genetic variation in g-protein-coupled receptors – consequences for g-protein-coupled receptors as...

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10.1517/14728222.9.6.1247 © 2005 Ashley Publications ISSN 1472-8222 1247

Ashley Publicationswww.ashley-pub.com

Oncologic, Endocrine & Metabolic

Genetic variation in G-protein-coupled receptors – consequences for G-protein-coupled receptors as drug targetsChih-Min Tang & Paul A Insel††University of California, San Diego, Departments of Pharmacology and Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0636, USA

G-protein-coupled receptors (GPCRs), including ‘orphan’ GPCRs whose naturalligands are unknown, comprise the largest membrane receptor superfamilyand are the most commonly used therapeutic targets. GPCR genetic loci har-bour numerous variants, such as DNA insertions or deletions and single nucle-otide polymorphisms that alter GPCR expression and function, therebycontributing to inter-individual differences in disease susceptibility/progres-sion and drug responses. In this article, the authors review examples of GPCRgenetic variants that influence transcription, translation, receptor folding andexpression on cell surface (by affecting receptor trafficking, dimerisation,desensitisation/downregulation), or perturb receptor function (by altering lig-and binding, G-protein coupling and receptor constitutive activity). In spite ofsuch effects, assessment for genetic variants is not currently applied to thedrug development and approval process or in the clinical use of GPCR drugs.Further insights will, the authors believe, alter drug discovery/development,therapeutics and likely provide new GPCR drug targets.

Keywords: classification, desensitisation, dimerisation, downregulation, G-protein-coupled receptor (GPCR), ligand binding, orphan receptor, single nucleotide polymorphism (SNP)

Expert Opin. Ther. Targets (2005) 9(6):1247-1265

1. Introduction

There are three principal types of membrane receptors: ion channel-coupled recep-tors, enzyme-coupled receptors and heterotrimeric GTP-binding protein (G-pro-tein)-coupled receptors (GPCRs). The superfamily of GPCRs represents the largestclass with > 700 members and may comprise an estimated 1 – 2% of the humangenome [1]. As GPCRs regulate a wide variety of physiological and metabolic proc-esses and are readily accessible from the extracellular space, it is perhaps not surpris-ing that GPCRs are commonly used as therapeutic drug targets and have providedthe most successfully targeted family in small-molecule drug discovery. It has beenestimated that > 50% of all marketed drugs, including, for example, antihistamines,neuroleptics and antihypertensives, are targeted against GPCRs; among the world-wide top 100-selling pharmaceutical products, 25% modulate GPCR activity [2].Therapeutic agents acting on GPCRs have been developed to treat numerous com-mon diseases or disease manifestations, including hypertension, congestive heartfailure, obesity, inflammation, depression and schizophrenia. In addition to theirwell-established clinical utility as drug targets, GPCRs offer potential for new drugdevelopment and for improved therapies. With the recognition that certain disor-ders show genetic alterations in GPCRs, it may be possible to selectively target‘abnormal’ GPCRs as a therapeutically rational approach.

1. Introduction

2. G-protein-coupled receptor

superfamily

3. Genetic polymorphisms

4. G-protein-coupled receptors

polymorphisms

5. Conclusion

6. Expert opinion

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Genetic variation in G-protein-coupled receptors – consequences for G-protein-coupled receptors as drug targets

1248 Expert Opin. Ther. Targets (2005) 9(6)

Sequencing of the human genome has been an importantlandmark for biology and has provided sequence informationfor ∼ 25,000 genes [3,4]. Data from the Human GenomeProject predict 367 non-sensory GPCRs that respond to adiverse variety of endogenous ligands, including peptides, lip-ids, neurotransmitters, nucleotides and ions, and thus aretermed ‘endoGPCRs’ [5]. The human genome is estimated tocontain an additional 380 or more chemosensory GPCRsthat recognise sensory signals such as odorants and photonsof light [5]. Of note, this latter number is substantially smallerin humans than other species, such as rat and mouse [6,7].Among the endoGPCRs, ∼ 210 bind natural ligands thathave been identified, whereas the remaining 160 are ‘orphanreceptors’ whose ligand and (patho)physiological function(s)are, as yet, unknown [5,8]. Concerted efforts have led to thedeorphanisation of many GPCRs that were initially identifiedas genes or cDNAs without known ligands [9]. As a relativelysmall fraction of known human endoGPCRs are targeted bycurrent prescription drugs, other members of this receptorfamily, in particular orphan GPCRs, seemed destined toemerge as drug targets.

2. G-protein-coupled receptor superfamily

2.1 GPCR structureAlthough the crystal structure of only one GPCR (rhodopsin)has been solved, this structure and the nucleotide and aminoacid sequence of other GPCRs has led to the inference of ageneral topological structure of GPCR [10]. A key feature isthe arrangement of seven transmembrane helices as a centralcore domain by which GPCRs intercalate into the cell mem-brane and that are connected by three extracellular and threeintracellular loops. GPCRs contain an extracellular N-termi-nus, generally with N-glycosylation sites and a C-terminalcytoplasmic domain, the latter, along with the third intracel-lular loop, as the principal sites for phosphorylation by secondmessenger kinases and by G-protein receptor kinases [11]. Inaddition to differences in the primary protein sequences,GPCRs vary greatly in length of the intracellular (and to alesser extent, extracellular) loops, as well as in length andfunction of the N- and C-termini. These distinct structuralproperties contribute to each individual receptor’s specificityin ligand binding, G-protein-coupling, homologous regula-tion (desensitisation) by agonists and heterologous regulationby other molecules.

There are two main requirements, one structural and onefunctional, for a protein to be classified as a member of theGPCR superfamily. The structural requirement relates toseven sequence segments: each ∼ 25 – 35 residues long, witha relatively high degree of calculated hydrophobicity, foldinginto α-helices that span the plasma membrane in an anti-clockwise manner. The functional requirement is the intra-cellular region required to couple the receptor toheterotrimeric G-proteins. Most GPCRs have been identi-fied based on their seven membrane-spanning α-helical

structure with extracellular N-termini and intracellularC-termini whereas interaction with G-proteins has not beendemonstrated for each putative member of the GPCR ‘fam-ily’. For this reason and because actions of the receptors mayoccur by mechanisms other than via G-proteins, somebelieve that it is more appropriate to refer to these receptorsas ‘7TM receptors’ as opposed to ‘GPCRs’ [12,13].

2.2 GPCR signal transductionGPCRs are stimulated by a wide diversity of ligands from dif-ferent chemical classes: these include cations, nucleosides,nucleotides, fatty acids and other lipid derivatives, amines,peptides and proteins, in addition to photons of light. Physio-logical ligands can be small, for example, divalent cations, bio-genic amines such as acetylcholine or noradrenaline, singleamino acids such as glutamate, or nucleotides such as ATP orUTP. Medium-size ligands include prostaglandins, small oli-gopeptides such as angiotensin II, bradykinin, and somatosta-tin, whereas larger ligands include chemokines, glucagon,neuropeptide Y, IL-8 and several glycoprotein hormones [12].

GPCRs interact with ligands that bind to the N-terminusand extracellular loops and/or the pocket formed by the fold-ing together of the 7TM segments. Ligand binding causesconformational changes of the receptor, transferring the signalinto transmembrane and cytoplasmic domains, thereby lead-ing to the interaction of heterotrimeric (α, β, γ) G-proteinswith the activated receptor. Binding of the G-protein pro-motes the exchange of GDP for GTP on the Gα subunit fol-lowed by the dissociation of this subunit from the Gβγ dimer.The activation of intracellular signalling effectors, such as ade-nylyl cyclase (AC), phospholipase C (PLC) or ion channels,depends on the type(s) of G-protein subunits activated andits/their pattern of linkage to effectors, which, in turn, regu-late production of second messengers [12]. Recent workemphasizes that different agonists interacting with a particularGPCR may selectively activate different G-proteins and sig-nalling pathways (termed ‘agonist trafficking’) [14,15]. Signaltransduction is terminated by hydrolysis of GTP to GDP viaGTPase activity of the Gα subunit and reassociation ofGDP-bound-Gα with Gβγ. This GTPase activity is enhancedby GTPase-activating proteins (GAPs) that have been termedregulators of G-protein signalling (RGS) proteins; in somecases, this enhancement occurs via effector molecules, but itmore commonly involves distinct RGS proteins [16,17].

The existence of multiple species of G-protein α-, β- andγ-subunits increases GPCR signalling complexity and specifi-city. Approximately 27 different heterotrimeric G-proteinswith 23 Gα, 5 Gβ, and 12 Gγ subunits have, thus far, identi-fied [16]. There are four main groups of Gα: Gs, Gi/o, Gq/11, andG12/13, based on their sequence similarities (Figure 1). The Gs

and Gi/o proteins classically cause the activation and inhibitionof AC, respectively, which, in turn, modulate the level of sec-ond messenger cyclic AMP (cAMP). Gq/11 activates PLC,resulting in the production of two second messengers: inositol1,4,5-trisphosphate (IP3), which increases intracellular Ca2+

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Expert Opin. Ther. Targets (2005) 9(6) 1249

concentration and diacylglycerol (DAG), which activates pro-tein kinase C (PKC). The G12/13 family is less well understood,but G12 appears to regulate signalling via the low molecularweight G-protein, Rho and by activation of phospholipase D(PLD), whereas G13 regulates Rho and in addition, theNa+-H+ exchanger [18-20].

Amplification of the activation of G-proteins transmitssignals to several distinct effector systems (Figure 1), forexample, AC/cAMP from Gs/Gi to the activation ofcAMP-dependent protein kinase A and other recently recog-nised signalling mechanisms: cyclic nucleotide-gated ionchannels and Epac, a regulator of the low molecular weightG-protein, Rap1 [21,22]. Gq/G11 that act via IP3 and DAG toPKC also can activate Ca2+/calmodulin-dependent proteinkinases [23]. Signal transduction by Gi/o family membersinvolves not only inhibition of AC activity, but also ability of

the Gβγ dimer to activate certain PLCs and to regulate othereffectors, such as certain ion channels. The G12/13 proteins,via the effector PLD, stimulate the production of DAG and,in turn, can activate various PKC isoforms. The phosphor-ylation cascades triggered by second messengers transfer sig-nals to proteins in the plasma membrane, cytoplasm and thenucleus, thereby regulating a diverse array of physiologicalfunctions that include neurotransmission, vision, smell, taste,secretion, metabolism, cell proliferation, differentiation anddeath in response to what began as an extracellular stimulus.

