The family of G-protein-coupled receptors - CiteSeerX

REVIEW

The family CATHERiNE

of G-protein-coupled D. STRADER,’

TUNG M1NG FONG, MICHAEL P. GRAZIANO,

Department of Molecular Pharmacology 07065, USA

and Biochemistry,

The family of G-protein-coupled receptors can be defined by their similar structural and functional characteristics. Although their primary sequences are quite diverse, these proteins share several common structural features that reflect their common mechanism of action. Mutagenesis and biophysical analysis of several of these receptors indicate that small molecule agonists and antagonists bind to a hydrophobic pocket buried in the tranamembrane core of the receptor. In contrast, peptide ligands bind to both the extracellular and transmeinbrane domains. The mechanisms by which these peptide and small molecule agonists cause receptor activation are being explored by various approaches, but are not yet well defined. A deeper understanding of structural basis for the functional activity of this large family of receptors will have important implications for drug design in a variety of therapeutic areas.-Strader, C. D., Fong, T. M., Graziano, M. P., Tota, M. R. The family of G-protein-coupled receptors. F.4SEB J. 9, 745-754 (1995)

ABSTRACT

Key Words transduction

amino

receptors

acid sequences #{149} peptide binding #{149} helices

site

#{149} signal

Merck Research

Laboratories,

AND MICHAEL

R. TOTA

Rahway, New Jersey

resides within the hydrophobic transmembrane domain, whereas the hydrophilic loop regions are more divergent. Upon detailed analysis of their sequences, GPCRs can he further classified into three subfamilies, termed the rhodopsinJ-adrenergic, secretin/vasointestinal peptide (VIP), and metabotropic glutamate (mGlu) receptor families. Members of the rhodopsin/-adrenergic subfamily constitute the majority of GPCRs identified to date. In addition to rhodopsin and the -adrenergic receptors (f3ARs), this family includes receptors for other small molecules such as dopamine and serotonin, as well as peptides such as substance P. bradykinin, angiotensin, and somatostatin, and glycoproteins such as lutropin. The structure-function relationships between ligand binding to the receptor and signal transduction to G-proteins have been extensively studied for several members of this family. The secretin/VIP and mGlu families share little primary sequence homology with each other or with the rhodopsin/f-adrenergic family of receptors, a!though their overall predicted tertiary structure is similar. These receptors will be discussed at the end of this article.

MOLECULAR

MODELING

OF RECEPTORS

#{149} musagenesis

The family of receptors that activate G-protein-coupled signal transduction pathways share characteristic structural features that presumably reflect their common mechanism of action (see ref 1 for review). In recent years, the deduced amino acid sequences of a large number (250) of G-proteincoupled receptors (GPCRs)2 have been elucidated. This explosion of primary sequence information is due, in large part, to the widespread application of molecular cloning techniques. The current challenge in the field is to apply and extend this molecular information to better understand the mechanism of action of these receptors and their downstream signaling events. GPCRs bind a variety of ligands ranging from small biogenic amines to peptides, small proteins, and large glycoprotein hormones (2). Despite the wide range of ligands that activate these receptors, the receptors themselves share a surprising amount of structural homology, both primary and tertiary. The overall structural features of the GPCR family are highly conserved: all of these receptors contain seven hydrophobic domains, postulated to span the plasma membrane, connected by hydrophilic extracellular and intracellular ioops. The majority of the primary sequence homology

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Because of the paucity of tertiary structural information on the family of GPCRs, investigators are combining biochemical, pharmacological, and genetic approaches with molecular modeling and biophysical analysis to identify key elements in the signaling mechanism of these receptors. Unfortunately, obtaining high-quality crystals for structural determination on membrane proteins remains extremely difficult. Until the structure of at least one GPCR is determined at atomic resolution, the structural insights inferred from mutagenesis studies can be combined with computer modeling to reconcile the various experimental data and suggest further experiments to refine or support the original model. For GPCRs, one major feature in the primary sequence is the occurrence of seven relatively hydrophobic

1To whom correspondence

and reprint

requests

should be addressed,

at:

Department of Molecular Pharmacology and Biochemistry, 80M 213, Merck Research Laboratories, Box 2000, Rahway, NJ 07065, USA. 2Ahbreviations: GPCR, G-protein-coupled receptor, YIP, vasointestinal peptide; mGlu, metabotropic glutamate; I3AR, 3-adrenergic receptor NK1, neucokinin-1; TRH. thyrotropin-reteasing hormone; GLR-1, glucagon-like peptide 1; PACAP, pituitary adenytate cyclase activating polypeptide; PTH, parathyroid hormone

