Receptors Coupling to G Proteins: Is There a Signal Behindthe Sequence?Florence Horn,1† Eleonora M. van der Wenden,2 Laerte Oliveira,3 Adriaan P. IJzerman,2 and Gerrit Vriend1,4*1BIOcomputing, European Molecular Biology Laboratory, Heidelberg, Germany2Division of Medicinal Chemistry, Leiden-Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands3Escola Paulista Medicina, UNIFESP, Sao Paulo, Brazil4CMBI, Nijmegen, The Netherlands

ABSTRACT Upon the binding of their ligands,G protein-coupled receptors couple to the heterotri-meric G proteins to transduce a signal. One receptorfamily may couple to a single G protein subtype andanother family to several ones. Is there a signal inthe receptor sequence that can give an indication ofthe G protein subtype selectivity? We used a se-quence analysis method on biogenic amine andadenosine receptors and concluded that a weaksignal can be detected in receptor families wherespecialization for coupling to a given G proteinoccurred during a recent divergent evolutionaryprocess. Proteins 2000;41:448–459.© 2000 Wiley-Liss, Inc.

Key words: receptor; GPCR; G protein selectivity;correlated mutations; sequence analy-sis

INTRODUCTION

The superfamily of G protein-coupled receptors (GPCR)plays an essential role in many biological processes and isinvolved in a wide range of diseases. GPCRs are integralmembrane proteins that contain seven transmembranehelices (Fig. 1). Upon the binding of an extracellularligand, they transduce a signal to a heterotrimeric Gprotein that activates or inhibits an effector, such asadenylyl cyclase, phospholipase, or an ionic channel. Stud-ies of point mutations, receptor chimeras and partiallydeleted receptors indicate that the sites for G proteincoupling and activation are mainly in the third intracellu-lar loop, but also in the second intracellular loop, and inthe C-terminal domain.1–3

The G proteins are composed of three subunits: a, b and g.The different G proteins are named after their a subunit.According to their sequence homology, the Ga subunits canbe classified into four main subtypes: as, ai/o, aq/11 and a12.4

Gi inhibits adenylyl cyclase and activates phospholipase Cand potassium channels. Go inhibits calcium channels andstimulates potassium channels and phospholipase C. Gs

stimulates adenylyl cyclase and calcium channels.There is much evidence that GPCRs are intrinsically

promiscuous in their G protein coupling. A whole GPCRfamily can couple to one particular G protein subtype (e.g.,all opioid receptors couple to Gi/o), but different membersof one GPCR family can also couple to different G proteinsubtypes (e.g., the muscarinic receptors couple to Gi/o or

Gq). It is also possible that some receptors exert theirfunction via several G protein subtypes. For example, theTSH receptor activates Gs, Gq, Gi and G12.5 Oliveira et al.6

published a theoretical study in which they interpret thehuge body of experimental data on G protein–GPCRcoupling. They find a relation between this promiscuityand sequence conservation of certain parts on the GPCRand the G protein that are likely to contact each other.This study does not indicate reasons at the atomic level forthe also observed, albeit often small, preferences of certainGPCRs for certain G proteins and vice versa.

A common route in protein research is to go fromsequence to structure to function. So far, crystallization ofthese membrane-bound proteins has not been successful.Many models for GPCRs have been published over the lastyears, mostly based on i) the three-dimensional structureof bacteriorhodopsin as a template, ii) the receptor se-quence, iii) structure-activity relationships and, if avail-able, iv) results of mutation studies.7–11 These models allhave limited accuracy because of the low sequence homol-ogy between bacteriorhodopsin and any GPCR (consider-ably less than the 25%, that according to the study bySander and Schneider28 is the lower limit for reliablemodeling). Additionally, the relative orientation of thehelices is rather different between bacteriorhodopsin andreceptors from the opsin family.12 Moreover, bacteriorho-dopsins do not even couple to G protein. The more recentmodels13,14 that are based on the opsin density map sufferfrom the low resolution of this data (around 7 Å resolu-tion). With these uncertainties about the structure ofGPCRs, it would be preferable to use the sequence as adirect source of information on the function of proteins.