2.3 GPCR classificationAlthough GPCRs display a common overall structure theyshare relatively limited sequence similarity and vary consid-erably in size of the extracellular N-terminus, cytoplasmicloops and C-terminus. On the basis of sequence homology,

Figure 1. GPCR-mediated stimulation of GTP-GDP exchange in the regulation of heterotrimeric G-proteins and effectors forthe major classes of G-proteins: Gs/Gi, Gq/11 and G12/13. Agonist binding induces conformational changes in GPCR and activates thereceptors to promote exchange of GTP for GDP on Gα subunits of heterotrimeric G-proteins. Activated GTP-bound Gα subunits dissociatefrom the Gβγ complex, facilitating the regulation of effectors by Gα and Gβγ and the resultant modulation of activity of 'downstream'signalling pathways. Examples of effector mechanisms for each G-protein family are shown. Signal transduction and the activation ofG-proteins is terminated by GTP hydrolysis via intrinsic GTPase activity of Gα and the augmentation of this activity by RGS proteins and bycertain effectors that function as GTPase activating-proteins. Gα and Gβγsubunits insert into plasma membrane via lipid modificationsindicated by squiggly lines.AC: Adenylyl cyclase; DAG: Diacylglycerol; GEF: Guanine-nucleotide exchange factor; GPCR: G-protein-coupled receptor; IP3: Inositol 1,4,5-trisphosphate;PA: Phosphatidic acid; PAP: Phosphatidic acid phosphohydrolase; PC: Phosphatidylcholine; PIP2: Phosphatidylinositol bisphosphate; PKA: Protein kinase A; PKC: Proteinkinase C; PLC: Phospholipase C; PLD: Phospholipase D; RGS: Regulators of G-protein signalling

Agonist

GPCRExtracellular

Intracellular

RGS, PLC

GTP GDP

Pi

βγ

βγ

γβ

γβG12/13

α-GTP

γβ

βγ

Gs Gi/o

AC cAMPATP PKA

PLC DAGPIP2PKC

IP3 Ca2+

PLD PAPC DAGCholine

PKC

Rho Rho kinase

PAP

RGS

GTP GDP

Pi

p115RhoGEF

GTP GDP

Pi

G s , G i/o

G q/11

G 12/13

Gq/11

α-GTP

Gs/Gi/o

α-GTP

G12/13

α-GDP

Gq/11

α-GDP

Gs/Gi/o

α-GDP

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functional domains, as well as ligand binding mode, thissuperfamily has been grouped into six families (A, B, C, D,E and F) that include GPCRs identified in both vertebratesand invertebrates (Figure 2). Three major families (A, B andC) encompass mammalian GPCRs whereas Families D andE identify fungal pheromone receptors and cAMP receptorsand Family F contains archaebacterial opsins [24]. Family A,the largest group, includes rhodopsin-like (or adrenergicreceptor-like) receptors that harbor short N-termini anddisplay highly conserved amino acids within each trans-membrane domain. Family A is further divided into threemajor subgroups (and three additional minor subgroups)according to their ligand size and binding site: the firstgroup includes receptors activated by small ligands such asbiogenic amines, nucleotides and small peptides that bind

to a transmembrane pocket. Receptors in the second grouprecognise oligopeptides and proteins such as IL-8, chemok-ines and thrombin. These ligands bind to the N terminus,the extracellular loops, and transmembrane regions that areclose to the extracellular loops. The third group containsreceptors stimulated by glycoprotein hormones such asluteinising hormone (LH), thyroid-stimulating hormone(TSH), and follicle-stimulating hormone (FSH). The lig-and binding region for this group is located in a relativelylarge extracellular N-terminus [25]. Family B receptors, char-acterised by longer N-terminus (> 100 residues) with sixconserved cysteine residues, are stimulated by peptide pro-teins such as glucagons, calcitonin, secretin, parathyroidhormone, or vasoactive intestinal peptide. Family C mem-bers, including metabotropic glutamate and γ-aminobutyric acid (GABA) receptors, have long N-termini (500 –600 residues) folded as a separate binding domain for theirrespective ligands.

A somewhat different classification system, proposed byBockaert and Pin (Figure 3), includes Families 1 – 5 in addi-tion to the cAMP receptor family that is found in the slimemold Dictyostelium discoideum [26]. Family 1 is the largest andfurther divides into three subfamilies (1a, 1b and 1c). Sub-family 1a contains receptors for small ligands, such as rho-dopsin and catecholamines. Subfamily 1b contains GPCRsfor peptides including IL-8 and thromobin. Subfamily 1ccontains receptors for glycoprotein hormones (LH, FSH andTSH). Family 2 comprises receptors for large peptides such asglucagons and secretin. Family 3 includes metabotropic gluta-mate receptors, GABA receptors, and a group of putative phe-romone receptors that couple to the G-protein Go (termedVRs and Go-VN). In contrast, pheromone receptors coupledto Gi and called VNs belong to Family 4. Both the Frizzledand the Smoothened receptors are grouped as Family 5.

Another classification system, the GRAFS system (Figure 4),organises only mammalian GPCRs [27]. Based on phylogeneticanalyses for > 800 human GPCR sequences, the receptors havebeen grouped into 5 major families: the ‘G’ family with15 glutamate receptor members; the ‘R’, Rhodopsin, familywith 701 members, equivalent to the Family A in the A–F sys-tem mentioned above. This largest family is further subdividedinto 4 groups (α, β, γ, δ) with an estimated 460 olfactoryreceptors in the δ group. The ‘A’ family is the adhesion recep-tor group whereas the ‘F’ family includes both the Frizzled andtaste receptors with 24 members. The final ‘S’ family is thesecretin receptor family consisting of 15 members.

A fourth classification scheme, one recently published bythe International Union of Pharmacology (IUPHAR) aspart of ongoing efforts in receptor nomenclature and classi-fication [8], omits sensory (7 opsin-like, 39 taste and ∼ 400olfactory) receptors and divides the ‘nonsensory’ receptorsinto 3 classes plus 11 Frizzled/Smoothened receptors: Class1, 2 and 3 with 276, 53 and 19 members, respectively. Thisscheme is akin to those used by Kolakowski [24] and by Vas-silatis et al. [5], the latter of which included a slightly larger

Figure 2. The A–F classification system of GPCRs. Theprototype receptors for each family are: Family A, β2-adrenergicreceptor; Family B, calcitonin receptor; Family C, metabotropicglutamate receptor; Family D, fungal pheromone receptor;Family E, Dictyostelium discoideum cAMP receptor; Family F,Halobacterium halobium bacteriorhodopsin. Family A consists ofsix subfamilies (A1-A6): A1, olfactory and adenosine receptors; A2,biogenic amine receptors; A3, neuropeptide receptors andvertebrate photopigments; A4, invertebrate photopigments; A5,paracrine/autocrine receptors; A6, other receptors. Family B hasthree subfamilies: B1, calcitonin-like receptors; B2, parathyroidhormone/parathyroid hormone related peptide receptors; B3,secretin-like receptors. Family D contains two subfamilies: D1,STE2; D2, STE3. The numbers in parentheses indicate the numberof members in each family/sub-family. Receptors inside the big bluecircle, including families A, B, and C, are mammalian receptors.GPCR: G-protein-coupled receptor.Adapted from data of Kolakowski LF [24].

F(6)E

(6)(5)

D1 D2D

(4) (5)D2

A

BC

(1)

(10)

(99) (211)

(118)

(33)(227)

(32)

(11) (4)B2B1

B3

A1 A2

A3

A4A5

A6

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number of receptors. IUPHAR plans to update its receptorlist every six months and this is accessible from theirwebsite [201].

3. Genetic polymorphisms

During completion of the Human Genome Project, it wasrecognised that GPCR loci possess a substantial number ofDNA variants, most of which occur within the coding and5′ untranslated regions (5′UTR). A genetic ‘polymorphism’,including nucleotide insertion or deletion or exchange of asingle nucleotide (i.e. single nucleotide polymorphism[SNP]), is defined as a genetic variant that occurs at a locuswith an allelic frequency of ≥ 1%. SNPs account for ∼ 90% ofall sequence variations and generally occur at a frequency∼ 1 in 1000 bases of coding/regulatory sequence [28]. In con-trast, the term ‘ mutation’ is generally used for a rare, geneticvariant that occurs as a germline-transmitted change orsomatic variation in isolated tissues.

Both somatic and germline genetic variants at GPCR locihave the potential to affect GPCR signalling and function.However, most polymorphic variants are unlikely to alterGPCR function because they occur in introns or in noncod-ing regions of the GPCR genes. SNPs in the coding regions ofGPCRs can either change encoded amino acids (i.e., are ‘non-synonymous’) or can be ‘silent,’ because they produce nochange in protein sequence (i.e., they are ‘synonymous’) oreven if sequence is changed, the alteration occurs in a regionnot essential for ligand binding or signal transduction.Another possibility is that synonymous variants might createor delete sites involved in binding and action of splicingenhancers of gene expression [27,28]. To date, most research has

focused on nonsynonymous SNPs as variants that potentiallyalter signalling and regulation of GPCRs [29-31].

Polymorphisms that alter GPCR function are likely respon-sible for inter-individual differences in receptor expression,ligand binding, and signalling. Such variants thus may helpexplain why specific individuals (or populations) fail to

Figure 3. The 1 – 5 system of GPCR classification. Family 1 divides into three groups and contains GPCRs for small ligands (Group 1a), forpeptides (Group 1b), and for glycoprotein hormones (Group 1c). Family 2 comprises receptors for large size hormones such as glucagon andsecretin with a possible link (indicated by dotted line) to the cAMP Family found in Dictyostelium discoideum. Family 3 includes metabotropicglutamate receptors. Fz, frizzled receptors. Smo, smoothened receptors. Ligand binding modes are shown for Families 1, 2 and 3.GPCR: G-protein-coupled receptor.Adapted from adendogram (established with Clustal W) by Bockaert and Pin [26].

1 2 31b 1c1a Fz/Smo

receptors

1

1b1a

54

family

cAMPPheromonereceptors

1b 1c

2 3

Figure 4. The GRAFS classification system of human GPCRs,as determined by phylogenetic relationships. Numbers inparentheses indicate members in each family/subfamily.* In R, the δ-subfamily refers to 58 nonolfactory and an estimated 460 olfactoryreceptors.GPCR: G-protein-coupled receptor; TAS2: Taste 2.Adapted from data in Fredriksson et al. [27].

Frizzled/TAS2

F

A Adhesion

S Secretin

(15)

GlutamateG

(15)

(24)

(24)

R Rhodopsin

(701)

S Secretin

(15)

GlutamateG

(15)

δ

β

α (24)

(35) (59)

(*)

γ

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respond to a particular drug or yield variable response to ther-apeutic agents, in particular via alteration in drug affinity andactivity, (i.e., the pharmacodynamics of drug response). Inaddition, genetic variation in GPCRs has the potential to con-tribute to inter-individual differences (‘risk factors’) in diseasesusceptibility and disease progression/complications.

4. G-protein-coupled receptors polymorphisms

It is currently generally believed that distinct conformationalstates of GPCRs are induced by different ligands and that, inturn, these different conformational states can promote differ-ential activation of G-proteins and their effectors, as well asdifferences in susceptibility to receptor desensitisation(including agonist-promoted internalisation) [32]. GPCRs cancomplex with each other to form functional homo- or het-erodimers or interact with other proteins involved in the regu-lation of GPCR targeting, function and turnover [33]. It is theauthors’ belief that understanding of the role of genetic vari-ants/polymorphisms in mechanisms of GPCR expression,binding, activation and regulation – and perhaps in the abilityof receptors to recognise and respond to particular pharmaco-logical agents – is likely to become important for the design,testing and approval of new drugs. In the remainder of thisarticle, the authors use selected examples to discuss currentlyknown, naturally-occurring variants that are involved inaspects of GPCR expression, function and protein interac-tions. The authors also comment upon their potential as ther-apeutic targets. The subsequent sections are divided accordingto the influence of genetic variants on receptor expression atthe transcription level, on receptor folding and expression oncell surface and receptor function. In terms of transcriptionalregulation of receptors, genetic variants have been found inpromoters, 5′UTR including 5′ leader cistrons, and 3′UTR aswell as polymorphisms that change RNA splicing or RNAediting. Other polymorphic alleles can impact on receptorfolding or expression of the cell surface by affecting receptortrafficking, dimerisation, desensitisation or downregulation.Still other variants can alter ligand binding, G-protein-cou-pling and second messenger formation. In addition, theauthors also discuss polymorphisms that modify receptor con-stitutive activity and those identified in certain orphanGPCRs. The current article expands upon previous informa-tion regarding genetic variants of GPCRs [29,31,32,34]. Table 1provides a list of sequence variants identified in humanGPCR genes.

