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CELLULAR AND MOLECULAR

Mapping the Binding Site of Six Nonpeptide Antagonists to the Human V₂-Renal Vasopressin Receptor

Departments of Biochemistry (R.M.-D., N.C., Z.X., N.W., M.S.) and Medicine (M.T.), Case Western Reserve University, Cleveland, Ohio

Received September 12, 2005; accepted October 14, 2005.

	Abstract

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Whereas arginine vasopressin binds to its receptor subtypes V₁R and V₂R with equal affinity of approximately 2 nM, nonpeptide antagonists interact differently with vasopressin receptor subtypes. The V₂R antagonist binding site was mapped by site-directed mutagenesis at six selected amino acid positions, K100D, A110W, M120V, L175Y, R202S, and F307I, predicted to be involved in antagonist binding differences between V₂ R and V₁R. These mutations did not alter the affinity for arginine vasopressin. However, the affinity for six nonpeptide receptor antagonists SR121463B [1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzenesulfonyl]-5-ethoxy-3-spiro-[4[(2 morpholinoethoxy)cy-clohexane]indoline-2-one, phosphate monohydrate cis-isomer], SR49059 [(2S)1-[(2R3S)-(5-chloro-3-(2 chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide], SSR149415 [(2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxyphenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2pyrrolidine carboxamide, isomer(-)], OPC21268 [1-[1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl]-3,4-dihydro-2(1H)-quinolinone], OPC41061 [(±)-4'-[(7-chloro-2,3,4,5-tetrahydro-5-hydroxy-1H-1-benzazepin-1-yl)carbonyl]-o-tolu-m-toluidide], and OPC31260, [(±)-5-dimethylamino-1-[4-(2-methylbenzoylamino)benzoyl]-1,2, 3,4,5-tetrahydro-1H-benzazepine monohydrochloride], was altered to varying degrees, resulting in differences up to 6000-fold. Replacement of the small alanine for the bulky tryptophan in position 110 resulted in a reduced affinity for all six antagonists. In contrast, replacement of the large methionine for the smaller valine in position 120 caused a dramatic increase in affinity, up to a K_i of 7 fM for OPC31260. Molecular modeling revealed that the binding sites for arginine vasopressin and the nonpeptide antagonists are partially overlapping. Whereas arginine vasopressin binds on the extracellular surface of V₂ R, the nonpeptide antagonists penetrate deeper into the transmembrane region of the receptor, in particular OPC21268. The mutagenesis data point to significant differences in the shape of the V₁R and V₂R antagonist binding pockets. The most important factor determining the specificity of nonpeptide antagonists seems to be the shape of the binding pocket on the receptor.

View larger version (56K): [in this window] [in a new window]   Fig. 1. Snake diagram of human V2R. Residues in white represent amino acids that are identical in human V1R and V2R, whereas shaded residues are the divergent amino acids. The two tandem cysteine residues in the C-terminal segment are palmytoylated, thus providing a membrane anchor. Arrows show selected mutations K100D, A110W, M120V, L175Y, R202S, and F307I. The box represents the putative ligand-binding pocket.

The AVP/oxytocin receptor family represents a suitable system to investigate structure-function relationships of receptor subtypes. Pharmacological and molecular cloning studies of the V₁, V₂, and V₃ AVP receptors, as well as the related oxytocin receptor, have shown that these peptide receptors display a great diversity in their functional properties despite high sequence homology. These receptors can bind not only the native hormone AVP but also potent and selective cyclic and linear peptide analogs, as well as nonpeptide antagonists (Cotte et al., 1998). Mutagenesis studies have indicated that the ligand-binding pocket includes residues located on the extracellular loops as well as in adjoining transmembrane helices of the receptors (Oksche et al., 2002).

