els || g protein-coupled receptors

G Protein-CoupledReceptorsJoel Bockaert, University of Montpellier, Montpellier, France

Among membrane-bound receptors that recognise reg-

ulatory messages (hormones, neurotransmitters, photon,

odours, etc.), the seven transmembrane receptors cou-

pled to G proteins (G protein-coupled receptors, GPCRs)

are the most numerous. They represent 3% of the total

number of genes in human genome. Following activation

by those messages, GPCRs activate one or several hetero-

trimeric G proteins (a, b and c subunits) by stimulating the

guanosine diphosphate/guanosine triphosphate (GTP)

exchange on the nucleotide binding site. The GTP form of

the subunits activate effectors such as enzymes (e.g. the

adenylyl cyclase) or channels. GPCRs can also trigger G

protein-independent signalling. GPCRs are targets for

more than 30% of the drugs used in human therapy.

Progress has been made recently on the structure and

activation of GPCRs, thanks to the crystallisation of more

than 60 GPCRs bound to agonists, antagonists and

inverse-agonists as well as a cocrystallisation between b2-

adrenergic receptors and its associated G protein.

Introduction

The evolution of multicellular organisms greatly dependson the capacity of their cells to communicate with eachother and with their environment. It has recently beenrecognised that membrane-bound receptors devised torecognise sensory messages from the environment (light,

odours, pheromones and gustative molecules) and inter-cellular messages (such as hormones, neurotransmitters(NTs), growth and developmental factors) are very similarand derive from common ancestral genes. These receptorsbelong to a small number of protein families that can beclassified based on their structure and function: (1) channelreceptors, (2) tyrosine kinase receptors, (3) guanylatecyclase receptors, (4) serine/threonine kinase receptors, (5)cytokine receptors and (6) receptors coupled to guanosinetriphosphate (GTP)-binding proteins (G protein-coupledreceptors,GPCRs). Themost common family is theGPCRfamily. In vertebrates, this family contains between 1000and 1400 members (more than 1–3% of the genomes)including more than 1000 coding for odorant and pher-omone receptors. Similarly, the Caenorhabditis elegansgenome encodes approximately 1100 GPCRs (5% of thegenome). In this organism, the GPCR family of genes hasmoremembers than any other family.GPCRs are certainlyamong the oldest devices devoted to signal transduction,being present before plants, and fungi. The first glutamate-like GPCR are found in sponge (Geodia cydonium) andslime mould (Dictyostelium discoideum). Fungi express themain classes of GPCRs and Saccharomyces cerevisiaeexpress also pheromone and glucose-sensing GPCRs.GPCRs had no evolutionary success in plants (Bockaertand Pin, 1999; Krishnan et al., 2012).

GPCRs are involved in the recognition and transductionof messages as diverse as light (for vision), Ca2+, odorants(for olfaction), small molecules, including amino acidresidues, sucrose and others taste molecules, nucleotidesand peptides, as well as proteins (Figure 1). In vertebrates,the more numerous GPCRs are those implicated inrecognition of odorants and pheromones (500–1000). Theendo-GPCRs (approximately 360) recognised hormones,NTs, growth factors, etc. Among them, more than 60 arestill ‘orphan’ because their ligands have not been identifiedyet. GPCRs have a ‘central core’ composed of seven ahelices transmembrane domains (TMI–VII) preceded by

Advanced article

Article Contents

. Introduction

. Transduction of the GPCR Signal: A Four Partner Affair

. Diversity and Structure of GPCRs

. A Touch of Eccentricity in Structure or Function of

GPCRs

. Desensitisation, Endocytosis and Downregulation of

GPCRs

. A New ‘Ligand-directed’ Signalling Concept

. Homo- and Heterodimerisation of GPCRs and

Interaction with GIPs (GPCR Interacting Proteins): New

Concepts in Targeting, Activation and Pharmacology

. GPCRs Viewed as Allosteric Molecules (AMs)

. Mutation of GPCRs in Pathology

. Summary

Online posting date: 15th April 2014

eLS subject area: Neuroscience

How to cite:Bockaert, Joel (April 2014) G Protein-Coupled Receptors. In: eLS.

John Wiley & Sons, Ltd: Chichester.

DOI: 10.1002/9780470015902.a0000118.pub3

eLS & 2014, John Wiley & Sons, Ltd. www.els.net 1

γ

(a)

Photons

OdorantsSmall endogenousmolecules

• Amino acids, amines

• PAF ...• Prostaglandins• Nucleotides, nucleosides

Proteins

NH2

• TSH• LH• FSH

• Wingless

Effector

• Enzyme • Channel

Effector

• Enzyme • Channel

• InterleukineNH2

COOH

COOH COOHG protein

COOH

GABA

NH2NH2

Glutamate

(b)

Ca2+ Sucrose,aspartame

Secondmessengers

Secondmessengers

β

α

βγ

α

G protein

Figure 1 GPCRs are homo- or heterodimers. GPCRs have seven TM domains, three extracellular loops (e1, e2, e3) and three intracellular loops (i1, i2, i3).

