©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Binding Site of Neuropeptide Vasopressin V1a Receptor
EVIDENCE FOR A MAJOR LOCALIZATION WITHIN TRANSMEMBRANE REGIONS (*)

(Received for publication, April 21, 1995; and in revised form, August 21, 1995)

Bernard Mouillac (1) Bice Chini (1)(§) Marie-Noëlle Balestre (1) Jack Elands (2) Susanne Trumpp-Kallmeyer (2) Jan Hoflack (2) Marcel Hibert (2) Serge Jard (1) Claude Barberis (1)(¶)

From the  (1)From Unité INSERM 401, Centre CNRS-INSERM de Pharmacologie-Endocrinologie, rue de la Cardonille, 34094 Montpellier cedex 05, France and the (2)Marion Merrell Dow Research Institute, 16 rue d'Ankara, 67009 Strasbourg cedex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To identify receptor functional domains underlying binding of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT), we have constructed a three-dimensional (3D) model of the V1a vasopressin receptor subtype and docked the endogenous ligand AVP. To verify and to refine the 3D model, residues likely to be involved in agonist binding were selected for site-directed mutagenesis. Our experimental results suggest that AVP, which is characterized by a cyclic structure, could be completely buried into a 15-20-Å deep cleft defined by the transmembrane helices of the receptor and interact with amino acids located within this region. Moreover, the AVP-binding site is situated in a position equivalent to that described for the cationic neurotransmitters. Since all mutated residues are highly conserved in AVP and OT receptors, we propose that the same agonist-binding site is shared by all members of this receptor family. In contrast, the affinity for the antagonists tested, including those with a structure closely related to AVP, is not affected by mutations. This indicates a different binding mode for agonists and antagonists in the vasopressin receptor.


INTRODUCTION

The neurohypophysial hormones arginine vasopressin (AVP) (^1)and oxytocin (OT) are two closely related nonapeptides that exhibit a high degree of functional diversity through interaction with specific receptors. Four different receptor subtypes have been characterized and possess different pharmacological and G protein coupling properties(1) . V2 vasopressin receptors are coupled to adenylyl cyclase and are responsible for the antidiuretic effect of AVP. V1a and V1b vasopressin receptors are both coupled to phospholipase C. V1a receptors are involved in blood pressure control and in all other known functions of AVP, except for the stimulation of corticotropin secretion by the adenohypophysis which is mediated via V1b receptor subtype. OT receptors are coupled to phospholipase C and are responsible for the galactobolic and uterotonic effects. Recently, cDNAs for rat, human, and pig V2 receptors (2, 3, 4) , rat and human V1a receptors(5, 6) , pig and human OT receptors (4, 7) , fish arginine vasotocin (AVT) receptor(8) , and human V1b receptor (9) have been cloned and sequenced. This confirmed that these receptors belong to the G protein-coupled receptor (GPCR) family, characterized by seven hydrophobic putative alpha-helical transmembrane regions. All subtypes cloned so far are similar to each other in both identity and size. The level of conservation is remarkable, not only in the transmembrane domains but also in the first and second extracellular loops. Despite this high degree of receptor identity and a strong structural homology between the two hormones, the receptor subtypes are distinguished on the basis of different pharmacological and functional profiles. Many potent selective agonists as well as peptidic and nonpeptidic antagonists have been developed and used as pharmacological probes for the characterization of AVP and OT receptor subtypes(10) .

