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J Biol Chem, Vol. 274, Issue 33, 23316-23327, August 13, 1999

Docking of Linear Peptide Antagonists into the Human V_1a Vasopressin Receptor
IDENTIFICATION OF BINDING DOMAINS BY PHOTOAFFINITY LABELING^*

Sylvie Phalipou, René Seyer^§, Nathalie Cotte, Christophe Breton, Claude Barberis, Marcel Hibert^¶, and Bernard Mouillac

From  U469 INSERM and ^§ UPR 9023 CNRS, CCIPE, 141 rue de la Cardonille, 34094 Montpellier cedex 5, France and ^¶ ERS 655 CNRS, Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch cedex, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A novel photoactivatable linear peptide antagonist selective for the V_1a vasopressin receptor, [¹²⁵I][Lys(3N₃ Phpa)⁸]HO-LVA, was synthesized, characterized, and used to photolabel the human receptor expressed in Chinese hamster ovary cells. Two specific glycosylated protein species at 85-90 and 46 kDa were covalently labeled, a result identical to that obtained with a previous photosensitive ligand, [¹²⁵I]3N₃Phpa-LVA (Phalipou, S., Cotte, N., Carnazzi, E., Seyer, R., Mahe, E., Jard, S., Barberis, C., and Mouillac, B. (1997) J. Biol. Chem. 272, 26536-26544). To identify contact sites between the new photoreactive analogue and the V_1a receptor, the labeled receptors were digested with Lys-C or Asp-N endoproteinases and chemically cleaved with CNBr. Fragmentation with CNBr, Lyc-C, and Asp-N used alone or in combination, led to the identification of a restricted receptor region spanning the first extracellular loop. The results established that sequence Asp¹¹²-Pro¹²⁰ could be considered as the smallest covalently labeled fragment with [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA. Based on the present experimental result and on previous photoaffinity labeling data obtained with [¹²⁵I]3N₃Phpa-LVA (covalent attachment to transmembrane domain VII), three-dimensional models of the antagonist-bound receptors were constructed and then verified by site-directed mutagenesis studies. Strikingly, these two linear peptide antagonists, when bound to the V_1a receptor, could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Despite divergent functional properties, these peptide antagonists could interact with a transmembrane-binding site significantly overlapping that of the natural hormone vasopressin.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Over the past few years, interest in locating ligand-binding sites in G protein-coupled receptors has increased exponentially. Indeed, identification of these binding sites is of prime importance both for a better understanding of the structure and the function of the G protein-coupled receptor superfamily and for facilitating rational design of potential therapeutic agents. Extensive mutational analysis and receptor three-dimensional molecular modeling have led to valuable information concerning "small ligand" and peptide/protein ligand receptor-binding sites (for review, see Refs. 1-6).

In 1995, we published the mapping of arginine-vasopressin (AVP)¹-binding site in the V_1a receptor subtype and described a major localization within transmembrane regions (TMR) in a position equivalent to that defined for the cationic neurotransmitters (7). Because all receptor residues potentially interacting with AVP are conserved in the different members of the AVP/oxytocin (OT) receptor family, we proposed that the binding pocket identified in the V_1a might be common to V₂, V_1b, and OT receptor subtypes. Extracellular residues responsible for receptor-selective and species-selective binding have also been identified (8-10). Unfortunately, these first analyses of AVP receptor structure/function relationships did not provide much information on AVP receptor antagonist-binding domains (Refs. 11 and 12, and for review see Ref. 13). The photoaffinity labeling technique is an essential complement to modeling and mutagenesis approaches and allows direct unambiguous identification of the contact regions between a receptor and its specific photoactivatable ligands (for review see Ref. 14). At the present time, very few photoaffinity labeling studies have led to the direct determination of labeled amino acid residues in peptide G protein-coupled receptor; remarkable results with bovine V₂ receptor (15), human NK1 tachykinin receptor (16), and rat type A cholecystokinin receptor (17) allowed identification of covalently labeled residues with photoactivatable agonist analogues of AVP, substance P, and cholecystokinin, respectively.

Very recently, a first radioiodinated photoreactive linear peptide antagonist has been used in our laboratory to photolabel the human and rat V_1a receptors (12, 18, 19). Our results have clearly indicated that covalent attachment of the [¹²⁵I]3N₃Phpa-LVA occurs in a restricted domain of the human receptor including TMR VII. Based both on this photolabeling result and on the hypothetical three-dimensional model of the human V_1a receptor, residues potentially involved in binding and affinity of the antagonist ligand have been targeted. An interaction between the hydrophobic N terminus of the [¹²⁵I]3N₃Phpa-LVA ligand and an aromatic cluster of residues in the TMR VI has thus been experimentally verified. However, because of the lack of structural and conformational data for this family of V_1a-selective compounds and because of their peptidic nature and highly variable linear structure, the determination of a single contact point between the peptide antagonist and the receptor does not provide enough information to propose a docking mode of the ligand into the receptor.

To allow a more complete location of the binding sites for this family of V_1a linear peptide antagonists and to generate meaningful information on receptor-antagonist interactions, we thus decided to label the human V_1a receptor with a second radioiodinated photoreactive antagonist. In the present study, properties of [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA, an antagonist containing an azido group at a position (side chain of lysine residue 8) likely to covalently bind another domain of the receptor are described. Combining photolabeling with this new ligand of the human V_1a receptor, cyanogen bromide cleavage and endoproteinase digestions of the receptor, a restricted photolabeled domain has been identified which spans the first extracellular loop from Asp¹¹² to Pro¹²⁰. Taking into account the present and previous photolabeling data, three-dimensional models of antagonist-bound receptors have been constructed and experimentally verified. Residues possibly involved in the interactions with the photoactivatable antagonists have been mutated, and affinities of the mutant receptors for the ligands have been estimated.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Synthesis of 4HO-Phpa-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(3N₃Phpa)-NH₂·CF₃CO₂H-- The peptide 4HO-Phpa-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys-NH₂·2CF₃CO₂H and the acid 3N₃Phpa were synthesized, purified by reverse phase HPLC, and characterized by fast atom bombardment mass spectrometry as described previously (18, 20). The peptide (7 mg, 5.2 µmol) was dissolved in anhydrous Me₂SO (100 µl) and mixed with an excess of azidoacid (1.9 mg, 10 µmol), PyBOP (5.2 mg, 10 µmol) (21), and (iPr)₂EtN (4 µl) and kept in the dark. After 1 h, monitoring of the reaction was done by injection of 0.2 µl of mixture onto the analytical HPLC (end-capped C₁₈ Merck Lichrosorb column, 4 × 250 mm, 5-µm particle size, 100 Å porosity, linear 1%/min CH₃CN gradient in water, all acidified by 0.1% CF₃CO₂H (v/v)) and eluted at 2 ml/min using a dual wavelength (214 nm and 254 nm) UV detection (22). The mixture was acidified using CF₃CO₂H, injected onto the semi-preparative HPLC (C₁₈ Vydac 218TP1022, 25 × 250 mm, 10-µm particle size, 80 Å porosity), and eluted at 10 ml/min using a 0.5%/min linear CH₃CN:0.1% CF₃CO₂H gradient. In these conditions, an impurity (noniodinatable) was eluted immediately before the expected compound and was poorly separated. The lyophilized preparation was injected onto two coupled semi-preparative columns (Vydac, as above, and Whatman Magnum 20, Partisil ODS-3, 25 × 500 mm, 10-µm particle size, 80 Å porosity) and eluted at 10 ml/min using a 0.2%/min CH₃CN:0.1% CF₃CO₂H gradient, running from 20 to 40%. In these conditions, the impurity eluted after the target compound and was well separated, as shown by the analytical screening of the fractions. The photoactivatable antagonist was lyophilized and characterized (see "Results").

