Structure and function of G protein coupled receptors

Pharmac. Ther. Vol. 50, pp. 425-442, 1991 Printed in Great Britain. All rights reserved

0163-7258/91 $0.00 + 0.50 © 1991 Pergamon Press pie

Specialist Subject Editor: C. W. TAYLOR

STRUCTURE A N D FUNCTION OF G PROTEIN COUPLED RECEPTORS TREVOR JACKSON Department of Biochemistry, AFRC Institute of Animal Physiology and Genetics Research, Babraham Hall, Babraham, Cambridge, CB2 4.4T, U.K. Al~tract--Application of a molecular genetic techniques has allowed the isolation and identification of more than 50 members of the G protein-coupled receptor family. Their specificities range from sensory receptors such as the opsins and odorant receptors through those for the amines, peptides and other small molecules to those for glycoprotein hormones. These studies make it clear that traditional pharmacological methods, often underestimate receptor diversity. G protein-coupled receptors share a common structure consisting of 7 transmembrane alpha helical segments. Receptor structure-function relationships are discussed in the light of results obtained by site-directed mutagenesis and the construction of chimeric receptors. Studies which have allowed the identification of ligand-binding domains, and of sequences defining G protein specificity as well as those involved in receptor desensitization and downregulation are also discussed.

CONTENTS 1. Introduction 2. G Proteins 3. The G Protein Receptor Family 4. G Protein-Coupled Receptor Structures 5. Structure-Function Relationships in G Protein-Linked Receptors 6. Receptor~ Protein Interactions: Specificity 7. Receptor-G Protein Interactions: Efficacy 8. Desensitization and Downregulation 9. Prospects Acknowledgements References

1. I N T R O D U C T I O N An essential property of any living cell is its ability to recognize and respond to external stimuli. Cell surface receptors function to transmit information of sensory, endocrine, paracrine or autocrine origin across the plasma membrane where they act to generate appropriate cellular responses. Some transmembrane signalling systems combine receptor and effector functions in a single protein as found in receptor tyrosine and serine/threonine kinases (Ullrich and Schlessinger, 1990; Hunter, 1991) or the receptor gnanylyl cyelases (Thompson and Garbers, 1990). Alternatively a multimeric complex may contain both receptor and effeetor functions as typified by ligand-gated ion channels such as the nicotinic acetylcholine, GABAA, glyeine and kaiuic glutamate receptors (Schofield and Abbott, 1989; Hollmarm et aL, 1989). A more complex form of transmembrane signalling was first suggested by Rodbell and coworkers (1971) when they found that separate receptor and effector proteins could communicate via a guanine

425 425 426 427 430 433 435 436 437 437 437

nucleotide-dependent regulatory protein (G protein). Binding of agonist to the receptor leads to activation of a G protein which in turn may regulate the activity of an effector enzyme or ion channel. The specificity of the receptor-G protein interaction then determines the nature of the effector mechanism(s) to which receptor activation is coupled. The advantages provided by such an inherently flexible signalling system may ~)ell be reflected in the finding that, amongst eukaryotes at least, the G protein-coupled receptor family is the most widespread and diverse of all cell surface receptors (Buck and Axel, 1991).

2. G PROTEINS There have been a number of excellent reviews on various aspects of receptor, effector and G protein interactions (Gilman, 1987; Neer and Clapham, 1988; Ross, 1989; Taylor, 1990). However, a simple interpretation of the mechanism of signalling employed by G protein-coupled receptors requires that the G protein itself cycles between an inactive GDP-bound

425

426

T. JACKSOr~

and an active GTP-bound form (see Fig. 1). Activation, the exchange of GDP for GTP, is catalyzed by an agonist-receptor complex (Fig. lb), whilst inactivation by hydrolysis of bound GTP is due to an intrinsic GTPase activity in the G proteins ~t-subunit (Fig. ld). GTP binding to the heterotrimeric G protein (,t, fl and y subunits) leads to both a decrease in affinity for the agonist bound receptor and also to dissociation of the G protein into free ct and fly subunits (Fig. lc). GTP hydrolysis (Fig. ld) allows reassociation of the heterotrimer which may in turn associate with a receptor molecule so completing one activation-inactivation cycle (Fig. la). There is now a considerable weight of evidence indicating that GTP-bound free ~t-subunits are capable of stimulating appropriate effector functions. Thus retinal rod cGMP phosphodiesterase is activated by GTP-ct-transducin (Fung and GriswaldPenner, 1989; Chabre and Dettere, 1989) as adenylyl cyclase is by the cq-GTP complex (Gilman, 1987). However though ct-GTP can itself inhibit adenylyl cyclase, free fly may produce a similar effect by binding to and inactivating Gjt (Gilman, 1987). Controversial claims have been made for both fly and Gk~ (G~ct) subunits as being responsible for muscarinic receptor activation of a cardiac K ÷ channel (Codina et aL, 1987; Lognthetis et al., 1987; Sternweis and Pang, 1990). In yeast there is convincing genetic evidence that mating factor receptor signalling is mediated by interaction of the free fly complex with a.

an as yet unidentified effector 0Vhiteway et al., 1989; Nomoto et ai., 1990). There is growing evidence that not all/~y complexes are equivalent (Cerione et al., 1987; Casey et al., 1989) and though not as yet experimentally demonstrated it is possible that functionally distinct dimers could result from alternative pairings of individual fl or y subunits (Table 1). Coupled with the extensive range of distinct G protein ~t subunits which have already been identified (Table 2), this could provide for an extraordinary diversity in the routes of signal transduction available to G protein-coupled receptors.

3. THE G PROTEIN RECEPTOR FAMILY The application of molecular genetic techniques to the isolation of G protein-coupled receptors has brought home the true scale of diversity exhibited by this family of signalling molecules. More than 50 individual G protein-coupled receptors have now been identified. Their functions range from sensory receptors capable of detecting a single photon through a wide range of amine, peptide, odorant and other small molecule receptors to those for a number of glycoprotein hormones (see Tables 3, 4 and 5). A wide number of surprises have emerged from the isolation of these receptors, chief amongst which is probably the finding that commonly accepted pharmacological estimates of receptor diversity fall short A

~7

°

/

b.