4.1 Polymorphisms involved in GPCR receptor expression4.1.1 Polymorphisms in promotersProbably the best-studied example of genetic variants in apromoter of a GPCR are the findings with the CC chemok-ine receptor-5 (CCR5) [35,36]. This receptor plays a crucialrole in HIV-1 pathogenesis, serving as a co-receptor for viralentry; based on this action, CCR5 promoter polymorphisms

influence progression of AIDS [37]. McDermott et al. identi-fied an A/G polymorphism at basepair 59029 in the CCR5promoter. Both alleles are common, 43 – 68% for 59029-A,depending on ethnicity. The 2 variants differ in their impacton in vitro promoter activity, 59029-G having 45% loweractivity than 59029-A. This difference has a clinicallyimportant impact: homozygous G/G subjects have slowerAIDS progression compared with A/A subjects followingHIV-1 infection, thus implying that CCR5 59029-G/G is aprotective allele [38]. This protective effect may also resultfrom reduced CCR5 mRNA production in subjects with the59029-G/G genotype. Such data have provided strong impe-tus for targeting CCR5 and its promoter polymorphic site indevelopment of new treatments for HIV-1 infection.

Several studies have concentrated on the possible contribu-tion of GPCR promoter polymorphisms in cardiovascular dis-eases. One example is the bradykinin B2 receptor. Erdmannet al. identified three SNPs in the promoter of this receptor inindividuals with cardiomyopathy: The -412C/G allele disruptsa binding site for the transcription factor Sp1; the -704C/Tvariant destroys a different nuclear protein binding site and the-78C/T mutation decreases the binding affinity of an unknownprotein [39]. All three polymorphisms affect the basal transcrip-tion level of the B2 receptor gene. Another example is angi-otensin II Type 2 receptor (AT2R) with a frequent variant,1334T/C, in the promoter region. This polymorphism is pre-dicted to perturb the binding of a transcriptional repressorCBF1 and is associated with hypertension in Chinese men [40].Alpha2C adrenergic receptors (α2C AR) provide an additionalexample: several promoter polymorphisms of α2C AR, togetherwith variants found in 5′UTR and 3′UTR, are organised intomultiple haplotypes, certain of which influence receptormRNA and protein expression and in addition, may contributeto cardiomyopathy [41,42].

4.1.2 Polymorphisms in 5′UTRGPCR genetic variants also occur in 5′UTRs. For example,5′UTR polymorphisms of two dopamine receptor genes(DRD2 and DRD4) can influence level of receptor expressionand contribute to individual variation in response to antipsy-chotic drug therapy [43-45]. Arinami et al. identified a -141Callele insertion/deletion polymorphism in D2 receptors, amajor target for neuroleptic agents used to treat schizophrenia[43]. The authors used reporter assays to show that the -141CDel polymorphism decreases the promoter activity; they alsoobserved an association of the -141C Ins polymorphism withschizophrenia in a case-control study in a Japanese population.There are ethnic differences in this effect because a case-con-trol study of schizophrenia in a Caucasian population showedthe reverse association [44]. Okuyama et al. identified a -521C/T polymorphism in the DRD4 gene and detected lower tran-scriptional activity from the -521T allele, as shown by anin vitro reporter assay [45]. In addition, the authors detected aweak association of the more transcriptionally active -521Cpolymorphism with schizophrenia. Previous findings have

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Table 1. A selection of known sequence variants identified in human GPCR genes with suggested or documented impact on signalling and/or disease

Receptor Wild type- > Variant Cellular mechanism/functional consequence/disease Reference

5-HT1A Gly22Ser Attenuated receptor downregulation and desensitisation [83]

-1019C/G Decreased binding of transcription repressorAssociated with predisposition to mental illness

[116]

5-HT1B Phe124Cys Increased binding affinity for agonist [117]

5-HT2A His452Tyr Blunted intracellular calcium mobilisationDecreased response to clozapineAltered kinetics of receptor desensitisation

[79,118,119]

-1438G/A Associated with food and alcohol intake [120]

Associated with energy and food intake [121]

5-HT2C Cys23Ser Decreased agonist binding affinityIncreased constitutive activityDecreased clozapine responseAssociated with psychotic symptoms in Alzheimer’s disease

[92,93,122]

Haplotype 3 Enhanced promoter activityAssociated with resistance to obesity and Type II diabetes

[123]

α2A AR Asn251Lys Enhanced agonist-dependent Gi coupling [124]

α2B AR Deletion (Del)301-303 Decreased GRK-mediated phosphorylationDecreased agonist-promoted desensitisation

[80]

α2C AR Del322-325 Decreased Gi couplingIncreased risk for CHFSynergistic increase of CHF risk with β1 AR Gly389Arg

[42,125]

3′UTR21bp insertion (Ins)/Del

Not associated with antipsychotic response [59]

β1 AR Ser49Gly Enhanced agonist-promoted downregulation [126]

Gly389Arg Enhanced basal and agonist-dependent Gs couplingSynergistic increase of CHF risk with α2C AR Del322-325

[42,127]

β2 AR -47T/C Altered receptor expression via translational effects [48]

Arg16Gly Enhanced agonist-promoted downregulation of receptorDecreased response to albuterol

[85,128]

Gln27Glu Resistance to downregulation, unless coupled with Gly16Reduced receptor desensitisationAssociated with propranolol-induced dyslipidaemia

[81,84,129]

Thr164Ile Altered ligand binding and Gs couplingBlunted receptor desensitisation

[82,130]

β3 AR Trp64Arg Associated with obesity [131]

Angiotensin II Type 1 receptor (AT1R)

3′UTR1166A/C

Associated with hypertensionAssociated with elevated blood pressure

[53-55]

AT2R 1334T/C Associated with hypertension in Chinese men [40]

Bradykinin B2 receptor -78C/T,-412C/G,-704C/T

Disrupted transcription factor bindingPossible association with cardiomyopathy

[39]

Calcium-sensing receptor

Lys47Asn Impaired calcium sensingAssociated with ADH

[95]

Glu604Lys Increased calcium sensingAssociated with ADH

[132]

Glu767Lys Associated with ADH [133]

5-HT: 5-Hydroxytryptamine; ADH: Autosomal dominant hypocalcaemia; AR: Adrenergic receptor; CaM: Calmodulin; CCK: Cholecystokinin; CCR: CC chemokine receptor; CHF: Congestive heart failure; D: Dopamine receptor; GPR: G-protein-coupled receptor; GRK: G-protein-coupled receptor kinase; HH: Hypogonadotropic hypogonadism; NDI: Nephrogenic diabetes insipidus; RP: Retinitis pigmentosa; SNP: Single nucleotide polymorphism; UTR: Untranslated region.

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Leu773Arg Increased calcium sensitivityAssociated with sporadic hypoparathyroidism

[76]

Phe788Cys Increased calcium sensitivityAssociated with familial hypoparathyroidism

[77]

CCK1 Val365Ile Decreased receptor expression and coupling efficiency [134]

CCK2 Val125Ile Enhanced binding affinity [134]

CCR2 Val64Ile Delayed progression of AIDS [135,136]

CCR5 59029A/G Reduced promoter activityDelayed progression of AIDS

[38]

CCR5 P1 Increased progression of AIDS [137]

CCR5 ∆32 Altered binding affinityResistance to HIV-1 infection

[38,138]

Chemo-attractant receptor expressed on TH2 cells (CRTH2)

G1544CG1651A

Decreased mRNA stabilityAssociated with expression and severity of asthma

[139]

D2 -141C Ins/Del Decreased promoter activity [43]

Val96Ala Reduced binding affinity for clozapine and dopamine [140,141]

Pro310Ser Altered receptor coupling to Gi [140]

Ser311Cys Decreased agonist binding affinity [140]

3′UTR52A/G Associated with depressiveness and anxiety [58]

D3 Ser9GlyPromoter SNPs

Haplotype associated with schizophrenia [49]

D4 -512C/T Reduced transcriptional activity [45]

Val194Gly Decreased agonist bindingDecreased sensitivity to dopamine and clozapine

[94]

Endothelin receptor B Cys109Arg Disruption of putative signal sequence [142]

Trp276Cys Altered receptor coupling to Gq [96]

Ser390Arg Altered G-protein coupling [142]

Follicle-stimulating hormone

Ala189Val Altered protein foldingInactivation of receptor

[143]

Phe591Ser Diminished agonist-stimulated cAMP production [98]

Thr307Ala Associated with infertility in women [144,145]

Asn680Ser Associated with female infertility [144,145]

Prostacyclin receptor Arg212His Decreased binding affinity at lower pHAbnormal activation at physiological and lower pH

[146]

Muscarinic receptor subtype 3

Promoter haplotype Possible association with asthma and atopy [147]

Mu opioid receptor Asn40Asp Increased affinity/potency of β-endorphin [71]

Arg260His Reduced basal G-protein coupling [148]

Arg265His Reduced basal G-protein couplingReduced CaM binding

[148]

Ser268Pro Reduced CaM binding [148]

Protease-activated receptor 2

Phe240Ser Altered ligand binding and effector activation [149]

Table 1. A selection of known sequence variants identified in human GPCR genes with suggested or documented impact on signalling and/or disease (continued)



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P2Y2 Arg334Cys Putative additional palmitoylation site [150]

P2Y12 CAdel at codon 240 Nonfunctional Gi-linked ADP receptorAssociated with bleeding disorder

[151]

Rhodopsin Thr4Lys Affects N-glycosylationAssociated with RP

[69]

Asn15Ser Affects N-glycosylationAssociated with RP

[70]

Phe52Tyr Associated with RP [152]

Gln344stop Autosomal dominant RP [153,154]

Thyroid-stimulating hormone

Ile630Leu Constitutive activity [91]

Phe631Leu Constitutive activity [90]

Vasopressin receptor Arg113Trp Reduced receptor transportIncreased ligand binding affinityAssociated with X-linked NDI

[72,155]

Arg137His Unable to couple to GsAssociated with X-linked NDI

[97]

Try128Ser Decreased ligand binding affinityAssociated with X-linked NDI

[72]

Lys44Pro, Trp164Ser,Ser167Lys, Ser167Thr

Defective processingAssociated with X-linked NDI

deltaV278 Blocked intracellular transportAssociated with X-linked NDI

[73]

GPRA Asn107Ile Asthma susceptibility [101]

GPR 40 Arg211His Variation of insulin secretory capacity [105]

Arg211His, Asp175Asn Not associated with Type 2 diabetes mellitus [104]

GPR 50 Thr532Ala Susceptibility to bipolar disorder in females [106]

Val606Ile Associated with depressive disorder in females [106]

∆502-505 Associated with bipolar disorder and depressive disorder [106]

GPR 54 Cys223Arg Impaired signallingAssociation with HH

[109]

Arg297Leu Association with HH [109]

155-bp del. Receptor truncationAssociation with HH

[107]

Leu148Ser, Arg331stop,stop399Arg

Loss of receptor functionAssociation with HH

[108]

GPR 75 Asn78Lys, Pro99Leu,Ser108Thr, Gln234stop

Possible connection with retinal pathology inage-related macular degeneration

[156]

GPR 154 HaplotypesH1, H5

Association with childhood allergy and asthma [102]

Table 1. A selection of known sequence variants identified in human GPCR genes with suggested or documented impact on signalling and/or disease (continued)



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shown increased expression of DRD4 in brain samples fromschizophrenic patients [46]; thus, the -521C/T allele may be acandidate gene that affects development/progression ofschizophrenia and response to treatment.

The 5′ leader cistron (5′LC) is a short open reading frame(ORF) found in the 5′ UTRs of eukaryotic mRNA that caninfluence the translational efficiency of the main ORF, pre-sumably by encoding peptides that influence translation[47,48]. Several studies have suggested that 5′LC polymor-phisms modulate GPCR expression [47-49]. For example, theD3 receptor gene has a putative 5′LC encoding a 36-residuepeptide in which changing A to G at position 204 upstreamof its main ORF causes a Lys→Glu substitution at residue 9.A cluster of SNPs including this 5′LC polymorphism andothers occurring in the promoter (as well as coding regions)may associate with susceptibility to schizophrenia [49].