Monoiodination of the probe: 4HO,3I-Phpa-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(3N₃Phpa)-NH₂·CF₃CO₂H-- Under HPLC monitoring, around 1 molar equivalent of ICl in MeOH (10² M, 50 µl) was added to a solution of the precedent compound (2 10⁴ M, 3.5 ml) in order to obtain the monoiodinated analogue (second peptide peak) as the major species. The three compounds were separated (Vydac column, 10 ml/min and 1%/min CH₃CN: 0.1% CF₃CO₂H, starting from 0%) as follows: noniodinated (41%), monoiodinated (44%), and diiodinated (47%). The monoiodinated compound (fast atom bombardment mass spectrometry, M+1 = 1412) was flash-frozen in liquid nitrogen, lyophilized, and used as HPLC standard for radioiodination.

Radioiodination of the probe: 4HO,3[¹²⁵I]Phpa-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(3N₃Phpa)-NH₂· CF₃CO₂H-- After synthesis, the photoactivatable linear peptide antagonist was radioiodinated at position 1 on the phenolic substituent in the presence of the oxidant 1,3,4,6-tetrachloro-3,6-diphenyl-glycouril (Iodo-Gen^®, Pierce) by means of 1 mCi of Na¹²⁵I (IMS 30, Amersham Pharmacia Biotech) as described (23). The radioiodinated peptide was purified on a HPLC column (Waters C₁₈ µBondapak) by two subsequent runs. The specific radioactivity of the monoiodinated antagonist was 2,200 Ci/mmol.

Cell Culture and Membrane Preparation-- The human V_1a receptor cDNA was a generous gift of Dr. M. Thibonnier (24), and CHO cells were transfected as described before (12). CHO cells stably expressing the human V_1a, V_1b, V₂, or OT receptor were maintained in culture in Petri dishes in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 4 mM L-glutamine, 500 units/ml penicillin and streptomycin, 0.25 µg/ml amphotericin B in an atmosphere of 95% air and 5% CO₂ at 37 °C. Depending on the experiment to be conducted, cells were treated overnight with 5 mM sodium butyrate to increase receptor expression (25, 26). Cells were harvested, washed two times in PBS without Ca²⁺ and Mg²⁺, polytron-homogenized in lysis buffer (15 mM Tris-HCl, pH 7.4, 2 mM MgCl₂, 0.3 mM EDTA) and centrifuged at 100 × g for 5 min at 4 °C. Supernatants were recovered and centrifuged at 44,000 × g for 20 min at 4 °C. Pellets were washed in Buffer A (50 mM Tris-HCl, pH 7.4, 5 mM MgCl₂) and centrifuged at 44,000 × 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 in binding assays and photolabeling experiments or stored at 80 °C.

Radioligand Binding Assays-- As described in previous papers (7, 12), binding assays were performed at 30 °C using [¹²⁵I]HO-LVA, [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA, [¹²⁵I]3N₃Phpa-LVA, or [³H]AVP as the radioligands and 1-3 µg (for assays with ¹²⁵I-labeled antagonists) or 10-15 µg (for assays with [³H]AVP) of membrane protein. Membrane were incubated in Buffer A with 1 mg/ml bovine serum albumin and with radiolabeled and displacing peptides for 30 min (with [³H]AVP) or 60 min (with radioiodinated antagonists). Affinities (K_d) for [¹²⁵I]HO-LVA, [¹²⁵I]3N₃Phpa-LVA, and [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA (concentrations from 10 to 600 pM), as well as for [³H]AVP (concentrations from 0.1 to 20 nM) were directly determined in saturation experiments. Affinities (K_i) for unlabeled ligands were determined from competition experiments using [³H]AVP (1-2 nM) or [¹²⁵I]HO-LVA (50-100 pM) as the radioligands. The concentrations of the unlabeled ligands varied from 1 pM to 10 µM. AVP (10 µM) and HO-LVA (400 nM) were used to determine nonspecific binding, respectively. Bound and free radioactivity were separated by filtration over bovine serum albumin presoaked Whatman GF/C filters. The ligand binding data were analyzed by nonlinear least squares regression using the computer program Ligand (27).

Inositol Phosphate Assays-- Inositol phosphate (IP) accumulation was determined as described (28). Briefly, CHO cells expressing the human V_1a receptor were plated and grown in 6-well dishes for 48 h in Dulbecco's modified Eagle's medium-supplemented medium and then labeled for 24 h with myo-[2-³H]inositol (10-20 Ci/mmol; NEN Life Science Products) at a final concentration of 1 µCi/ml in a serum-free, inositol-free medium (Life Technologies, Inc.). Cells were washed twice with PBS medium, equilibrated at 37 °C in PBS for 1 h, and then incubated for 10 min in PBS supplemented with 10 mM LiCl in the presence or absence of increasing concentrations of [Lys(3N₃Phpa)⁸]HO-LVA or 3N₃Phpa-LVA (from 10¹² to 10⁶ M). CHO cells were stimulated for 15 min with 10⁹ M AVP (a concentration close to the K_act value determined in CHO cells). After stopping the reaction with perchloric acid, total IPs were extracted and purified by anion exchange chromatography column (Dowex AG1x8, formate form, 200-400 mesh; Bio-Rad). For each sample, a fraction containing total IPs was collected and counted. K_inact constants were calculated as K_inact = IC₅₀/(1 + [AVP]/K_act), in which IC₅₀ is the concentration of antagonist leading to 50% inhibition, [AVP] = 1 nM and K_act is the concentration of AVP inducing half-maximal accumulation of IP (K_act = 0.32 nM in CHO cells expressing the wild-type human V_1a receptor (12)).