Previous molecular modeling studies of GPCR, based on the bacteriorhodopsin structure(11) , suggest that a similar binding pocket is present within all these receptors(12, 13, 14) . This site has already been explored in detail in the case of the cationic neurotransmitter receptors(12, 15) . Concerning GPCR for neuropeptide ligands, a model in atomic detail of the thyrotropin-releasing hormone within its receptor has been developed(16) . In this case, the tripeptide interacts with transmembrane domains (TM) of the receptor. At present, for peptides with a size much larger than three residues, the docking of these naturally occurring hormones and neurotransmitters has not yet been defined. The AVP and OT receptor family could constitute an interesting system to study hormone-receptor interactions in combining three-dimensional modeling and mutagenesis approaches. The present study aims at delineating residues in the vasopressin receptors responsible for the high affinity binding of AVP. Therefore, we have defined a computer-generated 3D model of the rat V1a vasopressin receptor subtype which allowed us to propose residues likely to be involved in agonist binding. Among these residues, several were selected for site-directed mutagenesis on the basis of putative direct interactions with bound ligands. Our experimental results validated the 3D model of the ligand-receptor interactions. This model proposes a major anchoring of the hormone within transmembrane regions of the receptor. Large polar residues such as glutamine and lysine, common to all members of AVP and OT receptors, could be involved in hormone binding. Preliminary results supporting our conclusions have been presented in 1994 at the 76th Annual Meeting of Endocrine Society in Anaheim (California) and at the XIIth International Congress of Pharmacology in Montreal (Quebec, CANADA).


EXPERIMENTAL PROCEDURES

Three-dimensional Modeling

The initial 3D model of the V1a vasopressin receptor has been defined following the same strategy and methods as described previously(12) . Sequences of rat, pig, and human V2 receptors(2, 3, 4) , rat and human V1a receptors(5, 6) , pig and human OT receptors(4, 7) , AVT receptor(8) , and human V1b receptor (9) have been analyzed in order to identify TM region boundaries.

Recently, a low resolution map of bovine rhodopsin has been published (17) . These data confirmed the seven helix topology of GPCR, but the relative positioning and tilt angles of the helices seemed to be somewhat different from the ones in bacteriorhodopsin. Therefore, the existing V1a model was refined by repositioning the seven TM helices. An electron density map at 9 Å was calculated from the refined V1a receptor model with the program Xplor and compared with experimentally derived footprint of bovine rhodopsin. The refined model showed a good match with the bovine rhodopsin projection map. Finally, the extracellular and intracellular loops were built, and the disulfide bridge between Cys and Cys was added. Side chain conformations were altered manually in order to optimize inter-residue interactions between the side chains. The model was energy minimized with the ``AMBER All Atom'' parameter set installed in SYBYL 6.04 to relax the structure and to remove bad steric contacts.

In the next step, AVP was docked into the proposed ligand-binding site. A first attempt to use a structure of AVP derived from the crystallographic structure of deamino-oxytocin (18) by simple side chain exchange and energy minimization did not lead to satisfying receptor-ligand interactions. Therefore, the minimized coordinates of the vasopressin structure were used as a starting point for a 300-ps molecular dynamic simulation run using the ``AMBER All Atom'' parameter set installed in SYBYL. The shake algorithm was used with an integration step of 1 fs and with a residue based ``cutoff'' of 8 Å. In the first 10 ps, the system was heated from 10 to 300 K with 30 K rise per 1000 steps by randomly assigning velocities from the Bolzmann distribution. The system was then allowed to equilibrate for 50 ps and the simulation continued for another 240 ps. The results from the last 240 ps were collected every 5 ps. A set of 48 low energy conformations of AVP was generated from molecular dynamic simulation and subsequent energy minimization and was used in further docking experiments. AVP was manually docked into the V1a receptor in order to optimize the structural and physicochemical complementarity. The criteria which were applied to choose among the different docking possibilities were the following: 1) the side chain of Arg^8 had to lay in the neighborhood of the first extracellular loop of the receptor in order to account for the photoaffinity labeling data(19) ; 2) the hydrophobic side of the cyclic part of AVP (Cys^1, Cys^6, Tyr^2, and Phe^3) had to fit into a hydrophobic pocket while the more polar side (Gln^4 and Asn^5) had to fit into a polar zone of the receptor; 3) the ligand-receptor hydrogen bonding network had to be optimal. The docking procedure was repeated several times with different initial orientations of the ligand and of the side chains. Only one way to fullfill the above criteria was found (see ``Results''). This ligand-receptor complex was finally energy minimized for 2000 steps with a dielectric constant of 5.