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REVIEW regions, and the periodic distribution of hydrophobicity is consistent with an a-helical conformation (3). This feature provides a rationale for modeling GPCRs after bacteriorhodopsin, which contains seven transmembrane helices and whose structure has been elucidated by electron diffraction (4). Although bacteriorhodopsin is not a GPCR, its primary sequence can be aligned with rhodopsin of the GPCR family, and both are activated by photoisomerization of retinal. Several groups have generated model of GPCRs by using the bacteriorhodopsin as a template and orienting the seven hydrophobic regions in GPCRs in the same fashion as bacteriorhodopsin (reviewed in ref 2), although alternative models have also been proposed (5). A projection map of rhodopsin has been reported (6), and suggests that the helical orientation may differ slightly between the two proteins (7). More recently, experimental data from receptor mutagenesis have led to further refinement of the original model (see below). For example, the vertical position and lateral orientation of the transmembrane helices in the AR can be realigned to allow simultaneous binding of the functional groups of the catecholamine to the residues shown by mutagenesis studies to be involved in ligand binding to the receptor. A model of the neurokinin-1 (NK1) receptor based on this AR model can then be further refined to modify the orientation of helices 4-7 to accommodate the small molecule antagonist site predicted from the mutagenesis studies of the NK1 receptor (see below). These exercises constrain the interresidue and interhelical distances to the limits of flexibility of the small molecule ligands. The current 3-dimensional models of GPCRs will continue to be modified as additional experimental data become available. Meanwhile, receptor modeling can provide a visualization tool with which to formulate testable hypothesis that can be verified by receptor mutagenesis and/or new ligand design.

SMALL MOLECULE

BINDING

SITE

The fAR, which binds the endogenous catecholamine agonists epinephrine and norepinephrine and activates the C-protein Gs to stimulate adenylyl cyclase, was the second GPCR to be cloned: after rhodopsin. The homology of this receptor with rhodopsin that was noted at the time was the first indication that these proteins form a structurally, as well as functionally, related family of receptors (8). Initial scanning deletion mutagenesis experiments indicated that the hydrophilic loop regions of the receptor were not important for agonist or antagonist binding, suggesting that the ligand binding domain of the 3AR must involve residues within the hydrophobic transmembrane domain (9). The analogy with rhodopsin was again apparent from these experiments in that the binding site for retinal in rhodopsin had previously been demonstrated to be buried within the core of that protein (10). More detailed analysis of the binding site relied on single residue substitutions within the transmembrane core of the AR. To identify specific molecular interactions between

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ligands and receptors, we have combined receptor mutagenesis with analysis of structurally modified analogs of its ligands in an approach that we term 2-dimensional mutagenesis. By looking at the additivity of the effects of modifying functional groups on the ligand and on the receptor, this “2-dimensional” approach allows the dissection of individual interactions between the receptor and the ligand. For the AR, the design of the mutations was based on pharmacophore maps that had been derived by medicinal chemical analysis of receptor-ligand interactions (ii). A detailed description of the ligand binding site of the receptor could be inferred from the structure-activity relationships developed for f-adrenergic agonists and antagonists. The genetic approach made possible by the availability of the DNA encoding the receptor then allowed the receptor pharmacophore to be tested and refined by making mutations in the receptor to probe residues that could potentially correspond to contact points according to the pharmacophore map. For example, all adrenergic agonists and antagonists are basic amines, suggesting that there is an acidic residue in the receptor that can provide the counterion for the amine on the ligand. Systematic mutagenesis of the negatively charged residues in the transmembrane domain of the AR implicated Aspi 13 in transmembrane helix 3 as the counterion for the basic amines in both agonists and antagonists. Subsequent analysis indicated that whereas this ionic interaction is a major source of binding energy between the ligand and the receptor, it is not critical for receptor activation. In fact, both agonists and antagonists interact with the side chain of Aspi 13 in the receptor

(12). In contrast, agonist activation of the AR was determined to involve an interaction of the catechol ring of the agonist with residues in transmembrane helices 5 and 6 (13). The aromatic catechol-containing aromatic ring appears to interact with the side chain of Phe290 in transmembrane helix 6, whereas the catechol hydroxyl groups of the agonist form hydrogen bonds with the side chains of two Ser residues, at positions 204 and 207 in transmembrane helix 5. The specific interactions between the ligand and the receptors at these positions were elucidated using 2-dimensional mutagensis, in which the activation of these mutant receptors by analogs of isoproterenol having each of the hydroxyl groups replaced with a hydrogen were analyzed. These studies indicated that there is a specific hydrogen bond linking the side chain of Ser204 to the meta-hydroxyl group of the ligand and a second specific hydrogen bond linking Ser207 in the receptor to the para-hydroxyl group of the agonist (13). Molecular modeling predicts that these two Ser residues would be located one helical turn apart in the fifth transmernbrane helix of the receptor, suggesting that the simultaneous interaction of the catechol ring with these two 5cr hydroxyls and Phe290 in helix 6 would serve to orient the agonist very specifically in the binding pocket of the receptor. Further analysis of several GPCRs in various laboratories has indicated that the third intracellular loop connecting helices 5 and 6 plays a role in receptor-C-protein interactions (2). Thus, the binding of the agonist to a

