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Molecular Endocrinology, doi:10.1210/me.2006-0202
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Molecular Endocrinology 21 (2): 512-523
Copyright © 2007 by The Endocrine Society

Mapping the Binding Site of Arginine Vasopressin to V1a and V1b Vasopressin Receptors

Jordi Rodrigo1, Ana Pena1, Brigitte Murat, Miguel Trueba, Thierry Durroux, Gilles Guillon and Didier Rognan

Bioinformatics of the Drug (J.R., D.R.), Centre Nationale de la Recherche Scientifique-Université Louis Pasteur (CNRS-ULP), Unité Mixte de Recherche (UMR) 7175-LC1, F-67401 Illkirch, France; and Institut de Génomique Fonctionnelle (A.P., B.M., M.T., T.D., G.G.), CNRS UMR 5203, Institut National de la Santé et de la Recherche Médicale Unité 661, Universités Montpellier I et II, F-34094 Montpellier Cédex 5, France

Address all correspondence and request for reprints to: Dr. Didier Rognan, Centre Nationale de la Recherche Scientifique Université Louis Pasteur Unité Mixte de Recherche 7175-LC1, 74 Route du Rhin, F-67401 Illkirch, France. E-mail: didier.rognan{at}pharma.u-strasbg.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Starting from the 2.8-Å resolution x-ray structure of bovine rhodopsin, three-dimensional molecular models of the complexes between arginine vasopressin and two receptor subtypes (V1a, V1b) have been built. Amino acid sequence alignment and docking studies suggest that four key residues (1.35, 2.65, 4.61, and 5.35) fine tune the binding of vasopressin and related peptide agonists to both receptor subtypes. To validate these predictions, a series of single or double mutants were engineered at V1a and V1b receptor subtypes and tested for their binding and functional properties. Two negatively charged amino acids at positions 1.35 and 2.65 are key anchoring residues to the Arg8 residue of arginine vasopressin. Moreover, two amino acids (V4.61 and P5.35) delineating a hydrophobic subsite at the human V1b receptor are responsible for the recognition of V1b selective peptide agonists. Last, one of the latter positions (5.35) is hypothesized to explain the pharmacological species differences between rat and human vasopressin receptors for a V1b peptide agonist. Altogether these refined three-dimensional models of V1a and V1b human receptors should enable the identification of further new selective V1a and V1b agonists as pharmacological but also therapeutic tools.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ARGININE VASOPRESSIN (AVP) IS a neurohypophysial nonapeptide hormone that exerts major physiological roles upon binding to three receptor subtypes regulating blood pressure (V1a subtype), ACTH release, stress and anxiety (V1b subtype also named V3), and water reabsorption in the kidney (V2 subtype) (1, 2). Both receptor subtypes have been cloned in various species and belong to the wide family of G protein-coupled receptors (GPCRs) characterized by a typical heptahelical transmembrane (TM) domain (3). AVP receptors are prototypes of peptidergic GPCRs (4) able to accommodate significantly different binding modes peptide or nonpeptide ligands (agonists and/or antagonists) and thus are particularly interesting for determining fine molecular features responsible for ligand binding (5). The combination of molecular modeling, covalent labeling, and site-directed mutagenesis (5, 6, 7, 8, 9, 10, 11, 12, 13, 14) has shed light on hypothetical binding modes of AVP or close analogs [oxytocin (OT), vasotocin] to their specific receptors, although a clear consensus on the exact binding mode is still missing for two main reasons. First, most recognition models further investigated by site-directed mutagenesis studies have been proposed from either an ancient x-ray structure of bacteriorhodopsin (6, 9), which is not a GPCR, or a low-resolution map of bovine rhodopsin (7, 8, 10, 11, 12, 14). Second, three-dimensional models based on the more recent high-resolution x-ray structure of bovine rhodopsin are still lacking experimental validation (15, 16). Although previous models may probably not be used for accurate structure-based design, they have proven their utility to partially map the binding site of AVP. AVP is a nonapeptide (CYFQNCPRG-NH2) exhibiting a 20-membered tocin ring resulting from a disulfide bridge between both cysteine residues. The conclusion of most studies is that the hydrophobic part of the molecule is accommodated by an hydrophobic pocket (Met3.36, Trp6.48, Phe6.51) lying deep in the 7-TM cavity between TMs III, V, VI, and VII, whereas conserved glutamine residues (Gln2.61, Gln3.32, Gln4.60, Gln6.55) located at the rim of the cavity H bonds to the polar part of the peptide hormone (main chain atoms as well as Gln4 and Asn5 side chains) (8, 9). Last, the C-terminal amidated tripeptide probably projects toward a region between TMI and the second extracellular loop (8, 9). Therefore, there is a consensus about the critical receptor residues important for AVP recognition and binding but not on the precise molecular interactions developed by each of the AVP amino acids. Starting from recent three-dimensional (3-D) models of both V1a and V1b receptors that are able to accurately predict the binding mode of nonpeptide antagonists (17, 18, 19), we herewith present a high-resolution model of AVP in complex with each of its two receptor subtypes. Proposed 3-D models, compatible with most known experimental data, have been successfully challenged by site-directed mutagenesis focusing on yet undisclosed peptide-receptor interactions. Binding and functional properties of six mutants of the V1a or the V1b human receptor were investigated using either AVP or the recently described d[Cha4]AVP analog exhibiting a nice V1b selectivity profile for human vasopressin receptor subtypes (20).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
3-D Molecular Modeling
Aligning the amino acid sequences of vasopressin/OT receptors with that of bovine rhodopsin is rather straightforward because rhodopsin-like fingerprints (21, 22) are common to all entries (Fig. 1Go). The modeled structure of V1a and V1b receptor subtypes, although simulated in an explicitly hydrated phospholipid membrane, remains quite close to the x-ray structure of bovine rhodopsin [root mean square deviation (rmsd) on backbone TM residues between 1.2 and 1.3 Å]. AVP adopts a rather similar binding mode to both V1a and V1b receptor models (Fig. 2Go and Table 1Go), which recalls earlier binding modes described by Mouillac et al. (8) and Thibonnier et al. (9). With respect to the starting conformation taken from the structure of neurophysin-bound OT (23), a ß-turn is still observed in AVP but shifted from positions 2–5 to positions 3–6. Interestingly, the receptor-bound conformation of AVP to V1a and V1b receptor subtypes is similar to that recently proposed for density functional theory (DFT)-optimized Zn2+-bound OT (24) (rmsd of 0.8 Å on backbone atoms of the tocin ring). Carbonyl moieties at positions 2, 3, 6, and 8 converge toward a common region that could accommodate a dicationic ion, as proposed for OT (24).


