Systematic Analysis of the Entire Second Extracellular Loop of the V1a Vasopressin Receptor

The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V1a vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe189, Trp206, Phe209, and Tyr218 are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp206 and Phe209 project into the binding crevice. Indeed, Phe209 is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe189 and Tyr218, located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues.

G-protein-coupled receptors (GPCRs) 5 exhibit a common tertiary structure comprising seven transmembrane helices (TMs) linked by extracellular loops (ECLs) and intracellular loops. The atomic detail of this generic GPCR protein fold has been reported for bovine rhodopsin (bRho). This confirmed that the chromophore 11-cis-retinal is covalently linked to TMVII and projects into a binding pocket formed within the TM bundle where it interacts with amino acid side chains and water molecules (1,2). Likewise, the binding pocket for small biogenic amine neurotransmitters such as acetylcholine and norepinephrine is buried deep within the TM bundle (3). Despite this buried location of the ligand binding site, the exofacial domains of bRho are highly structured and interact with each other and with the TM helices. In particular, ECL2 of bRho forms a twisted ␤-hairpin structure that plunges down into the TM helical bundle to form a plug over the chromophore. There is also evidence that this ECL2 fold is not restricted to bRho and occurs in other GPCRs (4). In addition, the orientation of ECL2 in the majority of GPCRs is restrained by a conserved disulfide bond between ECL2 and the top of TMIII (1,2).
The neurohypophysial peptide hormones vasopressin (AVP) and oxytocin (OT) are structurally related nonapeptides that generate a wide range of physiological effects, including vasopressor, antidiuretic, and uterotonic actions (5,6). The effects of AVP/OT are mediated by a family of receptors (V 1a R, V 1b R, V 2 R, and OTR), which, together with the receptors for vasotocin, mesotocin, and isotocin from lower vertebrates, constitute a sub-family of the rhodopsin/␤-adrenergic receptor class of GPCRs (Family A). The V 1a R is widely distributed and mediates nearly all of the actions of AVP with the exceptions of antidiuresis (V 2 R) and ACTH secretion (V 1b R). Activation of the OTR stimulates contraction of the uterine myometrium during labor and mammary myoepithelium to elicit lactation. The V 1a R, V 1b R, and OTR couple to phospholipase C thereby generating inositol 1,4,5-trisphosphate and diacylglycerol as second messengers, whereas the V 2 R stimulates adenylyl cyclase. In addition to the characteristic architecture of GPCRs, mem-bers of the neurohypophysial peptide hormone receptor family share certain sequence motifs and exhibit related pharmacologies (6 -8). The hormone binding site of these receptors includes residues from the TM bundle (9,10), ECL1 (11)(12)(13), and the N terminus (14 -16).
Overall, the roles of residues located within the ECL domains of GPCRs are not well understood compared with residues in the TM domain. Nevertheless, extracellular residues are important for binding amine (17) and peptide ligands (18), binding allosteric modulators (19), human immunodeficiency virus coreceptor activity (20), switching agonist/antagonist properties (21), and modulating agonist-induced receptor internalization (22). For GPCRs in general, interest in the extracellular domains has focused on ECL2 in particular, because it projects into the binding crevice and there is direct evidence that its conformation changes upon receptor activation (23). The aim of this investigation was to provide a comprehensive pharmacological characterization defining the role of all the individual residues comprising the entire ECL2 domain of a peptide-GPCR. Systematic substitution of the V 1a R by Ala generated a series of mutant receptors that were subsequently analyzed with respect to ligand binding (agonist/antagonist and peptide/ non-peptide) and intracellular signaling. Our results establish that key residues located in ECL2 of the V 1a R are required for normal receptor function, identifying Phe 189 , Asp 204 , Cys 205 , Trp 206 , Phe 209 , and Tyr 218 as essential for high affinity agonist binding and receptor activation.