Fro. 1. Signal transduction by G proteins. A simple model of receptor--G protein and --effector interactions. (a) The inactive GDP bound heterotrimer may associate with the unoccupied receptor. (b) Agonist binding to receptors catalyzes exchange of GDP for GTP on the O-proteins = subunit. (c) GTP binding to G-~ allows dissociation of the heterotrimeric complex; free ~y or GTP-~, subunits may then activate effector proteins. (d) GTP hydrolysis leads to inactivation and allows reassociation of the heterotrimeric G-protein. A---agonist; E----effector;R--receptor.

G protein coupled receptors T ~ L ~ 1. Identified G Protein ~ and 7 Subunits

Name

Reference

Mammalian:

"/~2

#3

}

Gilman (1987)

fit ~'t

Fong et al. (1986) Hurley et al. (1984)

Drosophila:

// Enhancer of split (fl-like) Yeast: Ste4 (,8) Stel8 (y)

Yarfitz et al. (1988) Hartley et al. (1988) Whiteway et al. (1989)

of that evinced by molecular biology. One example of this is the finding that beneath the pharmacological definitions of M 1, M 2 and glandular M 2 muscarinic acetylcholine receptors (as determined by selective antagonist affinities) there are at least five individual receptor molecules having distinct pharmacological profiles and tissue distributions (Bonner, 1989). Similarly whilst molecular genetics has confirmed the identities of pharmacologically defined ill- and //2" adrenergic receptors, it has also demonstrated the existence of a previously highly controversial class of 'atypical' ~3-adrenergic receptors (Emorine et al., 1989). These approaches have identified other novel amine receptors including: D3-, D4- and Ds-dopamine receptors having pharmacologies and distributions distinct from those of D : and D2-receptors (Sokoloff et al., 1990; Van Tol et al., 1991; Sunahara et al., 1991); two alternatively spliced forms of the D2dopamine receptor with identical pharmacology but potentially different signalling pathways (Dal Toso et al., 1989); and at least three genes for distinct human ~2-adrenergic receptors having unique phar-

macologies, tissue distributions and chromosomal locations (Lomasney et ai., 1990). Such discoveries have not been confined to the amine receptor subfamily. The cloning of the luteinizing hormone receptor (LH-CG-R) has allowed identification of alternatively spliced products lacking potential transmembrane segments which may serve other extracellular functions such as hormone transport or clearance (Loosefelt et al., 1989). Bombesin receptors preferring either gastrin-releasing peptide or neuromedin B have been isolated (Table 4) and multiple pharmacologically distinct receptors are now known to exist for the tachykinins (Shigemoto et al., 1990) and the endothelins (Arai et al., 1990; Sakurai et al., 1990). The cloning of two independent receptors for f-MLP suggests that this is also the case for the chemotactic peptides (Boulay et aL, 1990; Thomas et al., 1990). It may also be true for the angiotensins. The m a s - o n c o g e n e has been identified as an angiotensin receptor having both a novel distribution of expression and a unique pharmacology (Jackson et al., 1988).

4. G PROTEIN-COUPLED RECEPTOR STRUCTURES The cloning of the fl2-adrenergic receptor (Dixon 1986) confirmed that a 'classical' ligandbinding receptor had considerable sequence identity with the G protein-coupled 'light receptor' rhodopsin (Nathans and Hogness, 1984). By utilizing data on the physical structure from studies of bacteriorhodopsin (Henderson and Unwin, 1975) and rhodopsin itself, it has been possible to propose a canonical structure for the G protein-coupled recepet al.,

TABLE2. Identified G Protein a-Subunits MW (kDa)

CTx

Ptx

52 52 45 45 45 40 40 40 39 39 39

+ + + + + ? ? ? ? + +

sa sa sa sa sa + + + + + +

~/z

40

?

sa

?

gq ~'ll ~2 g~3

42 42 ? ?

? ? ? ?

sa sa ? ?

PLC + ? ? ?

Smrcka et aL (1991) Strathmann and Simon (1990) Strathmann et al. (1989) Strathmann et al. (1989)

PLC + ?

Quan et al. (1989) Provost et al. (1988) Yoon et aL (1989) Lee et aL (1990) Parks and Wieschaus (1991)

Name Mammalian: ~,l ~a • ,3 ~ "o~r • it 0q2 ~3 ~zo ~tl ~t2

Effector

Reference

cAMP + cAMP + cAMP + cAMP + cAMP + cAMP c A M P - "~ cAMP Ca2+/K + cGMP cGMP -

)

Bray et aL (1986) Reed and Jones (1989) Nukada et al. (1986) Itoh et al. (1988) Tanabe et al. (1985) Lochrie et al. (1985) Matsuoka et al. (1988); Fong et al. (1988)

Drosphila: oq O~o

,,q cta

Yeast: SCG 1/GPA 1

41.3 53

? ?

sa sa

427

Miyajima et al. (1987); Dietzel and Kurjan (1987)

428

T. JACKSON TABLE 3. The Amine Receptors

Receptor Adrenergic

Dopaminergic

Histaminergic Muscarinic

Octopamine Serotinergic

Subtype

Species

at a,/RDC5 a2"C2 1 a2-C4 ~ a2-Cl0J •1 /~2 //2 P3 Pt Dt Dh/D2b D3 De D~ H2 MI M2 M3 Me M5

hamster dog

Reference Cotecchia et al. (1988) Libert et al. (1989)

human

Lomasney et al. (1990)

human human hamster human turkey human/rat human rat human human dog human/pig/rat human/pig/rat human/rat human/rat human/rat

Frielle et al. (1987) Emorine et al. (1989) Dixon et al. (1986) Emorine et al. (1989) Yarden et al. (1986) Zhou et al. (1990) Dal Toso et al. (1989) Sokoloff et aL (1990) Van Tol et al. (1991) Sunahara et al. (1991) Gantz et al. (1991)

M

Drosophila Drosophila

Shapiro et al. (1989) Arakawa et al. (1990) Fargin et al. (1988) Witz et al. (1990) Julius et al. (1988) Pritchett et al. (1988) Saudou et al. (1990)

