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

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

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 pharma cology 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, GABAB, dopamine, and chemokines Several chapters are devoted to the key signaling features of GPCRs The authors take time to detail the importance of the pathophysiologi-cal 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 peutics and the British Pharmacological Society

Thera-Dr Graeme Milligan is Professor of Molecular Pharmacology at the University of Glasgow He is actively involved in numerous associa-tions, such as the Biochemical Society and the British Pharmacologi-cal Society Dr Milligan was awarded the Ariens Award for Pharmacol-ogy from the Dutch Pharmacological Society in 2006

G Protein-Coupled Receptors

struCture, siGnalinG, and PhysioloGy

Sandra SiehlerNovartis Institutes for BioMedical ResearchGraeme Milligan

University of GlasgowEdited by

São Paulo, Delhi, Dubai, Tokyo

Cambridge University Press

The Edinburgh Building, Cambridge CB2 8RU, UK

First published in print format

ISBN-13 978-0-521-11208-6

ISBN-13 978-0-511-90991-7

© Cambridge University Press 2011

2010

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Published in the United States of America by Cambridge University Press, New York

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eBook (NetLibrary) Hardback

Part i: advanCes in GPCr Protein researCh

1 The evolution of the repertoire and structure

Torsten Schöneberg, Kristin Schröck, Claudia Stäubert,

and Andreas Russ

2 Functional studies of isolated GPCR-G protein complexes

in the membrane bilayer of lipoprotein particles 32

Adam J Kuszak, Xiao Jie Yao, Sören G F Rasmussen,

Brian K Kobilka, and Roger K Sunahara

Part ii: oliGomerization of GPCrs

3 GPCR-G protein fusions: Use in functional dimerization analysis 53

Graeme Milligan

4 Time-resolved FRET approaches to study GPCR complexes 67

Jean Phillipe Pin, Damien Maurel, Laetitia Comps-Agrar,

Carine Monnier, Marie-Laure Rives, Etienne Doumazane,

Philippe Rondard, Thierry Durroux, Laurent Prézeau, and Erin Trinquet

5 Signaling of dopamine receptor homo- and heterooligomers 90

Ahmed Hasbi, Brian F O’Dowd, and Susan R George

6 Functional consequences of chemokine receptor dimerization 111

Mario Mellado, Carlos Martínez-A., and José Miguel Rodríguez-Frade

Part iii: GPCr siGnalinG features

7 G protein functions identified using genetic mouse models 125

Stefan Offermanns

8 Kinetics of GPCR, G protein, and effector activation 145

Peter Hein

9 RGS-RhoGEFs and other RGS multidomain proteins as effector molecules in GPCR-dependent and GPCR-independent cell signaling 159

José Vázquez-Prado and J Silvio Gutkind

10 Adenylyl cyclase isoform-specific signaling of GPCRs 189

Karin F K Ejendal, Julie A Przybyla, and Val J Watts

11 G protein-independent and β arrestin-dependent GPCR signaling 217

Zhongzhen Nie and Yehia Daaka

12 Assays to read GPCR modulation and signaling 231

Ralf Heilker and Michael Wolff

Part iv: liGand PharmaColoGy of GPCrs

13 Assessing allosteric ligand-receptor interactions 247

Ivan Toma Vranesic and Daniel Hoyer

Terry Kenakin

Part v: PhysioloGiCal funCtions and druG tarGetinG of GPCrs

15 β-Adrenoceptors in cardiovascular and respiratory diseases 287

Michele Ciccarelli, J Kurt Chuprun, and Walter J Koch

16 Role of metabotropic glutamate receptors in CNS disorders 321

Richard M O’Connor and John F Cryan

17 S1P receptor agonists, a novel generation of immunosuppressants 380

Rosa López Almagro, Gema Tarrasón, and Nuria Godessart

18 Wnt/Frizzled receptor signaling in osteoporosis 398

Georges Rawadi

Color plates follow page 32

1.1 Evolutionary occurrence of the different GPCR families

1.2 Signatures of positive selection during human evolution

1.3 Phylogenetic trees of primate TAAR3 and TAAR4 subtypes 23

2.1 Illustration of reconstituted HDL particles 34

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)

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

3.1 Reconstitution of function by co-expression of a pair of

inactive dopamine D2-Gαo1 fusion proteins: unaltered

4.1 Conditions for energy transfer to occur between two fluorophores 70

4.2 Emission spectra for CFP-YFP FRET (A), luciferase-YFP BRET (B),

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 Eu3+ cryptates TBP (black)