In this work, we set out to determine key residues responsible for the differential nonpeptide ligand binding specificity to V₂R versus V₁R. Six nonconservative single amino acid differences between the V₂R and the V₁R sequences (K100D, A110W, M120V, L175Y, R202S, and F307I) were selected for this investigation because of their location within the putative ligand binding pocket. The amino acid in position 202 is particularly interesting, because an R202C mutation has been identified in patients suffering from NDI (Morello and Bichet, 2001). Dissociation constants of the six nonpeptide receptor antagonists for each mutant V₂R were determined. The data were interpreted by molecular modeling of antagonist docking to wild-type and mutant receptors. The results point to differences between the V₁R and V₂R antagonist binding sites. The most important factor in determining the specificity of nonpeptide antagonists seems to be the shape of the binding pocket on the receptor.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Saturation binding curve of [3H]AVP to wild-type V2R. The concentration of free AVP was calculated from the difference between total and bound AVP, as described under Materials and Methods.

View larger version (73K): [in this window] [in a new window]   Fig. 3. A, chemical structure of AVP. B, surface representation of AVP docked onto the model of V2R. The view is from the outside of the cell looking down at the receptor. The extracellular loops have been omitted for clarity. The receptor surface is set to 50% transparency. AVP and residues selected for mutagenesis are in ball-and-stick representation. Nitrogen is blue, oxygen is red, and sulfur is orange, and carbon is magenta on the ligand, yellow on the mutated side chains, and green on the rest of the receptor. The amino acid one-letter code of AVP and V2R residues is in lowercase and uppercase, respectively. This figure was generated with program PyMOL.

	Materials and Methods

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Construction of Receptor Expression Plasmids
Wild-Type V₂R-GFP. The human V₂R cDNA (comprising the coding region, nucleotides 219-1354 of the human cDNA sequence, GenBank accession number 4895106) was isolated from pcDNA plasmid by XbaI/BamHI digest and subcloned into the XbaI/BamHI-cut pEGFP-N1 plasmid (BD Biosciences Clontech, Palo Alto, CA) for comparative expression analysis.

Mutant V₂R-GFP. Plasmids encoding mutants K100D, A110W, M120V, L175Y, R202S, and F307I were generated with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) following manufacturer's instructions. The wild-type V₂R-GFP plasmid was used as template. Sense and antisense primers encoding single amino acid changes were purchased from Invitrogen (Carlsbad, CA). The sequence of each construct was checked by Cleveland Genomics, Inc. (Cleveland, OH).

Cell Culture and Transfection
Chinese hamster ovary-K1 cells (American Type Culture Collection, Manassas, VA) were grown in F-12K medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and 500 units/ml penicillin/streptomycin (Invitrogen) in an atmosphere of 95% air and 5% CO₂ at 37°C. Stable transfection was done using the Lipofectamine 2000 transfection kit (Invitrogen) following the manufacturer's instructions. The stable cell lines of wild type and mutants were selected by flow-cytometry sorting (Cancer Center Core Facility, Case Western Reserve University) and G418 (Invitrogen) selection up to 8 or 12 µg/ml.

Radioligand Binding Assays
The binding of [³H]AVP to intact Chinese hamster ovary cells was performed as described previously (Thibonnier et al., 2000). In brief, the cells were seeded at a density of 1.75 x 10⁵/well in 12-well plates. Twenty-four hours after plating, the cells were washed twice with binding buffer (10 mM MgCl₂, 0.2% bovine serum albumin in 1x Dulbecco's phosphate-buffered saline, pH 7.4). For saturation binding analysis, the cells were then incubated with increasing concentrations of [³H]AVP diluted in the same buffer in the presence (nonspecific binding) or absence (total binding) of 100 nM unlabeled AVP for 30 min at 30°C in a shaking water bath. For competition binding analysis, cells were incubated with 2 nM [³H]AVP in the presence of increasing concentrations of unlabeled AVP or nonpeptide antagonist for 30 min at 30°C in a shaking water bath. After washing three times with ice-cold phosphate-buffered saline, the cells were lysed with 0.1 N NaOH and 0.1% SDS. The lysates were then transferred to scintillation vials, and 4 ml of ReadyProtein (Beckman Coulter, Inc., Fullerton, CA) scintillation cocktail was added. Radioactivity was determined in a liquid scintillation counter (LS6000; Beckman Coulter). K_d and inhibition constant (K_i) values were calculated using standard equations and program Prism 4.0 (GraphPad Software, Inc., San Diego, CA) (Thibonnier et al., 2000).