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REVIEW pocket formed by three residues in transmembrane helices 5 and 6 suggests that these interactions may serve to trigger the active conformation of the receptor. Examination of the sequences of related GPCRs reveals that an Asp residue is located at the position analogous to that of Aspi 13 in the third transmembrane helix of every GPCR for which the endogenous ligands are biogenic amines, suggesting that this ionic receptor-ligand interaction is conserved across the family of receptors. Mutagenesis analysis of several other GPCRs in this class, including a2-adrenergic (14), muscarinic (15), and histamine (16), have directly demonstrated the importance of this Asp residue in ligand binding to those receptors as well. In addition, affinity labeling of the binding site of the muscarinic acetylcholine receptor with propylbenzylcholine mustard resulted in specific labeling of the analogous AsplOS in the third transmembrane domain of that receptor, providing direct evidence for an interaction between this residue and a biogenic amine ligand (17). A Phe residue is conserved at the position analogous to Phe290 of the fAR in the sixth transmemhrane helix of all other GPCRs that bind aromatic biogenic amine ligands, but not in the muscarinic receptors, for which the endogenous agonist is an acetylated aliphatic amine. In the muscarinic receptor family, this Phe is substituted by a Tyr residue. Replacement of this TyrSO6 residue in the m3 muscarinic receptor with a Phe residue resulted in a decrease in the affinity of the receptor for the agonists acetylcholine and carbamylcholine with no effect on antagonist binding (18). Although the nature of the interaction between the agonists and Tyr506 has not yet been explored, these data would be consistent with an interaction between the hydroxyl group of Tyr507 and a hydrogen bond donor or acceptor on the ligand, most likely a portion of the ester moiety. In addition, Ser residues analogous to Ser204 and 5er207 are located in helix 5 of other receptors that bind catecholamines (ai-, a2-, -dopamine), and 5er204 (but not 5er207) is conserved among the serotonin receptor family, which binds mono-hydroxytryptamine as its agonist. These correlations strongly suggest that the helix 5-6 region evolved specifically to bind the polar aromatic portion of the biogenic amine in this subfamily of GPCRs. In the muscarinic receptor family, a Thr residue is located at the position analogous to Ser204 in the AR, and 5er207 is replaced by an Ala. Substitution of this Thr234 residue in the m3 muscarinic receptor with an Ala resulted in a decrease in the affinity of the receptor for agonists but not antagonists, again suggestive of a hydrogen bonding interaction between the hydroxyl side chain of Thr234 and a functional group on the agonist (18). In the histamine receptor, these two Ser residues are replaced by Asp and Thr, respectively. Substitution of these residues independently or together resulted in a decrease in the affinity and efficacy of the interaction of the receptor with agonists (16). Taken together, these data suggest that the binding triad of the two Ser residues in transmembrane helix 5 and the Phe in transmembrane helix 6 that interact with the catechol

G-PROTEN-COUPL.ED

RECEPTORS

ring of 13-adenergic agonists are representative of a similar binding pocket that is critical for the binding and agonist activation of all biogenic amine receptors, with the specificity of agonist recognition determined by the chemical nature of the side chains at these three positions. Now that -40 different biogenic amine GPCRs have been cloned, it is possible to determine, by simple inspection of the primary sequence of a GPCR, whether the natural ligand for that receptor is a biogenic amine. The presence of an Asp at the position analogous to 113 in transmembrane helix 3, along with the pattern of residues in helices 5 and 6 described above, is diagnostic of a biogenie amine receptor (Fig. 1), and can be used to identify such a receptor from a group of orphan receptors whose ligands are not known. In fact, such a strategy was instrumental in identification of the histamine receptor from the cloned cDNA (19) and of the orphan RDC4 as a receptor for the biogenic amine serotonin (20, 21).

PEPTIDE

BINDING

SITE

The endogenous ligands for many GPCRs are peptides, ranging from 3 to 40 amino acid residues in length. The design of small molecule ligands to activate or inhibit these peptidergic receptors remains a challenge for modern drug discovery. Understanding the interaction between small peptides and their receptors is critical for establishing the link between small molecule binding and peptide binding. In addition, such studies will provide a model that may be extended to the larger peptides and proteins that also serve as ligands for GPCRs. Mapping of peptide-receptor interactions has relied heavily on the development of structure-activity relationships for substituted peptide analogs and

Phe290 Aspll3

Ser207 Ser204

Figure showing

1.