4.6 Testing the proximity between cell surface proteins using anti-tag

antibodies conjugated with TR-FRET compatible fluorophores 77

4.7 TR-FRET between antibody-labeled GABAB subunits measured

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

4.11 GABAB oligomers revealed with snap-tag-TR-FRET 85

8.1 Sample FRET traces for early signaling processes 151

8.2 Kinetics of different steps of signal transduction 154

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 161

9.2 In migrating fibroblasts, Rho activation is important to promote

the removal of focal adhesions at the trailing edge in response to

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

10.1 Adenylyl cyclase is a membrane-bound enzyme that contains

an intracellular N-terminus, followed by a membrane-bound

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

12.1 HCS to monitor GPCR ligand binding, internalization, and

12.2 HCS to monitor GPCR-modulated second messenger responses

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

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

14.1 Two opposing views of receptor activation 271

14.2 Vectorial flow of allosteric energy 276

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

15.3 Inotropic support in patients with the Arg389Gly-;β1AR polymorphism 302

16.1 Schematic representation of the general structure of metabotropic

16.2 Schematic representation of the metabotropic glutamate receptor

16.3 General synaptic localization of the different metabotropic

17.2 G proteins coupled to the S1P receptors and downstream

18.1 Current general view of Frizzled receptor dependent Wnt signaling 399

18.2 Schematic representation of frizzled and secreted frizzled

18.4 Canonical Wnt signaling effect on bone metabolism 405

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

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

16.1 Pharmacological Evidence Implicating Group I mGLuRs in Anxiety 328

16.2 Pharmacological Evidence Implicating Group I mGLuRs

16.8 Pharmacological Evidence Implicating Group II mGLuRs in Anxiety 345

16.9 Pharmacological Evidence Implicating Group II mGLuRs

16.12 Pharmacological Evidence Implicating Group II mGLuRs in Epilepsy 354

16.13 Pharmacological Evidence Implicating Group II mGLuRs

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

Rosa López Almagro, Ph.D

Research and Development CenterAlmirall

Barcelona, Spain

J Kurt Chuprun, Ph.D.

Center for Translational MedicineThomas Jefferson UniversityPhiladelphia, PA

Michele Ciccarelli, MD

Center for Translational MedicineThomas Jefferson UniversityPhiladelphia, PA

Laetitia Comps-Agrar, Ph.D.

Department of Molecular Pharmacology

Institut de Génomique FonctionnelleMontpellier, France

John F Cryan, Ph.D

Senior LecturerSchool of PharmacyDepartment of Pharmacology and Therapeutics

University College CorkCork, Ireland

Yehia Daaka, Ph.D.

Department of Urology

UF Prostate Disease CenterUniversity of FloridaCollege of MedicineGainesville, 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 AssociateDepartment of Medicinal Chemistry and Molecular PharmacologySchool of Pharmacy and Pharmaceutical SciencesPurdue University

West Lafayette, IN

Susan R George

ProfessorDepartment of Pharmacology and Toxicology

University of TorontoToronto, Ontario, Canada

Nuria Godessart, Ph.D.

Head of Autoimmunity DepartmentAlmirall Laboratories

Llobregat, Spain

J Silvio Gutkind, Ph.D.

Oral & Pharyngeal Cancer Branch

National Institute of Dental and

Department of Molecular and Cellular

Pharmacology and Psychiatry

University of California at San

Department of Biological Reagents

and Assay Development

Adam J Kuszak

Department of PharmacologyUniversity of MichiganAnn Arbor, MI

Carlos Martínez-A., Ph.D.

ProfessorDepartment of Immunology and Oncology

Centro Nacional de BiotecnologiaMadrid, Spain

Damien Maurel, Ph.D.

ScientistEcole Polytechnique Fédérale de Lausane

Lausane, Switzerland

Mario Mellado, Ph.D.

Research ScientistDepartment of Immunology and Oncology

Centro Nacional de BiotecnologiaMadrid, Spain

Graeme Milligan, Ph.D.

ProfessorNeuroscience and Molecular Pharmacology

University of GlasgowScotland

Carine Monnier, Ph.D.

Department of Molecular Pharmacology

Institut de Génomique FonctionnelleMontpellier, France

Zhongzhen Nie, Ph.D.

Department of Urology

UF Prostate Disease CenterUniversity of FloridaCollege of MedicineGainesville, FL

Department of Medicinal Chemistry

and Molecular Pharmacology

School of Pharmacy and

Centro Nacional de BiotecnologiaMadrid, Spain

Philippe Rondard, Ph.D.

Department of Molecular Pharmacology

Institut de Génomique Fonctionnelle

Montpellier, France

Andreas Russ, Ph.D.

Department of BiochemistryUniversity of Oxford

Oxford, United Kingdom

Torsten Schöneberg, Ph.D.

Molecular BiochemistryInstitute of BiochemistryUniversity of LeipzigLeipzig, Germany

Kristin Schröck, Ph.D.

Molecular BiochemistryInstitute of BiochemistryUniversity of LeipzigLeipzig, Germany

Sandra Siehler, Ph.D

Research Investigator IICenter for Proteomic ChemistryNovartis Institutes for Biomedical Research

Basel, Switzerland

Basel, Switzerland

Val J Watts, Ph.D.

Department of Medicinal Chemistry and Molecular PharmacologySchool of Pharmacy and Pharmaceutical SciencesPurdue University

Xiao Jie Yao

Research AssociateDepartment of Molecular and Cellular Physiology

Stanford UniversityStanford, CA

Sandra Siehler and Graeme Milligan

This book provides a comprehensive overview of recent discoveries and the current 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 subcellu-lar expression profiles Chapter 1 of this volume illustrates the evolutionary his-tory 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 recep-tors 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

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 transac-

G12/13), which link GPCRs to effector cascades Chapter 7 explains functions of mammalian G proteins elucidated using subunit- and tissue-specific gene target-ing Besides effector cascades involving G proteins, non-G protein-mediated sig-naling 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 trans-regulatory fashion GPCRs form intra- and juxtamembrane signaling complexes

comprising not only G proteins, but also other GPCRs, ion channels, membrane and cytosolic kinases and other enzymes, G protein-modulatory proteins, and

and hetero-oligomerization features of GPCRs including receptors for glutamate,

not only with other subtypes in the same receptor family, but also with

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 fluores-cence 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 follow-ing receptor internalization have been described Receptor hetero-oligomers can co-internalize, and activation and internalization of one partner can therefore

fea-tures 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

con-trolling, 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

found to regulate AC activity as well Arrestins are known to bind to stimulated phosphorylated GPCRs and promote endocytosis Novel functions

agonist-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

screening allows the simultaneous capture of multiple signals, in both temporal and spatial fashion The pharmacological complexity of orthosteric and allos-teric GPCR ligands in the context of both receptor-G protein complexes and acti-