Heterotrimeric G proteins have three subunits: a, b and g. b are always associated. a on one hand and bg on the other hand are covalently bound to lipids.

These lipids allow the association of a and bg with the membrane. The effectors are enzymes, channels, transporters, etc. (a) Class 1 (or A) GPCR. The

diversity of ligands of GPCRs is illustrated: photons, odorants, small endogenous molecules such as amino acids, nucleotides, nucleosides, prostaglandins,

platelet-activating factor (PAF) and proteins such as thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH).

(b) Class 3 (or C) GPCRs. The dimmer can be a homodimer (e.g. the glutamate-metabotropic receptors, mGluRs) or a heterodimer (e.g. the GABAB

receptors). The diversity of the ligands is illustrated. It can be Ca2+, glutamate, GABA, sucrose and aspartame.

eLS & 2014, John Wiley & Sons, Ltd. www.els.net2

G Protein-Coupled Receptors

an extracellular N-terminal domain and followed by anintracellularC-terminal domain. Three extracellular (e1, e2and e3) and three intracellular loops (i1, i2 and i3) connectthe TM helices. GPCRs are dimeric structures (Figure 1),most of them are homodimers (two identical monomers),whereas some of them are heterodimers (two differentmonomers). Among the latter (Figure 1b), some are veryimportant in physiology. We can give, for example, thereceptor for sweet molecules (sucrose, aspartame) com-posed ofT1R2 andT1R3, the receptor for theUmami taste(glutamate taste typical of Asian food) composed of T1R1and T1R3 and the receptor for the major inhibitory NT: g-aminobutyric acid (GABA), the GABAB receptor (Pinet al., 2004). Recent crystallisation of dimers of GPCRs(CXCR4 chemokine receptor, m et k opiates receptors) hasbeen described (Granier and Kobilka, 2012). GPCRscontrol the activity of enzymes, ion channels and transportof vesicles, transcription and traduction via the catalysis of

theGDP/GTP exchange on heterotrimericGproteins (Ga,Gb and Gg) (Figure 1).

Transduction of the GPCR Signal: AFour Partner Affair

In the absence of GPCR activation, G protein subunits areclosely associated (state 1, Figure 2; Bockaert et al., 2002).The a subunit binds GDP. b and g subunits stick togetherforming a G bg complex. The only role of GPCRs is tocatalyse the GDP/GTP exchange on the a subunit. GPCRsare exchange factors similar to guanyl nucleotide exchangefactors (GEFs) or GDP-releasing factors of monomeric Gprotein such as ras, rho and rac. State 2 (Figure 2) is char-acterised by an empty a subunit, and a tight interactionbetween theGPCRand theGprotein (high-affinity state for

γβ

α γβ(State 1)

GDP GDP

GPCR +

+

Pi

Regulator ofG proteinsignalling (RGS)

GTP

α(State 2)

GTP

(State 3)

αγβ

Effectors

Adenylate cyclases (II) (+) L-type Ca2+ channels (+)

Effectors

Adenylate cyclases (all) (+)αs

αi family

αtα-Gust

cGMP-phosphodiesterase (+)

Adenylate cyclases (I, V, VI) (–)αi, 1, 2, 3

Adenylate cyclase (I) (–)αo Ca2+ channels (–)

αq family

Phospholipases C (β1, β3) (+)αq, α11

α12 family

Rac-cdc 42 (+)α12, α13

Phospholipases A2 (+)

Na+/H+ exchanger (+)

α14–16

GIRK (+)Phospholipase C (β2) (+)

Effectors

Ca2+ channels (N/P) (–)

Adenylate cyclase (I) (–)

Phospholipase A2 (+)

β-AR kinases (+)

MAP-kinases (+)

Figure 2 The cycle of activation of heterotrimeric G proteins, the role of GPCRs as GEF and the effectors. at, a-transducin; a-Gust, a-gustducin; Rac and

cdc42 are small G proteins; GIRK, G protein-regulated inward rectifying K+ channels; MAP kinases, mitogen-activated protein kinases and b-AR kinase, b-

adrenergic receptor kinase.



agonists). In the cell, this state has a very short life. GTP,which has a high cellular concentration compared to GDP,rapidly binds to the a subunit leading to the dissociation ofthe partners. a-GTP, on one hand and bg on the other,activate their effectors that are either different or identical(Figure 2). G proteins (17 genes coding for Ga, 5 for Gb and14 for Gg) are classified into four families, according to thenature of the a subunit (as, ai, aq and a12/13) and theirspecific effectors are summarised in Figure 2. GTP hydrolysisonGawill stop the interaction between the a subunit and itseffector, andprovides away for it to reassociatewithGbg. Ithas been known for a long time that the spontaneoushydrolysis ofGTPdeterminedonpurifiedGproteinwas tooslow to account for in vivo physiological deactivation pro-cesses. Transducin (at-GTP), the G protein activated byrhodopsin in the phototransduction system, hydrolysesGTP in vitro in the 1920s, which is 100 times slower than thedisappearance of the visual signal in vivo. In fact, a proteinfamily implicated in the activation of theGTPhydrolysis ona-GTP does exist. They are called regulators of G proteinsignalling (Figure2) andplaya similar role to thatofGTPase-activating proteins in monomeric G protein systems.See also: G Proteins; Rhodopsin

Diversity and Structure of GPCRs

There are threemainGPCRs families or classes (1, 2 and 3)that can be easily recognised when their sequence simila-rities are compared (Figure 3; Bockaert et al., 2002).