V

Model for the ligand binding site of the -adrenergic key interactions with the agonist isoproterenol.

receptor

747

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V

UVV

truncated

peptide

fragments

mutagenesis

has been

determining

receptor-ligand

(22).

widely

More

adopted interactions.

recently,

receptor

as an approach The

to

combination

of receptor mutagenesis with the analysis of peptide analogs allows simultaneous mapping of the peptide binding site from the perspective of both the receptor and the ligand. As reviewed in the previous section, catecholamines and related biogenic amines bind primarily within the transmembrane domain of their receptors. However, most peptide ligands are substantially larger than catecholamines, leading to the proposal that the peptide binding site may also involve the extracellular region of the receptor. The relationships between the transmembrane and extracellular regions in receptor binding and activation have been extensively studied in recent years. For example, site-directed mutagenesis of the NK1 neurokinin receptor, which binds the undecapeptide substance F, has demonstrated the involvement of residues in both the transmembrane and extracellular portions of the receptor in binding peptide agonists (23). Three residues in the first extracellular segment and at least three residues in the second extracellular segment are specifically required for the binding of substance P and related peptides. In contrast, nonpeptide antagonist binding is not affected by amino acid substitutions or deletions at those positions (23-25). These data suggest that residues in the first and second extracellular segments of the NK1 receptor either interact directly with peptide agonists or play a critical role in maintaining the local conformation

of the peptide

binding

site.

Involvement of the extracellular domains in peptide binding has also been demonstrated by mutagenesis studies of several other classes of GPCRs, including C5a, fMLP, bradykinin, and angiotensin (26-31). In particular, the acidic NH2-terminal regions of the IL-8 and C5a receptors have been determined to be critical for the binding of chemokine

peptides.

tor is important

The

NH2-terminus

in conferring

both

of the IL-8

high-affinity

recep-

binding

of

the peptide and in determining the specificity of related chemokine peptides for various receptor subtypes (26, 27). For the C5a receptor, the Asp-rich NH2-terminus of the receptor contributes approximately half of the binding energy for the 74-residue peptide C5a and appears to play a role in stabilizing the active conformation of the ligand (28). The 2-dimensional mutagenesis approach to the interaction between the decapeptide GnRH and its receptor indicated that the carboxylate side chain of G1u301 in the fourth extracellular loop of the receptor interacts directly with Arg8 in the peptide ligand (29). The extracellular regions of glycoprotein hormone receptors have been shown to play a role in high-affinity binding of these protein ligands (30, 31). However, the glycoprotein hormone receptors contain a very large NH2-terminal domain (300-400 amino acids), and the NH2-terminal region of the lutropin receptor alone exhibits high affinity and specificity for luteinizing hormones (32). Whether the transmembrane region of this receptor also participates in peptide binding remains to be determined. The thrombin receptor presents a unique role for the NH2 terminus of a

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class of GPCRs that are activated by proteases. This receptor is activated by thrombin-mediated cleavage of the NH2 terminus of the receptor; the newly generated NH2 terminus then serves as a tethered ligand to activate the receptor (Liu, 1991). The specificity of the receptor for the tethered ligand is determined by the extracellular loop regions, whereas the protease specificity is determined by the sequence in the NH2-terminal cleavage site (33, 34). In addition to residues in the extracellular domain, the transmembrane region is critically involved in peptide binding to many GPCRs. In the NK1 neurokinin receptor, several residues in the transmembrane region have been identified that are required for peptide agonist binding but not for binding of the nonpeptide antagonist L-703,606 (35, 36). Substitution of Asn85, Asn89, Tyr92, or Asn96 in the second transmembrane domain of this receptor results in a decrease in the affinity of the receptor for peptide agonists such as substance P. These four residues would be predicted by modeling studies to be positioned on the same face of the second transmembrane a-helical region, suggesting that their side chains form a hydrogen bonding surface projecting into the ligand binding site. Likewise, Tyr287 in ti-ansmembrane helix 7 is required for high-affinity binding of peptides to the NK1 receptor. Two-dimensional mutagenesis analysis suggests that the COOH terminus of substance P binds at or near Asn85 in helix 2 of the receptor, based on a comparison of the binding of the wild-type and N85A mutant receptors to the amidated peptide substance P and its methyl ester analog (36). Residues within the transmembrane regions of other GPCRs have also been shown to be important for the binding of peptide ligands to these receptors. Several residues in the transmembrane domain of the receptor for the tripeptide thyrotropin-releasing hormone (TRH) have been determined to be critical for the binding of the peptide (37, 38). Two-dimensional mutagenesis experiments involving the binding of analogs of TRH to mutant TRH receptors suggest that the NH2-terminal pyroglutamate residue of TRH binds to a pocket between TyrlO#{243} and Asni 10 in transmembrane helix 3 of the receptor. The ring carbonyl moiety of the pyroglutamate appears to interact with the hydroxyl side chain of Tyr106 on the receptor (37), with the ring nitrogen of the pyroglutamate interacting with the side chain of AsnilO (38). This region of the TRH receptor is located in a position similar to that of the Asp residue that interacts with the amine moiety of the biogenic amine ligands in that class of receptors. This region of transmembrane helix 3 has been shown to play a role in the binding of larger peptides to other GPCRs, as well. In the endothelin receptor family, a Lys residue is located at the position analogous to that of Aspi 13 in the 3AR. Substitution of this Lys with Asp decreased the affinity of the receptor for endothelin peptides, demonstrating a role for this residue in the interaction between the peptide agonists and the receptor (39). In addition, Tyri29 in transmembrane helix 2 of the endothelin receptor has been determined to be critical for the potency and specificity of peptide agonists and antagonists, although the specific in-