Photoaffinity Labeling Experiments-- These experiments were conducted as described before for the previous photoactivatable antagonist [¹²⁵I]3N₃Phpa-LVA (12). Briefly, membranes (500 µg) were resuspended in 4 ml of binding Buffer A containing bovine serum albumin (0.5 mg/ml) and were incubated for 1 or 3 h in the dark in the presence of [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA (1-2 nM) with or without vasopressin (10 µM) to define specific labeling. Membranes were separated from unbound ligand by two subsequent centrifugations (20 min, 44,000 × g, 4 °C) and washed with Buffer A. The final pellet was resuspended in 1 ml of Buffer A and irradiated with UV light (254 nm) for 1 min on ice. After photolysis, membranes were washed twice (2 × 1 ml of Buffer A) and finally resuspended in Laemmli buffer (29). Samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) using 1-mm-thick 12% cross-linked gels. Gels were fixed in glacial acetic acid:methanol:Me₂SO:water (16:40:2:42), dried, and exposed to Kodak XAR-5 film at 80 °C. In order to evaluate the yield of covalent binding, dried gels were also cut into slices, and radioactivity contents in the slices were determined. Covalent binding was calculated as percentage of the total number of receptor pmol expressed in the membrane preparation.

Electroelution of the Photolabeled V_1a Receptors-- Photolabeled membranes were subjected to SDS-PAGE using 12% cross-linked gels. The labeled bands were excised from the preparative gel and the human V_1a vasopressin receptor was electroeluted with electroeluter model 422 (Bio-Rad) in Tris/glycine running buffer (25 mM Tris, 182 mM glycine, pH 8.3, 0.1% SDS). Samples containing "partially purified" V_1a receptors were washed and concentrated using Microcon-30 (Amicon). Deglycosylation with N-glycosidase F and fragmentation with cyanogen bromide and endoproteases were performed on these concentrated samples.

Deglycosylation of the Photolabeled V_1a Receptors-- The photolabeled CHO membranes or the partially purified receptors were resuspended in deglycosylation buffer (100 mM Na₂HPO₄, pH 8.0, 10 mM EDTA, 1% digitonin, 1% 2-mercaptoethanol, 5 µg/ml leupeptin, 0.1% SDS) and digested with 2 units of N-glycosidase F (Roche Molecular Biochemicals) for 2 days at 37 °C. Deglycosylated receptors were analyzed by SDS-PAGE using 12% cross-linked gels.

Cyanogen Bromide and Endoproteinase Digestions-- The electroeluted receptors were subjected to digestion with CNBr, Lys-C protease, or Asp-N protease. Double digestions were also performed (first digestion with CNBr followed by a second one with Lys-C or Asp-N protease). CNBr (a few crystals) cleavage of the electroeluted V_1a receptors was carried out on samples in a 100-µl volume of 70% (v/v) formic acid. The mixture was incubated in the dark for 24 h at room temperature under argon, and the reaction was then stopped by adding 500 µl of water. Sample volume was reduced under vacuum, and solvent exchange (removing formic acid) with water was accomplished. Endoproteinase Lys-C (sequencing grade from Lysobacter enzymogenes, Roche Molecular Biochemicals) was used at 0.2 µg/assay in a final 50-µl volume. The digestion was performed in 25 mM Tris-HCl, pH 8.5, 1 mM EDTA, 0.1% SDS at 37 °C for 16-24 h and stopped by addition of Laemmli buffer. Endoproteinase Asp-N (sequencing grade from Pseudomonas fragi, Roche Molecular Biochemicals) was used at 0.1 µg/assay for 48 h at 25 °C in a final 50-µl volume of 25 mM Na₂HPO₄, pH 7.8. To perform double fragmentations, the CNBr digests were washed several times with water, and volume sample was reduced before dilution in Lys-C or Asp-N buffer. Results of CNBr and protease digestions were analyzed by a Tricine discontinuous SDS-PAGE system (10-16.5% cross-linked gels) applied to the separation of small molecular mass species (30). Gels were then fixed in glacial acetic acid:methanol:Me₂SO:water (10:50:2:38), dried, and exposed to Kodak XAR-5 film at 80 °C.

Computer Three-dimensional Molecular Modeling and Docking of Photoactivatable Antagonists-- The three-dimensional model of the human V_1a vasopressin receptor hosting AVP was constructed using the procedure already extensively described in a previous publication for the rat V_1a receptor (7). Briefly, the transmembrane part of the V_1a receptor was constructed by using the three-dimensional model first developed on the bacteriorhodopsin experimental structure (31, 32) and refined on the bovine rhodopsin footprint (33). The extracellular regions of the receptor were then built in an acceptable conformation. The rat residues were exchanged for the corresponding human residues. Transmembrane domain VII was rotated of about 20° to bring residues Thr710 and Ala711,² pointing both toward the core of the binding cleft.³ The whole receptor structure was then energy minimized in order to relax the structure and to remove unfavorable steric constraints. AVP was manually docked as reported and validated in previous publications (7, 9). In the next step, [¹²⁵I][Lys(3N₃ Phpa)⁸]HO-LVA was docked into the binding cleft. First, the ligand was built using Sybyl 6.3 (TRIPOS Associates, Inc.) facilities and energy minimized in a fully extended conformation. The backbone and side chain of the photoactivatable ligand have then been superimposed on the corresponding features of AVP docked into the V_1a receptor, as already published. The conformations of the ligand and of the side chains have been manually controlled to optimize the electrostatic and steric interactions with the receptor walls. The ligand-receptor complex has then been minimized without constraints (TRIPOS force field, Gasteiger-Hückel charges, 5,000 iterations). Alternative binding modes have been tried, but none of them was more satisfactory than the AVP-based model, on a physicochemical and energetical point of view. The same practical procedure has been followed to dock the previously published photoaffinity labeling agent, [¹²⁵I]3N₃Phpa-LVA (12).