Figure 1
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  		Fig. 1. Amino Acid Sequence Alignment of Human and Rat Vasopressin/OT Receptors (V1AR_HUMAN: human V1a receptor, V1BR_HUMAN: human V1b receptor, V2R_HUMAN: human V2 receptor, OXYR_HUMAN: human OT receptor, V1AR_RAT: rat V1a receptor, V1BR_RAT: rat V1b receptor, V2R_RAT: rat V2 receptor, OXYR_RAT: rat OT receptor) to Bovine Rhodopsin (OPSD_BOVIN)

Transmembrane helices (TMI–TMVII) are delimited by gray boxes. Amino acids residues mutated in this work are enclosed by white boxes. Down arrows indicate residues pointing inwards the TM binding cavity (37 ). Position 50 in Ballesteros numbering (27 ) of each TM is marked by a vertical bar.

 

Figure 2
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  		Fig. 2. Proposed Binding Mode of AVP to Human V1a (Left Panel) and V1b (Right Panel) Receptors

The seven TM helices of each receptor are displayed by cylinders and labeled from I–VII. The AVP main chain and side chain atoms are displayed by a dark tube and ball-and-sticks, respectively. AVP residues are labeled from 1–9. This figure (as well as Figs. 3Go and 7Go) has been prepared with MOLSCRIPT (35 ) and rendered with Raster3D (36 ).

 

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  		Table 1. List of Human V1a and V1b Receptor Residues in Close Contacts with AVP

 
A full description of intermolecular interactions between AVP and both receptor subtypes is listed in Table 1Go. A very tiny network of hydrogen bonds (16–20 H bonds) is used to anchor AVP to its receptor subtypes. Most residues proposed to participate in AVP binding have been shown to be of crucial importance for agonist peptide binding (8, 9, 10, 11). Key features of the proposed models involve hydrophobic interactions of Tyr2 side chain with a first apolar pocket between TMVI and TMVII, and of Phe3 aromatic ring with an apolar pocket delineated between TMIII and TMVI. Important hydrogen bonds are established between Gln4 side chain and Q4.60, Asn5 side chain, and Q6.55 for anchoring the tocin ring to the transmembrane cavity (Table 1Go). By opposition to previously described models (8, 9), a well-defined negatively charged subsite (E1.35, D2.65) perfectly accommodates the positively charged Arg8 side chain (Fig. 3Go, A and B). It should be noted that neither Y1152.68 (V1a) nor Y982.68(V1b) was found to interact with Arg8 side chain, as proposed in earlier 3-D models (8, 10).


Figure 3
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  		Fig. 3. Close-Up View of the Interaction of Arg8 Residue of AVP with Human V1a (Left Panel) and V1b Receptors (Right Panel)

The two TM helices (I and II) as well as the first extracellular loop (e1) at the vicinity of Arg8-contacting residues are displayed by gray ribbons and tube, respectively. The Arg8 main chain and side chain atoms of AVP are displayed by a dark tube and ball-and-sticks, respectively. V1a amino acids are labeled according to the SwissProt numbering with the Ballesteros numbering indicated in superscript.