In the final interaction complex, AVP possesses a hydrogen bonding pattern which is different from the x-ray structure in the related deamino-oxytocin. In this conformation, the carbonyl oxygen of Tyr^2 forms a hydrogen bond with the amide proton of Cys^6. Furthermore, Phe^3, Gln^4, and Asn^5 form a -turn with a hydrogen bond from the carbonyl oxygen of Phe^3 to the amide proton of Asn^5. A similar conformation has been found by NMR studies of [Lys^8]-vasopressin in Me(2)SO(20) . In view of the limited accuracy of the model of the receptor and the limited number of AVP conformations investigated, the possibility that AVP might bind in a different conformation/orientation to the receptor cannot be ruled out.

Site-directed Mutagenesis of the Rat V1a Vasopressin Receptor

Point mutations were introduced in the rat V1a vasopressin receptor cDNA (5) using oligonucleotide-directed mutagenesis. The phosphorothioate technique was used to select the mutant cDNA (Amersham Sculptor kit) and the mutations were verified by direct dideoxynucleotide-sequencing. All residues selected for mutagenesis were replaced by alanine. The wild-type and mutant receptors cDNAs were then subcloned into an eukariotic expression vector (pCMV)(21) .

Expression of Receptors and Cell Culture

Wild-type and mutant rat V1a vasopressin receptors were transiently expressed in COS-7 cells by electroporation. Cells (10^7/0.3 ml) suspended in electroporation buffer were incubated with plasmid DNA (20 µg of carrier DNA (pCMV expression vector without any insert) and 0.075 µg of expression vector containing receptor cDNA) for 10 min at room temperature before being pulsed (280 V, 960 microfarads, Bio-Rad Apparatus). Cells were then plated in Petri dishes or 6-well plates depending upon the experiment to be conducted. Cells were maintained in culture in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 4 mML-glutamine, 500 units/ml penicillin and streptomycin, and 0.25 µg/ml amphotericin B in an atmosphere of 95% air and 5% CO(2) at 37 °C. Cells were harvested 72 h after transfection either for measurement of phospholipase C activity or for radioligand binding assays.

Membrane Preparations and Radioligand Binding Assays

Binding experiments were performed on membrane preparations. Briefly, 72 h after transfection, cells were harvested, washed two times in phosphate-buffered saline without Ca and Mg, polytron-homogenized in lysis buffer (15 mM Tris-HCl pH 7.4, 2 mM MgCl(2), 0.3 mM EDTA), and centrifuged at 100 times g for 5 min at 4 °C. Supernatants were recovered and centrifuged at 44,000 times g for 20 min at 4 °C. Pellets were washed in a Buffer A (50 mM Tris-HCl, pH 7.4, 5 mM MgCl(2)) and centrifuged at 44,000 times g for 20 min at 4 °C. Membranes were suspended in a small volume of Buffer A and protein contents were determined. Aliquots of membranes were used immediately or stored at -80 °C. Binding assays were performed at 30 °C as described previously (22) using I-HO-LVA as the radioligand and 1-5 µg of membrane proteins. Affinities for I-HO-LVA were determined in saturation experiments using concentrations ranging from 10 to 600 pM. Affinities for other ligands were determined from competition experiments using 50-70 pMI-HO-LVA as the radioligand. The concentrations of the unlabeled ligands varied from 100 µM to 1 pM. Nonspecific binding was determined in the presence of 200 nM HO-LVA. The ligand binding data were analyzed by nonlinear least-squares regression using the computer program LIGAND (23) . All assays were performed in triplicate on at least three separate batches of electroporated cells.