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

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

mol-ecule 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

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

a few unique examples for intracellular binding sites for drugs have emerged

Despite the high drugability and importance of this target class, drug

discov-ery 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,

bio-physical, structural, and cellular approaches, which enable a detailed

under-standing 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

stud-ies 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

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

in complex with an antagonist one year later A novel approach for receptor

stabilization uses targeted amino acid mutations in order to thermostabilize the

adreno-ceptor in complex with an antagonist in 2008 All GPCR structures available

to date are derived from class A GPCRs and resemble inactive receptor

confor-mations More GPCR structures are expected to become public soon and will

enable structural drug discovery approaches including fragment-based ing 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.

screen-We thank all the authors for their comprehensive and professional tions, 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

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 of genomic regions containing GPCR genes 19

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 tionships and the physiological relevance of individual GPCRs makes sense only

rela-in the light of evolution Until recently, the study of natural selection has largely been restricted to comparing individual candidate genes to theoretical expecta-tions 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

1 the evolution of the repertoire and structure

of G protein-coupled receptors Torsten Schöneberg, Kristin Schröck, Claudia Stäubert, and Andreas 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 veri-fying 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 genesThe GPCR superfamily comprises at least five structurally distinct families/

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 mam-mals, 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 harvest-ing 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

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

ques-tion about the evoluques-tionary 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

GPCRs is present in yeast/fungi,13 plants,14 and primitive unicellular

complex must have evolved before the plant/fungi/animal split about 1.2 billion

promi-nent 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 sig-

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

receptors are present in D discoideum17 , 18 and the sponge Geodia cydonium,19 , 20

ligand-binding domain of glutamate-receptor-like receptors, also known as the “Venus fly trap” domain, is distantly related to the prokaryotic periplasmic-binding pro-

acids act at glutamate-like receptors as either direct-acting orthosteric

ago-nists or allosteric modulators of receptor activity In contrast to Dictyostelium

plants amoebo zoa fungi sponges cnidaria arthropods nematodes molluscs sea urchins tunicats amphioxus fugu zebrafis h amphibians reptiles birds primates rodents mammals

amniotes tetrapods

ve rtebrate s chordates ray finned fishes

deuterostomes bilater ia

protostomia

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)

glutamate-like receptors, the sponge receptor did show weak activation by millimolar concentrations of glutamate This suggests that glutamate activa-tion of glutamate-like receptors may have arisen early in metazoan evolution, with the high glutamate affinity seen in the resurrected ancestral receptor fully

glutamate-receptor-like proteins in Dictyostelium suggests that a prototypical

receptor structure might predate the origin of metazoa

adhesion-GPCR subfamily were present before the onset of metazoan evolution Sequences

however, the homology is modest and mainly based on alignment of tive 7TM regions Clear evidence for the ancient origin of the adhesion-GPCR subfamily comes from the analysis of the genome of a single-cell eukaryote

considered to be the closest relative to metazoans, the choanoflagellate Monosiga brevicollis.25 , 26 The Monosiga genome encodes proteins with the GPS (GPCR pro-

pathway Thus, like glutamate-receptor-like proteins, the signaling module used

in adhesion-GPCRs might predate the origin of metazoa

Frizzled-like receptors are identified in sponges,27 , 28 jellyfishes (Cnidaria),29 , 30

A recent phylogenetic study suggests that secretin-like GPCRs descended from the

(corticotropin-releasing factor receptor, calcitonin/calcitonin gene-related peptide

Monosiga brevicollis, and Dictyostelium This suggests an evolutionary age of more

than 550 Myr, concurrent with the evolution of bilaterial animals

protos-tome Bilateria (insects, mollusks, nematodes, vertebrates, etc.) and in jellyfishes

(Cnidaria), which suggests that rhodopsin-like receptors appeared ~570–700 Myr

ago34 – 37 (Figure 1.1) Within the rhodopsin-like GPCRs, glycoprotein hormone receptors and serotonin receptors appear to be among the oldest, as suggested

Rhodopsin is the name-giving GPCR in this family Recent reports suggested that opsins diverged at least before the deuterostome-protostome split about 550 Myr

indicat-ing that prototypical opsins may have existed before divergence of Cnidaria

G-protein signaling evolved Given that G-protein coupling to 7TM proteins evolved before the plant/fungi/animal split (about 1.2 billion years ago), and that the first rhodopsin-like receptors appeared early in metazoan evolution, one must consider the retinal-based photosensory system to be a “reinvention” (convergent evolutionary model)

expansion of GPcr genesThe recent completion of many vertebrate and nonvertebrate genome projects has enabled us to obtain a complete inventory of GPCRs in these species and,

by a comparative genomics approach, to analyze the evolution of the GPCR subfamilies Comparison of the repertoires of GPCRs in insects (fly, mosquito, beetle) and protochordate (Ciona) to that in vertebrates (mammals, birds, fish) reveals a high level of orthology This indicates that nonvertebrates contain the

num-ber of GPCRs in most sequences of nonvertebrate genomes (exceptions are the chemokine receptors in worms) is substantially lower than that in vertebrate

most abundant GPCR family in vertebrates when compared to nonvertebrates

Most modern rhodopsin-like GPCR subfamilies expanded in the very early tebrate evolution Still, many nonvertebrate GPCR clusters evolved about 500 Myr ago during a time called the “Cambrian Explosion.” There are interesting theories about what triggered the enormous gain of species, functionalities, and genes It was proposed that vision triggered the Cambrian Explosion by creating a new world of organismal interactions, the evolutionary consequence

ver-of which was a race in the invention ver-of attracting, attacking, and defending

including several genes of the phototransduction, were traced to the very early vertebrate evolution

