxford
Oxford, United Kingdom
Torsten Schöneberg, Ph.D.
Molecular Biochemistry
Institute of Biochemistry
University of Leipzig
Leipzig, Germany
Kristin Schröck, Ph.D.
Molecular Biochemistry
Institute of Biochemistry
University of Leipzig
Leipzig, Germany
Sandra Siehler, Ph.D
Research Investigator II
Center for Proteomic Chemistry
Novartis Institutes for Biomedical
Research
Basel, Switzerland

xvi

Contributors
Claudia Stäubert, Ph.D.
Molecular Biochemistry
Institute of Biochemistry
University of Leipzig
Leipzig, Germany
Roger K. Sunahara, Ph.D.
Associate Professor
Department of Pharmacology
University of Michigan
Ann Arbor, MI
Gema Tarrasón, Ph.D.
Research and Developement Center
Almirall
Barcelona, Spain
Erin Trinquet, Ph.D.
Cisbio Bioassays
Parc technologique Marcel Boiteux
Bagnols/Cèze, France
José Vázquez-Prado, Ph.D.
Professor
Department of Pharmacology
Center for Research and Advanced
Studies
National Polytechnic Institute
Mexico

Ivan Toma Vranesic, Ph.D.
Neuropsychiatry
Neuroscience Research
Novartis Institutes for BioMedical
Research
Basel, Switzerland
Val J. Watts, Ph.D.
Department of Medicinal Chemistry
and Molecular Pharmacology
School of Pharmacy and
Pharmaceutical Sciences
Purdue University
West Lafayette, IN
Michael Wolff, Ph.D.
Department of Lead Discovery
Boehringer Ingelheim Pharma GmbH
& Co. KG
Biberach, Germany
Xiao Jie Yao
Research Associate
Department of Molecular and
Cellular Physiology
Stanford University
Stanford, CA


Introduction
Sandra Siehler and Graeme Milligan

This book provides a comprehensive overview of recent discoveries and the
­c urrent understanding in the G protein-coupled receptor (GPCR) field.
A plethora of distinct GPCRs exist on the cell surface of every cell type and
generate signals inside cells to regulate key physiological events. The human
genome contains between 720 and 800 GPCRs with specific tissue and subcellular expression profiles. Chapter 1 of this volume illustrates the evolutionary history of GPCRs based on genomic information available from distinct ­species and
ancient genomic information. Many GPCRs are involved in olfactory/­sensory
mechanisms. Three hundred sixty-seven non-sensory human GPCRs are known
or predicted to be activated by native ligands; endogenous ligands for 224 human
GPCRs are described currently, but remain to be identified for 143 orphan receptors. Three hundred sixty-seven ligand-activated non-sensory GPCRs consist of
284 class A (rhodopsin-like) receptors, 50 class B (secretin-like) receptors, 17 class
C (metabotropic receptor-like) receptors, and 11 belong to the atypical class of
frizzled-/smoothened receptors. Polymorphisms (e.g., of β adrenoceptors, see
Chapter 15) and alternative splicing (e.g., of metabotropic glutamate receptors,
see Chapter 16) further increase the variety of GPCR proteins. Posttranslational
modifications such as N-linked glycosylation or carboxyterminal palmitoylation
can influence their function.
GPCRs are integral membrane proteins containing an extracellular amino
terminus of widely varying length, seven transmembrane α-helical stretches,
and an intracellular carboxy terminus. The molecular understanding of GPCRs
developed with the cloning of the β 2 adrenoceptor in 1986 and appreciation
that it was related to the photon receptor rhodopsin. The majority of signaling
events originate at the inner face of the plasma membrane and involve transactivation of one or more members of the four G protein families (Gs, Gi/o, Gq/11,
G12/13), which link GPCRs to effector cascades. Chapter 7 explains functions of
­mammalian G proteins elucidated using subunit- and tissue-specific gene targeting. Besides effector cascades involving G proteins, non-G protein-mediated signaling has been described for various GPCRs. Moreover, the activity of G proteins
can be regulated by non-GPCR proteins such as receptor tyrosine kinases. The
activity of GPCRs is further modulated by cellular signals in an auto- and transregulatory fashion. GPCRs form intra- and juxtamembrane signaling complexes
1