Inositol Phosphates Assays

Measurements of inositol phosphates accumulation were performed as described(24) . Briefly, COS-7 cells were electroporated, plated in supplemented Dulbecco's modified Eagle's medium using 6-well plates, and myo-[2-^3H]inositol (DuPont NEN) was added to the medium for the last 2 days of culture (final concentration = 2 µCi/ml). Before the experiment, medium was replaced by inositol- and fetal calf serum-free medium for 30 min. Cells (5 times 10^5 cells/well) were then washed two times in phosphate-buffered saline and incubated for 10 min at 37 °C in phosphate-buffered saline supplemented with 10 mM LiCl. Finally, cells were stimulated for 15 min with or without AVP in a range of concentrations from 10 to 10M. After stopping the reactions, inositol phosphates were extracted, recovered on strong anion-exchange chromatography columns, and counted. All assays were performed in triplicate on at least three separate batches of electroporated cells.


RESULTS

Three-dimensional Model of Rat V1a Vasopressin Receptor

In the final model, the receptor is oriented in the cell membrane in such a way that helices 4-7 are perpendicular to the membrane and helices 1-3 are tilted by approximately 15°. The presence of proline residues in TM regions 2 and 4-7 leads to slight kinks in the corresponding alpha-helices after optimization. Recalculation of the theoretical electron density map of the V1a receptor model at 9-Å resolution leads to a good match with the experimental bovin opsin footprint (data not shown). This orientation is similar to the helix bundle arrangement proposed by Baldwin(25) . The extracellular loops were added in reasonable, stable conformations, and the disulfide bond connecting Cys (the number refers to the primary sequence) on the second extracellular loop and Cys (in primary sequence) on the top of TM3, was introduced. It is important to note that no experimental information is available on the conformation of amino acids in the non-transmembrane part of the receptor. The merit of the model described above is to show that using bacteriorhodopsin and the experimental opsin footprint as a basis, a 3D structure for the complete V1a receptor can be built which contains all experimental restraints (disulfide bond, length of loops connecting the helices) and which presents all amino acid residues in a reasonable conformation.

In the final model, the extracellular loops surround the entrance of a central cavity in the transmembrane bundle, which appears as a 15-20-Å deep cleft defined essentially by helices 2-7 (Fig. 1). The bottom of the cleft is mainly hydrophobic in nature, closed by aromatic and hydrophobic residues: Val, Ala, and Met on TM3, Phe on TM5, Tyr, Trp, Phe, and Phe on TM6, Ala and Ala on TM7 (see legend of Table 1for the numbering of the residues). In contrast, the entrance of the TM region and the cleft itself contain predominantly hydrophilic residues: Gln and Gln on TM2, Lys and Gln on TM3, Ser and Gln on TM4, Thr and Thr on TM5, Cys and Gln on TM6, and Ser on TM7. Interestingly, this dual polarity is also found back in the endogenous ligand AVP: the exocyclic tripeptide Pro^7-Arg^8-Gly^9 and one side of the hormone cycle (Gln^4, Asn^5) are mainly hydrophilic, while the other part of the cycle (Cys^1, Cys^6, Tyr^2, and Phe^3) is essentially hydrophobic in nature. We attempted to dock AVP in the receptor cavity taking into account this dual polarity in both the ligand and the receptor cleft. The final docking result is given in Fig. 1. In this model, the cyclic part of AVP is embedded within the TM region of the receptor, filling the whole of the cleft up to the extracellular loops. The presence of the Arg^8 side chain close to the first extracellular loop is in agreement with photoaffinity labeling studies by Kojro et al.(19) . This hypothetical model allowed specific residues involved in hormone binding to be proposed. In Table 1, a review of putative interactions including a series of aromatic/aromatic, hydrogen bonding and ionic interactions likely to stabilize the receptor-hormone complex are listed. We concentrated on amino acid residues in the TM regions of the V1a receptor likely to be involved in ionic and hydrogen bonding interactions with AVP. Based on the 3D model, some of these amino acids were selected and mutated into alanine. All the residues chosen for site-directed mutagenesis, except Thr, are conserved in all members of AVP and OT receptor family cloned so far (Fig. 2).