Site-directed Mutagenesis of the Human V_1a Receptor-- The construction of mutants W613A, F616V, and F617L (see legend of Fig. 8 and Table IV for numbering) has been reported in a previous paper (12). Point mutations Y225D, Q218A, K308A and Q413A were introduced in the human V_1a vasopressin receptor using the QuickChange site-directed mutagenesis kit (Stratagene). These substitutions were directly done on the eukaryotic expression vector pCMV (34) and verified by direct dideoxynucleotide sequencing (T7 Sequencing^TM kit, Amersham Pharmacia Biotech). All the mutant receptors were transiently expressed in COS7 cells. In all cases, cell membrane preparations and radioligand binding assays were conducted as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemical, Pharmacological, and Functional Properties of the Photoactivatable Linear Peptide-- Considering firstly that all V_1a structurally related AVP linear peptide antagonists probably bind the V_1a receptor in the same way and secondly that introduction of an arylazido group at the N terminus of the ligand led to covalent binding into the TMR VII of the V_1a receptor (12), we designed, synthesized, and characterized a novel photoactivatable linear peptide compound (Fig. 1) containing an azido group at a position likely to covalently bind another domain of the receptor. Among the different possibilities in the series of linear peptide antagonists (35), we chose a peptide with a phenylpropionyl substitution at position 1, on the basis that this alkyl chain length confers a high affinity for the receptor (18). Instead of an Arg⁸ as in the highly potent V_1a antagonist HO-LVA (23), we introduced a Lys⁸ derivatized with a 3-azidophenylpropionyl group in order to photolabel the V_1a receptor. The peptidic part of the molecule was synthesized on p-methylbenzhydrylamine resin (18), and the arylazido group was attached in solution with PyBOP under a dual wavelength reverse phase HPLC monitoring (22), allowing the identification of the target compound [Lys(3N₃Phpa)⁸]HO-LVA (Fig. 2). This ligand was purified by semi-preparative reverse phase HPLC and characterized by mass spectroscopy (fast atom bombardment mass spectrometry, M+1 = 1287) and UV spectroscopy (azido characteristic peak at 250 nm,  = 7000 cm¹ M¹, destroyed by 254-nm UV irradiation). As for the HO-LVA, this compound can be iodinated on its phenolic N-terminal blocking group (4HO-Phpa) using ICl and purified by HPLC in order to obtain the nonradioactive probe as a chromatographic reference (fast atom bombardment mass spectrometry, M+1 = 1412). As shown in Fig. 3A, the corresponding radioiodinated peptide [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA exhibited a high affinity for the human V_1a receptor stably expressed in CHO cells; K_d mean value was 141.5 ± 25 pM (n = 4). This affinity was equivalent to that measured (K_d = 137 ± 37 pM) for [¹²⁵I]3N₃Phpa-LVA (see Figs. 3D and 1 for comparison of the ligand structures) and close to that of its parent compound [¹²⁵I]HO-LVA (K_d = 38 ± 7 pM). As reported in Table I, affinities (K_i) of the unlabeled [Lys(3N₃Phpa)⁸]HO-LVA for other human AVP/OT receptors expressed in CHO cells (V_1b, V₂, and OT subtypes) have been calculated from competition binding experiments using the [³H]AVP as the radioligand and compared with that deduced for the V_1a subtype. As observed in Fig. 3B, displacement of [³H]AVP with the photoactivatable peptide allowed the measurement of K_i values of 11, 574, and >2000 nM for human OT, V_1b, and V₂ receptor subtypes, respectively. These affinities were 60-10,000-fold lower than that measured for the human V_1a AVP receptor (0.18 nM), establishing the [Lys(3N₃Phpa)⁸]HO-LVA as a selective ligand. This photosensitive ligand displays competitive pharmacological properties equivalent to those of the 3N₃Phpa-LVA antagonist (Table I and Fig. 3E for comparison of the selectivity profiles of these two peptides). As shown in Fig. 3C, the photoactivatable peptide potently inhibited the IP accumulation induced by AVP (1 nM, a concentration producing a half-maximal response) in CHO cells in a concentration-dependent manner. The average K_inact calculated from experimental IC₅₀ values was 80 ± 25 pM (n = 3), a value close to that determined for 3N₃Phpa-LVA (130 ± 40 pM (n = 3); see Fig. 3F for comparison). This K_inact value is also in agreement with the K_i determined (180 pM; Table I) in binding experiments, a result equally obtained for 3N₃Phpa-LVA (130 pM for K_inact versus 240 pM for K_i). Moreover, no residual agonistic activity of the [Lys(3N₃Phpa)⁸]HO-LVA was detected (accumulation of IPs measured in the presence of 10⁶ M of the photoactivatable peptide was equivalent to that of basal level). Both peptides behave equivalently toward the human V_1a vasopressin receptor. Taken together, these results indicate that they competitively inhibit AVP binding and block the AVP-induced signal generation (for review see Ref. 6). In conclusion, the novel linear photoactivatable peptide [Lys(3N₃Phpa)⁸]HO-LVA could be considered as a potent and selective antagonist for the human V_1a receptor and appeared to be a valuable tool to further investigate its covalent binding sites in the receptor.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Structure of the photoreactive antagonist [125I][Lys(3N3Phpa)8]HO-LVA: comparison with [ 125I]3N3Phpa-LVA. The photoreactive azido group of [125I][Lys(3N3Phpa)8]HO-LVA is in the meta position on the aromatic ring of the Phpa moiety at the side chain of residue Lys8. As for its parent compound HO-LVA (23), the octapeptide was radioiodinated on the phenolic substituent of the phenylpropionyl moiety considered as position 1. Residues 2-7 are common to both photoactivatable linear peptide antagonists.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Reverse phase HPLC monitoring during coupling of the arylazido group to the [Lys8]HO-LVA peptide. An aliquot of the reaction mixture was injected onto the analytical column and eluted at 2 ml/min, in linear gradient mode, by CH3CN in water (1% min, shown by the dotted line), both acidified with 0.1% CF3CO2H. The compounds were detected by 214 nm (upward) and 254 nm (downward) UV absorption and characterized by their percentage of elution (corrected for the void volume of the apparatus) and their 214 nm/254 nm ratio. Hydroxybenzotriazole came from PyBOP. If needed, arylazido acid and PyBOP were added in order to substitute a maximum of peptide [Lys8]HO-LVA. The peak of target compound is shaded.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Pharmacological and functional properties of both linear peptide ligands [Lys(3N3Phpa)8]HO-LVA and 3N3Phpa-LVA. A and D, dose-dependent specific binding of [125I][Lys(3N3Phpa)8]HO-LVA (A) and [125I]3N3Phpa-LVA (D) to the human V1a receptor expressed in CHO cell membranes. The preparations were incubated for 60 min at 30 °C in Buffer A with 1 mg/ml bovine serum albumin and increasing concentrations of the radiolabeled peptides. Nonspecific binding was determined in the presence of 250-1000-fold excess of corresponding unlabeled peptides. Data were analyzed using the Ligand progam (27). The values represented in the graphs are taken from one experiment. They are representative of at least three independent experiments done in triplicate. B and E, selectivity of the peptides for the V1a receptor. Competition experiments were conducted with membrane preparations from CHO cells expressing the V1a, OT, V1b, or V2 receptor subtypes at 30 °C for 30 min in the presence of [3H]AVP and unlabeled [Lys(3N3Phpa)8]HO-LVA (B) or 3N3Phpa-LVA (E) peptides. Affinities (Ki) for both photoactivatable compounds were determined by displacement of radiolabeled AVP and data (see also Table I) were analyzed with the Ligand program (27). All values in the graphs are taken from representative experiments that were done at least three times each in triplicate. C and F, antagonistic properties of the peptides. myo-[3H]Inositol-prelabeled CHO cells expressing the human V1a receptor were incubated 10 min at 37 °C in PBS/10 mM LiCl medium with or without increasing concentrations of [Lys(3N3Phpa)8]HO-LVA (C) or 3N3Phpa-LVA (F) ranging from 1012 to 106 M. Cells were then stimulated with AVP 109 M (a concentration close to the AVP Kact in this system) for 15 min. After stopping the reaction, total inositol phosphates were extracted, counted, and expressed as dpm/well. For example, in panel C, basal activity of phospholipase C was 280 dpm/well (50,000 cells); half-maximal IP accumulation with AVP 109 M was 6450 dpm/well; phospholipase C activity in the presence of 1 nM AVP and 1 µM Lys(3N3Phpa)8]HO-LVA was 293 dpm/well, phospholipase C activity in the presence of 106 M Lys(3N3Phpa)8]HO-LVA alone was 302 dpm/well. All values in each graph are taken from one experiment and are representative of three independent experiments each done in triplicate. Data were analyzed using the software KaleidagraphTM. Kinact constants were calculated as described under "Experimental Procedures."