The information inherently present in the sequences ofGPCRs can be harvested by analyzing their evolutionaryrelationships.15,16 During evolution, parts of receptorsinvolved in the same function are under the same evolution-ary pressure. Different functions of the receptor, such asligand binding or G protein coupling can, however, evolve

Grant sponsor: EC; Grant number: PL96-0224.†Present address: Cellular & Molecular Pharmacology, UCSF, San

Francisco, California USA*Correspondence to: Gerrit Vriend, CMBI, Toernooiveld 1, 6525 ED

Nijmegen, The Netherlands. E-mail: Vriend@CMBI.KUN.NL

Received 17 December 1999; Accepted 6 July 2000

PROTEINS: Structure, Function, and Genetics 41:448–459 (2000)

© 2000 WILEY-LISS, INC.

almost independently. Furthermore, very different physico-chemical constraints apply to the transmembrane helicesand the loop regions. The helices that form the binding sitefor the ligand are embedded in lipid, in contrast to theaqueous environment of the loops that couple G proteins.

Functionally important residues tend to stay conservedduring evolution, but if they mutate, they tend to do it ingroups so as to maintain a common function; these compen-satory mutations of residues that are important for thesame function have to occur almost simultaneously on anevolutionary time scale. The tendency of sequence pat-terns to remain conserved or to mutate pair wise is calledcorrelated mutational behavior. Correlated mutation anal-ysis (CMA) can be applied to detect residues that deter-mine certain functions,17 or that are involved in intermo-lecular interactions.18 Several studies have been publishedthat use multiple sequence alignments and correlationbetween sequence and function to analyze properties ofGPCRs. Kuipers et al.19 have studied interactions betweenendogenous and exogenous ligands and receptors for bio-genic amines. Correlation mutation analysis has beenapplied to determine residues potentially involved inligand binding in olfactory receptors20 and in secretin-likefamily receptors.21 In the latter article, we proposed amodel for receptor activation in which the first sevenresidues of the N-terminal part of the ligand dock betweenthe receptor helices, whereas the rest of the ligand is incontact with external loops and the N-terminus of the

receptor. A similar method was used to study ligandbinding by muscarinic receptors.22 Oliveira and co-workers used CMA to investigate the common sequencemotifs involved in G protein coupling.17,23 They haveshown the importance of the highly conserved DRY motiffor G protein binding. The arginine residue in the middleof the DRY sequence is fully conserved in all rhodopsin-like GPCRs. It was therefore suggested that this residueplays a pivotal role in the signal transduction process.23

The authors have proposed an ‘arginine switch,’ in whichthe arginine moves from a polar pocket in the transmem-brane region (signal ‘off’) to the cytosol (signal ‘on’) to favorcoupling to a G protein.

In this article, we describe how in some cases CMA candetect residues in GPCRs that are involved in G proteinselectivity.

METHODS

Sequences of GPCRs were obtained from the GPCRDB24

(http://www.gpcr.org/7tm/), that holds sequence data im-ported from the Swiss-Prot and the TrEMBL databanks.25

Mutant data was obtained from the literature and fromthe snake-like plots provided by the GPCRDB. Residues inthese figures are hyperlinked to the tinyGRAP GPCRmutation databank.26

We numbered the residues as described by Oliveira etal.,17 that is similar to the method of Hibert and co-workers7,11 and now widely used in the GPCR literatureand throughout the GPCRDB database. Residues arenumbered in such a way that the first digit indicates thehelix number. In each helix the number of the mostconserved residue gets a round number. For example, Asnof the GN motif in helix I (see also Fig. 4, 6 and 8) wasnumbered 130, Arg of the DRY motif at the end of helix III340, the conserved Pro residues in helix V, VI and VII werenumbered 520, 620 and 720, respectively.