5HTta/G21 5HT t 5HTt¢ 5HT2

Tyramine

human Drosophila

rat rat Drosophila

tors (Finlay and Pappin, 1986). The receptors' core consists of a single polypeptide chain containing seven stretches of hydrophobic amino acids, each capable of forming a membrane-spanning a-helical segment of 18-30 residues in length. The seven

Bonner (1989)

transmembrane segments are connected by a series of hydrophilic intra- and extracellular loops, with free amino (N-terminal) and carboxy terminal (C-terminal) domains located in the extracellular space and the cytoplasm respectively. The structures adopted by

TABLE4. Peptide Receptors Receptor

Subtype

Species

Reference

Angiotensin Bombesin

III/mas GRP GRP NmB ETa ETb fMLPI FC3R

human mouse mouse rat cow rat human human rat rat cow human rat rat mouse human

Ste/a-mating factor

S. kluyveri S. cerevisiae S. cerevisiae

Jackson et al. (1988) Spindel et al. (1990) Battey et al. (1991) Wada et al. (1991) Arai et al. (1990) Sakurai et al. (1990) Boulay et al. (1990) Thomas et al. (1990) Tanaka et al. (1990) Shigemoto et al. (1990) Masu et al. (1987) Gerard et al. (1990) Yokoto et al. (1989) Yokota et al. (1989) Straub et al. (1990) Vu et al. (1991) Marsh and Herskowitz (1988) Burkholder and Hartwell (1985) Hagen et al. (1986)

Receptor

Polypcptide receptors Species

Reference

human rat pig rat human human dog

Gerard and Gerard (1991) Sprengel et al. (1990) Loosefelt et al. (1989) McFarland et al. (1989) Frazier et al. (1990) Frazier et al. (1990) Paramentier et al. (1989)

Endothelin fMLP Neurotensin Tachykinin

NmK SK SK SK SP

TRH Thrombin Ste2/a-mating factor

Anaphylotoxin/C5a FSH LH-CG TSH

G protein coupled receptors

429

TABLE5. Other G Protein-coupled receptors Receptor

Species

Adenosine-A2/RDC8 cAMP Cannabinoid Glutamine-metabotropic PAF Thromboxane A 2 Opsin

Name EDG1 Fc5R Frizzled

HCMVUS27 } HCMVUS28 HCMVUL33 RDC1 RDC4 (5HTla-like) ~7 RTA1 Odorant

Subtype

dog D. discoideum

rat rat guinea-pig human Opsins Species

Rhodopsin Rhodopsin blue- | green-/~ red- J

human cow

Reference Nathans and Hoguess (1984) Nathans and Hoguess (1983)

human

Nathans and Hoguess (1986)

Rh l / NinaE

Drosophila

Rh2 Rh3 Rh4

Drosophila Drosophila Drosophila

Zucker et al. (1985); O'Tousa et al. (1985) Cowman et al. (1986) Zucker et al. (1987) MonteU et al. (1987)

Unidentified receptors Species Reference Hla and Maeiag (1990) human Eva et al. (1990) rat Vinson et al. (1989) Dropsophila CMV

Chee et al. (1990)

dog

Libert qt al. (1989)

rat rat

Ross et al. (1990) Buck and Axel (1991)

these loops are as yet unknown, short sequences such as the first intracellular loop may form simple flturns, whereas longer stretches such as those between helices V and VI include both u-helical and fl-turn structures. Individual receptor species show considerable variation in the lengths of their intra- and extraeellular loops: in particular in the size of the third cytoplasmic loop and of the N- and C-terminal domains. However, as the recently isolated Drosophila 5HT,-receptor contains an additional Nterminal hydrophobic sequence which may be capable of forming a transmembrane helix 0Vitz et al., 1990), the term 7 transmembrane segment receptor may no longer be an accurate description for all members of the G protein receptor family. It is possible to align the primary sequences for all the currently identified G protein-coupled receptors. This task is made considerably easier by aligning them against a template composed of highly conserved residues such as that presented by Chee et al. 0989) and in a modified form in Table 6 (also see Fig. 2). The highly conserved residues are almost entirely restricted to the hydrophobic core of these receptors (Fig. 2). Amongst them are a number of conserved proline residues whose effect as helix-breaking residues must introduce kinks into the transmembrahe 0c-helices. These distortions may be critical in generating a basic structure which is capable of both binding agonist and transferring the energy of this interaction to activation of associated G proteins. wr S0/3--K

Reference Maenhaut et al. (1990) K l ~ n et al. (1988) Matsuda et al. (1990) Masu et al. (1991) Honda et al. (1991) Hirata et al. (1991)

A cysteine residue located in the second extracellular loop, between helices IV and V, is highly conserved amongst G protein-coupled receptors. It is implicated in forming an essential disulfide bond with another cysteine at the top of transmembrane segment III in both rhodopsin (Karnik et al., 1988) and the p2-adrenergic receptor (Dixon et al., 1988). This second residue is not conserved in the m a s / angiotensin III and cannabinoid receptors (Matsuda et al., 1990). However, in both of these receptors a cysteine residue located at the extraeellular end of transmembrane helix IV may be able to form a disulfide bond with its more highly conserved T AaLE 6. Residues Showing Maximal Conservation o f ldentity Amongst G Protein-Coupled Receptors

Location

Sequence

TMI GNxx0 TMII L A/S x x x 0 x x x x x x x x P/A/N EC II C TMIII 0xxLxx0xxD/E.R* TMIV WxxxxxxxxP TM V Y/F/W Y/F/W x P L x x x x x x Y TMVI 0xxxxxxFxxCWxP TMVII NPxxY TM--transmembrane helix; EC---extraceilular loop; x-any residue; 0---hydrophobic/nonpolar residues. A / separates alternative substitutions at one positon. The standard single letter code for amino acids is used. *D/E.R is located at cytoplasmic end of transmembrane helix.