Processes of creating new genes using preexisting genes as raw materials are well characterized, such as exon shuffling, gene duplication, retroposition, gene fusion, and fission GPCR gene expansion in vertebrates is mainly the result

of a combination of species-specific gene duplications and gene or genome duplication events Two rounds of whole-genome duplications are thought to have played an important role in the establishment of gene repertoires in verte-

urochordate and cephalochordate lineages but before the radiation of extant

multiplication and may trigger evolutionary adaptation One copy or even both members of a gene pair may mutate and acquire unique functionality without risking the fitness of the organism, which is ensured by the homolog Further, gene duplications often retained overlapping expression patterns and preserved partial-to-complete redundancy consistent with a role in boosting robustness

or gene doses On the other hand, if not advantageous, continuous lation of mutations (neutral evolution) will eliminate one of the duplicated genes As for other genes, disadvantageous mutations in GPCRs are removed from a population through purifying selection Therefore, many evolutionarily old GPCR genes, including the rhodopsins, display strong features of purifying

Current evidence suggests that an additional whole-genome duplication occurred in the teleost lineage after it split from the tetrapod lineage, and that

Support of these findings comes from sequence analysis of coelacanth, one of the nearest living relatives of tetrapods The two modern coelacanth species that

are known, Latimeria chalumnae and Latimeria menadoensis, are remarkably

simi-lar to their fossil relatives, showing little morphological change over 360 Myr Genomic sequence analyses show no evidence of whole-genome duplication, consistent with the explanation that the coelacanth genome has not experienced

contribute to GPCR expansion in tetrapode evolution However, polyploidy is common in fishes and has been determined in sturgeons up to ploidy levels of

been suggested, for example, trace amine-associated (TAAR) and (purinergic)

Whole-chromosome duplications have been made responsible for parallel duplications of more related GPCR For example, parts of chromosome 4 and 5 show a very similar order of paralog receptor genes It is assumed that a chro-mosomal duplication gave rise to dopaminergic receptor paralogs, DRD5 and DRD1, and adrenoceptor paralogs, ADRA2C and ADRA1, at chromosomes 4 and

ver-tebrate genomes and are often arranged in a tandem-like fashion The numbers

of functional receptor genes and pseudogenes of these GPCR subfamilies vary enormously among the genomes of different vertebrate species Much of the vari-ation in these receptor repertoires can probably be explained by the adaptation

of species to different environments For example, the platypus, a semiaquatic monotreme, has the largest repertoire of vomeronasal receptors in all vertebrates surveyed to date, with more than 300 intact genes and 600 pseudogenes in this

variety is generated by genomic drift, which probably also has an important role

The molecular mechanisms of gene amplification and genomic clustering are

genome replication due to an unequal sister strand exchange, producing two adjacent identical copies of a region (the amplicon) that can undergo homologous recombination Another mechanism, termed circle-excision and reinsertion, involves creation of a circular DNA molecule and its subsequent recombination with another DNA molecule to form the duplication Alternatively, a rolling-circle mechanism may account for instances of very rapid gene amplification There

is evidence that similar mechanisms are responsible for gene amplifications in

cluster formation, for example, are not yet known

the loss of GPcr functionsCurrently, most genome analyses rely on sequence information from extant species As more than 99 percent of all species that ever lived on earth are extinct, most of the information about receptor repertoires and their evolution-ary history cannot be studied However, recent advances in DNA extraction and

it possible to retrieve substantial amounts of ancient DNA sequences and even

sequences of extant organisms contain valuable information about past tions and gene evolution For example, pseudogenes are considered as genomic fossils and increasingly attract attention in GPCR research In addition, selection

func-of favorable gene variants had left footprints that can be identified by suitable bioinformatic methods (see further in this chapter)

Pseudogenes are inheritable and characterized by a homology to a known gene

are released from selective pressure Therefore, compared to functional genes, pseudogenes, if old enough, display a ratio of nonsynonymous to synonymous

mutations Depending on the mechanism by which they evolved, the ity of mammalian pseudogenes can be classified as duplicated pseudogenes or retrotransposed pseudogenes (also called processed pseudogenes) The latter are generated by the reverse-transcription of mRNAs, followed by genomic inte-gration The human gonadotropin releasing hormone GnRH type II receptor homolog is one well-characterized example of GPCR pseudogenization caused

unequal crossing-over (see the subchapter on expansion of GPCR genes earlier

in this chapter) Thus, they often retain the original exon–intron structures of the parental genes However, duplication or retrotransposition events are not always mechanistically necessary for pseudogenization Inactivating mutations

in former functional genes can cause loss of functionality, as demonstrated in

several million years that obvious signatures of inactivation (premature stop codon, frameshift) become fixated in the coding sequence In primate TAAR, for example, rough estimates suggest that 7–10 Myr are required to obtain and fix-ate at least one of such obvious signatures of receptor inactivation (unpublished data) On the other hand, signatures of the original sequence will gradually disappear over time As a consequence, a pseudogene may escape present-day detection depending on the date and mechanism of its pseudogenization Rough estimates suggest that signatures of genes can be detected for more than 80 Myr

of neutral evolution For example, the neuropeptide Y receptor type 6 (Y6R) is a pseudogene in all primate genomes investigated so far by an inactivating dele-tion mutation, which occurred in the common ancestor of primates Large dele-tions can remove informative sequences in an even shorter period, though, as

in the Y6R gene that disappeared in rats after the mouse/rat split 14–16 Myr ago

of GPCR pseudogenes within a genome can only be an estimate

sig-natures from only thirty nonolfactory rhodopsin-like GPCR pseudogenes have

Furthermore, some apparently intact human OR genes lack motifs that are very highly conserved in their mouse orthologs, suggesting that not all human OR genes with complete open reading frames encode functional OR proteins By contrast, in the mouse genome, only about 20 percent of the OR are pseudo-genes, giving mice more than three times as many intact OR genes as humans

specu-lated that the evolution of trichromatic color vision in hominoids and other Old World monkeys has relaxed the functional constraints for many taste, odorant,