 
E1.35 and D2.65 Are Key-Anchoring Residues in V1a and V1b Receptor Subtypes
The herein proposed molecular models were first challenged by probing the yet undisclosed molecular partner of residue Arg8 of AVP. Three V1b receptor mutants (E37A1.35, D95A2.65, Y98A2.68) have been engineered and tested for their binding properties for [3H]AVP (Fig. 4Go). As illustrated in Table 2Go, the three V1b mutants expressed on Chinese hamster ovary (CHO) membranes exhibited maximal binding capacities (Bmax) around 0.5 pmol [3H]AVP specifically bound per mg protein. These Bmax values were lower than those found for wild-type V1b receptor. As predicted, mutation of E37A1.35 dramatically decreased the affinity of the V1b receptor for [3H]AVP by nearly a factor of 50, thus confirming its utmost importance for AVP recognition. Mutation of the secondary Arg8-contacting residue (D952.65) was less spectacular but nevertheless still significant (4-fold decreased affinity, Table 2Go). Finally, the Y98A2.68 mutation, occurring at TMII-e1 (first extracellular loop) junction, did not changed the affinity of the mutant for [3H]AVP with respect to the wild-type receptor, confirming thus that the latter residue is not involved for AVP recognition to the V1b receptor. Competition experiments using [3H]AVP as radiolabeled ligand and a set of selective V1b unlabeled analogs showed that mutating E371.35 by alanine profoundly affected the pharmacological profile of this V1b mutant. Its affinity for the two peptidic V1b agonists tested (d[Cha4]AVP, dDAVP) was decreased by at least 135-fold. Moreover its affinity for the nonpeptidic V1b antagonist SSR149415 also dramatically decreased (Ki >10,000 nM). D95A2.65 mutation had a weaker effect on the pharmacological profile of this mutant. Whereas the affinity of d[Cha4]AVP was decreased by a factor of 9, that of dDAVP was increased by a factor of 2 (Table 3Go). As previously noted for AVP, mutation of Y2.68 did not strongly affect the pharmacological profile of this mutant for either peptide agonists or nonpeptide antagonists tested herein. Only slight increases of Ki for d[Cha4]AVP, dDAVP, and SSR149415 were observed as compared with V1b wild-type receptor.


Figure 4
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  		Fig. 4. Binding Properties of V1a and V1b Wild-Type and Corresponding Mutated Vasopressin Receptors for [3H]AVP

Membrane from CHO cells transiently transfected with wild-type or mutated vasopressin receptors were incubated with increasing amounts of [3H]AVP as described in Materials and Methods. Scatchard representation of saturation binding curves illustrated were representative of at least three distinct experiments each performed in triplicate. B/F, Bound/free; dpm, disintegrations per minute.

 

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  		Table 2. Binding Properties of Wild-Type (wt) and Mutated Human Vasopressin Receptors for [3H]AVP

 

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  		Table 3. Binding Properties of Wild-Type (wt) and Mutated Human Vasopressin Receptors for Selective AVP Analogs

 
The functional properties of E371.35A and D952.65A V1b mutants transiently expressed in CHO cells were further evaluated by testing their abilities to stimulate inositol phosphate (IP) accumulation. Functional properties of both V1b mutant receptors mirrored their binding properties with a parallel decrease of their affinity constant (Kact) for AVP (373 and 41 nM for E371.35A and D952.65A mutants, respectively) compared with V1b wild-type receptor (Kact= 0.88 nM for AVP, Table 4Go).


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  		Table 4. Coupling Properties of Wild-Type (wt) and Mutated Human Vasopressin Receptors to Phospholipase C

 
Residues 4.61 and 5.35 Control the Fine Specificity of d[Cha4]AVP to V1a and V1b Receptors
Our 3-D models were then used to find a plausible explanation to the previously described nice selectivity profile of d[Cha4]AVP, a desamino-cys-1 analog of AVP bearing a cyclohexylalanine side chain at position 4 (20). In contrast to AVP, which does not distinguish V1a from V1b subtype with regard to binding affinity, d[Cha4]AVP exhibits a much stronger affinity for the human V1b receptor than for the human V1a subtype (Table 3Go). A previous report demonstrated that a hydrophobic side chain at position 4 is clearly responsible for the favored binding to the human V1b subtype (20). Modeling the complex between the latter AVP analog and V1a/V1b receptor subtypes did not reveal major changes in the binding mode with respect to the previously described recognition model of endogenous AVP. A close-up view of amino acids contacting the Cha4 side chain provides a reasonable explanation for the selectivity profile of d[Cha4]AVP. When bound to the human V1b receptor, the cyclohexylalanine side chain is nicely embedded in a hydrophobic subsite delimited by K3.29, V3.33, A188 (e2 loop), Q4.60, V4.61, P5.35, and Y5.38 (see Fig. 8Go). Among these seven residues, five are fully conserved across human V1a/ V1b subtypes (Fig. 1Go) and therefore cannot be invoked to explain the peculiar binding properties of d[Cha4]AVP to the V1b receptor. The last two residues (V4.61 and P5.35) are mutated into more hydrophilic amino acids (Y4.61 and S5.35) in the human V1a receptor (Fig. 1Go) and may provide a molecular explanation for the preferred binding of d[Cha4]AVP to the human V1b subtype.


Figure 8
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  		Fig. 8. Close-Up View of the Interaction of Cha4 Residue of d[Cha4]AVP with the Human V1b Receptor

The three TM helices (III, IV, V) as well as the second extracellular loop (e2) at the vicinity of Cha4-contacting residues are displayed by gray ribbons and tube, respectively. The Cha4 main chain and side chain atoms of AVP are displayed by a dark tube and ball-and-sticks, respectively. V1b amino acids are labeled according to the SwissProt numbering with the Ballesteros numbering indicated in superscript.