430

T. JACKSON

a. ~AR

b.Rhodol~n

-

NH:

•

(

Q_

Q

NH:

c~

) C~H

FI~. 2. Residues implicated in receptor structure and ligand binding in G protein-coupled receptors. Schematic representations of (a) the fl2-adrcnergic receptor and (b) rhodopsin. The location of residues which show conservation of identity in all G protein coupled receptors are indicated by black letters on a white circle. Residues which have been implicated in interactions with agonists (a) or the retinal chromophore (b) arc shown as white letters on a black circle. Conserved cysteine residues implicated in forming a critical disulfide bond in each receptor arc indicated as a white C on a black diamond. In the fl2-adrenergic receptor (a) residues which may interact with antagonists only are shown simply as black letters. A number indicates the position of an individual residue in the linear sequence of the unmodified protein, these may be used to identify corresponding positions in the helical wheel projections in Fig. 3. neighbor and thus place appropriate constraint on the structure of the second extracellular loop. A helical wheel projection indicating the relative positions of individual side chains around each helical segment (based on one for rhodopsin presented by Nakayama and Khorana, 1991) indicates that some conserved hydrophobic residues are available for interaction with lipids on the outer face of the protein (e.g. Leu TM (TM III) and Trp 161 (TN IV) of rhodopsin). However, many actually lie at potential sites of interhelix contact leading to speculation that their interactions may actually involve pairing of hydrophobic residues (Fig. 3). The conserved Cys and Asn residues in helices VI and VII might also be involved in intramolecular interactions. Though their ability to form hydrogen bonds may also allow a structural role for Asn residues as hefix breakers (e.g. Asn 55 rhodopsin and Asn 51 in fl2-adrenergic receptors Fig. 3). Thus this pattern of conserved residues may actually define a basic structure utilized in the activation of all G protein-coupled receptors.

5. S T R U C T U R E - F U N C T I O N RELATIONSHIPS IN G P R O T E I N - L I N K E D RECEPTORS A complete understanding of the mechanism of receptor activation must eventually require investi-

gation of both agonist bound and unligated receptors by crystallographic or similar means. However, the manipulation of identified G protein receptor structures, through deletion or substitution mutagenesis, has provided a number of insights into the roles of individual domains and their component residues in receptor function. A series of gross deletions in the ~2-adrenergic receptor suggested that the location of the bound ligand is analogous to that of the retinal chromophore in rhodopsin, lying in a pocket formed between the transmembrane ~-helices (Dixon et al., 1987). Conserved aspartate and glutamate residues in helices II and III of the amine receptor subfamily have been proposed as potential counterions for the cationic amine moiety of their ligands (Dixon et al., 1988). Similarly mutagenesis studies on the hamster /~,-adrenergic receptor have suggested a role for Asp ]13 (TM III) in both agonist and antagonist binding (Strader et al., 1989), whilst Asp 79 (TM II) has been specifically implicated in agonist binding (see Fig. 4) (Dixon et al., 1988, Chung et ai., 1988). Substitution of potential hydrogen bond donors in the transmembrane domains of the hamster /~adrenergic receptor has implicated 3 residues; Set~ , Ser2°7 and Ser319 (TM V, TM V and TM VI respectively) in agonist but not antagonist binding. Similarly mutational analysis of a number of aromatic residues

G protein coupled receptors &.

~-AR

b. Rhodopsin

FI6. 3. Helical wheel projection indicating the relative locations of residues implicated in receptor structure and ligand binding in G protein receptors. Schematic representations of (a) the ~2-adrenergic receptor and Co) Rhodopsin. Each transmembrane helix is represented as a circle on which the relative angular positions of individual residues are displayed. Alternate helices run in opposing directions starting with helix one which runs anticlockwise into the membrane. The positioning of hcliees relative to one another and the determination of the starting position of each helix are as those proposed for rhodopsin by Nakayama and Khorana (1991). Residues showing conservation of identity in all G protein-coupled receptors are shown as black letters on a white circle. Residues which have been implicated in interactions with agonists (a) or the retinal chromophore Co) are shown as white letters on a black circle. Numbers identify position of individual residues in the linear sequence of unmodified proteins. Possible locations of the ligand's adrenalin (a), or c/s-retinal Co) are shown. The helices are oriented as if looking from the cytoplasm to the exterior.

431

432

T. JAClCSO~ . NH

2

'1

C_ COOH

FIG. 4. Regions of the fl2-adrenergic receptor implicated in interactions with G proteins. A schematic representation of the human ~2-adrenergic receptor. The location of residues which show conservation of identity in all G protein-coupled receptors are indicated by black letters on a white circle. Residues whose substitution or deletion leads to alteration or abolition of fl-adrenergic receptor Gs coupling are indicated as white letters on black. These include the identified site of palmitylation at Cys34~ in the receptors' cytoplasmic tail. A number indicates the position of an individual residue in the linear sequence of the unmodified protein; these may be used to identify corresponding positions in Fig. 2a. potentially responsible for interaction with the catechol ring of adrenergic agonists has suggested roles for Phe 2a9 and P b e ~ from helix VI and Tyr 3~6 from helix VII (Dixon et al., 1988). This study also implicated Tyr 326 (TM VII) in catecholamine binding; however, the conservation of this residue in most identified receptors suggests, rather, a role in the maintenance of the agonist binding sites structure. However, the helical wheel projection of the fl2-adrenergic receptor (Fig. 4) suggests that of those residues proposed only Scr319 (TM VII), Phe 289 (TM VI) and Tyr 3~ (TM VII) are actually likely to be available for interaction with ligands in the receptors' core. This projection supports the role proposed for Asp 79 (TM II) as a countedon for a cationic amine moiety. It aiso suggests that residues projecting into the ligand binding pocket from transmembrane helix III, such as Cys ns or Thr "8, may also participate in interaction with catccholamine ligands. Superimposition of the primary sequence of ovine rhodopsin on structural co-ordinates determined for bacteriorhodopsin has led Findlay and Pappin (1986) to suggest roles for Glu t~3 (conserved in an identical position in TM III in bovine rhodopsin) and Asp 8a