The program WHAT IF27 (http://www.cmbi.kun.nl/whatif/) was used for sequence alignments, to make phylo-genetic trees, and to apply the CMA method. The align-ment method within WHAT IF is iterative and profile-based, similar to the method described by Sander andSchneider.28 In this method, sequences are aligned againsta profile that is composed from the sequences in thealignment set rather than against all other sequences, asis done in most other multiple sequence alignment proce-dures. The alignment profiles are available on the GPCRDBserver.

WHAT IF employs a nearest neighbor-joining algorithmto construct phylogenetic trees. The aligned sequenceswere used to create dendrograms. The ancestry of recep-tors and the similarity of receptors and receptor regionswere assessed using these dendrograms.

The correlated mutation analysis options from WHATIF were used to compare the receptor sequences with theirG protein coupling selectivity. Correlated mutation analy-sis identifies pairs of sequence positions where residuesremained conserved or mutated in tandem. A detailedexplanation of the CMA method has been published else-where,17–21 and can also been found at http://www.gpcr.org/7tm/htmls/CMA.html. This method was extended to detect

Fig. 1. Schematic representation of a G protein-coupled receptor.(Figure kindly provided by T. Schwartz).

G PROTEIN SELECTIVITY OF GPCRS 449

correlations between residue patterns and characteristicsof the whole GPCR. For this purpose, a pseudo-residuerepresents a characteristic of the receptor, such as thebinding of a ligand or coupling to a G protein. Thispseudo-residue can be correlated with real residues. Thispseudo residue represents a property that some sequenceshave and others do not. Pseudo residues can also representthe class of a molecule (e.g., ‘q’ for Gq, ‘i’ for Gi/o coupling,etc.). These methods are further explained and illustratedby Kuipers et al.,19 but in summary, this method impliesthat the property of a sequence is encoded in its firstresidue, and this first (pseudo) residue is treated like anyother normal residue. A correlation between this first(pseudo) residue and normal residues indicate that thosecorrelating normal residues might be responsible for theproperty encoded in the pseudo residue.

RESULTS

Earlier observations made it clear that CMA works bestif the external factor for which the sequence patterns areanalyzed co-evolved with the sequences.19 This impliesthat receptors should have evolved from a common ances-tor and that the differences among the family members aremainly due to the selectivity for G proteins. Figure 2 showsa phylogenetic tree for the amine receptors. It can be seenthat muscarinic acetylcholine receptors originate from acommon ancestor; such a clear common ancestor cannot befound for any of the other families.

For example, the a1-adrenergic receptors are more ho-mologous to the serotonin 5HT1 receptors than to the otheradrenergic receptors and the histamine H1 and H2 recep-tors seem to have evolved from different ancestors to bindthe same endogenous ligand. The phenomenon of special-ization from an ancestral muscarinic receptor to musca-rinic receptor subtypes that couple to distinct G proteinswill be called ‘ligand-diverging evolution.’ This in contrastto the histamine receptors, for example, that we will call‘ligand-converging evolution.’

In view of the G protein promiscuity mentioned above, itdid not come as a surprise that there were no residues thatcorrelated perfectly with G protein selectivity when weanalyzed all GPCRs, all class A GPCRs, or even all aminereceptors. It is likely that all class A receptors evolved froma common ancestor, and virtually certain that all aminereceptors did. It is also likely that GPCRs learned how tocouple to G proteins in a very early stage of their evolution,but there is no reason to believe that the GPCRs co-evolvedwith G proteins all along their evolutionary path. On thecontrary, the fact that there are very many GPCRs andonly a few Ga proteins, in addition to the highly diverse Gapreferences within GPCR families (Table I and Fig. 2),strongly indicates that in many cases the G proteinselectivity was obtained via a convergent evolutionaryprocess.