Another example is the 5′LC found in the β2-AR, which islocated 102 nucleotides upstream of the main coding regionand encodes a putative 19-residue peptide. McGraw et al. [48]

identified a polymorphic T/C allele at position -47, whichresults in either Cys (codon TGC) or Arg (codon CGC) atresidue 19 with allelic frequencies of 0.37 and 0.63, respec-tively. The 5′LC Cys19 is associated with higher β2-AR recep-tor density via an effect on translational regulation of receptorexpression as amounts of the Arg19 and Cys 19 mRNA tran-scripts are similar. The 5′LC Cys19 variant is in linkage dise-quilibrium with two nonsynonymous SNPs (Arg16 andGln27) in the coding region and is part of a receptor haplo-type that influences receptor expression and contributes tointer-individual variation in β-AR responsiveness [50]. Yamadaet al. found the 5′LC-Arg19 variant is significantly associatedwith obesity and diabetic status [51].

4.1.3 Polymorphisms in 3′UTRThe 3′UTR plays a pivotal role in the post-transcriptional reg-ulation of gene expression via mechanisms that regulatemRNA stability, translation rate, nuclear transport and polya-denylation status [52]. Numerous studies have focused on a3′UTR polymorphism of the angiotensin II Type 1 receptorgene (AT1R) at nucleotide 1166 with A or C allele. Szombathyet al. [53] observed an association of the C allele and elevatedsystolic and diastolic blood pressure in overweight subjectswith hypertension. Bonnardeaux et al. and Wang et al. [54,55]

detected increased frequency of 1166C in Caucasian subjectswith essential hypertension, suggesting that this polymorphismis involved in the regulation of blood pressure. Miller et al. [56]

demonstrated the association of the C allele with lower base-line renal function in healthy Caucasian subjects. Alvarez et al.[57] reported that the 1166C polymorphism of AT1R, togetherwith a deletion polymorphism of angiotensin-convertingenzyme, increases the risk for coronary artery disease, while noassociation was found between the 1166 A/C alleles of AT1Rand early coronary disease. It was, therefore, proposed that thispolymorphism may contribute to other cardiovascular diseasesin addition to essential hypertension.

A 3′UTR polymorphism, an A/G allele in exon 8, 52 basepairs downstream of the stop codon, has been identified in thedopamine D2 receptor gene (DRD2) [58]. Finckh et al. reportedthat homozygous A/A was associated with increased depression,anxiety, and suicidal tendency in German alcoholics challengedwith a dopaminergic agonist, leading the authors to postulatethat the exon 8 A/A genotype results in a reduced level of DRD2expression. Another example of a 3′UTR variant occurs in α2C

AR, with a 21 base pairs Ins/Del 70 bp downstream of the stopcodon: ‘Ins’ identifies individuals who have two copies of the 21nucleotide sequence and those with ‘Del’ have only one copy; noassociation was noted for this polymorphism and antipsychoticdrug response in schizophrenic subjects [59].

4.1.4 Polymorphisms in splicingAlternative splicing contributes to molecular diversity inGPCRs. For example, the ETB receptor gene can generatewild-type and splice variants that have similar binding proper-ties; upon agonist stimulation; however, the ETB splice variantfails to activate PLC due to defective G-protein coupling [60].Because GPCR isoforms may vary in pharmacological responsesand can have differential expression according to tissue type anddevelopmental stage, it is important to consider alternativesplicing in therapeutic drug design. For instance, undifferenti-ated keratinocytes only express the full-length calcium-sensingreceptor (CASR), whereas a shorter spliced variant of CASRlacking exon 5 and without normal function replaces thefull-length receptor in differentiated keratinocytes [61]. A splicevariant of the 5-HT4 receptor identified only in human brain, incontrast to wild-type 5-HT4 receptor with expression in multi-ple tissues, has a shorter C-terminus and possesses constitutivereceptor activity [62]. It has been proposed that the action ofclinically used 5-HT4 receptor agonists that show ‘tissue-specificresponses’ (i.e., functioning as super agonists in neurons, but aspartial agonists in other tissues) may be explained by differentialexpression of splice variant receptors [63].

4.1.5 Polymorphisms in RNA editingRNA editing is a post-transcriptional modification mecha-nism that alters the primary sequence of a mRNA transcriptand thereby generates functionally different GPCRs [64].Burns et al. have reported that the 5-HT2C receptor tran-scripts, via RNA editing, generate multiple isoforms, one ofwhich has a 10 – 15-fold reduction in G-protein-couplingefficiency [65]. The brain expresses two different forms of thehuman 5-HT2C receptors and the edited forms, as well as thenon-edited form, differ in their constitutive activity [66]. Thenon-edited form can activate PLD via G13 (Figure 1), whereasone of the edited forms lacks such ability [67].

4.2 Polymorphisms involved in receptor folding/formation4.2.1 Polymorphisms in receptor traffickingProper receptor folding and trafficking to the plasma mem-brane depend, at least in part, on accurate glycosylation and

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processing of glycosylation sites, which, in turn, controlGPCR expression and activity on the plasma membrane[68]. Several polymorphisms change N-glycosylation inGPCRs. Two polymorphic alleles found in rhodopsin,Asn15Ser and Thr4Lys, alter N-glycosylation of rhodopsindirectly (codon 15) or indirectly (codon 4 affecting codon 2Asn) and have been linked to different forms of retinitispigmentosa [69,70]. The Asn40Asp variant of the µ-opioidreceptor exhibits a higher binding affinity for β-endophinand has significantly higher frequency in Hispanic non-opi-oid-dependent subjects compared to other ethnic groups[71]. Several polymorphisms have been identified in theV2-vasopressin receptor and appear to contribute toX-linked nephrogenic diabetes insipidus (NDI): Lys44Pro,Trp164Ser, Ser167Lys, and Ser167Thr [72]. These variantsabolish glycosylation, and one in-frame deletion polymor-phism, deltaV278, causes retention within the pre-Golgicompartment [73].

4.2.2 Polymorphisms in receptor dimerisationRecent data indicate that GPCRs can exist as homo- or het-erodimers and that such organisation is critical for receptorexpression and function on the cell surface [33]. A prerequisiteto demonstrate if polymorphisms alter receptor dimerisationis to clearly define the dimer interaction domains on bothreceptor partners. Limited data are available regarding this keyfeature, although short peptides encoding the putative dimer-isation motifs compete with the full-length receptor, presuma-bly disturbing homodimerisation and leading to a reductionin signal transduction [74].

A putative dimerisation domain (residues 770 – 782)was identified in CASR, along with several variants aroundthis region that are predicted to influence receptor dimeri-sation [75]. One such variant, Phe788Cys, was discovered ina Japanese patient with severe familial hypoparathyroidism,and another subject with sporadic hypoparathyroidism washeterozygous for Leu773Arg [76,77].

4.2.3 Polymorphisms in receptor desensitisationSubsequent to their rapid activation of signalling events,GPCRs generally undergo desensitisation, which occurs viamultiple mechanisms that include receptor uncouplingfrom heterotrimeric G-proteins, receptor internalisationfrom the plasma membrane and receptor downregulationvia various proteolytic mechanisms [78]. Numerous geneticvariants of GPCR have been shown to alter receptor desen-sitisation. For example, a common polymorphism,His452Tyr, of the 5-HT2A receptor results in altered desen-sitisation kinetics and diminished signalling downstream ofreceptor activation and has been proposed as contributingto psychiatric disease [79]. A polymorphism in the α2B-ARinfluences receptor desensitisation and occurs with anallelic frequency 31% in Caucasians: a deletion of threeglutamic acids (‘Del301-303’) shows ∼ 50% of agonist-pro-moted phosphorylation compared with wild-type receptor

and a loss of agonist-promoted receptor desensitisation [80].Another example with altered receptor desensitisationoccurs in the β2-AR: Gln27Glu and Thr164Ile slow recep-tor desensitisation and render subjects (Glu27 homozygotesor Thr164Ile heterozygotes) with altered cardiac β2-ARresponses, as determined by terbutaline infusion-inducedincreases in heart rate and contractility [81,82].

4.2.4 Polymorphisms that alter agonist-promoted downregulation of GPCRsThe Gly22Ser variant of the 5-HT1A receptor attenuatesagonist-induced downregulation without an alteration inligand binding [83]. It has been postulated that individualswith the Ser22 variant have altered response to drugs used totreat depression because treatment using serotonin re-uptakeinhibitors depends on the efficiency of 5-HT1A receptordownregulation [83].

Two nonsynonymous polymorphisms, Arg16Gly and theGln27Glu, in β2-AR influence agonist-promoted downregu-lation of receptor number: the Arg16Gly and Gln27Glu vari-ants enhance or blunt, respectively, such downregulation, butshow no difference in agonist binding affinity or G-proteincoupling [84]. The impact on downregulation of coexistingGly16 and Glu27 is similar to that of the Gly16 variant,implying that the Gly16 allele dominates functional responserelative to the Glu27 allele [85]. Moreover, becauseArg16Glu27 is an extremely rare diplotype [86,87], theenhanced downregulation of Gly16 ‘dominates’ in vivo. Ofnote, individuals homozygous for Gly16 display slower bron-chodilation response upon β2-AR agonist stimulation com-pared with Arg16 homozygotes [88]. These different in vivoresponses may result from the less efficient downregulation ofthe Arg16 β2-AR, thus implicating Arg16Gly as a drug targetfor asthma therapy and perhaps to stratify patients withrespect to use of β2-AR bronchodilators based on expressionof this variant [88].

4.3 Polymorphisms in receptor constitutive activationIncreasing evidence documents the existence of ligand-inde-pendent (‘constitutive’) activity of GPCRs [89]. Compoundswith preferential binding to inactive conformations of GPCRshave been termed ‘inverse agonists,’ based on their ability tostabilise GPCRs in their inactive state, thereby decreasingconstitutive activity. Several GPCR variants that enhance oractivate constitutive activity have been linked to genetic disor-ders. For example, two polymorphisms, Phe631Leu [90] andIle630Leu [91], of the TSH receptor that function constitu-tively and diminish response to TSH have been isolated frompatients with congenital hyperthyroidism and toxic multinod-ular goiter, respectively. Such variant alleles are usually domi-nant and have been observed in a number of rare humandiseases; thus, selective design of inverse agonists has thepotential to provide useful treatments for these conditions,albeit because of their rarity, they will likely prove to be‘orphan diseases’.

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4.4 Polymorphisms involved in receptor structure and function4.4.1 Polymorphisms that influence receptor-ligand bindingA Cys23Ser variant of the 5-HT2C receptor displays loweragonist binding affinity than wild-type receptors [92]. An asso-ciation study evaluated schizophrenic patients in theirresponse to clozapine treatment and noted that patients withCys23 genotype are ‘good responders’ to clozapine comparedto subjects with Ser23 [93]. Another example is the Val194Glypolymorphism found in the dopamine D4 receptor. This vari-ant disrupts the receptor’s ligand binding domain and causesreduced sensitivity to dopamine or clozapine [94].

A Lys47Asp polymorphism, located in a residue at the N-ter-minus that is required for ligand binding, has been implicatedin calcium sensing of the CASR and has been identified in anautosomal dominant form of hypocalcaemia [95]. Two variants,Arg113Trp and Tyr128Ser, of the V2 vasopressin receptor thataffect ligand binding in a positive and negative fashion, respec-tively [72], have been linked to NDI, suggesting that therapeu-tics design for NDI or other diseases should consider ligandbinding properties altered by such polymorphisms.