Class 1 includesmostGPCRs, in particular receptors forodorants and small NTs. They contain several highlyconserved signatures:

. one residue (D in TMII) and the tripeptide DRY (apeptide sequence composed ofD, aspartate; R, arginine;Y, tyrosine) (orERWapeptide sequence composedofE,glutamate; R, arginine; W, tryptophane) at the N-terminal part of i2, which are involved in receptor acti-vation and coupling to G proteins;

. a disulphide bond between e1 and e2 and hydrophobicresidues in TMVI and VII.

Class 1a contains GPCRs for small ligands includingrhodopsin and b-adrenergic receptors. The binding site islocalisedwithin the sevenTMs.Class 1b contains receptorsfor peptideswhose binding site includes theN-terminal, theextracellular loops and the superior parts of TMs. Class 1ccontains GPCRs for glycoprotein hormones. This ischaracterised by a large extracellular domain and a bindingsite that is mostly extracellular but has contact withextracellular loops. See also: Adrenergic Receptors; Pep-tide Neurotransmitters and HormonesClass 2 GPCRs have a similar morphology to group 1c

GPCRs but they do not share sequence similarities. Theirligands include high molecular weight hormones such asglucagon, secretine, VIP and pituitary adenylate cyclase-activating polypeptide (PACAP). A subclass 2 is named‘adhesion-GPCRs’ and contains GPCRs having long N-terminal domains containing many repetitive modularadhesion domains such as epithelium growth factor-like

Family 1

COOH

NH2

D

DR Y

SS

P

F

PN

RetinalOdorantsCatecholaminesAdenosineATP, OpiatesEnkephalinsAnandamide

COOH

NH2

D

DR Y

SS

P

F

PN

PeptidesCytokinesIl8Formyl Met-Leu-Phe(fMLP)-peptidePAF-acetherThrombin

COOH

NH2

D

DR Y

SS

P

F

PN

Glycoproteinshormones(LH, TSH, FSH)

COOH

NH2 Calcitoninα-LatrotoxinSecretinPTHVIPPACAPGnRHCRF

Family 2

Glutamate(metabotropic)Ca -sensing receptor2+

GABA (GABAB)UmamiSweet molecules

COOH

Family 3

(a)

(b)

(c)

Figure 3 The three most important familles of GPCRs. CRF, corticotropin releasing hormone; FSH, follicle-stimulating hormone; GnRH, gonadotropin

releasing hormone; IL8, interleukin 8; LH, luteinizing hormone; PACAP, pituitary adenylate cyclase activating polypeptide; PAF-acether, platelet-activating

factor; PTH, parathyroid hormone; VIP, vasoactive intestinal peptide and TSH, thyroid-stimulating hormone.



http://dx.doi.org/10.1038/npg.els.0000657



http://dx.doi.org/10.1002/9780470015902.a0000063.pub2


repeats and thrombospondin-like repeats among others.The black widow spider toxin, a-latrotoxin binds to suchan ‘adhesion-type GPCR’ called Latrophilin. See also:Insulin and Glucagon; Toxin Action: MolecularMechanismsClass 3 has been the most intriguing one and for a

long time because it only contains mGluRs and the Ca2+-sensing receptors present in parathyroid glands, kidneyand brain (Bockaert et al., 2002). Other members ofthis class include GABAB receptors, a group of putativepheromone receptors (V2R), receptors for sweet molecules(T1R2/T1R3) and Umami taste (T1R1/T1R3) (Pin et al.,2004). The most striking feature of this class is that theyare obligatory homo- or heterodimers. The first segment ofthe N-terminal external domain of class 3 GPCRs sharesweak but significant identities with the bacterial peri-plasmic-binding proteins (PBPs) (Figure 1b). In the peri-plasmic space, PBPs shuttle and specifically bind nutrientssuch as amino acids, saccharides, vitamins, anions andcations. The captured substrate is then delivered to a per-mease of the inner bacterial membrane. X-ray crystal-lography has shown that different PBPs fold to a similarthree-dimensional structure consisting of two lobes inter-connected by hinge regions that close on the ligand, like aVenus fly trap (Figure 1b). Based on detailed amino acidsequence comparisons and multiple alignments, a similartwo-lobed structure has been proposed for the mGluR-binding site localised within the N-terminal domain. Thecrystal analysis of the N-terminal domain of mGluRs hasconfirmed this Venus fly trap structure (Muto et al., 2007).See also: GABAB Receptors; Metabotropic GlutamateReceptorsClass 4 includes the ‘frizzled’ and the ‘smoothened’