(TM II) respectively as a counterion and wavelength regulator for the retinal chromophore. The position of Asp aa corresponds with that of Asp 79 in the fl2adrenergic receptor (see Figs 2 and 3). However, the helical wheel projections suggest that neither rhodopsin's Glu 113 nor the fl2-adrenergic receptors Asp m is actually located in the ligand-binding pocket: though it is possible that a kink in this helix could shift these residues into a position more favorable for ligand binding. Crosslinking and mutagenesis studies have implicated another residue on helix III, Trp 126, in interactions with the chromophore's ionone ring. The site of retinal attachment, Lys296 in helix VII of rhodopsin, is conserved in all identified opsins: and though its immediate neighbors vary their nature does appear to be limited by constraints on size of side chain (Findlay and Pappin, 1986). The position of Lys~ corresponds with that of Tyr ~l~ in the hamster fl2-adrenergic receptor which with its neighboring residues Asn m and Ser 3~9has been implicated in agonist binding (Dixon et aL, 1988). A common pattern of ligand-receptor interactions may be proposed for both rbodopsin and the

G protein coupled receptors ~2-adrenergic receptor involving residues situated on helices II, III, VI and VII. Significantly residues in corresponding positions show conservation of identity in receptors which are otherwise divergent in sequence though able to recognize common ligands (Table 7). This model is clearly distinct from that proposed by Dixon et aL (1988) which suggested that the fl2-adrenergic receptors agonist-binding site was actually formed by helices III, IV, V and VI. Consistent with this model is the observation that in putative odorant receptors regions of maximal divergence of identity lie on helices III, IV and V (Buck and Axel, 1991). However receptor chimeras between ~2- and fl2-adrenergic receptors demonstrate that a region inclusive of helices VI and VII is responsible for determining the specificity of agonist and antagonists for these receptors, thus lending support to the model outlined above. Photoaffinity labelling of the turkey fl-adrenergic receptor (flt-adrenergic receptor) has identified a tryptophan residue at the extraceUular end of helix VII as part of an antagonist binding site (Wong et al., 1988; Ross et al., 1988). Dixon et al. (1987) have found that deletion of residues in the extracellular loop between helices VI and VII reduces binding of fl-adrenergic receptor antagonists but not agonists. Thus an antagonist binding site appears to involve residues on helices III and VII as well as some in the third extracellular loop. Binding of antagonists to these residues may effectively block access to sites of agonist interaction located in the pocket formed by the receptors' transmembrane helices. The glycoprotein hormone receptors (those for thyroid-stimulating hormone, TSH; luteinising hormone-coriogonadotropin, LH-CG, and follicle stimulating-hormone, FSH; see Table 4) differ from other G protein-coupled receptors in that it is their large N-terminal domain which is responsible for ligand binding. Substitution of potential sites of N-linked glycosylation in the N-terminal domain of the TSH receptor prevents ligand binding suggesting a role for glycosylation in either stabilization of receptor structure or directly in ligand recognition (Russo et al., 1991). This is in direct contrast with the fl2-adrenergic receptor in which loss of glycosylation has no effect on ligand binding (Boege et al., 1988).

433

The presence of a large N-terminal domain with homology to the kainic-giutamate receptor in the metabotropic-glutamate receptor has led to speculation that these domains may also provide binding sites for small molecules (Masu et aL, 1991). Whether such large N-terminal domain proteins couple ligand binding to G protein activation by the same pathway utilized by the smaller receptors awaits investigation.

6. RECEPTOR--G PROTEIN INTERACTIONS: SPECIFICITY Using a series of receptor chimeras Kobilka et al. (1988) defined a region between helices V and VI of the human fle-adrenergie receptor as being sufficient to confer the ability to stimulate Gs to an ~2-adrenergic receptor. This region encompasses sequences at both N- and C-terminal ends of the third intracellular loop implicated in Gs coupling by deletion mutagenesis of the hamster fl2-adrenergic receptor (Strader et al., 1987; see Table 8 and Fig. 4). Muscarinic receptor subtypes M~, M3 and M5 couple to stimulation of inositol phosphate production via a pertussis toxin-insensitive G protein, whilst Me and M4 receptors couple to inhibition of adenylyl cyclase. M2 and M4 receptors also weakly stimulate inositol phosphate production, but through a pertussis toxin-sensitive species (Bonner, 1989). Chimeras between M~ and M2 receptors support the location of sequences responsible for selective determination of effector coupling in the third intracellular loop (Kubo et ai., 1988). M2/M3 receptor chimeras prepared by Lechleiter et al., (1990) have further defined the region of selective effector coupling. They found that transfer of a nine amino acid section from the N-terminal of the M3 receptor's third cytoplasmic loop to a corresponding position in the M e receptor was sufficient to confer M3-1ike responses. Surprisingly, substitution of the equivalent sequence from M: into the M3 receptor failed to generate Me-like responses. However, transfer of a 21 residue section including these residues and their N-terminal neighbors was able to confer full M2-1ike responses to the M3 receptor (see sequences in bold in Tables 9 and 10).

TABLE7. Conservation of Residues Implicated in Ligand Binding in Receptors for Related Agonists

II III VI VII con LAxx*L *xSI*xL* CWxP* x*xxxxxNP ETa LALGDL GITVLNLC CWFPL ATMNSCINP ETb LALGDL GITVLSLC CWLPL ASLNSCINP NmB LAAGDL VFTLTALS CWFPN SFSNSCVNP GRP LALGDL VFTLTALS C W L P N AFTNSCVNP Fc3R L A M A P A YSGILLLA C W L P Y GFLHSCLNP fMLP LAVADF FGSVFLIA C S W P Y AFFNSCLNP d5HT1 LALSDL TASILNLC C W L P F GYANSLLNP 5HTta LAVTDL TSSILHLC CWLPF GYSNSLLNP The positions of putative ligand binding residues are indicated in the consensus sequences by *. x--any residues. ET,---endothelin,; ETb--endothelinb;NmB--neuromedin B/bombesin;GRP-gastrin-releasing peptide/bombesin; Fc3R--fMet--Leu--Phe; fMLP-fMet-Leu-Phe; d5HTI--Drosophila 5HTfi 5HTt,--human 5HTmareceptor. For references see Tables 3 and 4.