 
Again, this hypothesis was experimentally challenged by engineering human V1a mutants (Y186V4.61, S213P5.35) bearing V1b residues at the proposed 4.61 and 5.35 positions. As observed for the V1b mutants, the V1a mutants were correctly expressed on CHO cell membranes and exhibited nanomolar affinities for [3H]AVP and maximal binding capacities ranging from 0.8–3.1 pmol [3H]AVP specifically bound·mg–1 protein (Table 2Go and Fig. 4Go). Yet, none of the single mutations significantly altered their binding affinity for d[Cha4]AVP (Table 3Go and Fig. 5Go). However, the V1a double mutant Y186V4.61/S213P5.35 resembles much more the V1b receptor in its binding properties for d[Cha4]AVP with a much higher affinity of the peptide analog for the double mutant than for the wild-type V1a receptor (Table 3Go). The Y4.61V mutation also weakly, but significantly, increased the affinity of the selective V1b antagonist SSR149415 to the V1a mutant. Strikingly, the S5.35P change affected SSR149415 binding only when presented together with the Y4.61V mutation. Last, the binding properties of the selective V1a agonist F 180 or antagonist SR49059 were almost unchanged by any of the two mutations either alone or in combination (Table 3Go).


Figure 5
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  		Fig. 5. Binding Properties of V1a and V1b Wild-Type and Corresponding Mutated Vasopressin Receptors for Selective Vasopressin Analogs

Membrane from CHO cells transiently transfected with wild-type or mutated vasopressin receptors were incubated with 1.5 nM to 15 nM [3H]AVP according to the receptors considered: vehicle (control), increasing amounts of unlabeled vasopressin analogs (total binding), or with 1 µM unlabeled AVP (nonspecific binding). Specific binding were calculated in each experimental condition and expressed as percent of control specific binding. Results are the mean ± SEM of at least three distinct experiments, each performed in triplicate.

 
Mutating residues 4.61 and 5.35 affected more the functional properties of the human V1a receptor as compared with binding parameters. Both single mutants were much more efficient than the wild-type V1a receptor in stimulating phospholipase C by AVP or d[Cha4]AVP. Yet these effects were of weak amplitude (Table 4Go, Fig. 6Go). In contrast to our previous observation concerning d[Cha4]AVP binding properties, the double mutant did not confer any significant functional gain with respect to the single mutants.


Figure 6
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  		Fig. 6. Coupling Properties of V1a and V1b Wild-Type and Corresponding Mutated Vasopressin Receptors to Phospholipase C

CHO cells transiently transfected with wild-type or mutated vasopressin receptors were labeled with 1µCi myo-[3H]inositol as described in Materials and Methods. Cells were further incubated with either increasing amounts of AVP or d[Cha4]AVP. Total IPs accumulated were extracted, counted, and expressed as percent of the maximal response induced by 1 µM AVP. Results are the mean ± SEM of at least three independent experiments each performed in triplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
A precise mapping of molecular interactions between peptide ligands and their receptors is a prerequisite for designing nonpeptide agonists for therapeutic applications. Taking advantage of the high-resolution structure of bovine rhodopsin (3), the only G protein-coupled receptor crystallized to date, we have developed homology models for two AVP receptor subtypes (V1a, V1b) in an explicit hydrated phospholipid environment and docked AVP and a recently described analog (d[Cha4]AVP) to each receptor model. AVP is proposed to interact quite similarly with V1a and V1b receptor subtypes (Fig. 2Go, A and B) in agreement with previously described site-directed mutagenesis-guided mapping of the AVP binding site in the latter receptors (8, 9, 14). To experimentally challenge the yet undisclosed molecular partners of arginine 8 residue of AVP, the two negatively charged residues (E1.35 and D2.65) in close contact with arginine 8 (Fig. 3Go, A, B) were mutated to alanine in the human V1b receptor. As predicted, binding of [3H]AVP to the mutant receptors was significantly decreased both for the E1.35A and to a lesser extend for the D2.65A mutations (Table 2Go). Because both residues are fully conserved in all V1a and V1b receptor subtypes (Fig. 1Go), it is likely that the herein predicted interaction is a common feature of AVP recognition by the latter subtypes. The present model and its experimental validation also exclude the possibility that the neighboring Y2.68 amino acid in V1a and V1b receptors directly interacts with Arg8, as previously reported in earlier recognition models (10), because the Y2.68A mutation has no influence on AVP binding to the V1b mutated receptor (Table 2Go). This negatively charged spot delineated by E1.35 and D2.65 is also used by other peptide agonists, especially E1.35, which seems to be a common anchor to all ligands tested herein whatever their selectivity for individual subtypes (Tables 3Go and 5Go). Lysine vasopressin for which the arginine 8 has been replaced by a lysine is also sensitive to both mutations, which can be easily explained by lysine’s potential to develop ionic interactions with the two negatively charged residues. To further evidence the direct interaction between arginine 8 and E1.35/D2.65, we then tested an AVP analog (Phe3OT: CYFQNCPLG-NH2) for which arginine has been replaced by a leucine at position 8 (Table 5Go). As expected from the present 3-D model, the E1.35A and D2.65A mutations have much less consequences on the binding affinity of the latter analog to the mutant V1b receptor. First, the binding affinity of Phe3OT to the wild-type V1b receptor is considerably decreased (Ki, ~100 nM) illustrating the significant contribution of arginine 8 to the overall binding affinity (recall Kd value of [3H]AVP, Table 2Go). Second, both mutations have only a weak effect on Phe3OT binding, confirming that residues 1.35 and 2.65 of the receptor and 8 of the ligand are in probable close proximity.