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REVIEW teractions between these residues and the peptide ligands have not yet been identified (40, 41). A dramatic demonstration of the role of the transmembrane domain in the binding of peptide ligands to GPCRs is provided by the GnRH receptor, in which the positions of two highly conserved residues are switched. Most GPCRs contain an Asp or Glu residue at a conserved position in transmembrane helix 2 and a conserved Asn residue in transmembrane helix 7; for the GnRH receptor, the positions of these two residues are exchanged, so that Asn87 is located in helix 2 and Asp318 in helix 7. Mutagenesis of these two residues showed that replacement of Asn87 with an Asp abolished detectable binding of agonists and antagonists to the receptor, whereas a double mutant in which the two residues were switched (Asp87+Asn318) restored high-affinity ligand binding (42). These data support a role for the transmembrane domain in binding the peptide GnRH to its receptor and also provide convincing evidence for the proximity of transmembrane domains 2 and 7 in the 3-dimensional structure of the receptor, as predicted by molecular modeling studies.

SIGNAL

TRANSDUCTION

The binding of an agonist to a GPCR causes a conformational change in the receptor, leading to the formation of a high-affinity agonist-receptor-C-protein complex, initiating the signal transduction cascade (1). Thus, the high-affinity agonist-receptor interaction reflects both the intrinsic binding energy between the agonist and the receptor and the initial isomerization step in the agonist-mediated signaling pathway. In the interpretation of mutations in which the agonist affinity for the receptor is reduced, it can be difficult to dissociate these two interrelated potential mechanisms for the observed reduction in affinity. The isomerization constant cannot be measured directly for GPCRs, and so the issue can only be addressed qualitatively by comparing the apparent binding affinity, activation potency, and maximal activation response of the wild-type and mutant receptors (43). For example, most of the amino acid substitutions that have been observed to decrease agonist affinity for the NK1 neurokinin receptor also result in an increase in the activation constant for functional responses with little change in the maximal response, suggesting an impairment in agonist binding rather than receptor activation. In contrast, two amino acids in this receptor have been identified (Glu7S in transmembrane helix 2 and Tyr205 in helix 5) for which substitution leads to a modest reduction in the binding affinity of agonists accompanied by a complete loss of the ability of the agonists to mediate a signal transduction response. These data suggest that Glu78 and Tyr205 play a role in the receptor activation process rather than in the initial binding step. An Asp/Glu residue in helix 2 and a Tyr/Phe residue in helix 5 are highly conserved in most GPCRs. Several other studies have implicated the conserved Asp/Glu of helix 2 in activation of other GPCRs (12, 44,

G-PROTEN-COUPLED

RECEPTORS

45). Thus, these two conserved amino acids, located in the inner half of the transmembrane domain, might comprise part of a multiresidue network that transduces the binding energy arising from the interaction of the agonist with the extracellular and outer transmembrane domain of the receptor into conformational changes in the intracellular domain to trigger the interaction with the C-protein. The role of the intracellular regions of the GPCRs in G-protein activation has been reviewed elsewhere (46).