Radioligand Binding Assays-A washed cell membrane preparation of HEK 293T cells, transfected with the appropriate receptor construct, was prepared as previously described (24), and the protein concentration was determined using the BCA protein assay kit (Pierce) using bovine serum albumin as standard. Radioligand binding assays were performed as previously described (25)  Binding data were analyzed by non-linear regression to fit theoretical Langmuir binding isotherms to the experimental data using Prism (GraphPad, San Diego, CA). Individual IC 50 values obtained for competing ligands were corrected for radioligand occupancy as described (27) using the radioligand affinity (K d ) experimentally determined for each construct.
Determination of Cell-surface Expression Using Enzymelinked Immunosorbent Assay-All receptor constructs incorporated an hemagglutinin epitope tag in the N terminus, which enabled cell-surface expression to be determined by enzymelinked immunosorbent assay as described previously (28). Results were normalized against a wild-type control processed in parallel. Non-transfected cells were used to determine background. All experiments were performed in quadruplicate.

RESULTS
Functional Importance of Individual Residues in ECL2 of the V 1a R-The individual residues comprising ECL2 of the V 1a R, plus the residues at the extracellular boundary of TMV, are presented in Fig. 1. Overall, this segment of the extracellular face of the V 1a R encompassed 30 residues, from Phe 189 to Tyr 218 inclusive. To assess the importance of these residues in V 1a R function, each residue was substituted individually by Ala (Ala 207 and Ala 217 were substituted by Gly) and then pharmacologically characterized using the natural agonist AVP and three structural classes of antagonist:  Table 1, corrected for radioligand occupancy. The majority of mutant receptors exhibited wild-type pharmacology. Consequently,  (Table 1), indicating that the receptor protein was folded appropriately. The wild-type V 1a R and these mutant receptors were all expressed at the same level of 1-2 pmol/mg of protein. Furthermore, the intracellular signaling capability of these mutants was also essentially wild type (Table 2), consistent with the wild-type ligand binding profile of these receptor constructs.

Second Extracellular Loop Functional Role
in Table 1 to provide a complete study. In addition to the marked decrease in AVP affinity noted above, [Y218A]V 1a R also had decreased affinity compared with wild type, for CA (2000-fold) and SR49059 (50-fold (Table 2), with the degree of perturbation being dependent on the locus of the mutation (Fig. 3)

DISCUSSION
The ECL2 domain of bRho forms a ␤-hairpin that plunges down into the TM bundle, forming a lid over the bound retinal, which shields it from the extracellular milieu. This protein fold positions residues in the second ␤-strand (␤4) of this ECL2 hairpin structure in close proximity to the chromophore. Consequently, Glu 181 , Gly 188 , Ile 189 , and Tyr 191 of bRho all make contact with retinal (1). Given the unusual nature of the   covalently bound ligand in bRho, the variation in sequence and length of ECL2 within Family A GPCRs, and the requirement for reversible ligand access from the extracellular medium, it is perhaps possible that this ␤-hairpin fold is a unique feature of opsins. Indeed, the two-dimensional nuclear magnetic resonance structure of some synthetic ECL2 peptides has resulted in GPCR models that incorporate a different conformation. For example, it has been proposed that ECL2 of the thromboxane A 2 receptor contains two ␤-turns and extends away from the TM bundle (32). Alternatively, a helical conformation preceding the conserved Cys, or central to the ECL2 loop, was suggested for the -opioid receptor (33) and the neurokinin-1 receptor (34,35), respectively. However, using the substitutedcysteine accessibility method to identify residues contributing   to the water-accessible binding site crevice of the D 2 dopamine receptor (D 2 R), Shi and Javitch concluded that the ECL2 loop of the D 2 R adopted a similar conformation as the corresponding loop in bRho (4). In addition, this conclusion is consistent with ECL2 site-directed mutagenesis data for other Family A GPCRs (reviewed in Ref. 17). The aim of this study was to systematically define the role of the individual residues that comprise the ECL2 domain of the V 1a R. The majority of these mutant receptor constructs, 24 in total, had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues were not important for normal receptor function. In contrast, substitution of Cys 205 ablated both ligand binding and signaling. This Cys is part of the disulfide bond between ECL2 and the top of TMIII (Cys 3.25 ), 6 which is conserved in nearly all Family A GPCRs, and contributes to structural integrity of the receptor. Consistent with this structural role, cell-surface expression of [C205A]V 1a R was only 40% of wild type. Receptor function was also disrupted when the corresponding Cys was mutated in bRho (36), M3 muscarinic acetylcholine receptor (37), ␤ 2 -adrenergic receptor (␤ 2 -AR) (38), NK 1 receptor (39), and the gonadotropin-releasing hormone receptor (40).