T. JACKSON

434

TABLE 8. Residues Proposed to Determine Specificity of Coupling in Receptors which Stimulate Adenylyi Cyclase fit huff1 huff2 haft2

R V

huff3 huDi caH2 dr5HTl hu5HTl^ CaAJRDC8 Cons

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

Y F F F

R R Q Q

E E E V

A A A A

K Q K K

E K R R

Q Q Q Q

I V L L

R K Q Q

K K K K

I I I I

D D D D

R S K K

C C S S

E E E E

G R G G

R R R R

F F F E

F Y F F F F

V R K R R L

V I <5> A A A

A A A A A A

T Q K R R R

R K R R E R

Q Q R Q

L I I I I L

R R H V R K

L R H L K Q

L I M E T M

R A G E V E

G A S K K S

E L W R K Q

L E K A V P

G R A Q E L

R A A T K P

F A T H T G

A

+

+

Q

0

+

+

0

-

+

V V V

R R R

N N N

P P P

E H T

L N I

M P V

A G S

T D S

N K S

F/Y +

huLH/CG huTSH raFSH

Y Y Y

F I L

A T T

K S

D D D

+ T T T

K K K

I I I

Numbers above sequences refer to positions on helical wheel projection (Fig. 5). Sequencesaligned by use of conserved proline and tyrosine (or acceptable substitutions) in transmembrane helix V (see Table 6). Residues conserved in more than 50% of receptor sequences are listed and their relative positions are plotted in Fig. 5. Mutagenesis of sequences in bold type is known to influence G protein coupling. Sequences in the lower panel contain potentially helix breaking proline residues and so were omitted from assignment of conserved positions, ca---canine; dr--Drosophila; ha--hamster; hu--human; ra--rat. and 17. That such amphipathic structures may be involved in G protein coupling was first suggested by findings that Mastoparan, a wasp venom toxin which may form a similar structure, is capable of directly stimulating G protein activation (Higashijima et al., 1988). A surprising finding of this analysis is that the receptors which couple to inositol phosphate production appear to fall into at least two separate groups; suggesting that each group may actually couple through a distinct G protein (Tables 10 and 11). Different G proteins need not necessarily bind to residues at identical positions on this structure so that on any given receptor an overlap of G protein specificities may be possible. Both Mrmuscadnic fl~-adrenergic receptor and certain M2/M 3 musearinic chimeras were capable of coupling to multiple G proteins (Wong et al., 1990; Lechleiter et al., 1990). This may not be merely a feature of artificial receptor hybrids: both the D2-dopamine and a2-adrenergic receptors have been reported to show coupling to different effectors according to the cell type expressing them (Valler et al., 1990; Cotecchia et al., 1990b)

Studies utilizing 0q-adrenergic receptor or M r muscarinic///2-adrenergic receptor chimeras, respectively, have implicated overlapping sequences in the third intracellular loop of each receptor in determination of G protein specificity (Wong et al., 1990; Cotecchia et al., 1990a). Loss of Gs coupling after deletion of a similar overlapping section of the fieadrenergic receptor was reported by Dixon et al. (1988; bold type in the hamster fl2-adrenergic receptor Table 8 and equivalent human sequences Fig. 4). The conservation of sequences including this region in muscarinic receptors with common mechanisms of effector coupling has been noted by Bonner (1989). Using the database of sequences now available it is possible to discern conserved patterns of both charged, neutral-polar and nonpolar residues in alignments of this region (Fig. 5 and Tables 8-11). A potential structure for this region can be imposed on an ~t helix of up to 18 residues in length, having conserved nonpolar/aromatic residues at positions 1 and 8, and a generally polar surface located between residues 12 and 6 (Fig. 5). Residues which may confer specificity upon G protein interactions then appear to lie on a surface between positions 11

TABLE 9. Residues Proposed to Determine Specificity of Coupling in Receptors which Inhibit Adenylyl Cyclase huM2 huM4 raCR hu,v2- 2 hu"2- l0 hu~t2- 4 raD2 drTYR

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

H I S

R

A

S

K

S

K

I

K

K

D

K

K

E

P

V

A

N

S

L

A

S

R

S

R

V

H

K

H

R

P

E

G

P

K

E

L Y Y

W L Q

K I I

A A A

H K K

S R R

H S R

A N T

V R R

R K V

M G P

I P P

Q R S

R A R

G K R

T G G

Q G P

K P D

Y Y

R I

V V

A L

K R

R K

R R

T R

R K

T R

L V

S N

E T

K K

R R

A S

P S

V R

F

V

A

T

R

R

R

L

R

E

R

A

R

A

N

K

L

N

Q

Cons Y/F 0 A/S + + + 0 + + +/Numbers above sequences refer to positions on helical wheel projection (Fig. 5). Sequencesaligned by use of conserved proline and tyrosine (or acceptable substitutions) in transmembrane helix V (see Table 6). Residues conserved in more than 50% of receptor sequences are listed and their relative positions are plotted in Fig. 5. Mutagenesis of sequences in bold type is known to influence G protein coupling, dr--Drosophila; hu--human; ra--rat.

G protein coupled receptors

435

TABLe 10. Residues Proposed to Determine SpecOfcity of Coupling in Type I Receptors which Stimulate Phospholipase C 1

2

3

4

5

6

7

8

9

10

11

drM~

W

R

E

T

K

K

R

Q

K

D

huM, huM 3

Y Y

R K

E E

T T

E E

N K

R R

A T

R K

E E

huM s raNT ra5HT2 ra5HTic

Y A Y Y

R K F F

E L L L

T T T T

E V I !

K M K Y

R V S V

T H L L

K A Q R

D E K R

Con

Y

+

+

+

0

+

-

E/L T

12

13

14

15

16

17

18

L

P

N

L

Q

A

G

K

L L

A A

A G

L L

Q Q

G A

S S

E G

L Q E Q

A G A T

D R T L

L V L M

Q C C L

G T V L

S V S R

D G D G

L/Q A

0

Numbers above sequences refer to positions on helical wheel projection (Fig. 5). Sequences aligned by use of conserved proline and tyrosine (or acceptable substitutions) in transmembrane helix V (see Table 6). Residues conserved in more than 50°/, of receptor sequences are listed and their relative positions are plotted in Fig. 5. Mutagenesis of sequences in bold type is known to influence G protein coupling, dr--Drosophila; hu--human; ra--rat.

suggesting an authentic overlap of G protein specificities.