View this table: [in this window] [in a new window]   Table I Affinities of [Lys(3N3Phpa)8]HO-LVA and 3N3Phpa-LVA for human oxytocin and vasopressin receptor subtypes Affinities (Kd) of wild-type receptor subtypes for [3H]AVP were all determined in saturation experiments using 10-15 µg of membrane proteins from CHO cells expressing each subtype of receptor and increasing concentrations of tritiated AVP from 0.1 to 20 nM. AVP 105 M was used to determine nonspecific binding. Affinities (Ki) for the [Lys(3N3Phpa)8]HO-LVA and 3N3Phpa-LVA were determined in competition assays by displacement of [3H]AVP used at 1-2 nM. Data were analyzed by nonlinear least squares regression with the Ligand program (27). All values in this table are expressed as the means ± S.E. calculated from three independent experiments, each done in triplicate.

Photoaffinity Labeling of the Human V_1a Receptor-- [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA covalently labeled the human V_1a receptor, as demonstrated by autoradiography of SDS-polyacrylamide gels used to separate proteins after binding to CHO cell membranes and UV irradiation (Fig. 4A). The photolabeled receptor migrated as two broad bands at approximately 85-90 and 46 kDa, respectively (lane 1). The labeling of the receptor was completely suppressed with an excess of AVP (lane 2), indicating that the photolabeling of both protein bands was specific for the human V_1a receptor expressed in the CHO cell line. The covalent binding yield of the probe [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA to the receptor was calculated, and the fraction of specifically receptor-bound radioactivity recovered in the labeled bands reached 17-18% (multiple determinations). This covalent binding yield was high and exceeded the one reported before for [¹²⁵I]3N₃Phpa-LVA (13-14%). As described in a previous study (12), the 46-kDa photolabeled band likely corresponds to a proteolytic truncated form of the entire receptor migrating at an apparent 85-90 kDa molecular mass. Indeed, the relative abundance of the 46-kDa species (50% in lane 1, Fig. 4A) could be significantly reduced (20-25%) in incubation conditions (1 h at 4 °C in the presence of ZnCl₂ and protease inhibitors leupeptin, benzamidine, and soybean trypsin inhibitor) reducing the action of endogenous proteases present in cell membrane preparations (data not shown). This observation confirmed that the V_1a receptor was proteolyzed during incubation with the [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA probe.

View larger version (76K): [in this window] [in a new window]   Fig. 4.   Photoaffinity labeling of the human V1a receptor with [125I][Lys(3N3Phpa)8]HO-LVA. A, photolabeled V1a receptor. 500 µg of membranes from CHO cells expressing the V1a receptor were photolabeled as described under "Experimental Procedures." To define nonspecific photolabeling of membranes, AVP (105 M) was added (lane 2) or not (lane 1). Irradiated and washed membranes were separated onto a 12% cross-linked SDS-PAGE. Equivalent amounts of proteins (10 µg) were loaded into the wells. B, deglycosylation of the photolabeled V1a receptor. CHO cell membranes (500 µg) expressing the receptor were photolabeled and then solubilized in deglycosylation buffer in the presence (lane 2) or not (lane 1) of 2 units of N-glycosidase F. Equivalent amounts of radioactivity (600,000 cpm) were loaded in each lane, and proteins were separated by SDS-PAGE using 12% cross-linked gels. The gels were dried and exposed overnight (A) or 5 h (B) at 80 °C to Kodak XAR-5 film. Molecular mass markers are indicated (kDa) on the left. The autoradiograms of dried gels are representative of at least three distinct experiments.

Deglycosylation of the Photolabeled Human V_1a Receptor-- As seen in Fig. 4B, the treatment of photolabeled membranes with N-glycosidase F before SDS-PAGE reduced apparent molecular masses of the 85-90- and 46-kDa protein bands (lane 1) to approximately 50 and 34 kDa, respectively (lane 2), indicating that they were both glycosylated and contained at least one N-glycosylated site. As shown in Fig. 5, potential N-glycosylation sites (Asn-Xaa-Ser/Thr) are located at Asn¹⁴, Asn²⁷, and Asn¹⁹⁶. 50 kDa is a molecular mass very close to the theoretical mass of the receptor core deduced from the cDNA sequence (48.2 kDa, including the 1.4-kDa antagonist mass), whereas 34 kDa is significantly smaller. This observation confirmed once again that the photolabeled glycosylated protein band at 46 kDa effectively corresponds to a proteolytic truncated form of the receptor and that the photolabeled species at 85-90 kDa likely represents the native glycosylated state of the receptor expressed in the CHO cell system. Looking at the primary sequence of the receptor (Fig. 5) and at the localization of the different N-glycosylation sites (Asn¹⁴, Asn²⁷, and Asn¹⁹⁶), a deglycosylated 34-kDa protein (or the glycosylated 46-kDa species counterpart) could only account for a large truncated receptor fragment including Asn¹⁹⁶ and spanning the protein to the C terminus. Taking into account both the present results of deglycosylation and sensitivity of the receptor to endogenous membrane protease degradation, we concluded that proteolytic cleavage must occur at a site located between Asn²⁷ and Asn¹⁹⁶. As mentioned before (12), sequence Phe¹⁰³-Gln¹⁰⁸ in the V_1a receptor (Fig. 5) corresponds to a potential metalloproteinase cleavage site. This enzyme has been clearly shown (36) to digest the bovine renal V₂ receptor in membrane preparations between Gln⁹² and Val⁹³ (Gln¹⁰⁴ and Val¹⁰⁵ in the V_1a sequence). The 34-kDa molecular mass of the deglycosylated photolabeled band is consistent with that of a proteolytic fragment that could be generated from such a cleavage in this receptor region. We also concluded that photolabeling of the V_1a receptor with [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA occurred at residue(s) distal to this proteolytic cleavage site.