Figure 1: Vasopressin docked into the rat V1a vasopressin receptor. A, view of the complex from the extracellular surface of the receptor, in a direction perpendicular to the membrane. B and C, side views from a direction parallel to the cell membrane surface. B shows in detail the interactions between the receptor and the hormone. C shows that the binding pocket for the neuropeptide is localized in the upper part (first third) of the transmembrane regions: white arrows indicate upper and lower limits of the lipidic bilayer. In all panels, the positioning of transmembrane domains 1-7 is anticlockwise. The C-chain trace of the transmembrane regions is displayed in magenta. The vasopressin C backbone is displayed in green. The carbon skeleton of the side chains of residues chosen for site-directed mutagenesis are displayed in white. Oxygen atoms are in red, nitrogen atoms are in dark blue, and sulfur atoms are in yellow. Only hydrogen atoms likely to be involved in hydrogen bonds are displayed (light blue). Except for tyrosine 115 (receptor primary sequence numbering) located in the first extracellular loop, residues are labeled according to the following code: the left digit indicates the number of the transmembrane alpha-helix, the next two digits indicate the rank of the residue in this transmembrane region as numbered in the alignment in Fig. 2(e.g. Lys is the 8th residue of helix 3).






Figure 2: Alignment of the seven putative TM domains of vasopressin and oxytocin receptors. The considered receptors are as follow: pV2, pig V2; hV2, human V2; rV2, rat V2; rV1a, rat V1a; hV1a, human V1a; hOT, human OT; pOT, pig OT, fAVT, fish AVT; and hV1b, human V1b. Residues selected for site-directed mutagenesis in the rat V1a receptor and conserved in other members of the receptor subfamily are highlighted in black.



Binding Properties of Wild-type and Mutant Receptors

The rat wild-type and mutant V1a vasopressin receptors were transiently expressed in COS-7 cells. Usually, 2-10 pmol of receptor/mg of proteins were obtained (Table 2). To study pharmacological properties of wild-type and mutated V1a receptors, a highly selective V1a receptor subtype antagonist I-HO-LVA, was used as the radioligand(26) . The binding affinities of agonists and antagonists for wild-type V1a receptor were calculated from saturation and competition experiments and their values are reported in Table 2. The I-HO-LVA bound to the V1a in a saturable fashion with a dissociation constant (K(d)) of 60 pM. The results obtained with three agonists, AVP, Phe^2Orn^8VT, and OT, and three antagonists, cyclic, linear and nonpeptide, specific for the V1a subtype, are characteristic of the cloned rat V1a receptor(5) .



For each mutated receptor, except for T505A, the substitution resulted in a substantial reduction in the affinity for the three agonists tested (Table 2). Substituting glutamine 214 and 218 by alanine resulted in 6-fold (Q214A) and 290-fold (Q218A) reduction in AVP affinity. For two other mutants, K308A and Q311A, loss of AVP affinity reached 60- and 40-fold, respectively. Most of the point mutants described here retained normal binding affinity for I-HO-LVA, d(CH(2))(5)[Tyr(Me)^2]AVP, and SR49059, suggesting that the chosen residues are not critical determinants for the antagonist affinities. By contrast, one mutant (Q413A) showed a substantial reduction in affinity for the antagonists (100-fold for d(CH(2))(5)[Tyr(Me)^2]AVP, 10-fold for HO-LVA and SR49059), which may indicate a perturbation of the antagonist/receptor interaction or a general perturbation of the receptor structure. The decrease in AVP affinity found with this mutant was the most pronounced (1220-fold). In contrast, substitution of threonine 505 in helix 5 (T505A) did neither affect the agonists nor the antagonists binding, suggesting that this residue does not play a critical role in ligand binding. The last substitution introduced in the V1a receptor concerns glutamine 620 situated in helix 6 (Q620A). This substitution resulted in a small decrease in affinity for the agonists tested (8-fold). For all the mutated receptors, the loss in affinity observed for AVP was accompanied by a similar decrease in affinity for the two other agonists Phe^2Orn^8VT and OT. The experimental results are in very good agreement with the proposed agonist-binding site in the 3D model and strongly support our AVP/V1a receptor interactions hypothesis. Moreover, as antagonist binding is in general not affected, this may indicate that agonist- and antagonist-binding sites of V1a vasopressin receptor are different.