The relatively recent arrival of the muscarinic receptorsubtypes suggests that they adjusted to two types of G

Fig. 2. Phylogenetic tree of thehuman G protein-coupled receptorsfor biogenic amines. The Swiss-Protaccession numbers and sequenceidentifiers are indicated. The corre-sponding G proteins are listed on theright. File names starting with 5H

correspond to serotonin receptors, Bfor b adrenergic receptors, A1 andA2 for a adrenergic receptors, D fordopamine receptors, HH for hista-mine receptors and ACM for musca-rinic receptors. The relative degreeof similarity of the receptor subtypesis indicated on the horizontal axis.

450 F. HORN ET AL.

proteins and perhaps co-evolved with them. We thereforedecided to analyze this family first. We then analyzed theadrenergic and the adenosine receptors, because the Gprotein preferences are well known for these classes.

Muscarinic Receptors

The muscarinic m1, m3 and m5 receptors couple to the Gq

subtype, whereas m2 and m4 receptors couple to Gi/o.29–31

The major split in the muscarinic phylogenetic tree (Fig. 3)corresponds to the subdivision m1, m3, m5 vs. m2, m4, andthus to the G protein preference. It’s clear that evolution

should have left a signature of this preference in thesequences.

The CMA on the muscarinic receptors revealed a singlegroup of 24 positions that correlate perfectly with Gprotein selectivity (Fig. 4), i.e., residues at these 24positions are 100% conserved in the m1, m3 and m5sequences but differ from the conserved residues at thesame positions in the m2 and m4 sequences. Of these 24residues, 18 are located in or close to the intracellularloops, mostly in the N- and C-terminal parts of the longthird loop. The other detected positions are found either inthe extracellular loops or in the transmembrane domains.

We compared the 24 correlated positions with resultsfrom receptor mutation studies using the tinyGRAP mu-tant database26 via the snake-like view provided in theGPCRDB.24 We have also checked whether mutations atother positions affect the G protein selectivity. We focusedour literature search on single and multiple substitutionmutations. Mutation data is available for 17 of the corre-lated positions (Fig. 4). Mutations at the other correlatedpositions (218, 314, 325, 417, 418, e2, 508 and 717;numbering according to Fig. 4) are part of large chimerasor deletions. It is therefore difficult to deduce their puta-tive role in G protein selectivity. Without further experi-mental evidence, we cannot decide what the correlations atthe extracellular side (314, e2 and 508) of the receptormean: are they involved in signal transduction, or doesthis noise result from an inadequate number of sequencesor some other spurious correlation?

Several groups have reported mutations in the receptorsthat resulted in a gain of G protein selectivity (see Table IIfor references). The combination of these experimentalresults highlighted twelve residues that could play a rolein G protein selectivity (positions i2a, i2b, i2c, 411, 530,i3a, i3b, i3c, 603, 604, 607, 608 in Fig. 4 and Table II). Ofthese 12 positions, 10 were detected by our method whereascontradicting results have been published for the othertwo positions (53032 and 60833). By a single amino acidsubstitution in the m3 receptor, Bluml et al.34 demon-strated that the position 530 is not essential for efficientactivation of the response mediated by the Gq subtype.Kostenis et al.35 have shown by means of site-directedmutagenesis of the human m2 muscarinic receptor that asingle point mutation at position 608 did not affect the Gprotein preference. The residues that were mutated with-out affecting G protein selectivity were not detected by oursequence analysis. Studies into the loss of G proteincoupling (Table II) confirmed the importance of 8 of thecorrelated mutations indicated in Figure 4.