View larger version (17K): [in this window] [in a new window]   Fig. 4. Chemical structure of the six nonpeptide antagonists used in this study.

Docking of AVP and Antagonists to the V₂R Model
Docking of AVP and antagonists was carried out with the program LIGIN (Sobolev et al., 1996). The docking was done for each compound and for each chimeric receptor separately followed by energy minimization with program CNS. Ligand-receptor distances were calculated with the program CONTACT within the CCP4 suite of crystallographic programs (Collaborative Computational Project, Number 4, 1994).

	Results

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Selection of Point Mutants. Previous studies have localized the binding site for AVP and its antagonists in a region delineated by the extracellular loops and the adjoining transmembrane regions of the V₂R (Thibonnier et al., 2002). To further map the binding site and to identify residues responsible for the specificity of antagonists binding to V₁ and V₂ receptor subtypes, point mutations were created by introducing into the V₂R sequence the corresponding residues from the V₁R sequence. The degree of sequence identity between V₂R and V₁R is 65, 44, and 21% for the extracellular loops el1, el2, and el3, respectively. The overall degree of sequence identity is 41%, high enough to assume that the overall fold of these two receptors is conserved. Because there are many sequence differences, we wanted to select mutations that potentially affect antagonist specificity. Twenty point mutations were initially selected within the putative ligand-binding region based on the nonconservative nature of the amino acid replacements. The number of point mutants employed in this study was eventually reduced to the following six-well expressed mutants: K100D, A110W, M120V, L175Y, R202S, and F307I. The criteria for selecting these mutations include charge reversal (K100D), large changes in the volume of the side chain (A110W, M120V, and R202S), and replacements of aliphatic to aromatic side chains or vice versa (L175Y and F307I). The location of these mutations in V₂R is shown in Fig. 1. Mutations K100D, A110W, R202S, and F307I are depicted on extracellular loops, whereas mutations M120V and L175Y are shown in transmembrane helices. However, there is some degree of uncertainty in assigning the boundaries between transmembrane regions and loops. For example, residue Phe-307 has been reported to be on transmembrane helix 7 (Mouillac et al., 1995).

Affinity of AVP for the V₂R Mutants. The affinity of AVP for the wild-type V₂R, as determined by saturation binding experiments with [³H]AVP, is 2.2 nM (Fig. 2), identical to the value we reported previously (see Table 1) (Thibonnier et al., 2001). None of the six point mutations affected AVP affinity for the V₂R in any significant way. In silico docking of AVP to V₂R explains these findings for each of the receptor mutant. Four of the six mutated V₂R residues make contact with AVP, namely Lys-100, Met-120, Leu-175, and Phe-307 (Fig. 3). Mutations at these positions do not affect the affinity, apparently for the following reasons. Residue 100 makes only backbone contacts with AVP; therefore, replacement of the side chain should has no effect on binding AVP. Mutations in the remaining three positions do not seem to alter interactions with AVP. Methionine and valine at position 120 both exhibit hydrophobic contacts with f3 and q4 of AVP. Leucine and tyrosine at position 175 both make hydrophobic contacts with backbone atoms of q4 and n5 of AVP. Phenylalanine and isoleucine at position 307 both make hydrophobic contacts with the AVP-disulfide bridge between c1 and c6 of AVP. The docking results provide an explanation for the lack of any significant effect of these amino acid replacements from V₂R to the corresponding residues in V₁R on the affinity for AVP. The concept of nonpeptide antagonists acting as molecular chaperones to restore agonist binding (Morello et al., 2000a) does not apply to these mutants as they all bind AVP with the same affinity as wild-type V₂R. However, these mutations greatly affect binding of nonpeptide antagonists.