7. R E C E P T O R - G PROTEIN INTERACTIONS: EFFICACY

+"~,:~:~+ +.++

2

NS ~ ~YIF

~

+/_

E~L~

÷

+

A Sl~ly 0

~+

Q

+

Fro. 5. Residues implicated in receptor-G protein interactions. Helical wheel projections show the relative positions of residues on a putative 18 amino acid a helix. Position 1 is designated from the position of a conserved tyrosine residue at the base of transmembrane helix V (Table 6). The consensus charge/polarity plotted for each group of receptors is that derived in their respective tables. Table 8: receptors stimulating adenylyl cyclase +cAMP. Table 9: receptors inhibiting adenylyl cyclase -cAMP. Table 10: receptors coupling to polyphosphoinositide-specific phospholipase C (type I) PLC I. Table 11: receptors coupling to polyphosphoinositide-speeitic phospholipase C (Type II) PLC II: For Table 12, the pattern for the c(3MP phosphodiesterase-coupled human opsins 0au Ops) is presented. 0, hydrophobic/nonpolar; + , positively charged; - , negatively charged residues.

The C-terminal section of the third cytoplasmic loop may also be involved in receptor-G protein interactions as, with the exception of some amine receptors, this entire loop often contains less than 30 amino acid residues. Conservative substitutions in this region actually increase the agonist sensitivity of the g2-adrenergic receptor (Cotecchia et al., 1990a). Cells expressing these mutant receptors show an enhanced basal level of inositol phosphate production which may be reduced by addition of antagonists. The effect of the substitution may have been to lock the receptor into an activated conformation but which may shift towards an inactive form on antagonist binding. Alternatively the receptors sensitivity may really be increased to the extent that low levels of catecholamines in serum free medium are sufficient to stimulate it. Intriguingly cells expressing the normal RDCS/A2-adenosine receptor show a similar pattern of high basal stimulation of adenylyl cyclase which may reflect the activity of a highly sensitive receptor subtype (Maenhaut et al., 1990). Substitution mutagenesis of the P2-adrenergic and TSH-receptors has also indicated roles for residues in both the first and second cytoplasmic loops in recept o r - G protein interactions (Dixon et al., 1988; Fraser et al., 1988; Chazenbalk et al., 1990; see Fig. 4). However, it is possible that such results could arise from allosteric rather than direct participation in receptor-G protein interactions. Similarly competition studies between antibodies against the second cytoplasmic loop of rhodopsin and Gt may reflect simply steric hinderance of G protein-receptor coupling rather than any specific interaction involving this region (Wiess et aL, 1988). Wong et al. (1990) found that whilst substitution of the second cytoplasmic loop from the M]-muscarinic receptor into the fitadrenergic receptor did not change G protein specificity, substitution with both second and third cytoplasmic loops from the muscarinic receptor gave

436

T. JACKSON

TABLE 11. Residues Proposed to Determine Specificity of Coupling in Type H Receptors which Stimulate Phospholipase C 1

boET^ raETe huF3R huAIII huC5a huThr moORP moTRH gpPAF

Y Y Y I Y Y Y Y I

Con

Y

ramGLU raSKR raSPR raNmK

T Y Y Y

Con

Y

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

L L L I F S P

M M R L I S V

T T T V L A E

C C L V L V G

E E F K R A N

M M Q V T N I

L L A R W R H

N R H K S S V

R K M N R K K

R K G T R K K

N S Q W A S Q

G G K A T R I

S M H S R A E

L Q R H S L S

K R

E Q

N P

S V

K K

M Q

W Q

K R

N N

D A

S E

I V

0

+/-

0

+

+

+

O

F

<6> < 12 > < 12 > I

L N

T T T T T S L

P H

S T

D L

P L

T

0

0

Y L

A T

F L

K W

T K

R R

N A

V V

P P

A R

N H

F Q

N A

E H

A G

K A

I I

T T

L L

W W

A G

S G

E E

I I

P P

G G

D D

S T

S C

D D

R K

Y Y

0

T

0

W

+

--

0

P

-

+

0

Y <4> < 4> < 4>

-

Numbers above sequences refer to positions on helical wheel projection (Fig. 5). Sequences aligned by use of conserved proliue and tyrosine residues (or acceptable substitutions) in transmembrane helix V (see Table 6). Residues conserved in more than 50% of receptor sequences are listed and their relative positions are plotted in Fig. 5. Sequences in the lower panel contain potentially helix breaking proline residues suggestive of a variant structure and so were omitted from assignment of conserved positions, bo--bovine; gp--guinea-pig; ha--hamster; hu--human; mo--mouse; ra--rat. a greater efficiency of coupling to phospholipase C than that provided by the third loop alone. Neither truncation mutations, which remove large portions of the cytoplasmic tail from ~-adrenergic or the yeast Ste2/~-mating factor receptors (Cotecchia et al., 1990a; Blumer et al., 1988) nor proteolytic removal o f a similar fragment of rhodopsin (Findlay and Pappin, 19886) have any effect on their ability to couple to G-proteins. However, substitution of a short sequence of the C-terminal tail adjacent to transmembrane helix VII in the fll-adrenergic receptor with a corresponding region of the fle-adrenergic receptor diminishes its coupling to phospholipase C (Cotecchia et al., 1990a). In a number o f receptors this proximal region of the C-terminai tail is proposed to form a fourth cytoplasmic loop following palmitoylation at conserved cysteine residues (Ovchinnikov et aL, 1988). Substitution of this cysteine in the fl2-adrenergic receptor reduces its coup-

ling to (3, ( O ' D o w d et al., 1989). However, as deletions in this region also dramatically reduce receptor stability (Dixon et aL, 1987) its importance may lie in maintaining an appropriate structural constraint rather than in direct coupling to a G protein.