The CMA method highlighted five positions (i1, i3d, i3e,i3f and C1 in Fig. 4) that were not yet identified byexperimental studies as playing a role in G protein selectiv-ity. No single point mutation has yet been made at thesepositions, and only results from multiple substitutions areavailable. A mutation at position i1 in the human m1receptor impaired the G protein coupling but the effect onG protein selectivity was not observed.36 Multiple substitu-tions at the two positions i3d and i3e in the third intracel-lular loop affect the response of the receptor.37–42 Somemultiple substitution mutants result in a slight decrease

TABLE I. G Protein Coupling Selectivity of Receptors forBiogenic Amines and Adenosine Receptors

Receptors Gi/o Gs Gq/11 Other

Adenosine A1 1 *A2 A2A 1

A2B 1A3 1 *

Adrenergic a1 a1A 1a1B 1a1C 1

a2 a2A 1 * *a2B 1a2C 1a2D 1

b b1 1b2 1b3 1

Dopamine D1 D1A 1D1B 1

D2 1D3 1 ?D4 D4S,

D4L

1

D5 1Histamine H1 1

H2 1H3 ?

Muscarinic M1 1M2 1M3 1M4 1M5 1

Serotonin 5HT1 5HT1A 15HT1B 15HT1D 15HT1E 15HT1F 1

5HT2 5HT2A 15HT2B 15HT2C 1

5HT3 nGPC5HT4 15HT5 5HT5B ?

5HT5A ?5HT6 15HT7 1

The following coding is used: 1 couples to; * alternative way ofcoupling shown to exist; ? G protein not known; nGPC non-G protein-coupling receptor. The information for this table was obtained fromWatson and Arkinstall.59

G PROTEIN SELECTIVITY OF GPCRS 451

Fig. 3. Phylogenetic tree of themuscarinic acetylcholine receptors.For details, see legend of Figure 2.

Fig. 4. Consensus sequence and correlated mutations of the musca-rinic receptors. All conserved amino acids are indicated. The residues thatcorrelate with the differences in G protein coupling, i.e., m1, m3, and m5 vs.m2 and m4, are depicted in grey and numbered (in bold) according to

Oliveira et al.17 when located in the helices. See Table II for thecorresponding amino acids. The correlation scores are calculated accord-ing to Singer et al.20

in the receptor potency43,44 at position i3f, located in the Cterminal portion of the third intracellular loop. All thesemutation studies, however, do not provide any experimen-tal clue about the role of i3d, i3e and i3f in G proteinselectivity, although they do not contradict our results.There is as yet no experimental data that shows theimportance of the position C1 in G protein selectivity.

To summarize, CMA detected 24 positions correlated toG protein preference. Ten were confirmed by experimentaldata, 8 are putative candidates that have not yet beenvalidated by mutation studies, and 6 positions can beconsidered as false positives as they are located far fromthe intracellular compartment. The CMA did not identifyany residues that were experimentally shown as notinvolved in G protein selectivity. In conclusion, the se-quence analysis firmly identified those residues of themuscarinic receptors that are important for G proteinselectivity, and suggests new candidates for experimentalverification.

Adrenergic Receptors

We applied the same approach to other families of aminereceptors whose members present a clear preference for

different G protein subtypes. The adrenergic receptorfamily consists of three main subtypes: a1, a2 and b. Thea1-adrenergic receptors couple to the Gq subtype, b-adren-ergic receptors to Gs, and the a2-adrenergic receptorsusually couple to Gi/o (Table I).

The major clusters in the phylogenetic tree of the adrener-gic receptors correspond to the three-way split a1-, a2-, andb-adrenergic receptors (Fig. 5). Because this split corre-sponds to G protein selectivity, it was expected that CMAcould detect residues responsible for G protein selectivity.

The analysis of the correlated mutations of adrenergicreceptors is more complex than that of the muscarinicreceptors. The CMA highlighted 11 correlated positions.Only two of these positions correlate perfectly with Gprotein coupling (604 and C1 in Fig. 6). The other corre-lated positions present a lower, albeit significant, degree ofcorrelation (Table III).