Coupling Properties of Wild-type and Mutant Receptors

The signal transduction of the rat wild-type V1a receptor in the transiently transfected COS-7 cells was controlled by measuring AVP-induced inositol phosphates accumulation (Table 3). AVP induced a dose-dependent increase in the formation of total inositol phosphates (5-fold for maximal stimulation). The concentration of vasopressin leading to half-maximum response (K) was 0.46 ± 0.06 nM (n = 15). This value is in accordance with the dissociation constant (K(d) = 0.9 ± 0.3 nM (n = 3)) determined for [^3H]AVP binding to membranes deriving from COS-7 cells expressing the wild-type receptor. These data suggest that the rat wild-type V1a receptor is indeed coupled to phospholipase C.



To verify whether the substitutions had any effect on the coupling properties of the receptors, AVP-induced inositol phosphates accumulation was also studied in COS-7 cells transfected with mutant receptors. Table 3shows that for each mutant the K is shifted to higher concentrations of AVP, except for T505A where the value is comparable to that of the wild-type. The loss in K value observed for each mutant receptor paralleled and confirmed the loss in AVP affinity observed in binding studies. Values for K(i)/K ratios (AVP) calculated for wild-type and mutant receptors are constant (6.1 < K(i)/K < 10.9), except for Q214A (ratio = 1.1), thus demonstrating that mutant receptors are coupled to phospholipase C with properties equivalent to that of the wild-type V1a receptor (Table 3). Q214 is located two turns above the Asp, a residue which plays a major role in coupling properties of the V1a receptor (see below). This proximity could explain a partial impairment of the coupling properties of mutant Q214A. In our 3D model, D207 has not been proposed to directly interact with AVP. However, since this residue is known to play a role in signal transduction in other GPCR(27, 28) , we decided to replace it by alanine. Results obtained with mutant receptor D207A are characteristic of an uncoupled receptor. Indeed, even with a measurable affinity for D207A (Table 2, K(i) AVP = 338 ± 61 nM), AVP was not able to stimulate inositol phosphates production, even with high concentrations such as 10M (basal accumulation, 4862 ± 486 disintegrations/min (n = 3), maximal concentration, 5443 ± 1700 disintegrations/min (n = 3)). In fact, this aspartic acid residue in the second transmembrane domain is highly conserved among GPCR. It is functionally important in biogenic amine receptors such as beta2-adrenergic receptor (27) and in peptide receptors such as AT1a receptor (28) where its substitution modifies ligand binding affinity and G protein coupling, respectively. It has also been implicated in the allosteric regulation of muscarinic receptors by intracellular sodium(29) . For the V1a vasopressin receptor, this aspartic acid also seems to play a crucial role in coupling to phospholipase C.


DISCUSSION

We have constructed a three-dimensional model of the rat V1a vasopressin receptor subtype and docked the endogenous ligand AVP. As in the previous cases studied(12, 13, 14, 15) , the complementarity in amino acid composition and physicochemical properties of the cleft in the receptor and the AVP structure is striking and represents a working hypothesis that we have experimentally verified. According to the model, the C-terminal carboxamide of AVP might form a strong hydrogen bond with Gln on TM2. Gln might also contribute to AVP binding by hydrogen bonding to the amino group of Cys^1 at the N terminus of AVP and to the carboxamide at the C terminus. The mutation of Gln and Gln lead to a decreased affinity for agonists. Structure-activity relationship studies show that the AVP terminal glycinamide is absolutely required for biological activity of the two hormones AVP and OT(30) . These data suggest that Gln and Gln could significantly contribute to the V1a receptor agonist binding. In contrast, both mutations Q218A and Q214A leave antagonists binding unaffected. These data are in agreement with the observation that in antagonists the carboxamide terminus of the peptides is not required for high affinity binding(31) .