View larger version (52K): [in this window] [in a new window]   Fig. 5.   Schematic representation of the human V1a receptor. The primary sequence of the receptor, deduced from the cDNA cloning (24), is shown as well as the possible arrangement of the protein through the cell membrane. The three potential N-glycosylation sites are shown at Asn14, Asn27, and Asn196. White squares indicate a consensus sequence (residues 103-108) spanning the cleavage site for a metalloproteinase (15, 36). Potential cleavage sites for Lys-C (gray triangles), Asp-N (gray squares), and CNBr (gray diamonds) are also indicated. As described previously (12), fragment photolabeled with the previous [125I]3N3Phpa-LVA antagonist including the TMR VII is shown (gray circles). The smallest photolabeled fragment with [125I][Lys(3N3Phpa)8]HO-LVA resulting from Asp-N digestion is represented by black circles and spans the first extracellular loop of the receptor.

Fragmentation of the Photolabeled Human V_1a Receptor-- To identify antagonist-binding domains covalently bound to the photoactivatable linear peptide [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA, photolabeled receptors were partially purified from a preparative SDS-PAGE by electroelution and then subjected to fragmentation with either CNBr, endoproteinase Lys-C, or endoproteinase Asp-N. Because CNBr, Lys-C, and Asp-N cleave proteins specifically at the C terminus of methionine and lysine residues and at the N terminus of aspartic acid residues, respectively (Met, Lys, and Asp in the V_1a receptor are shaded in Fig. 5), the photoactivatable peptide ligand itself is thus expected to be protected against these different fragmentations (Lys⁸ is modified and constitutes the amidated C terminus end of the antagonist). Because the 46-kDa photolabeled band derives from the 85-90-kDa photolabeled species, only the truncated receptor at 46 kDa was excised from gels, electroeluted, and subjected to the chemical cleavage and enzymatic digestions.

As seen in Fig. 6A, CNBr cleavage of the photoaffinity-labeled V_1a receptor yielded a major labeled fragment migrating at an apparent mass 5.5 kDa and a higher minor labeled band at 7.5 kDa. A similar cleavage pattern was observed when electroeluted 46-kDa fragment was first deglycosylated with N-glycosidase F before CNBr treatment (data not shown), indicating that radiolabeled fragments at 5.5 and 7.5 kDa do not contain N-glycosylation sites. This result eliminates fragments Arg²-Met⁸⁶ (a fragment that could also be eliminated on the basis that this sequence is not included in the truncated 46-kDa photolabeled receptor) and Ile¹⁹²-Met²²⁰ as the sites of covalent attachment of the [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA ligand (see primary structure of the V_1a receptor on Fig. 5). There are only three CNBr fragments of the V_1a receptor that could account for a 5.5-kDa molecular mass (including the 1.4-kDa mass of the antagonist itself): His⁸⁷-Met¹⁰⁹ (probably not entirely included in the 46-kDa truncated form of the receptor), Cys¹¹⁰-Met¹³⁵, and Thr¹⁴⁶-Met¹⁷⁰ (equivalent to the second intracellular loop).

View larger version (44K): [in this window] [in a new window]   Fig. 6.   Fragmentation of the human V1a receptor with CNBr and endoproteinases. CHO cell membranes (500 µg) expressing the V1a receptor were incubated with the [125I][Lys(3N3Phpa)8]HO-LVA for 3 h at 30 °C without ZnCl2 and protease inhibitors, a condition that favors the preferential accumulation of the 46-kDa receptor species. Membrane proteins were then separated on a preparative 12% gel, and the photolabeled 46-kDa species was electroeluted, washed, and concentrated as described under "Experimental Procedures." Equivalent amounts of electroeluted photolabeled receptors were used in each digestion or chemical cleavage assays (30,000 cpm). The samples were then loaded on discontinuous 10-16.5% Tricine gels. A, CNBr chemical cleavage. The partially purified receptor was treated (lane 2) or not (lane 1) with CNBr for 24 h in the dark at room temperature. B, Lys-C protease digestion. The 46-kDa species was treated (lane 2) or not (lane 1) with the protease for 24 h at 37 °C. C, Asp-N protease digestion. The partially purified receptor was treated (lane 2) or not (lane 1) with the enzyme for 48 h at 25 °C. The figure shows autoradiograms of dried gels exposed to Kodak XAR-5 film at 80 °C for 48 h. Molecular mass markers are indicated on the left in each panel. Each assay is representative of at least three distinct experiments.

As shown in Fig. 6B, digestion of the photolabeled 46-kDa receptor with Lys-C endoproteinase yielded a major fragment at 5 kDa and two minor higher labeled bands at 8 and 14.5 kDa. The smallest labeled fragment with an apparent 10.5-kDa molecular mass (Met²⁹²-Lys³⁷⁰), resulting from the Lys-C cleavage of the 46-kDa species photolabeled with a previously described photoactivatable peptide [¹²⁵I]3N₃Phpa-LVA (12), was not produced when the receptor was photolabeled with the [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA peptide antagonist. This indicated that fragment Met²⁹²-Lys³⁷⁰ cannot account for covalent binding of [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA. Only three fragments, Leu⁵³-Lys⁸² (obviously not present in the truncated 46 kDa species), Val¹⁰⁵-Lys¹²⁸ (assuming that the N terminus of this fragment would result from proteolysis of the receptor by a membrane protease during incubation with the photoactivatable ligand), and His¹²⁹-Lys¹⁵⁸ could be in agreement with a 5-kDa labeled band produced by Lys-C digestion of the receptor.

As seen in Fig. 6C, digestion of the radioiodinated antagonist-bound 46-kDa photolabeled species with Asp-N endoproteinase yielded an intense major 2-2.5-kDa labeled fragment and very minor higher labeled bands at 6, 12.5, and 18 kDa. Only one possible Asp-N fragment of the V_1a receptor could account for a labeled band with an electrophoretic migration at 2-2.5 kDa; this fragment, Asp¹¹²-Pro¹²⁰, spans the first extracellular loop of the V_1a receptor. The localization of this short receptor domain, as the site of covalent binding site for [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA, was consistent with the results of CNBr and Lys-C cleavages described above. In order to confirm this localization, CNBr cleavage was also used in combination with either Lys-C or Asp-N protease digestion. Successive treatment of the 46-kDa photolabeled receptor with CNBr and Lys-C (Fig. 7A, lane 2) generated a new fragment slightly smaller than the 5 kDa obtained with Lys-C alone (lane 1) or the 5.5 kDa produced with CNBr alone (lane 3). This labeled receptor fragment with a molecular mass of 4.5 kDa could correspond to Cys¹¹⁰-Lys¹²⁸ sequence. Successive treatment of the 46-kDa labeled species with CNBr and Asp-N endoproteinase (Fig. 7B, lane 2) demonstrated that fragments obtained with CNBr alone (lane 1) were converted into smaller ones with Asp-N protease to yield the smallest fragment with an apparent mass (2-2.5 kDa) equivalent to that generated with Asp-N protease alone (lane 3). The results of double fragmentations were consistent with those of CNBr cleavage and Lys-C or Asp-N proteinase digestions and confirmed the identity of the Asp¹¹²-Pro¹²⁰ fragment as the photolabeled receptor domain with [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA.