4.4.2 Polymorphisms in receptor G-protein coupling/second messenger formationVariants that regulate GPCR-G-protein coupling have beenidentified in interaction sites between numerous receptors andG-proteins. For example, a Trp276Cys polymorphism of theETB receptor changes its coupling to Gq and reduces ago-nist-stimulated Ca2+ levels [96]. This variant was associatedwith Hirschsprung’s disease in a gene dosage-dependent man-ner, such that homozygous individuals have higher risk todevelop this disease than heterozygotes [96]. A V2 receptor pol-ymorphism, Arg137His, retains normal binding affinity forvasopressin, but destroys Gs coupling [97]. A variant ofPhe591Ser found in FSH receptor eliminates AC activationvia Gs even though the variant receptors resemble wild type inligand binding affinities [98].

In summary, polymorphisms in noncoding regions (pro-moter, 5′UTR or 3′UTR) or at splice junctions can have pro-found effects on GPCR expression. Level of expression of agiven receptor is critical for determining response, especiallyreceptor affinity [99]. Therefore, such GPCR polymorphismsmight be predicted to contribute to interindividual variabilityin disease susceptibility and drug responses. By contrast,sequence variations in the coding region of GPCRs have thepotential to impact on multiple aspects of receptor function,including ligand binding, receptor stability, G-protein cou-pling, or cellular trafficking, all of which can result in lowerreceptor expression and/or activity on the cell surface. Despitesubstantial research efforts [33,100] on mechanisms of GPCRtransport, dimerisation, internalisation, ligand binding andG-protein coupling, much remains unknown. Discovery ofGPCR polymorphisms and the influence of genetic variantson various steps of GPCR signalling may thus help facilitate

understanding on the mechanisms of GPCR signal transduc-tion as well as improve drug therapy and understanding ofhuman diseases that involve GPCRs.

4.5 Polymorphisms of orphan GPCRsIn spite of the absence of information regarding their naturalligand(s) or physiological function, orphan GPCRs havebeen shown to have polymorphisms and certain of these var-iants have been implicated in human (patho)physiologicalconditions.

For example, the orphan GPCR GPRA (for ‘G-protein-cou-pled receptor for asthma susceptibility’) is proposed to increasepropensity for developing asthma [101]. GPRA contains a non-synonymous SNP, Asn107Ile, located in the first extracellularloop as part of the putative ligand-binding pocket, but there isno precise explanation for the genetic association with asthma.Another group identified seven polymorphisms of GPR154(an alternative designation for GPRA) and inferred seven hap-lotypes (H1–H7) in a case-control study of childhood allergyand asthma [102]. Haplotypes H1 and H5 were significantlyassociated with asthma whereas, H1, H5 and H6 were associ-ated with allergy. Such data suggest that GPRA may be a usefuldrug target, but more information is needed regarding theidentity of its natural ligand and the role of GPRA/GPR154 inphysiology and pathophysiology.

GPR40, an orphan GPCR with high level of expression inpancreatic islet β-cells, is activated by long-chain fatty acids[103]. A His211Arg variant in the third intracellular domainhas been linked to a variation of insulin secretory capacity inhealthy Japanese men, but not in nondiabetic Danish Cauca-sians or those who have Type 2 diabetes mellitus or alteredinsulin release [104,105].

An Ins/Del polymorphism of GPR50, GPR50∆502-505, hasbeen associated with bipolar affective disorder and majordepressive disorder [106]. The deletion variant lacking fouramino acids, Thr-Thr-Gly-His, shows increased risk for boththose disorders, in particular in female subjects.

Several ‘loss-of-function’ polymorphisms have been foundin GPR54, for which the peptide kisspeptin-1 is the putativeendogenous ligand [107-109]. Four nonsynonymous SNPs(Arg331stop, stop399Arg, Cys223Arg, Arg297Leu) and oneIns/Del variant (at intron 4-exon 5 junction) were isolatedfrom patients with hypogonadotropic hypogonadism, whichsuggests that GPR54 helps regulate puberty.

5. Conclusion

In addition to growing information regarding multiple aspectsof GPCR signal transduction, an ever-increasing number ofpublications has defined genetic variants of GPCRs, theirpotential importance in human biology, as well as their possi-ble pharmaceutical application. However, the complexity ofthis receptor superfamily, including structural heterogeneity,receptor multiplicity and redundancy in signalling pathways,makes the identification – and more importantly, proof of a

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contributing role – of polymorphisms of a given receptor in aparticular disease a difficult task.

GPCR sequence variations can influence receptor expres-sion and function via a variety of mechanisms and these var-iations could be the basis for differences in individualpathophysiology and therapeutic response to drugs. Majorquestions remain, such as: which GPCR genes predispose tospecific diseases? Which of those genes are linked to (andcan be used to predict) drug response? Which polymorphicallele(s) contribute(s) to these events? Pharmacogenomicstudies must, and certainly will, continue to explore mem-bers of the GPCR superfamily. Incorporation of studies ofgenetic variants into strategies for GPCR drug discoveryand clinical drug testing has, the authors believe, an impor-tant potential to improve efficacy and decrease toxicity ofnew (and old) drugs. Nevertheless, to date, no definitive evi-dence has been provided that would dictate the applicationof testing for GPCR variants, although some tentativeexamples have been proposed [42,88,110]. Of particular note,several articles have suggested that particular β1-AR geno-types may provide useful predictive information regardingclinical response to β-blockers (as recently discussed byMuszkat and Stein [111]).

6. Expert opinion

6.1 Polymorphisms regarding ethnicity or genderGenetic background in different populations (i.e., differentethnicities) may contribute to differences in impact of specificpolymorphisms in disease and drug response, as shown byexamples of the DRD2 and the AT1R polymorphisms. Otherdata indicate striking ethnic-specific differences in expressionof genetic variants in GPCRs [50,112,113]. Therefore, one mustconsider the contribution of GPCR variants to disease anddrug-responses in an ethnic-specific manner. Further data areneeded to test ethnic-specificity of GPCR variants, in particu-lar for multifactorial diseases such as asthma, hypertension,obesity or diabetes mellitus. Such data have the potential toguide new drug development so that therapy can target ‘goodresponders’ while avoiding ‘poor responders’ or those withincreased risk for side effects. Perhaps the application of thisconcept can be expanded to tailor therapy for gender-specific

phenotypes or for therapy of patients in different age groups(Rana et al., manuscript submitted).

6.2 GPCR haplotypesSeveral GPCRs, such as the V2-vasopressin, β2-AR, andα2-AR receptors and rhodopsin [114], express multiple poly-morphic alleles that are in linkage disequilibrium and there-fore yield different receptor haplotypes. In some cases(Table 1), haplotypes – but not individual SNPs – determinethe functional role and contribute to individual differencesin susceptibility to a given disease or in drug responses. Inthe case of receptor dimerisation (Section 4.2.2), haplotypesfrom SNPs within and between two GPCR genes may helpregulate receptor expression and function. The authorsbelieve that the impact of GPCR haplotypes will prove to bemore important than individual SNPs in developing newtherapeutic strategies.

6.3 Orphan GPCRs as drug targetsIdentification of novel GPCR targets for drug discovery bybioinformatic analyses has been fruitful in recent years. How-ever, the efforts to define the cognate ligands for orphanGPCRs have yielded limited (and slowing) rewards [9,115].Although about a quarter of GPCRs have been matched tospecific endogenous ligands, a large number of the 7TMreceptors are still orphan GPCRs [8,9]. Deorphanisation (i.e.,identification of the natural agonists for these orphanGPCRs) is a critical step not only to advance the understand-ing of their role in physiology and disease, but also to aid inthe development of new, improved therapeutic agents.Although it is possible to discover antagonists without know-ing endogenous agonists (e.g., by exploiting ability to blockconstitutively activated orphan GPCRs), the authors believethat it is important to define physiological agonist(s). Discov-ery of polymorphisms of orphan GPCRs will likely open newopportunities for drug design and perhaps new approaches toindividualise therapy.

Acknowledgements

Work in the authors’ laboratory on this topic has beensupported by grants from NIH.

BibliographyPapers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.

1. WANG X, TOMSO DJ, LIU X, BELL DA: Single nucleotide polymorphism in transcriptional regulatory regions and expression of environmentally responsive genes. Toxicol. Appl. Pharmacol. (2005) 207(2 Suppl.):84-90.

2. BOCKAERT J, DUMUIS A, FAGNI L, MARIN P: GPCR-GIP networks: a first step in the discovery of new therapeutic drugs? Curr. Opin. Drug Discov. Devel. (2004) 7(5):649-657.

3. VENTER JC, ADAMS MD, MYERS EW et al.: The sequence of the human genome. Science (2001) 291(5507):1304-1351.

•• One of the two original papers that initially described the sequencing of the human genome.

4. INTERNATIONAL HUMAN GENOME SEQUENCING CONSORTIUM: Finishing the euchromatic sequence of the human genome. Nature (2004) 431(7011):931-945.

•• Most recent and complete assembly of the human genome.

5. VASSILATIS DK, HOHMANN JG, ZENG H et al.: The G protein-coupled receptor repertoires of human and mouse. Proc. Natl. Acad. Sci. USA (2003) 100(8):4903-4908.

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• First detailed compilation of human endoGPCRs.

6. SCHIOTH HB, FREDRIKSSON R: The GRAFS classification system of G-protein coupled receptors in comparative perspective. Gen. Comp. Endocrinol. (2005) 142(1-2):94-101.

7. FREDRIKSSON R, SCHIOTH HB: The repertoire of G-protein-coupled receptors in fully sequenced genomes. Mol. Pharmacol. (2005) 67(5):1414-1425.

• A recent synthesis of information regarding GPCR families in various species.

8. FOORD SM, BONNER TI, NEUBIG RR et al.: International Union of Pharmacology. XLVI. G protein-coupled receptor list. Pharmacol. Rev. (2005) 57(2):279-288.

9. CIVELLI O: GPCR deorphanizations: the novel, the known and the unexpected transmitters. Trends Pharmacol. Sci. (2005) 26(1):15-19.

• A good overview regarding GPCR orphans.

10. SAKMAR TP, MENON ST, MARIN EP, AWAD ES: Rhodopsin: insights from recent structural studies. Ann. Rev. Biophys. Biomol. Struct. (2002) 31:443-484.

• A nice synthesis of structural information regarding rhodospin.

11. FERGUSON SS: Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol. Rev. (2001) 53(1):1-24.

• A comprehensive review of GPCR endocytosis.

12. KRISTIANSEN K: Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors: molecular modeling and mutagenesis approaches to receptor structure and function. Pharmacol. Ther. (2004) 103(1):21-80.

• Detailed analysis of several key aspects of GPCR structure and function.

13. LEFKOWITZ RJ: Historical review: a brief history and personal retrospective of seven-transmembrane receptors. Trends Pharmacol. Sci. (2004) 25(8):413-422.

•• A personal, but useful, history of GPCR biology.

14. MAUDSLEY S, MARTIN B, LUTTRELL LM: The origins of diversity and specificity in g protein-coupled receptor signaling. J. Pharmacol. Exp. Ther. (2005) 314(2):485-494.

15. VAUQUELIN G, VAN LIEFDE I: G protein-coupled receptors: a count of 1001

conformations. Fundam. Clin. Pharmacol. (2005) 19(1):45-56.

16. MCCUDDEN CR, HAINS MD, KIMPLE RJ, SIDEROVSKI DP, WILLARD FS: G-protein signaling: back to the future. Cell. Mol. Life Sci. (2005) 62(5):551-577.

17. RIDDLE EL, SCHWARTZMAN RA, BOND M, INSEL PA: Multi-tasking RGS proteins in the heart: the next therapeutic target? Circ. Res. (2005) 96(4):401-411.