2

Siehler and Milligan
comprising not only G proteins, but also other GPCRs, ion channels, membrane
and cytosolic kinases and other enzymes, G protein-modulatory proteins, and
interact with elements of the cell cytoskeleton. Chapters 3–6 describe homoand hetero-oligomerization features of GPCRs including receptors for glutamate,
GABA B, dopamine, and chemokines. Dopamine receptors can hetero-dimerize
not only with other subtypes in the same receptor family, but also with lessrelated GPCR members and ion channels such as NMDA or GABA A receptors. For
class C receptors, which contain a large extracellular domain, oligomerization is
mandatory for receptor function. For other GPCRs, oligomerization may result
in altered and/or novel ligand pharmacology. Methods applied to measure GPCR
complexes and oligomer signaling comprise GPCR-Gα protein fusion constructs
containing either a mutated receptor or Gα mutant, and time-resolved fluorescence resonance energy transfer (TR-FRET).
Downstream of the cellular plasma membrane, the complexity of intracellular
communication controlled by GPCRs increases dramatically. Ligand-activated
GPCRs often internalize, which mostly causes desensitization of signaling
events, although both prolonged signaling and even signaling initiated following ­receptor internalization have been described. Receptor hetero-oligomers can
co-internalize, and activation and internalization of one partner can therefore
silence the other interaction partner. Chapters 8–11 describe key signaling features of GPCRs better understood because of significant recent advancements.
These include understanding of kinetics of receptor activation and signaling
events studied using FRET and bioluminescent RET (BRET). Multiple related
proteins control GPCR-mediated cell signaling processes. For example four
RhoGTPase nucleotide exchange factors (Rho-GEFs) link G12/13 to pathways controlling, for example, contractile complexes of the cytoskeleton, whereas nine
mammalian adenylyl cyclases (ACs) are regulated by GPCRs in a receptor- and
tissue-specific manner. These enzymes are integral membrane proteins directly
regulated by Gs and Gi/o proteins, although Gq/11-coupled GPCRs also influence
AC activities via calcium and protein kinase C, and G12/13 proteins were recently
found to regulate AC activity as well. Arrestins are known to bind to agoniststimulated phosphorylated GPCRs and promote endocytosis. Novel functions
of arrestins include interactions with non-GPCR receptors or direct interaction
with signaling ­proteins including, for example, the ERK MAP kinases. Modern
assay technologies to assess GPCR signaling and ligand pharmacology are

described in Chapter 12. Multiplexing subcellular readouts using high content
screening allows the simultaneous capture of multiple signals, in both temporal
and spatial fashion. The pharmacological complexity of orthosteric and allosteric GPCR ligands in the context of both receptor-G protein complexes and activation state models, is illustrated in Chapters 13 and 14. Functional selectivity of
GPCR ligands due to receptor allosterism toward intracellular effector pathways
contributes to the complex pharmacological nature.
Dysregulated ligand concentration, GPCR protein level, coupling, and/or
­signaling are implicated in and often causative for many pathophysiological
conditions including central nervous system (CNS) disorders, cardiovascular and


Introduction
metabolic diseases, respiratory malfunctions, gastrointestinal disorders, immune
diseases, cancer, musculoskeletal pathologies, and eye illnesses. Targeting of
GPCRs is hence widely utilized for therapeutic intervention using small molecule weight ligands and, increasingly, therapeutic antibodies. About 30 percent
of marketed drugs target GPCRs. Pathophysiological aspects of β-adrenoceptors
in cardiovascular and respiratory diseases, of metabotropic glutamate receptors
in CNS disorders, of sphingosine 1-phosphate (S1P) receptors in the immune system, and of Wnt/Frizzled receptors in osteoporosis are described in Chapters 15–
18. Frizzled receptors possess a GPCR-like architecture, however, their coupling
to G proteins remains controversial. Drugability of GPCRs is generally high since
ligand binding pockets are found in the extracellular facing segments of GPCRs,
meaning that cell permeability is not a requirement. Exceptions exist regarding
drugability (e.g., for many chemokine receptors as elaborated in Chapter 6), and
a few unique examples for intracellular binding sites for drugs have emerged.
Despite the high drugability and importance of this target class, drug discovery technologies for GPCRs remained limited for a long time when compared to
other target classes such as kinases. Integrated lead finding strategies for ­cytosolic
kinases and intracellular parts of membrane kinases comprise biochemical, biophysical, structural, and cellular approaches, which enable a detailed understanding of mechanisms of actions of compounds. Lead finding for GPCRs, on
the other hand, was so far solely based on cellular approaches using recombinant
and native systems, and either intact cells or cell membranes. Reasons included
the challenges of purifying GPCRs in sufficient quantities, the stability of these
as isolated membrane proteins, and the lack of structural knowledge. All three