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  		Table 5. Binding Properties of Wild-Type (wt) and Mutated Human V1b Receptors, and of Human and Rat V2 Vasopressin Receptors for Selected Agonists

 
Quite surprisingly, both mutations also decrease the binding affinity of a nonpeptidic V1b antagonist (SSR149415, Table 3Go) the previously mapped binding site of which (18) only partially overlap with the herein defined AVP binding region and is far away from the E1.35-D2.65 spot (Fig. 7Go). The effect of both above reported mutations on the binding of SSR149415 is difficult to rationalize from the present interaction model because direct molecular interactions between ligand and mutated residues are unlikely. During this work, the same E1.35A mutation has been described for the V1a receptor subtype and reported to dramatically decrease AVP binding and signaling but also to have no influence on the affinity of the selective V1a nonpeptidic antagonist SR49059 (25). The binding site of SR49059 to the human V1a receptor has already been mapped by our laboratory (19) and demonstrated to largely overlap that of the V1b antagonist SSR149415 (18). At present, we have no clear explanation for the differential consequences of E1.35A mutation on V1a and V1b nonpeptidic antagonists. Among possible explanations, this precise mutation may result in subtle conformational changes remotely affecting the antagonist binding site in the V1b subtype.


Figure 7
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  		Fig. 7. Proposed 3-D Model of Interaction of AVP (Left Panel, This Work) and SSR149415 (Right Panel, Ref. 18 ) to the Human V1b Receptor

Only TM helices (labeled from I–VII) are shown as red ribbons for sake of clarity. Two negatively-charged residues (E1.35 and D2.65) the mutation of which decreases the binding affinity of the two ligands (Tables 2Go and 3Go) are shown by white (carbon) and red (oxygen) sticks. The ligand (blue sticks) is shown along with its molecular surface (magenta for AVP, green for SSR14915). The figure was prepared and rendered with VIDA2 (OpenEye Scientific Software, Santa Fe, NM).

 
To furthermore challenge the herein described 3-D models, we took advantage of the fine selectivity profile of a recently described V1b peptide agonist (d[Cha4]AVP), an analog of dAVP ([deamino-cys1]arginine vasopressin) (20) for which glutamine 4 has been changed to cyclohexylalanine (Cha) and which exhibits a 70-fold higher affinity for the V1a subtype than for the V1b subtype (Table 3Go). Our 3-D models suggest that the fourth residue of the peptide ligand is accommodated by a pocket delineated both by four conserved residues among the AVP receptor family (K3.29, V3.33, Q4.60, and Y5.38; Fig. 8Go) and two variable amino acids (V4.61 and P5.35). A comparative observation of the sequence alignment (Fig. 1Go) and 3-D models (Fig. 8Go) clearly shows that the above-described pocket is significantly more polar in V1a (Y4.61, S/T5.35) than in V1b receptors (V4.61, P5.35). Therefore, it is logical that AVP analogs exhibiting hydrophobic residues are the most selective for the V1b receptor subtype (20). To further validate this hypothesis, the two variable residues in the human V1a receptor were mutated to their V1b counterparts (Y186V, S213P). The two single and the double mutation had no effect on AVP binding (Table 3Go) because AVP does not discriminate between both subtypes, probably because of a tight hydrogen bond between Gln4 side chain and the conserved Q4.60 (Table 1Go). The Ki of the selective V1b agonist d[Cha4]AVP was also not really modified by single mutations, but a drastic gain in affinity was observed for the double mutant (Table 3Go), thus confirming our starting hypothesis. It is likely that single mutations in this plastic hydrophobic subsite are compensated by slight conformational rearrangements that do not occur upon double mutation. Interestingly, the coupling properties of the mutant receptors to phospholipase C were already affected by single mutations (Table 4Go) and much closer to that of wild-type V1b than wild-type V1a receptor. Contrary to our previous observations, the double mutation does not here confer a better V1b pharmacological profile to the V1a double mutant (Table 4Go).

The present sequence alignment (Fig. 1Go) also provides a good explanation for the d[Cha4]AVP pharmacological species differences previously observed for vasopressin V2 receptors (26). Hence, the only difference in the above-reported Cha4 binding pocket between human and rat V2 subtypes lies precisely at position 5.35 (Fig. 1Go). In the human V2 subtype, position 5.35 is a bulky and positively charged amino acid (Arg), which is probably not suited to accommodate a bulky hydrophobic side chain at position 4 of the peptide ligand. Mutation to a smaller and more hydrophobic residue (Leu) in the rat V2 receptor is therefore beneficial to the binding of d[Cha4]AVP (Table 5Go). Conversely, AVP analogs with smaller polar side chains (e.g. lysine vasopressin) do not distinguish human from rat V2 receptors (Table 5Go). It is difficult from the present data to explain the selective binding of d[Cha4]AVP to the human V1B receptor vs. human OT receptor (26). Strikingly, AVP itself does not distinguish between those receptors. These subtle differences are probably beyond the accuracy of the current homology models. It is still possible that the human OT receptor has slightly different orientation and kinks of the seven helices when bound to d[Cha4]AVP.