View larger version (63K): [in this window] [in a new window]   Fig. 7.   Double fragmentation of the human V1a receptor with CNBr/Lys-C protease or CNBr/Asp-N protease. Photolabeling of CHO cells membranes (500 µg) expressing the wild-type human V1a receptor, preparative 12% SDS-PAGE and electroelution of the 46-kDa photolabeled species were performed as described in legend to Fig. 6. Equivalent amounts of photolabeled receptors were used in each assay (60,000 cpm), and samples were loaded on discontinuous 10-16.5% Tricine gels. A, CNBr/Lys-C protease fragmentation. The radiolabeled receptors were treated with CNBr (lane 3) or Lys-C protease (lane 1) alone or in combination (CNBr first) (lane 2). B, CNBr/Asp-N protease fragmentation. The radiolabeled receptors were treated with CNBr (lane 1) or Asp-N protease (lane 3) alone or in combination (CNBr first) (lane 2). The figure shows autoradiograms of dried gels exposed to Kodak XAR-5 film at 80 °C for 60 h (Fig. 6A) or 72 h (Fig. 6B). Molecular mass markers are indicated on the left in each panel. Each assay is representative of at least three distinct experiments.

Docking of the Photoactivatable Linear Peptide Antagonists into the Human V_1a Receptor-- The putative models of [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA and [¹²⁵I]3N₃Phpa-LVA docked into the human V_1a receptor are displayed in Fig. 8. The residues putatively involved in their binding are listed in Tables II and III. The hypotheses presented here are based on the above photoaffinity labeling study and the previous photolabeling data obtained with [¹²⁵I]3N₃Phpa-LVA (12). The two linear ligands differ from AVP in a number of positions: no disulfide bridge; a substituted phenylpropionyl N-terminal; a O-methylated DTyr in position 2 instead of a Tyr; an Arg in position 6; and a modified C-terminal. However, they retain the same residues in positions 3, 4, 5, and 7. In the models, side chains of the four conserved residues and of the DTyr(Me)² could bind in a similar way compared with homologous features in AVP. Very interestingly, the Arg⁶ basic moiety of [¹²⁵I]3N₃Phpa-LVA is proposed to be located at the same position as the basic glycinamide terminus of AVP, whereas the alkyl part of the side chain occupies the same domain as Cys⁶ in AVP. Interestingly, the guanidinium group of Arg⁶ could also establish intramolecular interactions with the C-terminal amide group and the HO-Phpa group of [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA (Fig. 8C) or with the [¹²⁵I]Tyr⁹ and the Phpa group of [¹²⁵I]3N₃Phpa-LVA (Fig. 8A), thus stabilizing the linear peptides in a conformation similar to that of the cyclic AVP.

View larger version (47K): [in this window] [in a new window]   Fig. 8.   Docking of the two linear peptide photoactivatable antagonists in the three-dimensional model of the human V1a receptor. Side views of the upper regions of the V1a receptor are shown from a direction parallel to the cell membrane surface. In all panels, the positioning of TMR I to VII is anticlockwise, and their C backbone is displayed in purple. The antagonist C backbones are displayed in orange. The carbon skeleton of the side chains of residues potentially involved in the binding of the linear peptide antagonists are shown in white. Oxygen atoms are in red, nitrogen atoms are in dark blue, and [125I] atoms are in green. Receptor residues are labeled according to the modeling numbering: the left digit indicates the transmembrane -helix, the next two digits indicate the rank of the residue in this transmembrane domain (e.g. F616 represents the sixteenth residue in TMR VI). Panels A and C show the interactions between the receptor and [125I]3N3Phpa-LVA and [125I][Lys(3N3Phpa)8]HO-LVA ligands, respectively. Panel B highlights the residues of TMR VII in contact with the photoreactive azidophenyl moiety of the [125I]3N3Phpa-LVA (T710, A711, G714, S715, and possibly N717 and S718). Panel D highlights the residues of the first extracellular loop in contact with the photoreactive azidophenyl moiety of the [125I][Lys(3N3Phpa)8]HO-LVA (Y225, R226, F227, and R228).

The 3-azido-phenylpropionic moiety of [¹²⁵I]3N₃Phpa-LVA is found buried in a hydrophobic pocket of the binding cleft, in contact with TMR VII that has been found to be photolabeled (12). According to the present model, the residues that might be covalently bound are Thr710, Ala711, Gly714, Ser715, and possibly Asn717 and Ser718 (Fig. 8B). Phe616 is also found in the direct neighborhood in agreement with site-directed mutagenesis data (12). As in AVP (7), only Arg⁸ side chain protrudes toward the extracellular loop 1 of the human V_1a receptor in order to potentially interact with Tyr²²⁵.

In the model, the 4-hydroxy-3-[¹²⁵I]phenylpropionyl group of [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA is found in contact with residues of TMR VII, in a position equivalent to that proposed for the 3-azidophenylpropionic moiety of [¹²⁵I]3N₃Phpa-LVA. In contrast, the ninth residue is not present in [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA, and Arg⁸ has been replaced by the 3-azidophenylpropionylated lysyl. The backbone constraints are such that the modified lysyl side chain has to point toward the extracellular part of the receptor. Consequently, the azidophenyl moiety is found directly in contact with the first extracellular loop (Fig. 8D), in the neighborhood of residues Tyr225, Arg226, Phe227, and Arg228, in full agreement with the labeling data reported here.