COMPARISON NONPEPTIDE

BETWEEN PEPTIDE BINDING SITES

AND

The binding site for nonpeptide antagonists in the NK1 neurokinin receptor has been extensively characterized, using a 2-dimensional mutagenesis approach involving modification of both the receptor and the ligand. At least 6 residues in helices 4-7 (i.e., G1n165, Ser169, His197, His265, Tyr272, and Tyr287) define the binding site of nonpeptide substance P antagonists (36, 47-50). By combining ligand modification with receptor mutagenesis, several primary contacts have been assigned between the receptor and analogs of the quinuclidine antagonist CP96,345 (Fig. 2). Specifically, the benzhydryl moiety of the antagonist appears to interact with Hisi97 in transmembrane helix 5 through an amino-aromatic interaction (47). A second interaction of this substituent with Tyr272 in transmembrane helix 6 has also recently been proposed (48). The C3 heteroatom of the antagonist appears to form a hydrogen bond with the side chain of G1n165 in transmembrane helix 4 (49), whereas the substituted benzyl moiety of these antagonists interacts with His265 in helix 6 (50). The identification of four residues in three different

Figure 2. Model for the ligand binding Site of the neurokinin-1 receptor showing key interactions with the antagonist CP-96,345.

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transmembrane helices involved in direct interaction with quinuclidine antagonists allows the refinement of receptor models that can be used as a framework to facilitate the design of new antagonists. At least two other structural classes of NK1 antagonists also bind to the region defined by transmembrane helices 4-7, making slightly different sets of contacts with these key residues in the receptor. In interactions with the perhydroisoindole antagonist RP67580, Ser169, His197, His265, and Tyr287 are the most important residues identified to date (36, 49). For the tryptophan ester antagonist L732,138, His197, and His265 provide critical points of interaction with the receptor (51). His197 and His265 are located in approximately the same position, at the top of helices 5 and 6, in the NK1 receptor as are Ser204 and Phe290 in the 3AR. The transmembrane domain of the AT1 angiotensin receptor has also been identified as the key site of interaction of the nonpeptide antagonists typified by the imidazolebiphenytetrazole losartan and related analogs (52, 53). Several residues within helices 3-7 were identified as critical for the binding of small molecule antagonists to this receptor, although the specific interactions with functional groups on the ligand remain to be defined. Several of these key residues for the binding of AT1 antagonists are in positions similar to those residues determined to be critical for the binding of small molecule ligands to the I3AR and NK1 receptors. The fact that many small molecules bind to the transmembrane region, which is the region of highest homology among the family of GPCRs, may explain the occurrence of spurious cross-reactivity of compounds among receptors whose natural ligands are not related (54). For example, the 2-substituted benzodiazepine tifluadom is an opiate agonist and a CCK antagonist (55). Understanding the molecular basis of receptor-liganci interactions should help to minimize such unwanted crossreactivity during the course of drug discovery. Comparison of the binding sites for peptide agonists and nonpeptide antagonists reveals that only two residues in the transmembrane region of the NK1 receptor (Gln 165 and Tyr287) are known to be important for both agonist and antagonist binding. None of the residues in the extracellular region of the NK1 receptor that have been determined to be important for agonist binding are required for the binding of nonpeptide antagonists. Thus, the receptor contacts with peptide agonists and nonpeptide antagonists do not substantially overlap at the atomic level. Similar distinctions between the binding sites for peptides and small molecules have been observed for the AT1 angiotensin receptor (52, 53). Despite the minimal molecular overlap between the binding sites for peptide agonists and nonpeptide antagonists, all currently available NK1 antagonists and most AT1 antagonists have been pharmacologically characterized as competitive antagonists. Because only a few residues of the NK1 receptor are required for the binding of both agonists and antagonists, this observation implies that competitive antagonism arises primarily from a steric exclusion or allosteric effect rather than from competing interactions at the

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atomic level. Although an agonist bound to the receptor may not be positioned to interact directly with a residue that interacts with an antagonist, the presence of a bound agonist on the receptor is able to exclude the binding of antagonists, and vice versa. One residue, Asn295 in transmembrane helix 7, in the AT1 angiotensin receptor has been shown to interact differently with competitive and insurmountable antagonists of the losartan class (53), suggesting that key molecular interactions responsible for these different pharmacological profiles may be identified in the future. It is obvious that the potential exists for a continuous spectrum of relationships between an agonist/antagonist pair, ranging from complete structural overlap to complete allosteric exclusion. Based on mutational analysis of the NK1 receptor, the currently known antagonists all appear to share only a minimal number of receptor contacts with peptide agonists, such that the antagonist binding site is intercalated among helices 4-7, whereas the agonist binding site involves the extracellular domain and helices 2 and 7. This observation raises the possibility of developing new structural classes of NK1 antagonists that would bind to the “peptide site” region surrounded by helices 1, 2, and 7. In the case of CCK-B receptor, dipeptoid antagonists have been developed based on the minimal side chain elements of CCK (56). Whether these rationally designed antagonists actually bind to the region in the receptor that would be occupied by CCK itself could be determined by mutagenesis experiments.