18. NEVES SR, RAM PT, IYENGAR R: G protein pathways. Science (2002) 296(5573):1636-1639.

19. KUROSE H: Galpha12 and Galpha13 as key regulatory mediator in signal transduction. Life Sci. (2003) 74(2-3):155-161.

20. RIOBO NA, MANNING DR: Receptors coupled to heterotrimeric G proteins of the G12 family. Trends Pharmacol. Sci. (2005) 26(3):146-154.

21. MAILLET M, ROBERT SJ, CACQUEVEL M et al.: Crosstalk between Rap1 and Rac regulates secretion of sAPPalpha. Nat. Cell Biol. (2003) 5(7):633-639.

22. RANGARAJAN S, ENSERINK JM, KUIPERIJ HB et al.: Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor. J. Cell Biol. (2003) 160(4):487-493.

23. WONG LF, POLSON JW, MURPHY D, PATON JF, KASPAROV S: Genetic and pharmacological dissection of pathways involved in the angiotensin II-mediated depression of baroreflex function. FASEB J. (2002) 16(12):1595-1601.

24. KOLAKOWSKI LF, JR.: GCRDb: a G-protein-coupled receptor database. Receptors Channels (1994) 2(1):1-7.

• First comprehensive classification of GPCRs.

25. FAN QR, HENDRICKSON WA: Structure of human follicle-stimulating hormone in complex with its receptor. Nature (2005) 433(7023):269-277.

• Recent new information regarding GPCR structure.

26. BOCKAERT J, PIN JP: Molecular tinkering of G protein-coupled receptors: an evolutionary success. EMBO J. (1999) 18(7):1723-1729.

•• Useful and insightful review on GPCRs.

27. FREDRIKSSON R, LAGERSTROM MC, LUNDIN LG, SCHIOTH HB:

The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. (2003) 63(6):1256-1272.

• Redefinition of GPCR family relationships.

28. DORIS PA: Hypertension genetics, single nucleotide polymorphisms, and the common disease:common variant hypothesis. Hypertension (2002) 39(2 Pt 2):323-331.

29. SMALL KM, MCGRAW DW, LIGGETT SB: Pharmacology and physiology of human adrenergic receptor polymorphisms. Ann. Rev. Pharmacol. Toxicol. (2003) 43:381-411.

• A useful update on adrenergic receptor polymorphism data from the authors’ laboratory.

30. SADEE W, HOEG E, LUCAS J, WANG D: Genetic variations in human G protein-coupled receptors: implications for drug therapy. AAPS PharmSci. (2001) 3(3):E22.

• Historically useful compilation of GPCR genetic variants.

31. KIRSTEIN SL, INSEL PA: Autonomic nervous system pharmacogenomics: a progress report. Pharmacol. Rev. (2004) 56(1):31-52.

32. PEREZ DM, KARNIK SS: Multiple signaling states of G-protein-coupled receptors. Pharmacol. Rev. (2005) 57(2):147-161.

•• Recent comprehensive review regarding GPCR conformational states.

33. BULENGER S, MARULLO S, BOUVIER M: Emerging role of homo- and heterodimerization in G-protein-coupled receptor biosynthesis and maturation. Trends Pharmacol. Sci. (2005) 26(3):131-137.

• Up-to-date synthesis of data and ideas regarding GPCR dimerisation.

34. RANA BK, SHIINA T, INSEL PA: Genetic variations and polymorphisms of G protein-coupled receptors: functional and therapeutic implications. Ann. Rev. Pharmacol. Toxicol. (2001) 41:593-624.

35. O’BRIEN SJ, NELSON GW: Human genes that limit AIDS. Nat. Genet. (2004) 36(6):565-574.

• Good review on CCR5 and other genes that influence AIDS.

36. WINKLER C, AN P, O’BRIEN SJ: Patterns of ethnic diversity among the genes

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that influence AIDS. Hum. Mol. Genet. (2004) 13(Spec. No. 1):R9-R19.

37. COAKLEY E, PETROPOULOS CJ, WHITCOMB JM: Assessing chemokine co-receptor usage in HIV. Curr. Opin. Infect. Dis. (2005) 18(1):9-15.

38. MCDERMOTT DH, ZIMMERMAN PA, GUIGNARD F, KLEEBERGER CA, LEITMAN SF, MURPHY PM: CCR5 promoter polymorphism and HIV-1 disease progression. Multicenter AIDS Cohort Study (MACS). Lancet (1998) 352(9131):866-870.

39. ERDMANN J, HEGEMANN N, WEIDEMANN A et al.: Screening the human bradykinin B2 receptor gene in patients with cardiovascular diseases: identification of a functional mutation in the promoter and a new coding variant (T21M). Am. J. Med. Genet. (1998) 80(5):521-525.

40. ZHANG Y, ZHANG KX, WANG GL, HUANG W, ZHU DL: Angiotensin II Type 2 receptor gene polymorphisms and essential hypertension. Acta Pharmacol. Sin. (2003) 24(11):1089-1093.

41. SMALL KM, MIALET-PEREZ J, SEMAN CA, THEISS CT, BROWN KM, LIGGETT SB: Polymorphisms of cardiac presynaptic alpha2C adrenergic receptors: Diverse intragenic variability with haplotype-specific functional effects. Proc. Natl. Acad. Sci. USA (2004) 101(35):13020-13025.

• First detailed analysis of α2C haplotype.

42. SMALL KM, WAGONER LE, LEVIN AM, KARDIA SL, LIGGETT SB: Synergistic polymorphisms of beta1- and alpha2C-adrenergic receptors and the risk of congestive heart failure. N. Engl. J. Med. (2002) 347(15):1135-1142.

• First evidence of synergism between adrenergic receptor variants that influence heart failure.

43. ARINAMI T, GAO M, HAMAGUCHI H, TORU M: A functional polymorphism in the promoter region of the dopamine D2 receptor gene is associated with schizophrenia. Hum. Mol. Genet. (1997) 6(4):577-582.

44. BREEN G, BROWN J, MAUDE S et al.: -141 C del/ins polymorphism of the dopamine receptor 2 gene is associated with schizophrenia in a British population. Am. J. Med. Genet. (1999) 88(4):407-410.

45. OKUYAMA Y, ISHIGURO H, TORU M, ARINAMI T: A genetic polymorphism in the promoter region of DRD4 associated

with expression and schizophrenia. Biochem. Biophys. Res. Commun. (1999) 258(2):292-295.

46. STEFANIS NC, BRESNICK JN, KERWIN RW, SCHOFIELD WN, MCALLISTER G: Elevation of D4 dopamine receptor mRNA in postmortem schizophrenic brain. Brain Res. Mol. Brain Res. (1998) 53(1-2):112-119.

47. PAROLA AL, KOBILKA BK: The peptide product of a 5’ leader cistron in the beta 2 adrenergic receptor mRNA inhibits receptor synthesis. J. Biol. Chem. (1994) 269(6):4497-4505.

• Nice mechanistic assesement of role of 5′ leader cistron in GPCRs.

48. MCGRAW DW, FORBES SL, KRAMER LA, LIGGETT SB: Polymorphisms of the 5’ leader cistron of the human beta2-adrenergic receptor regulate receptor expression. J. Clin. Invest. (1998) 102(11):1927-1932.

• First evidence of 5′ leader cistron genetic variant in GPCR.

49. SIVAGNANASUNDARAM S, MORRIS AG, GAITONDE EJ, MCKENNA PJ, MOLLON JD, HUNT DM: A cluster of single nucleotide polymorphisms in the 5’-leader of the human dopamine D3 receptor gene (DRD3) and its relationship to schizophrenia. Neurosci. Lett. (2000) 279(1):13-16.

50. DRYSDALE CM, MCGRAW DW, STACK CB et al.: Complex promoter and coding region beta 2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness. Proc. Natl. Acad. Sci. USA (2000) 97(19):10483-10488.

• Useful definition of existence and biological role of β2-AR haplotypes.

51. YAMADA K, ISHIYAMA-SHIGEMOTO S, ICHIKAWA F et al.: Polymorphism in the 5’-leader cistron of the beta2-adrenergic receptor gene associated with obesity and Type 2 diabetes. J. Clin. Endocrinol. Metab. (1999) 84(5):1754-1757.

52. CONNE B, STUTZ A, VASSALLI JD: The 3’ untranslated region of messenger RNA: A molecular ‘hotspot’ for pathology? Nat. Med. (2000) 6(6):637-641.

• Good review of 3’UTR.

53. SZOMBATHY T, SZALAI C, KATALIN B, PALICZ T, ROMICS L, CSASZAR A: Association of angiotensin II Type 1 receptor polymorphism with

resistant essential hypertension. Clin. Chim. Acta. (1998) 269(1):91-100.

54. BONNARDEAUX A, DAVIES E, JEUNEMAITRE X et al.: Angiotensin II Type 1 receptor gene polymorphisms in human essential hypertension. Hypertension (1994) 24(1):63-69.

• Early description of GPCR variant that influence blood pressure.

55. WANG WY, ZEE RY, MORRIS BJ: Association of angiotensin II Type 1 receptor gene polymorphism with essential hypertension. Clin. Genet. (1997) 51(1):31-34.

56. MILLER JA, THAI K, SCHOLEY JW: Angiotensin II Type 1 receptor gene polymorphism predicts response to losartan and angiotensin II. Kidney Int. (1999) 56(6):2173-2180.

57. ALVAREZ R, REGUERO JR, BATALLA A et al.: Angiotensin-converting enzyme and angiotensin II receptor 1 polymorphisms: association with early coronary disease. Cardiovasc. Res. (1998) 40(2):375-379.

58. FINCKH U, ROMMELSPACHER H, KUHN S et al.: Influence of the dopamine D2 receptor (DRD2) genotype on neuroadaptive effects of alcohol and the clinical outcome of alcoholism. Pharmacogenetics. (1997) 7(4):271-281.

59. DE LUCA V, VINCENT JB, MULLER DJ et al.: Identification of a naturally occurring 21 bp deletion in alpha 2c noradrenergic receptor gene and cognitive correlates to antipsychotic treatment. Pharmacol. Res. (2005) 51(4):381-384.

60. ELSHOURBAGY NA, ADAMOU JE, GAGNON AW, WU HL, PULLEN M, NAMBI P: Molecular characterization of a novel human endothelin receptor splice variant. J. Biol. Chem. (1996) 271(41):25300-25307.

• Early description of endothelin receptor mutant.

61. ODA Y, TU CL, PILLAI S, BIKLE DD: The calcium sensing receptor and its alternatively spliced form in keratinocyte differentiation. J. Biol. Chem. (1998) 273(36):23344-23352.

62. CLAEYSEN S, SEBBEN M, BECAMEL C, BOCKAERT J, DUMUIS A: Novel brain-specific 5-HT4 receptor splice variants show marked constitutive activity: role of the C-terminal intracellular domain. Mol. Pharmacol. (1999) 55(5):910-920.

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63. BOCKAERT J, CLAEYSEN S, SEBBEN M, DUMUIS A: 5-HT4 receptors: gene, transduction and effects on olfactory memory. Ann. NY Acad. Sci. (1998) 861:1-15.

64. SCHMAUSS C: Regulation of serotonin 2C receptor pre-mRNA editing by serotonin. Int. Rev. Neurobiol. (2005) 63:83-100.

• Good update on mRNA editing of 5-HT2C receptors.

65. BURNS CM, CHU H, RUETER SM et al.: Regulation of serotonin-2C receptor G-protein coupling by RNA editing. Nature (1997) 387(6630):303-308.

66. NISWENDER CM, COPELAND SC, HERRICK-DAVIS K, EMESON RB, SANDERS-BUSH E: RNA editing of the human serotonin 5-hydroxytryptamine 2C receptor silences constitutive activity. J. Biol. Chem. (1999) 274(14):9472-9478.