(Smo) receptors involved in embryonic development and inparticular in cell polarity and segmentation.Other classes do exist. For example, the pheromone

receptors (V1Rs in vertebrates), pheromones receptors infungi, receptors for bitter taste (T2Rs in vertebrates), thecyclic adenosine monophosphate (cAMP) receptor familythat has only been found in lower invertebrates (not pre-sent in nematoda). It is particularly studied in D. dis-coideum (Krishnan et al., 2012). See also: SignalTransduction Pathways in Development: Wnts and theirReceptorsUntil very recently (2007), the high-resolution structure

had only been published for the light-sensitive rhodopsin,which is unique because of its covalently linked ligand, theretinal. In 2007, the crystal structure of b2-adrenergicreceptor occupied by the partial inverse agonist carazololhas been published (for reviews see Audet and Bouvier,2012; Venkatakrishnan et al., 2013). This was achieved byrigidification of the i3 intracellular loop with an antibody(resolution 3.4/3.7 nm) or the fusion with the T4 lysozymeprotein (resolution 2.4 nm). One of the surprises was tofind, in all these structures, that, in addition to the seven ahelices forming the TMdomain (TMI, II, III, IV, V,VI andVII), an additional intracellular a helix (VIII) runningparallel to the membrane does exist.

An explosive number of GPCR crystals has beenobtained since 2007, mainly within the rhodopsin family(Family1) but more recently crystals of family B GPCRs(glucagon and CRF-corticotropin releasing factor) as wellone crystal of smoothened receptor (family 4) have beenpublished (Hollenstein et al., 2013; Siu et al., 2013;Venkatakrishnan et al., 2013). Crystals of family1 GPCRsindicate that there are only few differences in TMs struc-ture. In contrast, many differences do exist in extracellularloops, particularly in e2 loop. Note the presence of a shorta-helice in extracellular e2 loop in the b2-adrenergicreceptor (Figure 4). In contrast, in rhodopsin and m opiatereceptor b-sheets are present in the e2 loop (Manglik et al.,2012). In some receptors, like rhodopsin and shingosine 1phosphate, the space within the sevenTMs is almost closedat its extracellular entry (the ligand should access this spacevia the hydrophobic membrane) whereas in other recep-tors, like the m opiate receptor, the space within the sevenTMs is largely opened at the extracellular entry. This mayexplain with the binding of opiates and naloxone (the mopiate antagonist used to treat overdoses) is very rapid. Inrhodopsin family, the binding sites within the seven TMshave different sizes. For a unique receptor, for example, theCXCR4 receptor implicated in Human immunodeficiencyvirus (HIV) cell penetration, several antagonists of differ-ent sizes (the CVX15 peptide and the small polycyclic IT1tcompound) blocked the receptor interacting with com-pletely different binding sites.Curiously, the crystal structures of family 1 GPCRs

occupied by agonists and antagonists do not show very

V

i2VI

VIII

II

III

IV

VII I

e1

e2

e3

i1

i3 loop

Carazolol(inverse-agonist boundto the receptor duringcrystal formation)

Figure 4 Structure of b2-adrenergic receptor. The seven TM a helices plus

the intracellular a helice eight (parallel to the membrane) are labelled with

roman numerals. The proline-induced kinks in TM VI and VII are clear. The

i3 loop is not figured. Note the a helice in extracellular loop e2. Structure at

2.4 nm. Modified from Kobilka and Schertler (2008).













important differences. Cocrystallisation of the b2-adrenergic receptor with Gs, with a ‘nanobody’ antibodymimicking the G protein stabilising effect or opsin with theC-terminal domain of Gt indicates that the ‘active’ struc-ture is only obtained when the agonist-bound receptor isassociated with the G protein (Audet and Bouvier, 2012).Important allosteric interactions do exist between theGPCRs and the G proteins.In such GPCR-G cocrystallisation experiments the

changes in TM orientations is more pronounced with alargemovement of TMVI outward (with the rupture of theionic lock in rhodopsin between the arginine (R135) of theDRY (in fact ERY in rhodopsin) sequence and the gluta-mate of TMVI (E247)) (Audet and Bouvier, 2012). Theionic lock is less tight in b2-adrenergic receptor than inrhodopsin which may explain the higher ‘constitutive’activity (activity in the absence of agonist) of the b2-adrenergic receptor. The ionic lock does not exist in the mopiate receptor. An inward movement of TMVII with apositioning of the tyrosine of its conserved NPXXYsequence in such a way that it is blocking the TMVI in itsoutward positioning. All these TMs’ movements allow theopening of intracellular space between the TMs and thecontacts of the intracellular domains of TM III, V and VIand of the i2 loopwith theGprotein. On theGprotein side,several domains are engaged in interacting with GPCRs.One of the most important ones is theC-terminus. The last4–5 residues of a subunits determine the specificity ofinteraction. Convincing proof for such a conclusion is thefact that it is possible to construct G protein chimaeras inwhich the last five residues of one a subunit (e.g. Gaq) hasbeen replaced by the corresponding residues of another asubunit (e.g. Gai) making a Gaq2i5 protein. It is easy toshow that receptors (such asmGluR2), which are naturallycoupled to Gai, and not to Gaq, can now activate theGaq2i5 chimaera (Blahos et al., 1998). When GDP dis-sociated from the a-subunit leaving the nucleotide siteempty, the a-helical domain of the a-subunit is making a1308 rotation largely opening the cleft between the a-helicaldomain and the ras domain of the a-subunit a structuralchange that highly facilitates the GTP access (Audet andBouvier, 2012).The fine tuning of GPCR coupling to G proteins can be