Reduction or even a loss of GPCR function can also be restricted to distinct populations of a species Differences in environmental conditions may relax the constraint and promote neutral evolution of a gene formerly under purifying selection The melanocortin type 1 receptor (MC1R) gene nicely represents this situation The MC1R controls pigmentation of melanocyte and is, therefore, a central component in determining hair, skin, and coat color Numerous studies have shown that even single amino acid mutations in MC1R can have profound effects on pigment phenotypes in vertebrates In many cases, these changes

in MC1R function, and the resulting pigmentation pattern, are thought to be

MC1R gene, whereas the great MC1R sequence diversity in European and Asian

variants show reduction or loss of functionality leading to pale skin color and

the maintenance of dark pigmentation in Africans and pigment variation in non-African populations It is still a matter of debate whether the loss of MC1R functionality in vertebrates is always due to a loss of constraint as a result of adaptation to habitats in which protection from sunlight is less relevant like

Reduced pigmentation may increase fitness The reduced MC1R activity in some

promoted vitamin D synthesis in skin under the extreme climate conditions during Pleistocene ice ages Further, reduced pigmentation due to loss of MC1R function can provide an advantage against predation Illustrating examples are

Both animals have lighter-colored coats than their mainland counterparts, driven by natural selection for camouflage against the pale sand dunes These examples demonstrate that not only the gain but also the elimination of a GPCR

function (pseudogenization) may have an evolutionary advantage and may also trigger adaptation.

structural evolutIon of GPcrs

The growing number of crystal structures of GPCRs not only allows the

available structural information forms the basis to generate models of other

how-ever, still requires refinements based on experimental data from comparative studies, mutagenesis, cross-linking, and/or nuclear magnetic resonance (NMR) studies, and even the best data sets are only based on mutations at a few selected positions and limited in vitro functional analyses Therefore, we are often unable to predict mutational effects from such models, and experimental assess-ment is still required Mining evolutionary diversity as an additional source of structural/functional information may direct GPCR modeling and mutagenesis studies This approach has been successfully applied since the very early stages

of GPCR structure/function analysis to predict the approximate arrangements

a given GPCR that participate in ligand recognition and signal transduction, sequence analysis has to be focused on the comparison of receptor orthologs and paralogs The basic concept of this approach is that the structural diversity between orthologs is the result of a long evolutionary process characterized by

a continuous accumulation of mutations The maintenance of vital functions

in an organism strictly requires enough structural conservation to ensure the functionality of the receptor protein The significance of such multisequence-based analyses increases with the number of orthologs included Studies based

on large numbers of orthologs analyzed the conservation and relative

structural shaping of the core of GPcrsProteins with a 7TM-core architecture are already present in prokaryotes, but it

is still a matter of debate whether these prokaryotic archaeal rhodopsins are the blueprint of the eukaryotic GPCRs Clear evidence for direct phylogenetic rela-

found because of the general difficulties in establishing meaningful netic relations between pro- and eukaryotic sequences However, recent studies showed some structural homology within the putative TM regions of haloar-chaeal rhodopsins, especially to bacteriorhodopsin and sensory rhodopsin, and

shuffling of preexisting TM domains that may have occurred in the evolution of

GPCR gene (“intron early” theory) The two long-standing alternative nations for the origin of introns, the “intron-early” and “intron-late” theories,

GPCRs that have orthologs in vertebrates and invertebrates contain introns within the coding region of the 7TM core and retained their intron/exon-struc-ture during evolution However, evolutionary gain and loss of introns, as found

in rhodopsin-like GPCRs, are frequently observed, and it was unclear why malian GPCR genes are characterized by a large proportion of intronless genes

mam-or a lower density of introns when compared with GPCRs of invertebrates A

A more detailed analysis showed that there was not a major loss of introns in mammalian GPCRs, but the formation of new GPCRs among mammals explains

mecha-nisms of intron loss are suggested including recombination or gene duplication

for an RNA intermediate mechanism, the four introns found in the ancestral vertebrate rhodopsin gene were simultaneously lost in the common ancestor

introns in vertebrate rhodopsin-like GPCR For example, GPR34 subtypes and

evolution-arily old teleostei (zebrafish) and tetrapods, gained an intron in more recent fish

dupli-cation occurred in the evolution of an ancestral gene, such that helixes 5–7 originated as duplicates of helixes 1–3, leading to intragenic as well as inter-genic similarities between helixes 1–3 and 5–7 of bacteriorhodopsin and various GPCRs Nevertheless, there is very little sequence homology between the five present-day GPCR subfamilies and haloarchaeal rhodopsins, but also between the five families This makes it impossible to reconstruct the GPCR prototype

Long evolutionary processes have shaped the TM core of the individual GPCR families over hundreds of millions of years, releasing specific structural deter-minants One key feature of the TM core is a highly conserved disulfide bridge between extracellular loops (ECL) 1 and 2 This disulfide bond is found in most members of the families rhodopsin/R, glutamate/G, adhesion/A, and secretin/S Numerous functional analyses of mutant rhodopsin-like GPCRs, in which these cysteine residues were replaced by other amino acids, have shown that this disul-

this disulfide bond is required to maintain more distinct functions Systematic mutagenesis studies of the conserved cysteine residues in several GPCRs showed that disruption of the disulfide bond does not influence the receptor’s ability to activate G proteins, but interferes with high affinity ligand binding and receptor

structure appears to remain intact, allowing receptor function Consistent with this notion, some GPCRs, for example, receptors for sphingosine 1-phosphate and lysophosphatidic acid, lack the conserved extracellular Cys residues.