View larger version (51K): [in this window] [in a new window]   Fig. 5. Superposition of the models of AVP and OPC21268 in ball-and- stick representation as docked onto the V2R model shown as a ribbon diagram. The loops are labeled el1, el2, and el3 for the extracellular loops and il1, il2, and il3 for the intracellular loops. The binding sites for agonist and antagonist are distinct with partial overlap. This figure was generated with programs MOLSCRIPT (Esnouf, 1999) and RASTER3D (Merritt and Murphy, 1994).

View larger version (27K): [in this window] [in a new window]   Fig. 6. Competition binding curves of [3H]AVP in the presence of antagonists. A, binding of [3H]AVP to wild-type V2R as a function of antagonist concentration. SR31260 and OPC21268 do not displace AVP from the receptor. B, binding of [3H]AVP to the A110W mutant as a function antagonist concentration. SSR14915 and OPC21268 do not displace [3H]AVP to any significant degree. C, binding of [3H]AVP to wild-type and mutant V2R as a function of OPC31260 concentration. This antagonist is able to completely displace [3H]AVP in the case of the A110W mutant. For the M120V and F307I mutants, the displacement is partial. OPC31260 does not displace [3H]AVP at all in the case of wild-type and mutants K100D, L175Y, and R202S.

Antagonist Binding to the K100D Mutant. Large differences in the affinity of this mutant are confined to SR49059 (14-fold weaker affinity) and OPC41061 (6-fold stronger affinity). Interestingly, docking followed by energy refinement did not show any direct involvement of residue 100 in the binding of these two compounds. It is possible that the K100D mutation causes a conformational change, which indirectly affects the binding of SR49059 and OPC41061.

Antagonist Binding to the A110W Mutant. A110W is the only mutation that dramatically weakens the binding of all six nonpeptide antagonists, particularly for SSR14915 and OPC21268. For these two antagonists, no binding at all could be detected. A likely explanation for the decrease in affinity is overcrowding due to the introduction of the bulky tryptophan in place of the small alanine side chain. To relieve steric strain, the nearby Leu-175 has to adopt a different conformation, which probably results in a loss of hydrophobic interactions of this residue with antagonists (Fig. 7). Tryptophan in position 110 may also affect the nearby Arg-202, which interacts with some of the antagonists. The decrease in affinity ranges from 42-fold for SR121463B to 1340-fold for OPC41061. The binding of all nonpeptide antagonists to this mutant is also weaker than the binding of AVP by a factor up to 200-fold.

View larger version (38K): [in this window] [in a new window]   Fig. 7. Mutagenesis of the small alanine at position 110 for the bulky tryptophan introduces overcrowding and narrows the binding site. To alleviate overcrowding, Leu-175 may readjust to a position where it no longer can interact with the antagonist. This figure was generated with programs MOLSCRIPT (Esnouf, 1999) and RASTER3D (Merritt and Murphy, 1994).

Antagonist Binding to the L175Y Mutant. Although unlabeled AVP could displace [³H]AVP from this mutated receptor, all six nonpeptide antagonists prevented the binding of [³H]AVP, even at antagonist concentration as low as femtomolar. The most likely explanation is that antagonist binding is so tight that AVP cannot displace it. The increased affinity may be due to hydrogen bonds of the tyrosine hydroxyl group as well as -stacking of the tyrosine ring with the aromatic moieties of the compounds.

Antagonist Binding to the R202S Mutant. This mutation has distinct effects on the binding characteristics of one SR and one OPC compound. There is no change in the affinity for OPC41061, whereas there is a 10-fold increase in the affinity for SR121463B. Docking of OPC41061 suggests that this antagonist makes no interactions with residue 202, whether it is an arginine or a serine. In contrast, docking of SR121463B suggests short contacts of this antagonist with the guanido group of Arg-202. Substitution of the large arginine for the smaller serine improves binding, apparently because of relief of overcrowding.