assumed by generation of splice variants differing in theirintracellular domains, the i3 loop in PACAPand dopamineD2 receptors, the C-terminal domains in mGluRs, ser-otonin types 4 and 7 receptors andprostaglandin receptors.More subtle regulations of GPCR coupling to G proteinshave been discovered. Transcripts encoding the serotonintype 2C (5-HT2C) receptor, a phospholipase C (PLC)-coupled receptor, undergo ribonucleic acid (RNA)-editingevents in which the genomically encoded adenosine resi-dues are converted into inosines by a double-strandedRNA adenosine deaminase(s) (Burns et al., 1997). Sevenmajor 5-HT2C receptor isoforms are predicted, encoded by11 distinct RNA species, differing in their second intra-cellular loops. See also: Alternative Splicing: Cell-type-specific and Developmental Control; Serotonin Receptors

A Touch of Eccentricity in Structure orFunction of GPCRsWewanted to highlight someGPCRs characteristics whichare slightly eccentric either in their structure or in theirfunctions (Figure 5). Some of them, such as the protease-activated receptors 1–4 (PAR1, PAR2, PAR3, PAR4),have a very original way of being activated. PAR1, PAR3and PAR4 are thrombin receptors. PAR2 mediates sig-nalling by trypsin and tryptase but not thrombin, whereasPAR4 is also activated by trypsin. These GPCRs arecharacterised by a tethered peptide ligand at their extra-cellular N-terminus that is unmasked on proteolysis bythrombin, trypsin or tryptase (Figure 5a). The tetheredpeptides, SFFLR (a peptide composed of S, serine; F,phenylalanine; L, leucine and R, arginine) in PAR1, areagonists of their corresponding receptors. See also:Thrombina-Latrotoxin from the black widow spider venom pro-

vokes a massive release of NTs in neurons and other exci-table cells, even in the absence of Ca2+. Although themechanism of such a release remained mysterious for 20years, some light appeared in 1998 when a GPCR calledlatrophilin was cloned. This GPCR mediates both theCa2+-dependent and -independent NT release (Figure 5b).In the nerve terminals the latrophilin receptors areexpressed together with a great number of receptors(GABAB, opiates, adenosine, mGluRs, etc.), which inhibitNT release via a Gi/Go pathway. See also: Calcium andNeurotransmitter ReleaseThe importance of cell–cell interaction in regulating

embryonic development has been known for many years,but it is only recently that some main actors have beenidentified. The hedgehog (Hh) family has been shown toplay a leading role. For example, the patterning of most ofthe vertebrate body, planned by two of the organisingcentres (the notochord and floorplate) is mediated by sonichedgehog. The Hh pathway involves two membrane-bound receptors: smoothened (Smo) and a 10 TMreceptor, patched (Ptc) (Figure 5c). The originality here isthreefold: (1) Smo can be considered as ‘constitutively’active (agonist-independent) in the absence of Ptc; (2) Ptcinhibits Smo in the absenceofHh; under these conditions, aprotease complex cleaves the transcription factor cubitusinterruptus (Ci-p155–Ci-p75) a repressor of gene tran-scription controlled by theHhpathway; (3)Hh, the agonistof Ptc, suppresses its inhibition of Smo, leading to aninhibition of the protease complex. Ci-p155 is then acti-vated to Ci-act. See also: Hedgehog Signalling; SignalTransduction Pathways in Development: Hedgehog Pro-teins and their ReceptorsHIV and related viruses have selected both CD4 and

GPCRs to infect their target cells (lymphocytes or mac-rophages) (Figure 5d). These GPCRs are mostly CCR5 andCXCR4 chemokine receptors. The importance of CCR5 inHIV infection is supported by the observation that allelemutation of CCR5 is highly efficient in protecting homo-zygotes against infection and confers partial resistance to













heterozygote individual (Samson et al., 1996). Themechanism of HIV entry involves the interaction ofthe envelope protein gp160 (cleaved to gp120 and gp41 bythe cell) with CD4, the association of gp120 with theGPCR, andfinally the dissociation of gp41 fromgp120 andits participation in the fusion of the virus with the cell(Figure 5d). Antagonists of CCR5 receptors are underdevelopment for therapy of HIV. See also: HIV Life Cycleand Inherited Co-receptors

Desensitisation, Endocytosis andDownregulation of GPCRs

The early events of signalling by GPCRs, for example, thegeneration of second messengers such as cAMP and ino-sitol-1,4,5-trisphosphate, are usually rapidly attenuated byregulatory processes known as receptor desensitisation.Two types of desensitisation are recognised. Homologousdesensitisation is mediated by agonist-dependent activa-tion of the same receptor, whereas heterologous desensi-tisation is mediated by activation of different receptors.Homologous desensitisation is generally initiated by a

unique class of serine/threonine protein kinases, namely Gprotein-coupled receptor kinase (GRK), whereas hetero-logous desensitisation is initiated by second messenger-dependent kinases, such as protein kinasesAorC.See also:Receptor Transduction MechanismsThe GRK family includes GRK1 (rhodopsin kinase),