OTHER FAMILIES OF GPCR The secretin/VIP family includes receptors for the peptides secretin, glucagon, glucagon-like peptide 1 (GLP-1), gastric inhibitory peptide, parathyroid hormone, VIP, pituitary adenlylate cyclase activating polypeptide (PACAP), calcitonin, and growth hormone releasing hormone. These receptors share 25-50% amino acid identity among themselves and little primary sequence homology with the rhodopsin/3adrenergic receptor family. Given this minimal level of sequence homology and the difficulty in isolating purified receptor from native tissues, it is not surprising that the initial cDNA clones identified in this family were discovered by expression cloning strategies (57-59). As the number of known receptor sequences in this family has increased, low stringency hybridization cloning techniques have been used to identify additional members of the family. In addition to their overall sequence homology, several structural features common to the members of the secretin/VIP family support their classification as a new subfamily of GPCRs. These receptors all contain an NH2-terminal hydrophobic domain, which is presumed to serve as a signal sequence followed by a relatively long (115-160 residues) hydrophilic domain, preceding the seven transmembrane domains. Within this NH2-terminal hydrophilic domain are a series of conserved cysteines,

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REVIEW presumed to play a role in determining the tertiary structure of this region. Like the other members of the GPCR superfamily, these receptors contain consensus sites of N-linked glycosylation, as well as a conserved cysteine residue in each of the first two extracellular loops. The sequence conservation in the NH2-terminal region of these receptors suggests a role for this region in mediating receptor-ligand interactions. This notion is supported by recent data obtained by site directed mutagenesis of the human and rat parathyroid hormone (P’I’H) receptors (60). These two species homologs have equal affinity for native PTH, whereas the human PTH receptor binds a series of PTH analogs with 50-fold higher affinity than does the rat receptor. A chimera in which the NH2-terminal extracellular domain of the rat receptor is replaced by the homologous human sequence displays considerably higher affinity for the PTH analogs tested than does the native rat receptor, although the affinity for native PTH is unchanged. These data suggest that divergent residues in the NH2-terminal region of the PTH receptor are important determinants of ligand binding affinity. Similar evidence for a key role for the hydrophilic NH2-terminal domain in determining ligand binding arises from analysis of chimeric glucagon/GLP1 receptors (M. P. Graziano, unpublished observations). Further studies will be needed to refine the regions involved in ligand binding and to determine whether these observations apply to other members of this receptor subfamily. All members of the secretin/VIP subfamily of receptors cloned to date are coupled to the G-protein G8, and upon activation lead to increases in levels of intracellular cAMP (57-59). In addition to coupling to C, many of these receptors (PTH, glucagon, GLP-i, calcitonin, PACAP) have been reported to activate other C-proteins, leading to increases in intracellular calcium levels and inositol phosphate hydrolysis (61, 62). Thus, it appears that the potential to signal through multiple second-messenger pathways may be a hallmark of this receptor subfamily. Mutagensis studies have implicated regions in the second and third intracellular loops or the COOH-terminal tail of the rhodopsin/3-adrenergic subfamily of GPCRs in the interaction with C-proteins (2). In the secretin/VIP family, mutagenesis revealed that removal of the COOH-terminal tail of the P1’H receptor does not alter its ability to activate adenylyl cyclase (58). In addition, a series of PACAP receptors that have altered amino acid sequences in third intracellular loop arising from alternative spicing display differences in their selectivity for various C-proteins (62). Although the physiological relevance of these splice variants remains unclear, these data implicate regions of the third intracellular loop as critical determinants of C-protein specificity, suggesting functional parallels between the Secretin/VIP and rhodopsin/f3-adrenergic receptor subfamilies. Metabotropic glutamate receptors form a third, highly specialized subfamily of CPCRs. mGlu receptors are present in the central and peripheral nervous systems, where they regulate a wide variety of functions in response to the agonist glutamate (for review, see ref 63). Like the Secretin/VIP subfamily, the initial member of this family

G-PROTEIN-COUPLED

RECEPTORS

(mGluRl) was cloned by expression, and its minimal primary sequence homology to other GPCRs was noted with surprise (64, 65). Subsequent homology-based approaches have revealed six closely related mGlu receptor subtypes (63). The mGlu receptors are considerably larger than typical members of the other GPCR subfamilies (mGluRl is 1199 amino acids in length). In addition to the seven hydrophobic transmembrane domains, these receptors are characterized by relatively long NH2-terminal extracellular and COOH-terminal intracellular regions. The NH2-terminal extracellular domain has been implicated in the binding of glutamate to the mGlu 1 receptor (66). Members of this family of receptors, like the secretin/VIP family, contain a number of conserved cysteines in their extracellular domains, suggesting a structural role for this region of the mGlu receptors, as well. The signal transduction pathways activated by the mGlu receptors are not yet well defined, but appear to overlap considerably with those activated by members of the rhodopsinfl3-adrenergic receptor family. Several splice variants of mGluRl have been isolated and appear to activate different G-protein systems. Expression studies involving these splice variants suggest that the COOH-terminal domain of the mGlu receptor contributes to C-protein coupling.