67. MCGREW L, PRICE RD, HACKLER E, CHANG MS, SANDERS-BUSH E: RNA editing of the human serotonin 5-HT2C receptor disrupts transactivation of the small G-protein RhoA. Mol. Pharmacol. (2004) 65(1):252-256.

68. DUVERNAY MT, FILIPEANU CM, WU G: The regulatory mechanisms of export trafficking of G protein-coupled receptors. Cell. Signal. (2005) 17(12):1457-1465.

69. BUNGE S, WEDEMANN H, DAVID D et al.: Molecular analysis and genetic mapping of the rhodopsin gene in families with autosomal dominant retinitis pigmentosa. Genomics (1993) 17(1):230-233.

70. SULLIVAN LJ, MAKRIS GS, DICKINSON P et al.: A new codon 15 rhodopsin gene mutation in autosomal dominant retinitis pigmentosa is associated with sectorial disease. Arch. Ophthalmol. (1993) 111(11):1512-1517.

71. BOND C, LAFORGE KS, TIAN M et al.: Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction. Proc. Natl. Acad. Sci. USA (1998) 95(16):9608-9613.

• First evidence for a µ-opioid receptor variant in opiate dependence.

72. OKSCHE A, ROSENTHAL W: The molecular basis of nephrogenic diabetes insipidus. J. Mol. Med. (1998) 76(5):326-337.

73. TSUKAGUCHI H, MATSUBARA H, TAKETANI S, MORI Y, SEIDO T, INADA M: Binding-, intracellular transport-, and biosynthesis-defective mutants of vasopressin Type 2 receptor in patients with X-linked nephrogenic diabetes insipidus. J. Clin. Invest. (1995) 96(4):2043-2050.

• Initial detailed description of V2 receptor mutation in NDI.

74. HEBERT TE, MOFFETT S, MORELLO JP et al.: A peptide derived from a beta2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J. Biol. Chem. (1996) 271(27):16384-16392.

• Early description of GPCR dimerisation and possible domains involved.

75. BAI M, TRIVEDI S, BROWN EM: Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells. J. Biol. Chem. (1998) 273(36):23605-23610.

• Early example of role of dimerisation in GPCR.

76. DE LUCA F, RAY K, MANCILLA EE et al.: Sporadic hypoparathyroidism caused by de novo gain-of-function mutations of the Ca(2+)-sensing receptor. J. Clin. Endocrinol. Metab. (1997) 82(8):2710-2715.

77. WATANABE T, BAI M, LANE CR et al.: Familial hypoparathyroidism: identification of a novel gain of function mutation in transmembrane domain 5 of the calcium-sensing receptor. J Clin. Endocrinol. Metab. (1998) 83(7):2497-2502.

78. GAINETDINOV RR, PREMONT RT, BOHN LM, LEFKOWITZ RJ, CARON MG: Desensitization of G protein-coupled receptors and neuronal functions. Ann. Rev. Neurosci. (2004) 27:107-144.

• Up-to-date review of GPCR desensitisation.

79. HAZELWOOD LA, SANDERS-BUSH E: His452Tyr polymorphism in the human 5-HT2A receptor destabilizes the signaling conformation. Mol. Pharmacol. (2004) 66(5):1293-1300.

80. SMALL KM, BROWN KM, FORBES SL, LIGGETT SB: Polymorphic deletion of three intracellular acidic residues of the alpha 2B-adrenergic receptor decreases G protein-coupled receptor kinase-mediated phosphorylation and

desensitization. J. Biol. Chem. (2001) 276(7):4917-4922.

81. BRUCK H, LEINEWEBER K, BUSCHER R et al.: The Gln27Glu beta2-adrenoceptor polymorphism slows the onset of desensitization of cardiac functional responses in vivo. Pharmacogenetics (2003) 13(2):59-66.

• A first example of a GPCR variant that influences rate, but not extent, of GPCR desensitisation.

82. BRUCK H, LEINEWEBER K, ULRICH A et al.: Thr164Ile polymorphism of the human beta2-adrenoceptor exhibits blunted desensitization of cardiac functional responses in vivo. Am. J. Physiol. Heart Circ. Physiol. (2003) 285(5):H2034-H2038.

83. ROTONDO A, NIELSEN DA, NAKHAI B, HULIHAN-GIBLIN B, BOLOS A, GOLDMAN D: Agonist-promoted down-regulation and functional desensitization in two naturally occurring variants of the human serotonin1A receptor. Neuropsychopharmacology (1997) 17(1):18-26.

84. GREEN SA, TURKI J, BEJARANO P, HALL IP, LIGGETT SB: Influence of beta 2-adrenergic receptor genotypes on signal transduction in human airway smooth muscle cells. Am. J. Respir. Cell Mol. Biol. (1995) 13(1):25-33.

85. GREEN SA, TURKI J, INNIS M, LIGGETT SB: Amino-terminal polymorphisms of the human beta 2-adrenergic receptor impart distinct agonist-promoted regulatory properties. Biochemistry (1994) 33(32):9414-9419.

• One of the first examples of a GPCR variant influencing function.

86. BUSCHER R, EILMES KJ, GRASEMANN H et al.: beta2 adrenoceptor gene polymorphisms in cystic fibrosis lung disease. Pharmacogenetics (2002) 12(5):347-353.

• First evidence implicating GPCR variants as modifying genes in cystic fibrosis.

87. BRODDE OE, LEINEWEBER K: Beta2-adrenoceptor gene polymorphisms. Pharmacogenet. Genomics (2005) 15(5):267-275.

88. ISRAEL E, CHINCHILLI VM, FORD JG et al.: Use of regularly scheduled albuterol treatment in asthma: genotype-stratified, randomised, placebo-controlled cross-over trial. Lancet (2004) 364(9444):1505-1512.

• Recent evidence for impact of β2-AR variant on clinical response to β-agonist.

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89. KENAKIN T: Efficacy as a vector: the relative prevalence and paucity of inverse agonism. Mol. Pharmacol. (2004) 65(1):2-11.

•• Nice synthesis of evolving ideas regarding efficacy at GPCRs.

90. KOPP P, VAN SANDE J, PARMA J et al.: Brief report: congenital hyperthyroidism caused by a mutation in the thyrotropin-receptor gene. N. Engl. J. Med. (1995) 332(3):150-154.

• Early example of a GPCR variant causing disease.

91. HOLZAPFEL HP, FUHRER D, WONEROW P, WEINLAND G, SCHERBAUM WA, PASCHKE R: Identification of constitutively activating somatic thyrotropin receptor mutations in a subset of toxic multinodular goiters. J. Clin. Endocrinol. Metab. (1997) 82(12):4229-4233.

92. OKADA M, NORTHUP JK, OZAKI N, RUSSELL JT, LINNOILA M, GOLDMAN D: Modification of human 5-HT(2C) receptor function by Cys23Ser, an abundant, naturally occurring amino-acid substitution. Mol. Psychiatry (2004) 9(1):55-64.

93. SODHI MS, ARRANZ MJ, CURTIS D et al.: Association between clozapine response and allelic variation in the 5-HT2C receptor gene. Neuroreport (1995) 7(1):169-172.

94. LIU IS, SEEMAN P, SANYAL S et al.: Dopamine D4 receptor variant in Africans, D4valine194glycine, is insensitive to dopamine and clozapine: report of a homozygous individual. Am. J. Med. Genet. (1996) 61(3):277-282.

95. OKAZAKI R, CHIKATSU N, NAKATSU M et al.: A novel activating mutation in calcium-sensing receptor gene associated with a family of autosomal dominant hypocalcemia. J. Clin. Endocrinol. Metab. (1999) 84(1):363-366.

96. PUFFENBERGER EG, HOSODA K, WASHINGTON SS et al.: A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung’s disease. Cell (1994) 79(7):1257-1266.

•• Classic description linking endothelin-B receptor mutants and Hirschsprung’s disease.

97. ROSENTHAL W, SEIBOLD A, ANTARAMIAN A et al.: Mutations in the vasopressin V2 receptor gene in families with nephrogenic diabetes insipidus and functional expression of the Q-2 mutant.

Cell. Mol. Biol. (Noisy-le-grand). (1994) 40(3):429-436.

98. KOTLAR TJ, YOUNG RH, ALBANESE C, CROWLEY WF, JR., SCULLY RE, JAMESON JL: A mutation in the follicle-stimulating hormone receptor occurs frequently in human ovarian sex cord tumors. J. Clin. Endocrinol. Metab. (1997) 82(4):1020-1026.

99. OSTROM RS, POST SR, INSEL PA: Stoichiometry and compartmentation in G protein-coupled receptor signaling: implications for therapeutic interventions involving G(s). J. Pharmacol. Exp. Ther. (2000) 294(2):407-412.

100. LEFKOWITZ RJ, SHENOY SK: Transduction of receptor signals by beta-arrestins. Science (2005) 308(5721):512-517.

•• Recent comprehensive review summarising roles of β-arrestins.

101. LAITINEN T, POLVI A, RYDMAN P et al.: Characterization of a common susceptibility locus for asthma-related traits. Science (2004) 304(5668):300-304.

• Novel observation regarding an orphan GPCR in asthma.

102. MELEN E, BRUCE S, DOEKES G et al.: Haplotypes of G protein-coupled receptor 154 are associated with childhood allergy and asthma. Am. J. Respir. Crit. Care Med. (2005) 171(10):1089-1095.

103. ITOH Y, KAWAMATA Y, HARADA M et al.: Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40. Nature (2003) 422(6928):173-176.

• First definition of biological activity of GPR40.

104. HAMID YH, VISSING H, HOLST B et al.: Studies of relationships between variation of the human G protein-coupled receptor 40 Gene and Type 2 diabetes and insulin release. Diabet. Med. (2005) 22(1):74-80.

105. OGAWA T, HIROSE H, MIYASHITA K, SAITO I, SARUTA T: GPR40 gene Arg211His polymorphism may contribute to the variation of insulin secretory capacity in Japanese men. Metabolism (2005) 54(3):296-299.

106. THOMSON PA, WRAY NR, THOMSON AM et al.: Sex-specific association between bipolar affective disorder in women and GPR50, an X-linked orphan G protein-coupled receptor. Mol. Psychiatry (2005) 10(5):470-478.

107. DE ROUX N, GENIN E, CAREL JC, MATSUDA F, CHAUSSAIN JL, MILGROM E: Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc. Natl. Acad. Sci. USA (2003) 100(19):10972-10976.

108. SEMINARA SB, MESSAGER S, CHATZIDAKI EE et al.: The GPR54 gene as a regulator of puberty. N. Engl. J. Med. (2003) 349(17):1614-1627.

• Defined clinical significance of GPR54.

109. SEMPLE RK, ACHERMANN JC, ELLERY J et al.: Two novel missense mutations in g protein-coupled receptor 54 in a patient with hypogonadotropic hypogonadism. J. Clin. Endocrinol. Metab. (2005) 90(3):1849-1855.

110. TATTERSFIELD AE, HALL IP: Are beta2-adrenoceptor polymorphisms important in asthma-an unravelling story. Lancet (2004) 364(9444):1464-1466.

111. MUSZKAT M, STEIN CM: Pharmacogenetics and response to beta-adrenergic receptor antagonists in heart failure. Clin. Pharmacol. Ther. (2005) 77(3):123-126.

112. TANG CM, HOERNING A, BUSCHER R et al.: Human adenosine 2B receptor: SNP discovery and evaluation of expression in patients with cystic fibrosis. Pharmacogenet. Genomics (2005) 15(5):321-327.

113. XIE HG, KIM RB, WOOD AJ, STEIN CM: Molecular basis of ethnic differences in drug disposition and response. Ann. Rev. Pharmacol. Toxicol. (2001) 41:815-850.