issues have been tackled, and recent successes become prominent. Expression,
solubilization, and purification methods of GPCRs using eukaryotic insect or
mammalian cells, prokaryotic bacterial cells, or in vitro expression systems
have been significantly improved. New methods are being applied to stabilize
­isolated membrane proteins in semi-native lipid environments like, for example,
recombinant high density lipoprotein (rHDL)-membrane discs. Functional studies of isolated GPCR-G protein complexes reconstituted in rHDLs are described
in Chapter 2 and deliver novel insights that cannot be obtained from cellular
systems.
The first crystal structures of a non-rhodopsin GPCR were published for the
human β 2 adrenoceptor in 2007 using either a T4 lysozyme fusion replacing
the third intracellular loop or a Fab antibody fragment binding to the third
intracellular loop, and with the receptor in complex with an inverse agonist
and stabilized in a lipid environment. The T4 lysozyme approach also facilitated
the identification of the crystal structure of the human A 2A adenosine receptor
in complex with an antagonist one year later. A novel approach for receptor
stabilization uses targeted amino acid mutations in order to thermostabilize the
receptor, and enabled crystal structure determination of the turkey β 1 adrenoceptor in complex with an antagonist in 2008. All GPCR structures available
to date are derived from class A GPCRs and resemble inactive receptor conformations. More GPCR structures are expected to become public soon and will

3


4

Siehler and Milligan
enable structural drug discovery approaches including fragment-based screening and ligand co-crystallizations. Stabilized purified GPCRs reconstituted in a
lipid environment facilitate not only biochemical, but also biophysical methods
such as surface plasmon resonance (SPR) or back-scattering interferometry (BSI)
measurements. These novel advances allow confirmation of direct binding of a
ligand – whether of competitive or allosteric nature – to a GPCR, and to directly

study mechanisms of actions of ligands and G protein activation to determine
pharmacological textures of GPCRs. This will boost further understanding of
GPCR biology, biomedical research, and ultimately translation of new therapies
into the clinic.
We thank all the authors for their comprehensive and professional contributions, and Amanda Smith, Katherine Tengco, Joy Mizan, Allan Ross and Monica
Finley from Cambridge University Press and Newgen for assistance, final editing
and formatting of the chapters, and printing of the book. From planning the
outline of the book to final printing, it has been a rewarding experience. We
hope the book will be exciting to read for both newcomers and professionals in
the GPCR field.


Part I: Advances in GPCR Protein Research

1 The evolution of the repertoire and structure
of G protein-coupled receptors
Torsten Schöneberg, Kristin Schröck, Claudia Stäubert,
and Andreas Russ

Introduction

5

Gain and loss of GPCR s

6

The origin of GPCR genes
Expansion of GPCR genes
The loss of GPCR functions

6
9
11

Structural evolution of GPCR s

13

Structural shaping of the core of GPCRs
13
Structural evolution of intra- and extracellular domains of GPCRs 15
Coevolution of GPCRs and their ligands/associated factors
17
Selection on GPCR genes

17

Genetic signatures of selection
Selection of genomic regions containing GPCR genes
Selection of individual GPCRs

18
19
21

In vitro evolution of GPCRs

22

Suggested reading

24

Introduction
With the advent of large, publicly available genomic data sets and the completion of numerous invertebrate and vertebrate genome sequences, there has
been much effort to identify, count, and categorize G protein-coupled receptor
(GPCR) genes.1,2 This valuable source of large-scale genomic information also
initiated attempts to identify the origin(s) and to follow the evolutionary history
of these receptor genes and families. Since all recent genomes have been shaped
by selective forces over millions of years, understanding structure-function relationships and the physiological relevance of individual GPCRs makes sense only
in the light of evolution. Until recently, the study of natural selection has largely
been restricted to comparing individual candidate genes to theoretical expectations. Genome-wide sequence and single nucleotide polymorphism (SNP) data
now bring fundamental new tools to the study of natural selection. There has
been much success in producing lists of candidate genes, which have potentially
been under selection in vertebrate species or in specific human populations.3–9
5


6

Schöneberg, Schröck, Stäubert, and Russ
Less effort has gone into a detailed characterization of the candidate genes,
which comprises the elucidation of functional differences between selected and
nonselected alleles, as well as their phenotypic consequences, and ultimately
the identification of the nature of the selective force that produced the footprint
of selection. Such further characterization creates a profound understanding of
the role and consequences of selection in shaping genetic variation, thus verifying the signature of selection obtained from genome-wide data. Since GPCRs
control almost every physiological process, several receptor variants are involved
in adaptation to environmental changes and niches. Consistently, genomic

scans for signatures of selection revealed a number of such loci containing GPCR
genes. This chapter sheds light on the origin(s), rise, and fall of GPCR genes and
functions, and focuses on recent advantages in elucidating selective ­mechanisms
(still) driving this process.