BIOPHYSICAL STUDIES Molecular biology has contributed a wealth of information about the structure-function relationships of C-protein-coupled receptors. However, as mentioned previously, the current molecular models of these receptors must rely on bacteriorhodopsin and rhodopsin as templates because of the lack of high-resolution structural information on other GPCRs. Other approaches are needed to examine the details of ligand-receptor interactions and the conformational changes that occur following ligand binding in order to activate the C-protein. The advent of high-level expression systems and improvements in optical techniques are making a variety of structural studies feasible. Studies of the I3AR using the fluorescent ligand carazolol support the working hypothesis that the ligand binding site for the adrenergic receptors is in the center of the seventransmembrane helical bundle (67). The situation with peptide ligands is not as straightforward. As mentioned earlier, the binding of peptides and small ligands may share some common points of interaction within the receptor binding pocket, but additional interactions have been proposed for the peptide ligands. Fluorescently labeled peptides may help address how peptides and small proteins interact with this family of receptors. Several fluorescein-labeled peptides have been described recently for structural studies of C-protein-coupled receptors. Fluorescein has a high extinction coefficient and high quantum yield and can be excited at wavelengths well separated from most fluorescent contaminants found in biological material, making it a useful probe in GPCR systems. Recently, several analogs of the formyl peptide were used to identify two microenvironments

751

REVIEW of the receptor, a tetrapeptide where quenching was caused by a hydrophobic interaction, and a pentapeptide were protonation was causing the quenching (68). A fluorescent analog of substance P ([fluorescein Lys3] substance P) was developed in order to probe the binding site of the NK1 receptor. A modest degree of quenching was observed upon binding, and the quenching was not seen at higher pH, suggesting a protonation event associated with binding (69). These data suggest that the microenvironment of the fluorescein at Lys3 in substance P in the NK1 receptor is similar to that surrounding the fluorescein of the pentapeptide analog of the formyl peptide in the fMLP receptor. The binding of fluorescein labeled peptides, such as [fluorescein Lys3] substance P. may be more easily observed by monitoring the increase in polarization that accompanies the association to the small peptide with the larger receptor (69). When the change in fluorescence intensity is small, then increases in polarization can be directly correlated with the amount of bound ligand if the polarization of the bound ligand is known. While fluorescently labeled peptide ligands can provide information about the environment of the fluorophore or serve as a tool to monitor the kinetics of ligand binding, they can also serve to introduce a chromophore for proximity studies using fluorescence energy transfer. Used in conjunction with labeled receptors or C-proteins, such probes could be used to address the topology of the receptor and perhaps address details of how the receptor interacts with C-proteins. Labeling of C-protein subunits has been reported (70) and has provided information on the arrangement of the C-protein subunits (71). Specific labeling of the receptor will best be accomplished by using site-directed mutagensis to introduce cysteines or other readily derivatized functional groups at the desired locations. Extrinsic labeled receptor could also be used to monitor conformational changes. Such experiments using spin label probes have been performed on rhodopsin and revealed a conformational change in the loop connecting transmembrane helices 3 and 4 associated with activation of the protein (72). Fluorescence and ESR studies should be useful for determining overall receptor topology, conformational changes, and provide some information on the nature of the ligand binding site. A detailed analysis of the ligand binding site will await X-ray diffraction studies. Crystallization and X-ray diffraction studies on CPCRs will require high-level expression systems and large-scale purification. Examples of successfully crystallized membrane bound proteins are rare. However, because of the widespread therapeutic importance of GPCRs, these problems are being tackled from a variety of approaches, and crystallography of CPCRs remains a central goal in the field.

mary sequence homology and are predicted by modeling to share a common tertiary structure. Mutagenesis studies have revealed several key interactions in the binding sites of these receptors, which can be further explored by biophysical analysis to provide a map of the ligand binding sites. Because of the wide range of physiological roles played by these various receptors, the development of an understanding of their common mechanism of action will have far-reaching implications for the development of better medicines in a variety of therapeutic areas.

CONCLUSION

19.

In summary, the family of GPCRs is functionally diverse and responds to a wide variety of ligands. Despite this physiological heterogeneity, these receptors share some pri-

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