Another hallmark of the GPCR structure is the high frequency of TM kinks, which commonly occur at proline residues The pattern of helical kinks appears

to be conserved within but differs between the individual GPCR families This implicates conserved differences in the fine arrangement of the TM core between

always require proline It was proposed that in evolution, a mutation to line initially induces the kink in a helix The resulting packing defects are later repaired by further mutation, thereby locking the kink in the structure even in

kinks due to proline residues within a TM also may have played a pivotal role in the structural evolution of subfamilies As a specific example, indels (insertion/

deletion of base pairs) shaped the relative positioning of a conserved proline in

Within their TM core, most GPCR families possess a number of highly served sequence motifs In the rhodopsin-like GPCRs, for example, the E/DRY motif at the transition of TM3 and ICL2 and the N/DP(X)nY motif in 7TM are preserved during more than 570–700 Myr of evolution (see the beginning

con-of this chapter on the origin con-of GPCR genes) and present in almost all ily members Numerous studies highlight the functional importance of the conserved E/DRY motif, and the crystal structures of (rhod)opsins implicate

fam-an intramotif salt bridge or multiple intramolecular hydrogen bonds of the

which the acidic residue (Asp, Glu) within this motif is naturally substituted

by His, Asn, Gln, Gly, Val, Thr, Cys, or Ser residues This fact questions a eralization of the structural arrangement of this motif found in rhodopsin structures The N/DP(X)nY motif within the 7TM near the cytoplasmic face

gen-of the plasma membrane is highly conserved The functional importance

of, for example, the Asn and the Pro within the N/DP(X)nY motif has been

Asn/Asp residue within the N/DP(X)nY is naturally replaced by Ser, Thr, Lys,

or His This all indicates that even with GPCR structures in hand, highly conserved motifs may have different structures and functions and need to be individually analyzed for the respective receptor

structural evolution of intra- and extracellular domains of GPcrsSeveral families (glutamate-like, adhesion-like GPCRs) and subfamilies (leucin-rich repeats [LRR] receptors of rhodopsin-like GPCRs) possess very long N and/or C termini The N termini are variable in length, up to 6,000-amino-acids-long in

ectodomains are often rich in glycosylation sites and proline residues, forming what has been described as mucin-like stalks, and are likely to participate in cell

adhesion.130 The module-like structures encoded by complex intron/exon loci suggest exon shuffling as the mechanism that formed these large ectodomains.

In rhodopsin-like GPCRs, LRR-containing GPCRs (LGR), such as the cal glycoprotein hormone receptors TSHR, LHR, and FSHR, and the LGR for relaxins, constitute a unique cluster of receptors with large N termini sharing a large LRR domain for hormone binding The early origin of LGRs is illustrated

classi-by the existence of a receptor related to human glycoprotein hormone receptors

large N-terminal extracellular domain involved in selective hormone binding that is composed of tandem arrays of LRRs motifs, N- and C-terminally flanked

that is, the number of LRRs, and the sequence similarity, LGRs can be classified

and the 7TM core, were preformed earlier and joined together by genomic rangement This hypothesis is further supported by the fact that the isolated ectodomain structurally assembles and crystallizes and binds the glycoprotein

immedi-ately N-terminal of the 7TM domain This GPS-7TM module is conserved from choanoflagellates to humans, suggesting that it represents a combination of core functional domains rather than an association that arose later by exon shuf-fling The GPS and N-terminal TM helixes are indeed typically encoded in a single exon

With increasing complexity of animal genomes, the adhesion-GPCR subfamily expanded to up to thirty-three members that comprise divergent long N-terminal

can be grouped into at least eight different classes again defined by their domain architectures The number of receptors in each adhesion-GPCR subclass is vari-able from species to species, indicating rapid evolution by duplication and muta-tion Consistent with this assumption, nearly 50 percent of adhesion-GPCR

Within the range of divergent N-termini, two highly conserved subgroups

of adhesion-GPCRs appear to be highly conserved in all bilateral animals The

is an essential component of planar cell polarity and tissue polarity pathways

where the N terminus contains an unusual domain related to rhamnose-binding

calci-um-independent receptor for latrotoxin, the neurotoxin found in the venom of

the Black Widow spider Latrodectus mactans Latrophilins have been suggested to

essential developmental functions The comparison of invertebrate and tebrate latrophilins provides a good illustration of domain shuffling, because

ver-vertebrate latrophilins have acquired an additional olfactomedin-like domain in

Many adhesion-GPCRs contain a hormone-binding (HRM) domain in their

N termini, which is homologous to the HRM of the secretin receptor subfamily

Because the 7TM regions of adhesion-GPCRs and secretin-type receptors also show significant homology, it is tempting to speculate that secretin-receptor-like

functional data supporting this hypothesis have not yet been presented

coevolution of GPcrs and their ligands/associated factorsComparative genomics is a valuable tool for deciphering coevolution of GPCRs and their (putative) ligands leading to novel insights in the evolutionary conservation of signaling systems As stated previously in this chapter, gene duplication provides redundancy and therefore robustness and an increase in gene doses, but gives the chance for developing new ligand/receptor systems without risking fitness of the organism There are a number of examples where the receptor and the ligand underwent coevolution upon gene duplications

events led to the expansion of both, the subtype A LGR (see the subchapter on structural evolution of intra- and extracellular domains of GPCRs earlier in this chapter) and glycoprotein hormone subunits in vertebrates Afterward, coevolu-tion shaped ligand and signaling properties of the subtypes In contrast to the