Gain and loss of GPCRs
The origin of GPCR genes
The GPCR superfamily comprises at least five structurally distinct families/
subfamilies (GRAFS classification) named:  Glutamate, Rhodopsin, Adhesion,
Frizzled/Taste2, and Secretin receptor families.2 Because there is very little
sequence homology among the five families, the evolutionary origin of GPCRs
and their ancestry remain a matter of debate.
The evolutionary success of the GPCR superfamily is reflected by both its
presence in almost every eukaryotic organism and by its abundance in mammals, but proteins that display a seven transmembrane (7TM) topology are
already present in prokaryotes. The prokaryotic light-sensitive 7TM proteins,
such as proteo-, halo-, and bacteriorhodopsins, facilitate light energy harvesting in the oceans, coupled to the carbon cycle via a non-chlorophyll-based
pathway. Further, there are prokaryotic sensory rhodopsins for phototaxis in
halobacteria, which control the cell’s swimming behavior in response to light.
As in ­rhodopsins of bilateral animals, prokaryotic rhodopsins contain retinal
­covalently bound to 7TM. Moreover, 7TM proteins with a structural ­similarity
to prokaryotic ­sensory rhodopsins are found in eukaryotes.10,11 These structural
and functional features shared by pro- and eukaryotic rhodopsins suggest a
common ancestry. However, despite these similarities, sequence comparisons
provide no convincing evidence of an evolutionary linkage between prokaryotic
rhodopsins and eukaryotic G protein-coupled rhodopsins.12 Therefore, the question about the evolutionary origin of eukaryotic GPCRs remains open. Currently,
all our insights into their evolutionary history are based on the analysis of the
GPCR repertoire of distantly related extant species.
Structural and functional data clearly show that G-protein signaling via
GPCRs is present in yeast/fungi,13 plants,14 and primitive unicellular eukaryotes, such as the slime mold Dictyostelium discoideum.15 This receptor-signaling

Evolution of G protein-coupled receptors

G

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t
R am
ho at
e
Ad dop -R
h si
Fr esio n-R
iz n
Se zled -R
cr -R
et
in
-R

7

rodents

mammals

primates

amniotes

birds

tetrapods

reptiles

e
v rtebrate s

amphibians

chordates

zebrafis h

ray finned fishes

fugu
amphioxus

deuterostomes

tunicats
bilater ia

sea urchins
molluscs
nematodes

protostomia

arthropods
cnidaria
sponges
fungi
amoebo zoa
plants

120

70

0

6 0

50 0

4

0

30

0

2

0

10 0

0

Myr ag r

Figure 1-1:  Evolutionary occurrence of the different GPCR families in eukaryotes.
GPCRs and their signal transduction probably evolved ~1.2 billion years ago, before plant/fungi/
animal split. Genomes of extant plants and fungi usually contain less than ten GPCR genes.
The first rhodopsin-like GPCRs, which compose the main GPCR family in vertebrates, appeared
~570–700 Myr ago. Expansion of rhodopsin-like GPCRs started ~500 Myr ago, giving rise to
over 1,000 members in some mammalian genomes. The relationships of some major lineages
are controversially discussed, hence a very simplified phylogenetic tree of eukaryotes together
with a raw time scale are shown. There is some sequence relation between adhesion receptors
and GPCRs in plants and fungi, but key features of adhesion receptors, such as the GPS domain
in the N terminus, are not present in plant and fungi GPCRs (23,24).

complex must have evolved before the plant/fungi/animal split about 1.2 billion
years ago (Figure 1.1). Signal transduction through G proteins is the most prominent and eponymous feature of GPCRs. However, one has to consider that GPCRs
signal not only via G proteins but also via alternative, non-G-protein-linked signaling pathways.16 Therefore, it remains open whether G proteins were involved
in GPCR signaling from the very evolutionary beginning or if the prototypes of
what we now call GPCRs initially fulfilled other functions.
In contrast to GPCR signaling as such, it is more difficult to ascertain the deep
evolutionary origin of the five prototypical receptor structures we know today.
Genomic data and functional evidence indicate that glutamate-receptor-like
receptors are present in D. discoideum17,18 and the sponge Geodia cydonium,19,20
which diverged more than 600 million years (Myr) ago (Figure 1.1). The ligandbinding domain of glutamate-receptor-like receptors, also known as the “Venus
fly trap” domain, is distantly related to the prokaryotic periplasmic-binding proteins involved in amino acid and nutrient transport in bacteria.21 Free amino
acids act at glutamate-like receptors as either direct-acting orthosteric agonists or allosteric modulators of receptor activity. In contrast to Dictyostelium