Five correlated positions (i1, 526, 604, 727, and C1) arelocated in or close to the intracellular loops. Single andshort multiple substitution mutant data is available forthe positions i1, 604 and C1. A point mutation at positioni1 results in decreased efficacy and potency of the receptoractivation.45 The position 604 has been intensively studied

TABLE II. Summary of Data for Correlated Positions in the Muscarinic Receptors

Position Location Ga/Gi Mutant data Gain of coupling Loss of coupling Selectivity

i1 IC 1 K/Q 32,36,83 no32 1218 bot TM2 L/F ?314 top TM3 A/V 2325 mid TM3 A/V 2i2a IC 2 S/C 32,36,83,84 yes32 111i2b IC 2 R/K 32,39,84,85 yes32 111i2c IC 2 R/P 32,39,84,85 yes32 111411 bot TM4 R/M 32,84 yes32 111417 bot TM4 G/A ?418 bot TM4 L/A ?e 2 EC 2 Y/F 2508 top TM5 I/V 2530 bot TM5 R/HQa 34,38,42,85 yes32/no34 no42 2i3a bot TM5 Y/S 32,34,36,38,42,83,85–89 yes32 yes34,36,42,83,86 111i3b IC 3 E/A 32,34,38,41–43,85,89 yes33 no36,42,43,83 111i3c IC 3 T/S 34,38,39,42,85,89–91 yes33 yes32,42,92,93 111i3d IC 3 L/E 38,39,41,42 1i3e IC 3 R/K 44 1i3f IC 3 K/R 43,44 no43 1603 bot TM6 A/V 32,35,44,87,88 yes33 yes35,92,93 111604 bot TM6 A/T 32,35,44,87,89,94 yes32,33 yes92,93 111607 bot TM6 L/I 32,35,44,87 yes32,33 yes92,93 111608 bot TM6 S/LFb 32,35,44,87,91 yes32,33/no35,87 2623 top TM6 I/V 84,95 2717 mid TM7 L/I 2C1 C term R/K 36,83 ?

The positions detected by the CMA are indicated in bold in the first column. The second column indicates theputative location of these positions. The third column lists the amino acids correlated to G protein subtype. Numbersin the next column correspond to the bibliographic references from which the single and multiple substitutionmutant data has been extracted. The eventual effect on the loss and the gain of G protein coupling is summarized inthe two following columns with the corresponding references. The last column indicates our confidence (based on themutant data) in the role of the position on G protein selectivity. Abbreviations: TM: transmembrane domain, bot:bottom, mid: middle, C term: C terminus, 111: correlated position confirmed by experimental data, 1: putativecandidates (not yet validated by experimental data), 2: putative candidates (no experimental data available), ?: notinvolved in G protein selectivity.aQ in the chick m2 receptor.bL in m2 receptors and F in m4 receptors.

G PROTEIN SELECTIVITY OF GPCRS 453

and most of the publications report that any substitutionat this position leads to the constitutive activity of the a1-,a2-, and b-adrenergic receptors.46–49 It has to be notedthat the residues at the same position in the muscarinicreceptors play a key role in G protein selectivity (position604 in Fig. 4). The available mutant data at positions 72750

and C151 does not give any clues on their role in G proteinselectivity.

Mutation data is also available for positions located inthe transmembrane domains. A double mutant at posi-tions 228 and 22952 had no effect on ligand binding, but theauthors did not look at the G protein coupling level.Mutation at position 33253 leads to a hyperactivity of thereceptor and could be involved in the stabilization of theresidues that form a polar pocket. Several studies haveshown that a mutation at position 625, located at the top ofhelix VI, not only altered agonist binding but also thesignal-transduction pathway.52,54–56

There are also residues that correlate perfectly with oneof the subtypes vs. the two others (Table III). In this case, itis much harder to assign the residues that are responsiblefor G protein selectivity. To eliminate the residues in-

volved in the a vs. b ligand selectivity, we only indicated inFigure 6 the correlated positions that were equal in thetwo subtypes b and a2, but differ in the third subtype a1.Four of these correlated positions residues are located in orclose to the intracellular loops (219, 527, C2 and C3 in Fig.6). Unfortunately, mutant data is only available for posi-tion C257 and does not allow us to confirm the role of thecorrelated positions in G protein selectivity.