more intramolecularly constrained during evolution in the chordate lineage In the case of human FSHR, for example, evolutional changes in the sequence of the 7TM core lead to a decreased basal receptor activity and, in addition, cause

a decrease in the sensitivity of FSHR to structurally related glycoprotein mones such as chorionic gonadotropin hormone (CG) and TSH Therefore, it has been suggested that an intramolecular constraint acquired during the evolu-tion of primates contributed to the protection of the FSHR against promiscuous

selectIon on GPcr Genes

Determining the molecular basis of evolutionary adaptation remains largely unresolved, and the respective roles of selection and demography in shaping the genomic structure are actively debated The recent availability of large panels of

search for signatures of recent selection and the mutations underlying variation

in complex traits, with the latter being investigated in genome-wide phenotype association (GWA) studies GPCRs, as one of the largest gene families with high relevance in almost every biological function, are highly involved in adaption processes It is therefore not surprising that GWA studies in humans

genotype-and animals revealed a number of GPCRs, at least at a statistical level, sible for previously unappreciated phenotype variations For instance, variations

respon-in the thyreotroprespon-in-releasrespon-ing hormone receptor gene and the GPR133 gene are

per-sonal communication), respectively This field in GPCR research just started, and the paragraphs that follow can only expose the potential power of these new methods

Genetic signatures of selection

As in other genes, disadvantageous mutations in GPCRs are removed from a ulation (purifying selection) Many evolutionarily old GPCR genes, including the rhodopsins, display strong purifying selection across their entire coding regions

shows that the N- and C termini and the intracellular loop regions of GPCRs are

we detect more subtle adaptive changes that improve gene performance?First, phylogenetic comparisons of several dozens of ortholog sequences can

dif-ferences in highly conserved determinants, which are unique for a species or species group, may be indicative of adaptive change (positive selection) The modules integrated into the PAML package are well-established bioinformatic

fixed differences are not necessarily the result of selection; therefore, testing for the functional relevance of the change is required

Second, population genetic models predict that rapid selection should leave

‘footprints’ in closely linked genomic regions (selective sweeps) Complete or near-complete fixation of an allele may be indicative of adaptive changes in populations of one species, especially when accompanied by signatures of a selective sweep There are several methods for detecting signatures of selective

human genome for signatures of recent positive selection on the basis of species

The combination of allele frequency analyses, geographic allele distribution data, and sequence comparison to closely related species has provided evidence for the presence of selective pressure on many GPCR genes (see the subchap-ter immediately following) The human bitter-taste receptor TAS2R16 is an illustrative example Sequence comparison between mammalian species and human populations revealed signatures of positive selection, as indicated by an

an increased sensitivity toward harmful cyanogenic glycosides The improved recognition of bitter natural toxins through taste may have conferred an impor-tant selective advantage and may have driven the increase of frequency of the

selection of genomic regions containing GPcr genesIdentifying regions in genomes that have been targets of positive selection will provide important insights into recent evolutionary history Methods used to identify regions of recent positive selection in a given genome are based on

a simple fact Alleles that are under positive selection will increase in lence in a population leaving distinctive patterns of genetic variation in the DNA sequence These signatures can be identified by comparison with the background distribution of genetic variation in the species, which is generally

Most studies are based on large-scale SNP data of selected human populations

as created by the International Haplotype Map (HapMap) and Perlegen Sciences

(1) reduction of genetic diversity, (2) frequency of derived alleles, (3) differences

in allele frequency between populations, and (4) long haplotypes (for further

are broadly not codon-based, hence carrying the potential to elucidate natural selection even on non-genic genome areas The signatures appear and disap-pear in different time scales so that with the same data set, the different meth-ods do not always produce the same results Further, a challenging problem

of such genomic scans of recent positive selection is yet to clearly distinguish selection from neutral effects because of genetic drift, demographic reasons, and

high proportion of function altering mutations (Ka/Ks)

reduction in genetic div ersity (CLR)high-frequency der iv ed allelespopulation differences (FST)long haplotypes (EHH)

75 ky a

250 ky a

6 my ahomo/panspilt

25 ky amajor migrationwav es out of Africa

Figure 1-2: Signatures of positive selection during human evolution and examples for tion methods

detec-The five main signatures of selection exist over variable time scales, thus covering different ods of human evolution The statistical tests named here are those applied in the genome-wide selection studies from Table 1.1 (adapted from Sabeti et al.7) Ka/Ks: rate of non-synonymous to the rate of synonymous substitutions, CLR: composite likelihood ratio, EHH: extended haplotype homozygosity, FST: fixation index, mya: million years ago, kya: kilo years ago

The meta-analysis of genome-wide studies with human SNP data revealed

a number of genomic regions with signatures of positive selection ing GPCRs Among them are several receptors that are implicated in immune

table 1-1 Potentially Selected GPCR Genes in the Evolution of Modern Human Populations Identified in Genome-wide Studies

HapMap1 (analysis restricted to chromosome 2)

africans: GPR34, GPR82, OR4C2P Ψ, OR4C3, OR4C4P Ψ, OR4C5 Ψ, OR5T2, OR8J2 Ψ, OR8K5, OR8V1P Ψ, OR10V2P

Ψ, OR10Y1P Ψeuropeans: OR2AK2, OR2L1P Ψ, OR2L2, OR2L5 Ψ, OR2L9P Ψ, OR2L13 asians: GRM3*, GRM8*

SNP data from HapMap1

iHS (EHH based) [9]