The proposed interaction models, however, should be valuable to design novel selective agonists, notably by focusing on some positions (5.42 and 7.39 for example) which vary across the AVP receptor family and which also explain the selective binding of nonpeptide V1b antagonists (18).

In conclusion, receptor modeling of human AVP receptors in complex with AVP and d[Cha4]AVP, a selective V1b agonist, shed light on putative molecular interactions between the peptide hormone and its receptors. Validation of the computational models by studying the binding and coupling properties of a few mutants on V1a and V1b receptor unambiguously demonstrate that arginine-8, a very important residue for ligand binding, interacts with a set of negatively charged amino acids (E1.35, D2.65) at V1a and V1b receptor subtypes. Moreover, a rational explanation to the V1b-selective binding of d[Cha4]AVP is proposed and confirmed by site-directed mutagenesis. Altogether, the refined 3-D models of V1a and V1b human receptors in complex with peptide agonists should facilitate the identification of selective and nonpeptidic V1a and V1b agonists as pharmacological but also therapeutic tools.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Residue Numbering and Nomenclature
The residue numbering proposed by Ballesteros et al. (27) was used throughout this manuscript. It allows an unambiguous comparison of TM residues for any class A GPCR by assigning position 50 to a fully conserved amino acid at each TM (Asn for TMI, Asp for TMII, Arg for TMIII, Trp for TMIV, Pro for TMs V–VII; see Fig. 1Go) and numbering other amino acids according to this reference residue. Residue x.y is thus the amino acid describing position y of TMx. For purposes of clarification, amino acids from the peptide ligands will be labeled using a three-letter code whereas receptor residues will be labeled using a single-letter code.

Alignment of Amino Acid Sequences
The amino acid sequences of human and rat AVP and OT receptor subtypes were retrieved from the Swiss-Prot database (accession nos: human V1a receptor, P37288; rat V1a receptor, P30560, human V1b receptor, P47901; rat V1b receptor, P48974; human V2 receptor, P30518; rat V2 receptor, Q00788; human OT receptor, P30559; rat OT receptor, P70536) and aligned to the sequence of bovine rhodopsin (accession no. P02699) using the in-house developed GPCRmod program (21) focusing on transmembrane (TM) domains only. The alignment of the amino and carboxy-terminal domains as well as of the intra- and extracellular loops was realized using ClustalW (28). A slow pairwise alignment using BLOSUM matrix series (29) and a gap opening penalty of 15.0 were chosen for aligning the amino acid sequences to the sequence of bovine rhodopsin.

Modeling the AVP-Bound Conformation of Human V1a and V1b Receptors
3-D ground-state models of human V1a and V1b receptors have recently been reported by our group (17, 18, 19). To achieve an agonist-bound model from an antagonist-bound model, we followed, for each receptor subtype, a five-step protocol as proposed by Bissantz et al. (17). In a first step, only the first and third extracellular loops between helices 2 and 3, and helices 6 and 7, respectively, were included in a preliminary model. In a second step, AVP was docked to this preliminary model using the Gold 2.1 program (30). For each of the 10 independent genetic algorithm (GA) runs, a maximum number of 1000 GA operations was performed on a single population of 50 individuals. Operator weights for crossover, mutation, and migration were set to 100, 100, and 0, respectively. To allow poor nonbonded contacts at the start of each GA run, the maximum distance between hydrogen donors and fitting points was set to 5 Å, and nonbonded van der Waals energies were cut off at a value equal to kij (well depth of the van der Waals energy for the atom pair i,j). To further speed up the calculation, the GA docking was stopped when the top three solutions were within 1.5 Å rmsd. If this criterion is met, we can assume that these top solutions represent a reproducible pose for the ligand. It should be observed that the starting conformation of AVP was modeled from the x-ray structure of neurophysin-bound OT (23). In a third step, the receptor-AVP complex was minimized with AMBER8.0 (31) using the AMBER03 force field to relax the structure and to remove steric bumps. In a fourth step, the extracellular loop 2 between helices 4 and 5 and the N-terminal region were added to the model through a simple knowledge-based loop search procedure as previously described (17). The disulfide bridge present between Cys3.25 and a conserved Cys in the extracellular 2 loop was kept unchanged with respect to the x-ray structure of bovine rhodopsin (3). After the heavy atoms were modeled, all hydrogen atoms were added, and the protein coordinates were then minimized again with AMBER. The minimizations were carried out by 1000 steps of steepest descent followed by conjugate gradient minimization until the rms gradient of the potential energy was lower than 0.05 kcal/mol·Å. A twin cutoff (10.0, 15.0 Å) was used to calculate nonbonded electrostatic interactions at every minimization step, and the nonbonded pair list was updated every 25 steps. A distance-dependent ({epsilon} = 4r) dielectric function was used. Last, the complex was embedded in a preequilibrated lipid bilayer consisting in 70 molecules of palmitoyloleoylphosphatidylcholine and solvated by 11,588 TIP3P water molecules (box dimensions: 86.6 * 93.1 * 78.0 Å) as recently described by Urizar et al. (32). A short minimization was applied to the complex embedded in the hydrated lipid bilayer using AMBER8.0 and applying a positional harmonic constraint of 50 kcal/mol·Å on C{alpha} carbons. A 1-nsec constant pressure molecular dynamics (MD) simulation was then applied to the entire system. Periodic boundaries and Particle Mesh Ewald summation were considered to treat electrostatic interactions. All bonds involving hydrogen atoms were frozen with the SHAKE algorithm, thus enabling the selection of a 2-fsec integration step. During the first 250 psec, the {alpha}-carbons were constrained as previously described, and the temperature was linearly increased from 0 to 300 K. In the productive part of the MD simulation (last 750 psec), the temperature was fixed to 300 K by coupling to a heat bath using a coupling constant of 0.2. All atoms were free to move, energies and coordinates were saved every 10 psec. The analysis of the MD trajectories was realized using the ptraj module of AMBER8.0.