View larger version (60K): [in this window] [in a new window]   Fig. 8. The ligand-binding site on the V2R. The view is from the extracellular side perpendicular to the membrane. Docked AVP is shown in ball-and-stick representation with dark gray bonds. Docked OPC41061 is shown as an example of a nonpeptide antagonist in ball-and-stick representation with light gray bonds. The N-terminal extracellular segment and the extracellular loops 1 and 2 have been omitted for clarity. The binding sites for AVP and the nonpeptide antagonists partially overlap. The overall size of the ligand binding pocket is 25 x 20 x 15 Å.

	Discussion

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Our findings suggest that AVP and the nonpeptide antagonists fit tightly into the binding pocket of the V₂R. Docking indicates an overall binding pocket of dimensions 25 x 20 x 15 Å, with the first dimension parallel to the surface of the membrane (Fig. 8). AVP binds to a subpocket of size 22 x 14 x 14Å. The nonpeptide antagonists occupy a subpocket of dimensions 12 x 20 x 10 Å. The investigated mutations affect only the subpocket for nonpeptide antagonists. Narrowing of this ligand-binding subpocket with the A110W mutation interferes with binding. In contrast, widening of the pocket by the M120V and the F307I mutations improves antagonist binding. These results point to a significant difference in the shape of the nonpeptide antagonist binding pocket between V₁R and V₂R. In V₁R, the single small-to- large mutation investigated in this work, A110W, is apparently compensated by three large-to-small mutations, K100D, M120V, and R202S. None of the single point mutants by themselves is responsible for nonpeptide antagonist specificity toward a particular receptor subtype. Rather, the cumulative effect of multiple mutations seems to determine receptor subtype specificity. These findings may help design more potent and selective nonpeptide AVP receptor antagonists.

	Acknowledgements

 
We thank Claudine Serradeil-Le Gal from sanofi-synthelabo for providing us with the nonpeptide antagonists SR121463B, SR49059, and SSR14915; Koji Komuro from Otsuko Pharmaceutical Company for providing us with the nonpeptide antagonists OPC21268, OPC41061, and OPC31260; Bryan Roth, Vincent Setola, and Jonathan Birkes for help with radioligand binding assays; and the Flow Cytometry Core Facility at Case Western Reserve University for expert assistance in cell sorting.

	Footnotes

 
This work was supported by National Institutes of Health Grant R01 HL-39757 (to M.S. and M.T.) and by a National Kidney Foundation of Ohio Research Grant (to N.W.).

doi:10.1124/jpet.105.095554.

ABBREVIATIONS: AVP, 8-arginine vasopressin; V₁R, V₁-vascular vasopressin receptor; V₂R, V₂-renal vasopressin receptor; NDI, nephrogenic diabetes insipidus; GFP, green fluorescent protein; OPC21268, 1-[1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl]-3,4-dihydro-2(1H)-quinolinone; OPC41061, (±)-4'-[(7-chloro-2,3,4,5-tetrahydro-5-hydroxy-1H-1-benzazepin-1-yl)carbonyl]-o-tolu-m-toluidide; OPC31260, (±)-5-dimethylamino-1-[4-(2-methylbenzoylamino)benzoyl]-1,2,3,4,5-tetrahydro-1H-benzazepine monohydrochloride; SR49059, (2S)1-[(2R3S)-(5-chloro-3-(2 chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide; SR121463B, 1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzenesulfonyl]-5-ethoxy-3-spiro-[4[(2 morpholinoethoxy)cy-clohexane]indoline-2-one, phosphate monohydrate cis-isomer; SSR149415, (2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxyphenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2pyrrolidine carboxamide, isomer(-); SR, SR49059 and SR121463B; OPC, OPC31260, OPC41061, and OPC21268.

¹ Current affiliation: Cleveland Clinic Foundation, Lerner Research Institute, Cleveland, OH.

Address correspondence to: Menachem Shoham, Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4935. E-mail: mxs10{at}case.edu

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This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.095554v1 316/2/564    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Macion-Dazard, R. Articles by Shoham, M. Search for Related Content PubMed PubMed Citation Articles by Macion-Dazard, R. Articles by Shoham, M.