There are a total of five aromatic residues within the extracellular segment of the V 1a R investigated in this study, Phe 189 , Trp 206 , Phe 209 , Trp 213 , and Tyr 218 . All five residues are highly conserved throughout members of the vertebrate neurohypophysial hormone sub-family of GPCRs cloned to date (Fig. 5). Furthermore, residues Trp 206 , Phe 209 , Trp 213 , and Tyr 218 are part of a sequence motif, DCWAXFXXPWGX(R/K)AY, which is highly conserved throughout this sub-family of GPCRs but is not a feature of Family A GPCRs in general. This family-specific conservation, plus the extracellular location of the motif in the receptor architecture, led to the hypothesis that residues in this motif may be candidates for ligand recognition (5,41). Consistent with this hypothesis, we show in the current study that mutation of the aromatic residues Trp 206 , Phe 209 , or Tyr 218 (shown underlined in the motif DCWAXFXXPWGX(R/K)AY) resulted in decreased affinity for agonist (Table 1) and impaired intracellular signaling (Table 2). This loss of AVP binding was not due to gross aberrant assembly of the mutant receptors, because the binding of at least one antagonist was wild type. Disruption of receptor function was not due to poor expression of the mutant receptors, because cell-surface expression of [W206A]V 1a R, [F209A]V 1a R, and [Y218A]V 1a R was 60 -100% of wild type (Fig. 4), and we have shown previously that a mutant receptor expressed at only 50% of wild-type V 1a R exhibited wild-type signaling (28). In common with Trp 206 , Phe 209 , and Tyr 218 , the residue Trp 213 is also part of the DCWAXFXX-PWGX(R/K)AY motif (shown underlined) and is highly conserved in neurohypophysial hormone receptors cloned to date, with the single exception of the vasotocin receptor from bull frog, in which Lys replaces the Trp. However, despite this high degree of conservation, Trp 213 is not important for receptor function, because [W213A]V 1a R exhibited essentially wild-type ligand binding and signaling.
Conserved residues within the TM bundle were used to establish a universal residue nomenclature system (43). It is difficult to directly compare ECL2 residues from different GPCRs, because there is a lack of sequence conservation and the loop length varies between receptors. However, ECL2 contains a highly conserved Cys, which is one half of the disulfide bond conserved in the majority of Family A GPCRs. This disulfide bond will spatially constrain relative movement between ECL2 and TMIII and therefore provide a point of reference for comparison between different GPCRs. We propose an indexing method for comparing aligned ECL2 residues in different GPCRs in which the conserved Cys is the reference point and other residues are indexed relative to this position. For example, for the rat V 1a R (Fig. 5), the residue preceding the conserved Cys is Asp (CϪ1) and the residue following the Cys is Trp (Cϩ1) .