GRK2 and GRK3 (b-adrenergic receptor kinases), whichalso phosphorylate many other GPCRs. GRK4,5,6 havedifferent tissue distributions and GPCR substrate specifi-city.Rhodopsinkinase is associatedwith themembrane viaa farnesylation of a cysteine at the C-terminus, whereasGRK2 and GRK3 are translocated to the membrane fol-lowing their interaction with bg released during receptoractivation. GRKs generally phosphorylate one or severalserine and threonine residues of theC-terminus. The kinaseactivity ofGRKs is not always necessary forGRK-induceduncoupling. Once phosphorylated, the GPCRs interactwith another family of proteins, the b-arrestins. See also:Protein Kinases: Physiological Roles in Cell SignallingAgonist-mediated endocytosis is another way of redu-

cing GPCR functions. The b-arrestin–GPCR complexesare internalised via clathrin vesicles that shed their clathrincoat and become early endosomes. Acidification of endo-somes and dephosphorylation of GPCRs allow their

COOH

NH2Proteolyticcleavage

PAR1 (thrombin receptor)

COOH

SFFLRN

Smoothened

SmoothenedPatched

Ci-p155

Proteasecomplex

Ci-p75

Ci-p75 Hh, Ptc

Ci-p155

Proteasecomplex

Ci-act

Hh, Ptc

Hh

α-Latrotoxin receptor (latrophilin)

Latrophilin

NT Gi/o

GABAB-ROpiates-RAdenosine-Retc…

CCR5-CXCR4

gp120

CD4

gp41

Virus (HIV)

CCR5CXCR4

Ci-act

(a)

(c) (d)

(b)

Figure 5 A touch of eccentricity in structure or function of GPCRs. CCR5-CXCR4, chemokine receptors; Ci, cubitus interruptus; Hh, hedgehog; NT,

neurotransmitter; PAR, protease-activated receptor and Ptc, patched receptor.







recycling to the membrane more or less rapidly dependingon the stability of the b-arrestin–GPCR complexes. SomeGPCRs, like the PAR receptors, are not recycled at all butdegraded following their passage from the early endosomesto the lysosomes. See also: Clathrin-coated Vesicles andReceptor-mediated EndocytosisFollowing very long receptor stimulation, down-

regulation of GPCRs occurs. Several mechanisms arecertainly involved, including lysosomal degradation, ubi-quitin-mediated proteolysis and destabilisation of mes-senger RNA (mRNA) coding for GPCRs. See also:Receptor Adaptation Mechanisms

A New ‘Ligand-directed’ SignallingConcept

GPCRs oscillate between an (or more likely several) active(R�) and inactive (R) conformations. In the absence of anyligand, the concentration of the R� can be sufficient toconfer a basal constitutive activity. The intrinsic efficacy of aligand is viewed as a geometric parameter that characterisesthe ligand, referring to its dominant interactionwitheitherRor R�, a full agonist bind essentially to R�, a full inverse-agonist to R and a neutral antagonist equally to R and R�.An inverse agonist is thus able to reverse the basal con-stitutive activity of a GPCR. A single GPCR receptormolecule can trigger several signalling pathways leading toactivation of not only differentGproteins but also of non-Gproteins (b-arrestins, Src or other GPCR interacting pro-teins, GIPs) (Bockaert et al., 2004b, 2010). One of the moststudied non-G signalling pathway is the activation, by theinternalised b-arrestin–GPCR complexes of the Src/extra-cellular receptor kinase (ERK) signalling pathway. Differ-ent ligands, acting on a single GPCR, may have differentintrinsic activities depending on the nature of the signallingpathway considered. For example, a b-adrenergic antago-nist, such as propranolol, can be antagonist, as expected, onthe b2-adrenergic receptors (b-AR)Gs/cAMP coupling and‘agonist’ on the non-G protein-mediated b-AR/b-arrestin-mediated ERK activation. The ‘ligand-directed signalling’concept proposes that the ligand, by stabilising ‘a’ particularR� conformation of the GPCR, triggers a ‘unique array’ ofsignalling.

Homo- and Heterodimerisation ofGPCRs and Interaction with GIPs(GPCR Interacting Proteins): NewConcepts in Targeting, Activation andPharmacology

The classical view of GPCR/G protein-coupling stoichio-metry was one receptor for one G protein. This is no longerthe case. The importance of GPCR dimerisation has been

well documented for class 3 GPCRs. These receptors,including mGluRs and the Ca2+-sensing receptor, arehomodimers, GABAB receptors are obligatory hetero-dimers. This latter receptor is constituted of two ‘sub-units’ sharing no sequence similarity (GABABR1 andGABABR2). GABABR1 bind GABA and is not able toactivate the G protein, whereas GABABR2 does not bindGABA and is coupled to G protein (Pin et al., 2004).GABABR2 is required for GABABR1 to reach the cellsurface. In class 1 GPCRs, the general view is also that theyform dimers and that 2 GPCR subunits interact with one Gprotein. However, the exact functional importance of thedimerisation of class 1 GPCR is still a matter of debate.Indeed, a single class 1 GPCR subunit is able to activate theG protein. The dimerisation may assure a better couplingefficacy or a better trafficking to themembrane. Finally, twopharmacologically different class 1GPCRs subunits or evena class 1 GPCR subunit and a class 3 GPCR subunit canform heterodimers. Such a heterodimer has been recentlyreported composed of one 5-HT2A receptor subunit andone metabotropic (mGluR2) receptor subunit. This com-plex is proposed to play a crucial role in the hallucinogenicaction of lysergic acid diethylamide and its regulation bymGluR2 agonists (Gonzalez-Maeso et al., 2008).