The interaction between a close analogue of AVP and the bovine V2 receptor subtype has been recently studied by photoaffinity labeling (19) . These results suggest that the exocyclic residues of AVP are located in the vicinity of the first extracellular loop. In our 3D model of the V1a receptor-hormone complex, the hormone Pro^7-Arg^8-Gly^9 backbone is located within the V1a receptor transmembrane domain, but the side chain of Arg^8 is in the direct neighborhood of tyrosine 225 located in the first extracellular loop (see Table 1; Tyr in primary sequence). This tyrosine has been shown to represent a key position responsible for agonist binding and selectivity(32) . Indeed, when this Tyr is replaced by the residues naturally occurring in the V2 or the OT receptor subtypes, the agonist selectivity of the V1a receptor switches accordingly.

The Q311A mutation leads to a decrease in AVP binding affinity while antagonist binding is again not significantly affected. Gln could play a role in stabilizing AVP in its active conformation and in positioning correctly AVP, OT, and structurally related agonists in the binding site by doing a hydrogen bond with the backbone carbonyl of Arg^8 in AVP. Gln could form additional hydrogen bonds with the amide carbonyl proton of Cys^6 and the amide proton of Gln^4 in AVP. It should be noted that Gln in AVP/OT receptors corresponds in sequence alignment to the Asp on TM3 which is highly conserved in cationic neurotransmitter receptors and plays a key role in their binding(27) . A similar structural role might be played by Lys which is located one helix turn above Gln. The substitutions of Lys and Gln by alanine probably make the hormone/receptor interactions less efficient with a consequent decrease in agonist binding. The observation that the affinity of peptidic antagonists is not changed by these mutations suggests different interactions of antagonists on V1a receptor.

The mutation Q413A is of special interest since it strongly influences agonist and antagonist binding. Furthermore, agonist and antagonist binding affinities are not affected to the same extent. Docking of AVP highlighted a hydrogen bond between Gln^4 in the hormone and Gln on TM4 in the V1a receptor. An additional hydrogen bond can be formed between Gln side chain and the carbonyl oxygen of Phe^3 in AVP. However, the decrease in AVP affinity is too high to be accounted for by the loss of two hydrogen bonds. One should note that Gln is adjacent in the receptor sequence to a proline residue. As discussed previously (12) , proline behaves as a hinge residue in a transmembrane alpha-helix. This allows such helices to wobble around an equilibrium position. Gln can stabilize the helix conformation by forming an intramolecular hydrogen bond via its side chain to the helix backbone. One might think that this local phenomenon plays a key role in the dynamics of the receptor, ligand binding, and functional coupling to the G protein. The dramatic decrease in AVP affinity observed in the Q413A mutant might be explained by a perturbation in both helix 4 dynamics and hydrogen bonds formation.

In our model, the carboxamide of Gln^4 side chain in the hormone makes an additional hydrogen bond with Thr, located on TM5. This possible interaction is of particular interest since this threonine residue corresponds to a serine residue conserved in catecholamine receptors. This serine has been shown to contribute to catecholamine binding(33) . However, the binding and efficacy of agonists and antagonists on this T505A mutant are not affected in V1a receptor. It seems that in V1a receptor this residue plays a much less important role in the recognition process. Moreover, this threonine 505 on TM5 is not conserved in V2 vasopressin receptors. Another possibility is that T505 forms a hydrogen bond with the ligand in the V1a receptor but, due to the large total number of interactions subsites, the net contribution of binding energy of this hydrogen bond is negligible.

With the exception of the D207A and Q214A mutations, there is a linear correlation between binding (K(i)) and efficacy (K) constants for all mutants tested. This suggests that the hydrophilic residues studied are specifically involved in the ligand binding process and do not modulate intrinsically the efficacy of the functional response.