Automated Docking of d[Cha4]AVP
The docking of d[Cha4]AVP to the two receptor models was done as described for AVP.

Site-Directed Mutagenesis
All the mutations were generated into the human V1a or V1b receptor sequence. The human V1a and V1b cDNA inserted into the pRK5 expression vector was kindly provided by Dr. B. Mouillac. Amino acid replacements were generated by PCR using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), as previously described (26). For single amino acid replacements of mutants E37A, D95A, and Y98A, cDNA of the wild-type human V1b receptor was used as the template with the appropriate mutated oligonucleotide primers. For replacements of Y186V and S213P mutants, cDNA from V1a receptor was used as the template. The double mutant Y186V/S213P was generated as described above using cDNA of the Y186V mutant as the template and mutated oligonucleotide primers corresponding to the S213P mutation. Each construct was transformed, amplified in the Escherichia coli DH5-{alpha} strain (Invitrogen, Cergy-Pontoise, France), and then purified with the QIAprep Spin Miniprep Kit (QIAGEN, Courtaboeuf, France). All the mutations were verified by sequencing (Genome Express, Meylan, France).

cDNA Expression of Wild-Type and Mutant Vasopressin Receptors and Cell Culture
The human V1a and V1b wild-type receptors were stably expressed in CHO cells as previously described (26). The mutants of the human V1a or V1b receptors were transiently expressed in CHO cells by electroporation. Briefly, the cells (107/0.3 ml) were resuspended in an electroporation buffer (50 mM K2HPO4; 20 mM C2H3KO2; 20 mM KOH; pH 7.4) with 20 µg of carrier DNA (pRK5 expression vector without insert) and 0.5–2.0 µg of the expression vector containing the mutated receptor cDNA and 40 mM MgSO4. They were then incubated for 20 min at room temperature before being pulsed [280 V, 950 microfarads; Bio-Rad Apparatus (Bio-Rad Laboratories, Hercules, CA)]. CHO cells expressing the wild-type and mutated vasopressin receptors were plated in 150-mm Petri dishes or 24-well plates depending upon the experiment to be conducted. Cells were cultured 48 h at 37 C in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 500 U/ml penicillin and streptomycin, and nonessential amino acids (1x) in an atmosphere of 95% air and 5% CO2. Sodium butyrate (3 mM) was added in the incubation medium for the last 24 h.

Membrane Preparation
CHO cells, stably or transiently transfected with wild-type or mutated receptors, were washed twice in PBS without CaCl2 and MgCl2, harvested in lysis buffer (15 mM Tris-HCl, pH 7.4; 2 mM MgCl2; 0.3 mM EDTA), polytron homogenized, and centrifuged at 44,000 x g for 20 min at 4 C. Pellets were washed in a Buffer A (50 mM Tris-HCl, pH 7.4; 3 mM MgCl2) and centrifuged at 44,000 x g for 20 min at 4 C. Membranes were resuspended in a small volume of Buffer A. Protein concentration was determined by the method of Bradford with the Bio-Rad protein assay kit and using BSA as a standard. Membranes were stored at –80 C before use.

Binding Experiments
Membrane binding assays were performed as previously described using [3H]AVP as radioligand. Membranes (5–10 µg protein) were incubated 60 min. at 30 C in a medium containing: 50 mM Tris-HCl, pH 7.4; 3 mM MgCl2; 1 mg·ml–1 BSA; and 0.01 mg·ml–1 leupeptin. Affinity (Kd) of [3H]AVP for the wild-type and the mutated vasopressin receptors was determined by saturation experiments using concentrations of labeled vasopressin ranging from 0.1 to 30 nM. For each concentration of radioligand, total and nonspecific binding was determined in absence or presence, respectively, of 1 µM unlabeled AVP. The affinities (Ki) of the unlabeled analogs were determined by competition experiments. Briefly, depending upon the receptors studied, 0.5 to 15 nM of [3H]AVP was added in the incubation medium with or without increasing amounts of the unlabeled analogs to be tested (total binding). Nonspecific binding was determined under the same experimental conditions in the presence of 1 µM unlabeled AVP. The radioactivity found associated with plasma membranes was determined by filtration through glass microfiber filters (GF/C Series; Whatman International Ltd., Maidstone, UK). Specific binding was calculated and expressed as percent of the maximal binding capacity determined without unlabeled analog. Ki values were calculated from the dose-displacement curves fitted with the Cheng and Prusoff equation.