8. D E S E N S I T I Z A T I O N A N D DOWNREGULATION Studies of both the yeast Ste2/a-mating factor and fl2-adrenergic receptors have highlighted a role for the more distal component of their C-terminal tails in stimulus-dependent uncoupling of the receptor from its signalling pathway. Many G protein-coupled receptors show desensitization; and it is known that for some including the ~¢-adrenergic (Lecb-Lundberg et al., 1987), fl2-adrenergic (Sibley et al., 1987),

TABLE 12. Residues Proposed to Determine Specificity of Coupling in Opsins which Stimulate Phospholipase C 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

drRhl drRh2 drRh3 drRh4

Y Y Y Y

<3> <3> <3> < 3>

I I V V

A A G G

A A H H

V V V V

S A F F

A A S S

H H H H

E E E E

K K K K

A A A A

M M L L

R R R R

E E D E

Q Q Q Q

A A A A

K K K K

Con

Y

A/S

H

E

K

A

0

R

-

Q

A

K

0 A/G

V

Residues Proposed to Determine Specificity of Coupling in Opsins which Stimulate cGMP Phosphodiesterase 1

2

3

4

5

6

7

8

9

I0

11

12

13

14

15

16

17

18

huG huR huB huRh

Y

< 3>

L

A

I

R

A

V

A

K

Q

Q

K

E

S

E

S

T

Y Y

< 3> < 3>

L R

A A

I L

R K

A A

V V

A A

K A

Q Q

Q Q

K Q

E E

S S

E A

S T

T T

Y

< 3>

F

T

V

K

E

A

A

A

Q

Q

Q

E

S

A

T

T

Con

Y

A

0

+

A

0

A

Q

Q

E

S

T

Numbers above sequences refer to positions on helical wheel projection (Fig. 5). Sequences aligned by use of conserved proline and tyrosiue residues (or acceptable substitutions) in transmembraue helix V (see Table 6). Residues conserved in more than 50% of receptor sequences are listed and their relative positions are plotted in Fig. 5. dr--Drosophila; hu--human.

G protein coupled receptors rhodopsin (Wilden and Kuhn, 1982) and Ste2/~t-mating factor receptors (Blumer et a/., 1988) that this process correlates with phosphorylation of the receptor itself. The kinases responsible for these events are unique in that they recognize only the activated state of the receptors; though they show some ability to phosphorylate other receptors but rhodopsin- and fl2-adrenergic receptor kinases (fl-ARK) appear to be separate entities (Benovic et al., 1986). The recent cloning of the gene for fl-ARK suggests that it is only one representative of a large family of related protein kinases (Benovic et al., 1989). The major site of phosphorylation of both rhodopsin and the Ste2/ct-mating factor receptor lies in their C-terminal tails (Benovic et aL, 1986; Blumer et aL, 1988). Deletion mutagenesis of the Ste2 product demonstrates that removal or alteration of this region generates cells which are hypersensitive to x-mating factor (Konopka et al., 1988; Reneke et al., 1988). Similarly deletion of a serine- and threoninerich domain in the fl2-adrenergic receptor's C-terminal tail, or their substitution, delays the onset of agonist-promoted desensitization (Bouvier et al., 1988). Inactivation of bleached rhodopsin following phosphorylation by rhodopsin kinase requires the receptors association with a 48 kDa protein called arrestin (Wilden et al., 1986). Though high concentrations of retinal arrestin can inactivate phosphorylated fl2-adrenergic receptors (Benovic et al., 1987), a homologous protein, fl-arrestin, having greater specificity for the fl-adrenergic receptor has recently been isolated by Lohse et al. (1990). It seems likely that this will prove to be a common mechanism of inactivation amongst the G protein-coupled receptor family. C-terminal deletions of the Ste2/~ mating factor receptor have also suggested a role for this region in the regulation of ligand-dependent endocytosis (Reneke et aL, 1988). A suggestion that C-terminal tyrosine residues might be involved in ligand induced sequestration of the t2 have not been supported by studies on mutants with substitutions at these positions (Valiquette et aL, 1990). Intriguingly though the flt-adrenergic receptor which normally fails to show this phenomenon may become sensitive to ligand-induced endocytosis following the loss of additional sequences at the end of its large C-terminal tail (Hertel et aL, 1990). Thus the role of C-terminal sequences in regulating receptor sequestration and agonist sensitivity in general must remain as prime areas for future investigation.

9. PROSPECTS The last five years have witnessed an astonishing expansion in our knowledge and understanding of the G protein receptor family. However, as always, one answer only generates a new plethora of questions. Despite the clues provided by mutagenesis and modciting, resolution of the precise mechanisms of ligand binding and G protein activation must await the application of more detailed means of structural investigation. The flexibility of the basic structure of these receptors is obvious from the variety of ligands which

437

members of the family are capable of selectively recognizing, including a diversity of peptides, biogenie amines and polypeptide-hormones in addition to the enormous range of molecules recognized by the odorant receptor subfamily. The identification of a novel mechanism of receptor activation; proteolytic cleavage of the thrombin receptor giving exposure of an agonist peptide integral to its N-terminal domain (Vu et al., 1991) only serves to further emphasize this flexibility. As yet there is little data on the regulation of expression of G protein receptors. One such receptor has been identified as an immediate-early response gene induced by phorbol esters (Hla and Maciag, 1990) suggesting that their selective expression may play a role in acutely determining cellular responses. A more long term role in determining cell fates is suggested by the identification of one such receptor as a tissue-polarity gene in Drosophila (Vinson et aL, 1989). Only a limited number of G protein receptors show the potential for differential splicing. However, those which do demonstrate both tissue specificity and differential coupling of their alternatively spliced products (Frazier et al., 1990; Dal Toso et al., 1989). A point mutation in human rhodopsin has now been identified as the underlying cause of one form of retinal degeneration (Dryja et al., 1990); whilst deletion or partial exchange of the visual pigment genes is recognized as a source of color blindness (Nathans and Hogness 1986). Similar aberrations or mutations in genes encoding receptors may lie behind other inherited disorders. The importance of a number of these receptors, adrenergic, dopaminergic and H2-histaminergic as targets for pharmacological intervention is already obvious. However the identification of G protein receptors as potential oncogenes (Jackson et al., 1988; Julius et al., 1989), and of receptor homologs in a human herpes-virus (Chee et aL, 1990) suggest that the therapeutic value of agents which act at such receptors has yet to be fully appreciated. thank L. A. C. Blair and P. T. Hawkins for many helpful suggestions regarding this manuscript. I thank The Royal Society for support through a Mr and Mrs J. Jaffe Research Donation Fellowship. Acknowledgements--I

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