GRM6, TAS1R1, GPR111*, PTGER4, OR2A14, OR5D18, OR51D1, OR52W1

gene coding regions of human and chimpanzee

Ka/Ks [188]

europeans: CXCR4*, CXCR7, NPY1R, GPR65, GPR151, DRD5

asians: BAI3*, GRM3*, GPR65

SNP data from HapMap2 and Perlegen Sciences project

Rsb (EHH based) [189]

europeans: GPR64*

asians: CELSR1*

SNP data from HapMap2

africans: BAI3*, OR52A1, OR52A4, OR52A5, OR4K1, OR4K5

europeans: GPR64*, GPR177, TACR3, OR4K1, OR4K5

asians: GPR111*, GPR115, OR4A47*

SNP data from HapMap2

LRH and/or iHS (EHH based)

[154]

africans: OR4P4asians: OR9K2, MC2R

SNP data from Perlegen Sciences project

PTGER3, GRM7, GRM8*, LGR7, BAI3*, P2RY2, ADORA2B, CELSR1*, OR52K2

SNP data from HapMap2

The following methods, mainly testing for positive selection, were applied: Ka/Ks… rate of non-synonymous

to the rate of synonymous substitutions, CLR … composite likelihood ratio, EHH … extended haplotype homozygosity, iHS … integrated haplotype score, Rsb … relative integrated EHH of a site between populations, XP-EHH … cross population EHH, LRH … long-range haplotype, FST … fixation index Please see Figure 1.2 for the signature of selection the statistical tests are based on *… selection candidates as determined by two or more different detection methods, Ψ … pseudogenes.

signatures of selection are GPCRs, indicating that there is no overrepresentation

of GPCRs in potentially selected genes It is of interest to note that several GPCR

s of the adhesion receptor family (BAI3, CELSR1, GPR64/HE6) are in selected

studies are now necessary to elucidate the functional relevance of the selected allele and the causality of selection

selection of individual GPcrs

As outlined earlier in the chapter, genome-wide scans can assist in identifying signatures of positive selection in a genomic sequence block It is even more challenging to proof selection of an individual gene Thus functional data and

an idea of the selective force that worked on a specific gene variant are tially required There are only a few reports where selection may have favored

essen-a functionessen-al GPCR vessen-ariessen-ant Chemosensory receptors, such essen-as odoressen-ant essen-and tessen-aste receptors, have been found under positive selection in human and vertebrate

example comes from Drosophila genetics Methuselah, a member of the sion receptor family in insects, has been proposed to have major effects on stress

high level of adaptive amino acid divergence concentrated in the intra- and extracellular loop domains of the receptor protein This suggests the histori-cal action of positive selection on those regions of the molecule that modulate

signal transduction Analysis of haplotypes in D melanogaster populations

pro-vided further evidence for contemporary and spatially variable selection at the

Domestication is an evolutionary process in which animals are exposed to cific selection pressures defined by humans The melanocortins and their receptors play a pivotal role in skin pigmentation and regulation of the energy homeostasis

spe-For that reason, they have been popular targets in domestication of farm animals

as seen in traits of domestic pigs that have been selected because of an increase in growth and food intake A partial inactivating mutation in the MC4R gene has been

phe-notypes in domestic pigs result from direct human selection and not via a simple

selec-tion contain pseudogenes of GPCR This raises the quesselec-tion of whether inactive GPCR variants may provide advantage and are, therefore, selected as shown for

receptor inactivation may provide an advantage under distinct environmental circumstances For example, the FY*O allele at the Duffy locus is at or near

antigen, which is a chemokine receptor, acts as a co-receptor for the cell entry of

Plasmodium vivax Mutation studies have shown that inactivation of Duffy leads

Selection on the null-allele is probably responsible for the evolution of the

mammalian genome more than 125–190 Myr ago, this receptor underwent pseudogenization in humans, other hominoids, and some rodent species Simultaneous pseudogenization in several unrelated species within the last 1 Myr caused by neutral drift appears to be very unlikely It was speculated that

a likely cause of GPR33 inactivation was its interplay with a specific pathogen Although selection of the GPR33 pseudogene is still hypo-thetical, this consideration is supported by the fact that rats and gerbils are

rodent-hominoid-frequently the host of zoonotic pathogens like hanta viruses and Yersinia pestis,

An even more impressive example was identified in TAARs TAARs form a cific subfamily of GPCRs in vertebrates and were initially considered neurotrans-mitter receptors Recent studies suggest that mouse and fish TAAR function as

TAAR3 and TAAR4, became independently inactivated in both great apes and some New World monkeys, obviously in a comparable evolutionary time scale,

occurred independently and in a number of species of unrelated orders an enous trigger is likely to be responsible for driving the inactivation of distinct TAAR These examples clearly show that evolutionary and population genetic data can conceptually enrich GPCR research even for those GPCRs that became inactivated during evolution

exog-In vItro evolutIon of GPcrs

All approaches described previously in this chapter extract only past ary information where the selective forces cannot be manipulated or remain unknown For many biomolecules such as RNA, DNA, enzymes, and antibodies,

evolution-in vitro evolution techniques have been developed, which guide structure and

one billion years of GPCR evolution, the unique TM architecture and fold combinations of other residues assure the evolutionary adaptability toward almost any chemical structure that may serve as ligand In keeping with the idea of a universal receptor backbone, artificial programming of the binding site

mani-by in vitro evolution approaches has led to the generation of GPCRs with new

of “designing“ GPCRs for special purposes, for example, as biosensors or to better study distinct GPCR signaling pathways As a first step toward this goal, so-

RASSLs are unresponsive to endogenous agonists but can be activated by molar concentrations of pharmacologically inert, drug-like small molecules.Until recently, most mutations converting pharmacological ligand properties were accidentally identified by site-directed mutagenesis approaches Although