Some GPCRs need to form heterodimers with one TMdomain proteins to be correctly folded, exported to themembrane and, in the case of calcitonin receptor-likereceptor (CRLR), to obtain its final identity. CRLR is avirtual receptor that will generate the calcitonin gene-related peptide receptor when associated with RAMP1(receptor activity-modifying protein) and the adrenome-dullin receptor when associated with RAMP2.In addition toG proteins, GPCR environment includes a

large number ofGIPs assembled into functional complexes.GIPs clearly control GPCR subcellular localisation, as wellas nature, kinetics, strength and fine-tuning of GPCR sig-nalling. Thus, in neurons, GIPs regulate not only GPCRtargeting to subcellular compartments but also their traf-ficking in and out of the plasma membrane, within endo-plasmic reticulum, Golgi apparatus, endosomal andlysosomal compartments during synthesis, endocytosis,recycling and degradation. GIPs can cluster not onlyGPCRs but also various proteins, thus coordinating posi-tive and negative feedback signals, creating molecularthreshold, graded or digital signals, as well as transient,sustained or oscillatory signalling. SomeGIPs trigger eventsthat are independent of G protein activation, as welldemonstrated for the b-arrestin. Several recent reviews havedrawn up the inventory of GIPs and their various functionsin cellular physiology (Bockaert et al., 2004a,b, 2010).

GPCRs Viewed as Allosteric Molecules(AMs)

Recently, a great number of AMs of GPCRs have beendescribed. AMs are ligands that interact with binding sites






that are topographically distinct from the orthosteric sitesrecognised by the receptor’ endogenous agonist. PositiveAMs (PAMs) and negative AMs (NAMs) (Pin et al., 2001)can be distinguished. The therapeutic advantage of AMs isthat they are only active in conjunction with the naturalligand. In contrast, orthosteric agonists or antagonists areacting independently of fluctuations of the natural ligandand are more likely toxic and for agonists able to desensi-tise the receptor. Many antagonists of peptidergic GPCRs(angiotensin, ocytocin, bradykinin, endothelin, neuro-tensin, etc.) have recently been developed. These are non-peptidergic antagonists that often bind to sites that do not(or not completely) overlap those of the natural agonists.Their advantages are a great metabolic stability and theirability to cross the blood–brain barrier. In class CGPCRs,PAM sansNAMs bindwithin the TMdomain far from theortosteric site that is within the external Venus fly trap. Thepotential for developing therapeutic allosteric drugs in thisclass is high. A PAM for the humanCa2+-sensing receptor(cinecalcet) is on the market for treating primary hyper-parathyroidism. See also: Drugs and the Synapse; Recep-tor Binding in Drug Discovery

Mutation of GPCRs in Pathology

Two types of GPCR mutations are responsible forpathologies (Vassart and Costagliola, 2011) The gain-of-function mutations in which the receptor becomes con-stitutively active and the loss-of-function mutations arelargely hereditary. Among the gain-of-function mutationsare the common sporadic and hereditary toxic thyroidhyperplasia observed in the absence of autoimmunityGraves’ disease and that is largely due to constitutivelyactive TSH receptors. One can also quote the congenitalnight blindness (constitutively active rhodopsin) or thefamilial male precocious puberty (constitutively active LHreceptors). Among the most common loss-of-functionmutations, more than 150 rhodopsin mutant alleles havebeen identified not only in retinitis pigmentosa but also innephrogenic diabetes insipidus inwhichmutations affectedthe vasopressin V2 receptors. Among other loss-of-function mutations, one can quote familial glucocorticoiddeficiency (adrenal corticoid hormone receptor), bleedingdisorder (thromboxane A2 receptor), male pseudo-hermaphroditism (LH receptor), familial hypocalciurichypercalcaemia, neonatal hyperparathyroidism (Ca2+

receptor) andHirschprung disease (endothelin B receptor).

Summary

Amongmembrane-bound receptors, GPCRs are by far themost diverse. They have been very successful during evo-lution in transducing messages as different as photons,organic odorants, nucleotides, nucleosides, peptides, lipidsand proteins. Indirect studies, as well as crystallisation ofrhodopsin andnowmore 60GPCRs, have led to amodel of

a common ‘central core’, composed of seven TMs and itsstructural modifications during activation. There are sev-eral families (or classes) of GPCRs having low sequencesimilarity. They use a remarkable number of differentdomains both to bind their ligands and to activate G pro-teins. The fine tuning of their coupling to G proteins isregulated by splicing, RNA editing and phosphorylation.Some GPCRs have been found to form either homo- orheterodimers with structurally different GPCRs. Theyinteract with a great number of GIPs including b-arrestins.They are implicated not only in their signalling but alsoin their trafficking and targeting. GPCRs are allostericproteins that can adopt a constitutively active state,especially following mutations. Mutated receptors areresponsible for many endocrinological diseases. Regula-tionofGPCRfunctions occurs via several processes nameddesensitisation.