• Good overview of ethnicity and GPCR genetic variations.

114. WILSON JH, WENSEL TG: The nature of dominant mutations of rhodopsin and implications for gene therapy. Mol. Neurobiol. (2003) 28(2):149-158.

115. TANG CM, INSEL PA: GPCR expression in the heart; ‘new’ receptors in myocytes and fibroblasts. Trends Cardiovasc. Med. (2004) 14(3):94-99.

• Review of GPCRs, in particular orphan GPCRs, expressed in cardiac myocytes and/or fibroblasts.

116. LEMONDE S, DU L, BAKISH D, HRDINA P, ALBERT PR: Association of the C(-1019)G 5-HT1A functional promoter polymorphism with antidepressant response. Int. J. Neuropsychopharmacol. (2004) 7(4):501-506.

Exp

ert O

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r. T

arge

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nly.



117. BRUSS M, BONISCH H, BUHLEN M, NOTHEN MM, PROPPING P, GOTHERT M: Modified ligand binding to the naturally occurring Cys-124 variant of the human serotonin 5-HT1B receptor. Pharmacogenetics (1999) 9(1):95-102.

118. OZAKI N, MANJI H, LUBIERMAN V et al.: A naturally occurring amino acid substitution of the human serotonin 5-HT2A receptor influences amplitude and timing of intracellular calcium mobilization. J. Neurochem. (1997) 68(5):2186-2193.

119. ARRANZ MJ, MUNRO J, BIRKETT J et al.: Pharmacogenetic prediction of clozapine response. Lancet (2000) 355(9215):1615-1616.

120. AUBERT R, BETOULLE D, HERBETH B, SIEST G, FUMERON F: 5-HT2A receptor gene polymorphism is associated with food and alcohol intake in obese people. Int. J. Obes. (2000) 24(7):920-924.

121. HERBETH B, AUBRY E, FUMERON F et al.: Polymorphism of the 5-HT2A receptor gene and food intakes in children and adolescents: the Stanislas Family Study. Am. J. Clin. Nutr. (2005) 82(2):467-470.

122. HOLMES C, ARRANZ MJ, POWELL JF, COLLIER DA, LOVESTONE S: 5-HT2A and 5-HT2C receptor polymorphisms and psychopathology in late onset Alzheimer’s disease. Hum. Mol. Genet. (1998) 7(9):1507-1509.

123. YUAN X, YAMADA K, ISHIYAMA-SHIGEMOTO S, KOYAMA W, NONAKA K: Identification of polymorphic loci in the promoter region of the serotonin 5-HT2C receptor gene and their association with obesity and Type II diabetes. Diabetologia (2000) 43(3):373-376.

124. SMALL KM, FORBES SL, BROWN KM, LIGGETT SB: An asn to lys polymorphism in the third intracellular loop of the human alpha 2A-adrenergic receptor imparts enhanced agonist-promoted Gi coupling. J. Biol. Chem. (2000) 275(49):38518-38523.

125. SMALL KM, FORBES SL, RAHMAN FF, BRIDGES KM, LIGGETT SB: A four amino acid deletion polymorphism in the third intracellular loop of the human alpha 2C-adrenergic receptor confers impaired coupling to multiple effectors. J. Biol. Chem. (2000) 275(30):23059-23064.

126. RATHZ DA, BROWN KM, KRAMER LA, LIGGETT SB: Amino acid 49 polymorphisms of the human beta1-

adrenergic receptor affect agonist-promoted trafficking. J. Cardiovasc. Pharmacol. (2002) 39(2):155-160.

127. MASON DA, MOORE JD, GREEN SA, LIGGETT SB: A gain-of-function polymorphism in a G-protein coupling domain of the human beta1-adrenergic receptor. J. Biol. Chem. (1999) 274(18):12670-12674.

128. MARTINEZ FD, GRAVES PE, BALDINI M, SOLOMON S, ERICKSON R: Association between genetic polymorphisms of the beta2-adrenoceptor and response to albuterol in children with and without a history of wheezing. J. Clin. Invest. (1997) 100(12):3184-3188.

129. ISAZA C, HENAO J, RAMIREZ E, CUESTA F, CACABELOS R: Polymorphic variants of the beta2-adrenergic receptor (ADRB2) gene and ADRB2-related propanolol-induced dyslipidemia in the Colombian population. Meth. Find. Exp. Clin. Pharmacol. (2005) 27(4):237-244.

130. GREEN SA, COLE G, JACINTO M, INNIS M, LIGGETT SB: A polymorphism of the human beta 2-adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor. J. Biol. Chem. (1993) 268(31):23116-23121.

131. MITCHELL BD, BLANGERO J, COMUZZIE AG et al.: A paired sibling analysis of the beta-3 adrenergic receptor and obesity in Mexican Americans. J. Clin. Invest. (1998) 101(3):584-587.

132. ALVAREZ-HERNANDEZ D, SANTAMARIA I, RODRIGUEZ-GARCIA M, IGLESIAS P, DELGADO-LILLO R, CANNATA-ANDIA JB: A novel mutation in the calcium-sensing receptor responsible for autosomal dominant hypocalcemia in a family with two uncommon parathyroid hormone polymorphisms. J. Mol. Endocrinol. (2003) 31(2):255-262.

133. UCKUN-KITAPCI A, UNDERWOOD LE, ZHANG J, MOATS-STAATS B: A novel mutation (E767K) in the second extracellular loop of the calcium sensing receptor in a family with autosomal dominant hypocalcemia. Am. J. Med. Genet. A. (2005) 132(2):125-129.

134. MARCHAL-VICTORION S, VIONNET N, ESCRIEUT C et al.: Genetic, pharmacological and functional

analysis of cholecystokinin-1 and cholecystokinin-2 receptor polymorphism in Type 2 diabetes and obese patients. Pharmacogenetics (2002) 12(1):23-30.

135. SMITH MW, DEAN M, CARRINGTON M et al.: Contrasting genetic influence of CCR2 and CCR5 variants on HIV-1 infection and disease progression. Hemophilia Growth and Development Study (HGDS), Multicenter AIDS Cohort Study (MACS), Multicenter Hemophilia Cohort Study (MHCS), San Francisco City Cohort (SFCC), ALIVE Study. Science (1997) 277(5328):959-965.

136. O’BRIEN TR, MCDERMOTT DH, IOANNIDIS JP et al.: Effect of chemokine receptor gene polymorphisms on the response to potent antiretroviral therapy. AIDS (2000) 14(7):821-826.

137. BREAM JH, YOUNG HA, RICE N et al.: CCR5 promoter alleles and specific DNA binding factors. Science (1999) 284(5412):223.

138. SAMSON M, LIBERT F, DORANZ BJ et al.: Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature (1996) 382(6593):722-725.

139. HUANG JL, GAO PS, MATHIAS RA et al.: Sequence variants of the gene encoding chemoattractant receptor expressed on Th2 cells (CRTH2) are associated with asthma and differentially influence mRNA stability. Hum. Mol. Genet. (2004) 13(21):2691-2697.

140. CRAVCHIK A, SIBLEY DR, GEJMAN PV: Functional analysis of the human D2 dopamine receptor missense variants. J. Biol. Chem. (1996) 271(42):26013-26017.

141. CRAVCHIK A, SIBLEY DR, GEJMAN PV: Analysis of neuroleptic binding affinities and potencies for the different human D2 dopamine receptor missense variants. Pharmacogenetics (1999) 9(1):17-23.

142. TANAKA H, MOROI K, IWAI J et al.: Novel mutations of the endothelin B receptor gene in patients with Hirschsprung’s disease and their characterization. J. Biol. Chem. (1998) 273(18):11378-11383.

143. GROMOLL J, SIMONI M, NORDHOFF V, BEHRE HM, DE GEYTER C, NIESCHLAG E: Functional and clinical consequences of mutations in the FSH receptor. Mol. Cell. Endocrinol. (1996) 125(1-2):177-182.

Exp

ert O

pin.

The

r. T

arge

ts D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cMas

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vers

ity o

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Tang & Insel


144. SIMONI M, NIESCHLAG E, GROMOLL J: Isoforms and single nucleotide polymorphisms of the FSH receptor gene: implications for human reproduction. Hum. Reprod. Update (2002) 8(5):413-421.

145. FALCONER H, ANDERSSON E, AANESEN A, FRIED G: Follicle-stimulating hormone receptor polymorphisms in a population of infertile women. Acta Obstet. Gynecol. Scand. (2005) 84(8):806-811.

146. STITHAM J, STOJANOVIC A, HWA J: Impaired receptor binding and activation associated with a human prostacyclin receptor polymorphism. J. Biol. Chem. (2002) 277(18):15439-15444.

147. DONFACK J, KOGUT P, FORSYTHE S, SOLWAY J, OBER C: Sequence variation in the promoter region of the cholinergic receptor muscarinic 3 gene and asthma and atopy. J. Allergy Clin. Immunol. (2003) 111(3):527-532.

148. WANG D, QUILLAN JM, WINANS K, LUCAS JL, SADEE W: Single nucleotide polymorphisms in the human mu opioid receptor gene alter basal G protein coupling and calmodulin binding. J. Biol. Chem. (2001) 276(37):34624-34630.

149. COMPTON SJ, CAIRNS JA, PALMER KJ, AL-ANI B, HOLLENBERG MD, WALLS AF: A

polymorphic protease-activated receptor 2 (PAR2) displaying reduced sensitivity to trypsin and differential responses to PAR agonists. J. Biol. Chem. (2000) 275(50):39207-39212.

150. JANSSENS R, PAINDAVOINE P, PARMENTIER M, BOEYNAEMS JM: Human P2Y2 receptor polymorphism: identification and pharmacological characterization of two allelic variants. Br. J. Pharmacol. (1999) 127(3):709-716.

151. HOLLOPETER G, JANTZEN HM, VINCENT D et al.: Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature (2001) 409(6817):202-207.

• Initial identification of P2Y12 receptors as the target of antithrombotic drugs such as clopidogrel and ticlopidine.

152. TENG Y, TIAN H, WANG H et al.: Mutation identification in a 5-generation pedigree with autosomal dominant retinitis pigmentosa. J. Huazhong Univ. Sci. Technolog. Med. Sci. (2003) 23(3):242-244, 253.

153. SUNG CH, DAVENPORT CM, HENNESSEY JC et al.: Rhodopsin mutations in autosomal dominant retinitis pigmentosa. Proc. Natl. Acad. Sci. USA (1991) 88(15):6481-6485.

154. YONG RY, CHEE CK, YAP EP: A two-stage approach identifies a Q344X

mutation in the rhodopsin gene of a Chinese Singaporean family with autosomal dominant retinitis pigmentosa. Ann. Acad. Med. Singapore (2005) 34(1):94-99.

155. BIRNBAUMER M, GILBERT S, ROSENTHAL W: An extracellular congenital nephrogenic diabetes insipidus mutation of the vasopressin receptor reduces cell surface expression, affinity for ligand, and coupling to the Gs/adenylyl cyclase system. Mol. Endocrinol. (1994) 8(7):886-894.

156. SAUER CG, WHITE K, STOHR H et al.: Evaluation of the G protein coupled receptor-75 (GPR75) in age related macular degeneration. Br. J. Ophthalmol. (2001) 85(8):969-975.

Website

201. http://www.iuphar-db.org/iuphar-rd IUPHAR receptor list (2005).

AffiliationChih-Min Tang PhD & Paul A Insel† MD†Author for correspondenceUniversity of California, San Diego, Departments of Pharmacology and Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0636, USATel: +1 858 534 2295; Fax: +1 858 822 1007;E-mail: [email protected]

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genetic variation in g-protein-coupled receptors – consequences for g-protein-coupled receptors as...

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