Results reported in this paper clearly indicate that the binding sites for agonists and antagonists considered do not coincide in the V1a receptor. Three different classes of antagonists, cyclic and linear peptides, as well as nonpeptide, have been tested. None of the mutations in this study significantly affected antagonist binding except Q413A and K308A. These data show that the structure of cyclic and linear peptide antagonists is not totally superimposed with the structure of agonists although these ligands have similar amino acid composition. Similar results have shown that binding sites for agonists and antagonists are different on other GPCR such as neurokinins receptors(34) .

Taken together, the experimental data reported here indicate that large, cyclic neuropeptides such as AVP and OT most probably penetrate and bind in a pocket located in the first third of the transmembrane region of the receptor, extending from the extracellular loops up to 15-20 Å deep in the transmembrane region. In the case of cationic neurotransmitter receptors, this similar binding cleft has been explored in details using site-directed mutagenesis and contains residues important for both agonist and antagonist binding and receptor activation(35) . Based both on a striking complementarity between residues in this cleft and the corresponding ligands, it was proposed that this region corresponds to the activation site in most GPCR(12, 14) .

Other peptide receptors share common properties with AVP and OT receptors. Although experimental data indicate that transmembrane regions are involved in ligand-receptor interactions in angiotensin (36) , endothelin(37, 38) , neuromedin(39) , neurokinin(40) , thyrotropin-releasing hormone(16) , and somatostatin (41) receptors, it appears that most peptides interact with residues widely distributed throughout the surface of their receptors(42) . In addition, in receptors for the neurokinins (peptides of size comparable to that of vasopressin), thrombin and chemiotactic peptide fMet-Leu-Phe, the extracellular domains are of crucial importance in the binding of either agonists or antagonists analogs(40, 43, 44) .

The docking of AVP in V1a receptor allowed us to identify functional domains underlying the binding of the hormone by introducing single amino acid substitutions in the receptor sequence. Site-directed mutagenesis was restricted to the rat V1a vasopressin receptor. Interestingly (see Fig. 2), all residues supposed to have a key role in agonist binding (glutamines 214, 218, 311, 413, and 620, lysine 308, and even amino acids which have not been mutated such as Trp and Phe) are highly conserved in all the AVP and OT receptors cloned so far. Therefore, we propose that the agonist binding pocket is common to all the different subtypes of this receptor family. Moreover, as predicted from early modeling studies, it appears that GPCR agonists with very different chemical structures such as cationic, low molecular weight neurotransmitters, linear peptides, cyclic peptides, retinal, and even glycoproteins have in common the property to interact with a similar region in their respective receptors, resulting from the transmembrane helix topology. However, as we already demonstrated(32) , part of the agonist selectivity of AVP/OT receptor family is not localized in the TM regions of these receptors but in the first extracellular loop.


FOOTNOTES

*
This work was supported by INSERM and CNRS. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of an INSERM ``poste vert.'' Present address: CNR Center of Cytopharmacology, Dept. Pharmacology, Via Vanvitelli 32, 20129 Milan, Italy.

To whom correspondence should be addressed. Tel.: 67-14-29-17; Fax: 67-54-24-32.

(^1)
The abbreviations used are: AVP, arginine vasopressin; I-HO-LVA, I-HO-phenylacetyl^1-D-Tyr(Me)^2-Phe^3-Gln^4-Asn^5-Arg^6-Pro^7-Arg^8-NH(2); OT, oxytocin; AVT, arginine vasotocin; GPCR, G protein-coupled receptor; TM, transmembrane; 3D, three-dimensional.


ACKNOWLEDGEMENTS

We are grateful to Dr. M. Manning for providing us with the HO-LVA antagonist compound, to Dr. A. Morel for providing us with the rat V1a receptor cDNA, to Dr. Herman Schrender for calculation of electron density maps, to Mireille Passama, Marion Chalier, and Laurent Charvet for help in the illustrations, to Dr. J. Marie and J. P. Pin for reading the manuscript.


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