References

AudetM and BouvierM (2012) RestructuringG-protein coupled

receptor activation. Cell 151: 14–23.

Blahos J II, Mary S, Perroy J et al. (1998) Extreme C terminus of

G protein alpha-subunits contains a site that discriminates

betweenGi-coupledmetabotropic glutamate receptors. Journal

of Biological Chemistry 273: 25765–25769.

Bockaert J, Claeysen S, Becamel C, Pinloche S and Dumuis A

(2002) G protein-coupled receptors: dominant players in cell-

cell communication. International Review of Cytology 212: 63–

132.

Bockaert J, Dumuis A, Fagni L andMarin P (2004a) GPCR-GIP

networks: a first step in the discovery of new therapeutic drugs?

Current Opinion in Drug Discovery & Development 7: 649–657.

Bockaert J, Fagni L, Dumuis A and Marin P (2004b) GPCR

interacting proteins (GIP). Pharmacology & Therapeutics 103:

203–221.

Bockaert J, Perroy J, Becamel C, Marin P and Fagni L (2010)

GPCR interacting proteins (GIPs) in the nervous system: roles

in physiology and pathologies.Annual Review of Pharmacology

and Toxicology 50: 89–109.

Bockaert J and Pin JP (1999) Molecular tinkering of G protein-

coupled receptors: an evolutionary success. EMBO Journal 18:

1723–1729.

Burns CM, Chu H, Rueter SM et al. (1997) Regulation of ser-

otonin-2C receptor G-protein coupling by RNA editing. Nat-

ure 387: 303–308.

Gonzalez-Maeso J, AngRL,YuenT et al. (2008) Identification of

a serotonin/glutamate receptor complex implicated in psy-

chosis. Nature 452: 93–97.

Granier S and Kobilka B (2012) A new era of GPCR structural

and chemical biology. Nature Chemical Biology 8: 670–673.

Hollenstein K, Kean J, Bortolato A et al. (2013) Structure of class

B GPCR corticotropin-releasing factor receptor 1.Nature 499:

438–443.

Kobilka B and Schertler GF (2008) New G-protein-coupled

receptor crystal structures: insights and limitations. Trends in

Pharmacological Sciences 29(2): 79–83.

Krishnan A, Almen MS, Fredriksson R and Schioth HB (2012)

The origin of GPCRs: identification of mammalian like






rhodopsin, adhesion, glutamate and frizzled GPCRs in fungi.

PLoS One 7: e29817.

Manglik A, Kruse AC, Kobilka TS et al. (2012) Crystal structure

of themicro-opioid receptor bound to amorphinan antagonist.

Nature 485: 321–326.

Muto T, Tsuchiya D, Morikawa K and Jingami H (2007) Struc-

tures of the extracellular regions of the group II/III metabo-

tropic glutamate receptors. Proceedings of the National

Academy of Sciences of the USA 104: 3759–3764.

Pin JP, Kniazeff J, Goudet C et al. (2004) The activation

mechanism of class-C G-protein coupled receptors. Biology of

the Cell 96: 335–342.

Pin JP, Parmentier ML and Prezeau L (2001) Positive allosteric

modulators for gamma-aminobutyric acid(B) receptors open

new routes for the development of drugs targeting family 3G-

protein-coupled receptors. Molecular Pharmacology 60: 881–

884.

SamsonM, Libert F, Doranz BJ et al. (1996) Resistance toHIV-1

infection in caucasian individuals bearing mutant alleles of the

CCR-5 chemokine receptor gene. Nature 382: 722–725.

Siu FY, He M, de Graaf C et al. (2013) Structure of the human

glucagon class BG-protein-coupled receptor.Nature 499: 444–

449.

Vassart G and Costagliola S (2011) G protein-coupled receptors:

mutations and endocrine diseases. Nature Reviews Endocrinol-

ogy 7: 362–372.

Venkatakrishnan AJ, Deupi X, Lebon G et al. (2013) Molecular

signatures of G-protein-coupled receptors. Nature 494: 185–

194.

Further Reading

Bourne H (1997) How receptors talk to trimeric G proteins.

Current Opinion in Cell Biology 9: 134–142.

Changeux J and Edelstein S (1998) Allosteric receptors after 30

years. Neuron 21: 959–980.

Granier S and Kobilka B (2012) A new era of GPCR structural

and chemical biology. Nature Chemical Biology 8: 670–673.

Lefkowitz RJ (2004) Historical review: a brief history and per-

sonal retrospective of seven-transmembrane receptors. Trends

in Pharmacological Sciences 25: 413–422.



els || g protein-coupled receptors

Documents