IP Assays
Total IP accumulation was determined as previously described. Briefly, CHO cells stably or transiently transfected with the wild-type and the mutated receptors were grown for 24 h in DMEM supplemented with 10% fetal calf serum. Cells were further incubated for another 24-h period in a serum and inositol-free medium supplemented with 1µCi·ml–1 myo-[3H]-inositol and 3 mM sodium butyrate. Cells were then washed twice with a Hanks’ buffered saline (HBS) medium, equilibrated at 37 C in HBS for 30 min, and incubated for 15 min in HBS supplemented with 15 mM LiCl, 1 mg·ml–1 glucose, 1 mg·ml–1 BSA and 2.1 g·liter–1 NaHCO3. Cells were further stimulated for 15 min with increasing concentrations of the analogs to be tested. Reaction was stopped by adding perchloric acid (5% vol/vol, final concentration). IPs accumulated were extracted and purified on Dowex AG1-X8 anion exchange chromatography column as previously described (26) and counted.

Chemicals
All reagents used were of analytical grade. Most standard chemicals were purchased from Sigma (St. Louis, MO), Roche Molecular Biochemicals (Mannheim, Germany), or Merck & Co., Inc. (Darmstadt, Germany), unless otherwise indicated. AVP came from Bachem (Bubendorf, Switzerland). [3H]AVP (60–80 Ci/mmol) was from PerkinElmer Life Sciences (Courtaboeuf, France). SSR149415 and SR49059 were from Sanofi-Aventis Laboratories (Toulouse, France). DMEM, penicillin-streptomycin, L-glutamine, and nonessential amino acids were purchased from Invitrogen (Cergy Pontoise, France). Inositol-free DMEM came from ICN Biochemicals (Orsay, France). Dowex AG1-X8 format form 200–400 mesh was purchased from Bio-Rad Laboratories, Inc. (München, Germany). d[Cha4]AVP was synthesized by Dr. M. Manning (College of Medicine, University of Toledo, OH).

Data Analysis
The radioligand binding data were analyzed by GraphPad Prism (GraphPad Software, Inc., San Diego, CA). The dissociation constants (Kd) of the radioligands were determined according to the Scatchard linearization of the saturation curve obtained (33). The inhibitory dissociation constants (Ki) for unlabeled analogs were calculated from binding competition experiments according to the Cheng and Prusoff equation (34): Ki = IC50 x (1+[L]/Kd), where IC50 is the concentration of unlabeled analog leading to half-maximal inhibition of specific binding, [L] the concentration of the radioligand present in the assay, and Kd its affinity for the receptor studied. Concentrations of analog leading to half-maximal stimulation (Kact) or inhibition (Kinact) of IPs accumulation were calculated from functional studies using GraphPad Prism. Results are expressed as the mean ± SEM of the number of distinct experiments indicated.


   ACKNOWLEDGMENTS
 
We thank Sanofi-Aventis for providing us selective nonpeptidic vasopressin antagonists. The Computational Centers at Montpellier (CINES, Centre Informatique National de l’Enseignement Supérieur) and Orsay (IDRIS, Institut du Développement des Ressources Informatiques Scientifiques) are gratefully acknowledged for allocation of computing time. D.R. thanks Dr. Michael T. Bowers for providing us the structure of Zn2+-bound OT. We also thank M. Passama for drawing the illustration and Dr. M. Manning for his generous gift of d[Cha4]AVP.


   FOOTNOTES
 
This work was supported by the European Commission for the Marie-Curie fellowship to J.R (HPMF-CT-2002-02141), the Basque Country University (Bilbao, Spain; Grant 9/UPV-00042.310-15852/2004), and "Secretaría de Estado de Educación y Universidades" (Madrid, Spain) for the fellowship to M.T.; A.P. received a fellowship from Sanofi-Aventis.

Current address for J.R.: Centre d’Etudes et de Recherche sur le Médicament de Normandie, Unité Propre de Recherche et de l’Enseignement Supérieur, Equipe d’Accueil 3915, Département de Modélisation Moléculaire, Université de Caen Basse-Normandie, Normandie, France.

First Published Online November 2, 2006

1 J.R. and A.P. contributed equally to this work. Back

Abbreviations: AVP, Arginine vasopressin; CHO, Chinese hamster ovary; 3-D, three dimensional; d[Cha4]AVP, [desamino-cys1]-cyclohexylalanine-4 arginine vasopressin; dDAVP, [desamino-cys1]-DArg-8 vasopressin; GA, genetic algorithm; GPCR, G protein-coupled receptor; HBS, Hanks’ buffered saline; IP, inositol phosphate; MD, molecular dynamics; OT, oxytocin; rmsd, root mean square deviation; TM, transmembrane.

Received for publication May 11, 2006. Accepted for publication October 24, 2006.


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A. Pena, B. Murat, M. Trueba, M. A. Ventura, G. Bertrand, L. L. Cheng, S. Stoev, H. H. Szeto, N. Wo, G. Brossard, et al.
Pharmacological and Physiological Characterization of d[Leu4, Lys8]Vasopressin, the First V1b-Selective Agonist for Rat Vasopressin/Oxytocin Receptors
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