Serotonin, Dopamine and Histamine Receptors

The CMA results for other biogenic amine receptorswere considerably less clear than those for the adrenergicreceptors. Especially for the serotonin receptors, a veryheterogeneous family of receptors (see Fig. 2), no residuescould be found that mutated in a correlated fashion with Gprotein selectivity. Conversely, the CMA method high-lighted a very large number of residues correlated to the G

TABLE III. Summary of Data for Correlated Positions inthe Adrenergic Receptors

Positions Location Gs/Ga/Gi Mutant data

i1 IC 1 R/H/AS 45,96

228 mid TM2 G/ST/A 52

229 mid TM2 L/FAS/T 52

319 top TM3 TI/A/L331 bot TM3 E/LMI/V332 bot TM3 T/SG/H 53

526 bot TM5 F/VA/LT604 bot TM6 L/A/T 46–48,68,96–109

625 top TM6 N/LM/Y 52,54–56,110

727 bot TM7 AG/C/S 50

C1 C term R//F 51

Gs/Gi/Ga219 bot TM2 S/N524 bot TM5 M/I527 bot TM5 V/MC2 C term D/E 57

C3 C term R/KGs/Ga, Gi

i1a TM1/IC1 I/V320 top TM3 S/A326 mid TM3 V/C 53

431 top TM4 I/L610 bot TM6 I/V719 mid TM7 N/F 111

Gs/Ga/Gii1b IC1 T/Q225 mid TM2 L/I236 top TM2 A/L318 top TM3 W/Y325 bot TM3 C/F 45,48,99,105,112,113

328 mid TM3 A/S518 mid TM5 Y/F 53,114

i3 IC3 V/I 115,116

603 bot TM6 A/F 96,103,104

606 bot TM6 T/V 103,104

The positions detected by the CMA are indicated in bold in the firstcolumn. The second column indicates the putative location of thesepositions. The third column lists the residues correlated to G proteinsubtypes. Numbers in the next column correspond to the bibliographicreferences from which the single and multiple substitution mutantdata has been extracted. Abbreviations: TM: transmembrane domain,bot: bottom, mid: middle, C term: C terminus.

Fig. 5. Phylogenetic tree of the adrenergic receptors. For details, seelegend of Figure 2.

454 F. HORN ET AL.

protein subtypes for the histamine receptors. So far,however, the histamine receptor family is composed of onlyten sequences and this is too small a dataset for reliableCMA analyses.

Adenosine Receptors

The correlated mutation analysis was also applied to anon-aminergic family of receptors that seem to have acommon ancestor, the adenosine receptors. The adenosineA2 receptor subtypes, A2A and A2B, couple to Gs proteins,the adenosine A1 and A3 receptors couple to both Gi/o andGq proteins. The dendrogram for the adenosine receptorsis shown in Figure 7.

CMA highlighted 12 positions that correlate with Gprotein selectivity, 8 of which are intracellular (134, i1,i2a, i2b, i2c, i3a, 13b, 602 in Fig. 8), mainly in the secondand third intracellular loop. So far, multiple substitutionmutant data is available for 5 of these positions. Olah58

has highlighted the important role of the N-terminal partof the third intracellular loop (including i3a) and theC-terminal part of the second intracellular loop (includingi2c) in conferring selective coupling to Gs for the humanA2A receptor.

Fig. 7. Phylogenetic tree of the adenosine receptors. For details, seelegend of Figure 2.

Fig. 6. Consensus sequence and correlated mutations of the adrenergic receptors. The residues thatcorrelate with the differences in G protein coupling, i.e., a1- vs. a2- vs. b-adrenergic receptors, are depicted ingrey. The shaded circles represent the amino acids that are identical in the two receptor subtypes b and a2 andare different but conserved in the third receptor subtype a1 (see text).