Trp 206(Cϩ1) and Phe 209(Cϩ4) in the V 1a R correspond to Gly 188(Cϩ1) and Tyr 191(Cϩ4) in bRho. In the bRho crystal structure, Gly 188(Cϩ1) and Tyr 191(Cϩ4) are in the ␤4 strand of the ECL2 hairpin and come within 5 Å of the retinal to form part of the chromophore binding pocket (1,44). Consequently, assuming that the tertiary fold of ECL2 in the V 1a R is similar to that of ECL2 in bRho, then Trp 206(Cϩ1) and Phe 209(Cϩ4) will be FIGURE 5. Comparison of the ECL2 sequence of neurohypophysial hormone receptors cloned from different species. The sequences of the ECL2 domain of the V 1a R, V 1b R, V 2 R, and OTR from different species have been aligned. The species of origin is indicated by a single letter code preceding the receptor subtype: r, rat; m, mouse; v, vole; s, sheep; h, human; p, pig; b, cow; d, dog; mk, rhesus monkey. ECL2 residues that are highly conserved throughout the neurohypophysial hormone receptor family are shown in bold. The highly conserved aromatic residues within ECL2, shown to be functionally important in this study, are boxed and numbered according to the rV 1a R sequence. Sequences cited were obtained from SwissProt and GenEMBL. The Cys in ECL2 which forms part of the disulfide bond conserved in Family A GPCRs is indicated by an asterisk. directed down into the binding cavity within the helical bundle. Such an orientation of these residues would be entirely consistent with the disruption of ligand binding and signaling observed for [W206A]V 1a R and [F209A]V 1a R. It is noteworthy that Cϩ1 and Cϩ4 residues in this "␤4 strand segment" of ECL2 can also be important for ligand binding to amine-GPCRs. Indeed, Phe 209(Cϩ4) is part of the Cys-X-X-X-Ar motif (where Ar is an aromatic residue) that is well conserved in both peptide-GPCRs (59%) and amine-GPCRs (17%). In addition, Gln 189(Cϩ1) of the 5-HT 1D receptor, corresponding to Trp 206(Cϩ1) of the V 1a R, contributes to the subtype selectivity of ketanserin (45), and Gln 177(Cϩ1) is part of a triad of ECL2 residues responsible for ␣ 1A AR versus ␣ 1B AR pharmacology (46). In peptide-GPCRs, Arg 197(Cϩ1) of the cholecystokinin-1 receptor makes direct contact with Tyr(SO 4 ) 2 of cholecystokinin (47) and mutation of Tyr 190(Cϩ4) in the CXCR4 chemokine receptor, resulted in impaired signaling (48).
Tyr 218(5.38) at the extracellular boundary of TMV is absolutely conserved throughout the neurohypophysial peptide hormone receptor sub-family of GPCRs and is part of the same conserved sequence motif (DCWAXFXXPWGX(R/K)AY, shown underlined) as Trp 206(Cϩ1) and Phe 209(Cϩ4) . Mutation of Tyr 218 (5.38) in the construct [Y218A]V 1a R disrupted both ligand binding and intracellular signaling suggesting that Tyr 218(5.38) is orientated into the ligand binding site. For the D 2 R, it has been shown that the corresponding residue Phe 5.38 points into the binding site crevice by using a substituted-cysteine accessibility method in conjunction with ligand protection (49). Likewise, mutation of Tyr 5.38 of the ␣ 1B -adrenergic receptor also disrupted agonist binding and signaling (50). Our conclusion that Tyr 218(5.38) orientates into the ligand binding crevice also provides a feasible mechanism for the naturally occurring "loss-offunction" mutation Y205C in the human V 2 R (equivalent to Tyr 218 in the V 1a R), which has been identified as a cause of nephrogenic diabetes insipidus in some families (51).
Phe 189 at the start of ECL2 is highly conserved throughout the neurohypophysial peptide hormone receptor family with the single exception of the cephalotocin receptor, found in octopus, where a Trp replaces the conserved Phe. This high level of conservation reflects functional importance, because [F189A]V 1a R exhibited a dramatic decrease in potency (ϳ150fold) of AVP-induced InsP signaling and severely disrupted ligand binding. The corresponding residue (Trp 175 ) in bRho packs against Phe 203 at the top of TMV (corresponding to conserved throughout the neurohypophysial hormone receptor sub-family of GPCRs.