G PROTEIN SELECTIVITY OF GPCRS 455

DISCUSSION

In the present study, it was assumed that receptors ofone family use similar regions for coupling to G proteins.We have suggested that this hypothesis holds for thereceptors that undergo ligand-diverging evolution. Somereceptors couple very selectively to G protein subtypes ofone family,59 but receptors can also be very promiscuous intheir coupling to G proteins.6,29,59–65 Promiscuity in Gprotein coupling does not only depend on the properties ofthe receptor, but also on the relative amounts of ligands,receptors and G proteins.41,64–70 It can even depend on celltypes and the G proteins present therein.71,72 In addition,the G protein-coupling specificity is determined by otherfactors than contact regions between receptors and Gproteins.73 Those factors include the cytoskeleton,74 thelipid composition,75 and proteins such as the regulators ofG protein signaling (RGS).72,76–79 Several groups alsosupport the idea that the G protein bg subunit is alsoinvolved in G protein recognition.80–82

The dendrogram of the complete class of biogenic aminereceptors (Fig. 2) showed that a distorted view could beobtained from dendrograms of separate receptor families.For example, studying only the serotonin receptors or onlythe histamine receptors, one could easily get the wrongimpression that a common ancestor exists, but Figure 2

shows that that is not the case for these two families. It istherefore better to display a family in the context of awhole class of receptors.

The separation of the muscarinic receptor into subtypesis a recent process on the evolutionary scale (Fig. 2). It is,for example, comparable in time with the subdivision ofb-adrenergic receptors into b1, b2, and b3 subtypes. Thus,the conserved residues and correlated mutations in themuscarinic receptors could represent a function such asselectivity in G protein coupling, but could also mean alack of time to mutate.

The receptor families described in detail (muscarinic,adrenergic, and adenosine) show different distributionpatterns for residues correlated with G protein selectivity.The common feature in these families is the presence ofcorrelated mutations in the third intracellular loop. Thesecond intracellular loop of muscarinic and adenosinereceptors contain several residues that may determineselectivity. The second intracellular loop, however, seemsto be of little importance for the selectivity of the G proteincoupling for the adrenergic receptors. Furthermore, theC-terminal tail seems to be involved in the G proteinselectivity for the adrenergic receptors, whereas its rolehas not yet be been verified experimentally for the othertwo families.

Fig. 8. Consensus sequence and correlated mutations of the adenosine receptors. For details, see legend of Figure 4.

456 F. HORN ET AL.

The CMA method has some restrictions such as the needfor large numbers of sequences and a divergent evolution-ary relationship between the factors that are being corre-lated. But, if these restrictions can be overcome, as in thecase of the muscarinic receptors, CMA becomes a verypowerful tool for understanding the intricacies of howGPCRs function.

In conclusion, the CMA method, that is a sequence-onlytechnique as no model building is needed, can be used tostudy receptor properties such as G protein selectivityprovided that the property studied has co-evolved diver-gently with the sequences. The residues responsible for Gprotein selectivity are not committed to certain positionsin the receptor, but can, nevertheless, be assigned unam-biguously in several cases. The question posed in the titleof this paper can therefore be answered in the affirmative.

ACKNOWLEDGMENTS

We thank Amos Bairoch, Margot Beukers, Robert Bywa-ter, Fabien Campagne and Øyvind Edvardsen for stimulat-ing discussions. We also thank J. Eckert and K. Krmoianfor technical assistance. F.H. and G.V. acknowledge finan-cial support from EC grant PL96-0224.

NOTE ADDED IN PROOF

After acceptance of this article, the structure of a GPCR,bovine rhodopsin, was published (K. Palczewski et al.Science (2000) 289:739–745). This structure is supportiveto our conclusions.

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