Site-directed Mutagenesis of the Human V_1a Receptor-- In order to verify the proposed docking of both linear peptide antagonists into the V_1a receptor, the role of some receptor residues (Tables II and III) potentially interacting with the ligands, was investigated. These residues were mutated, and binding properties of the mutant receptors transiently expressed in COS7 cells were studied (Table IV). Because we have already demonstrated important aromatic/aromatic interactions between [¹²⁵I]3N₃Phpa-LVA and the aromatic residue cluster (particularly Phe616) of TMR VI (12), affinities of [Lys(3N₃Phpa)⁸]HO-LVA for mutant receptors W613A, F616V, and F617L were measured. Mutation of Phe616 into a valine led to a dramatic loss in binding affinity (1266-fold reduction, 228 nM compared with 0.18 nM for the wild-type receptor). This result confirmed the crucial role played by Phe616 in defining high affinity of the V_1a-selective linear peptide antagonists. Consequences of Trp613 and Phe617 mutations in binding affinity were not significant. Conserved hydrophilic residues Gln218, Lys308, and Gln413 that have been demonstrated to control the binding of AVP in the rat V_1a receptor (7) were substituted with alanine residues. Binding properties of the mutants were measured using [¹²⁵I]HO-LVA as the radioligand (Table IV); a significant decrease in the affinity of [Lys(3N₃Phpa)⁸]HO-LVA and 3N₃Phpa-LVA for the three mutants was observed (5-28-fold reduction in K_i values). Finally, we decided to investigate a potential role for Tyr225, located in the first extracellular loop (corresponding to Tyr¹¹⁵ in the primary sequence), in the affinity and selectivity of the photoactivatable antagonist ligands because (i) this residue has been shown (9) to control receptor subtype selectivity and participate in agonist high affinity binding by interacting with hormone residue 8; (ii) photolabeling of the receptor with [¹²⁵I] [Lys(3N₃Phpa)⁸]HO-LVA (azido group at the side chain of residue 8) occurred in the first extracellular loop. Tyr225 in the human V_1a vasopressin receptor was mutated into an Asp, the residue naturally occurring at the same position in the V₂ receptor and the properties of the Y225D mutant studied. When compared with the wild-type V_1a receptor, displacement of [³H]AVP by [Lys(3N₃Phpa)⁸]HO-LVA led to a K_i increased from 0.18 to 0.9 nM (Table IV). This small reduction in binding affinity was emphasized for 3N₃Phpa-LVA, the previous photoactivatable antagonist ligand: in this case, K_i shifted from 0.24 to 4.9 nM (20-fold reduction in affinity). In conclusion, Tyr225 could participate in the binding of both [Lys(3N₃Phpa)⁸]HO-LVA and 3N₃Phpa-LVA peptides, but its role in the receptor-selective binding of these two ligands is only minor. Taken together, these mutagenesis data suggest that residues Gln218, Tyr225, Lys308, Gln413, and Phe616 contribute to the binding of 3N₃Phpa-LVA and [Lys(3N₃Phpa)⁸]HO-LVA and validate the proposed docking of the photoactivatable antagonists into the human V_1a receptor.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we have characterized [¹²⁵I][Lys(3N₃ Phpa)⁸]HO-LVA as a useful V_1a-selective photoaffinity ligand. Compared with [¹²⁵I]3N₃Phpa-LVA, the previous photoactivatable antagonist used for mapping peptide-binding domains of the rat and human V_1a vasopressin receptors (18, 19, 12), this novel antagonist analogue also combines high affinity, selectivity, possibility of radioiodination and high covalent binding yield. The novel ligand allowed us to photolabel the receptor which migrated on SDS-PAGE as two protein bands with apparent molecular masses of 85-90 and 46 kDa, respectively. This result is comparable with that previously observed with [¹²⁵I]3N₃Phpa-LVA (12), thus confirming the identity of the receptor and the specificity of the signal. Once again, the human V_1a vasopressin receptor was degraded during incubation with the ligand. As demonstrated in our previous study, the 46-kDa species deriving from the larger protein at 85-90 kDa is cleaved by endogenous proteases present in the CHO membrane preparations. Deglycosylation of the photolabeled receptors with N-glycosidase F confirmed this observation. Fragmentation of the photolabeled V_1a vasopressin receptor with a combination of chemical CNBr cleavage and Lys-C and Asp-N protease digestions led to the identification of a restricted receptor region that likely spans the first extracellular loop. Results of double fragmentations (CNBr followed by Lys-C or Asp-N proteases) were all consistent with that of single digestions or chemical cleavages and confirmed the identity of the Asp¹¹²-Pro¹²⁰ sequence as the smallest receptor fragment photolabeled with [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA.

Interestingly, chemical CNBr cleavage and endoproteinase Lys-C digestion of the photolabeled V_1a receptor yielded labeled fragments probably corresponding to Cys¹¹⁰-Met¹³⁵ and Val¹⁰⁵-Lys¹²⁸, respectively. Both receptor fragments contain Cys¹²⁴, a residue located in the TMR III and possibly involved in a disulfide bond with Cys²⁰³ in the second extracellular loop (for review see Refs. 13, 31, and 32). This observation prompted us to verify the presence of such a disulfide bridge in the human vasopressin V_1a receptor. Thus, preparation of samples for CNBr cleavage as well as migration on SDS-PAGE were then performed in nonreducing conditions in order to possibly generate a larger photolabeled fragment including both sequence Cys¹¹⁰-Met¹³⁵ and Ile¹⁹²-Met²²⁰ (fragment containing Cys²⁰³). The result of CNBr cleavage in this case was equivalent to that obtained in reducing conditions: only photolabeled fragments at 5.5 and 7.5 kDa were produced (data not shown), suggesting that disulfide bond between Cys¹²⁴ and Cys²⁰³ would be absent in the V_1a receptor. This conclusion is in agreement with those of several previous reports. Indeed, it has been demonstrated that affinity of [³H]AVP to V_1a receptors on rat liver membranes was not significantly reduced after treatment with sulfhydryl reagents such as N-ethylmaleimide (37, 38). This result suggests either that the disulfide bridge is missing or that it does not play any role in AVP binding. A different result has been obtained for the V₂ receptor. For this receptor subtype, it has been shown that one or more cysteine residues are essential for hormone binding (38).

The position of the photosensitive azido group in the [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA, at the side chain of residue lysine 8, was chosen in order to covalently bind a receptor domain different from the TMR VII labeled with [¹²⁵I]3N₃Phpa-LVA (12) and then to propose docking of these antagonists. The present results validate our strategy. The Asp¹¹²-Pro¹²⁰ sequence, identified as the smallest photolabeled fragment with [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA, spans the first extracellular loop of the V_1a receptor. This region has already been shown to constitute the site of covalent attachment in the bovine renal V₂ receptor with a photoactivatable agonist (15). In this case, the AVP analogue [³H]1-deamino[Lys⁸]vasopressin contained the photoreactive arylazido group at the side chain of Lys⁸. Determination of the first extracellular loop of the V₂ receptor as the site of interaction with AVP analogue residue 8 led to the identification of Asp¹⁰³ as the residue responsible for agonist binding specificity (8). Independently and at the same time, equivalent result was demonstrated in our laboratory for the V_1a receptor: Tyr¹¹⁵ (homologue to Asp¹⁰³ in the V₂) was shown to behave as a crucial residue for agonist high affinity binding and also for receptor selectivity (9). The position of the photoreactive moiety (azidophenyl) in the [¹²⁵I][Lys(3N₃Phpa)⁸]HO-LVA antagonist used in the present study is again at the side chain of peptide residue 8. Interestingly, both peptide agonist and antagonist photoaffinity ligands allow covalent attachment of the first extracellular loop in the V₂ and V_1a vasopressin receptors respectively when containing the photoactivatable group at the side chain of Lys⁸. However, residues in this extracellular region of vasopressin receptors responsible for agonist receptor subtype selectivity only play a minor role for antagonists. Other binding selectivity determinants for these compounds have to be discovered elsewhere in the receptor.

Photolabeling of the human V_1a receptor with two different selective linear photoactivatable and iodinatable peptide antagonists of AVP led to the identification of two receptor regions in close proximity to the bound photoligands (Ref. 12 and the present study). Taking into account these photoaffinity labeling results, three-dimensional models for the antagonist peptide-binding sites of the V_1a vasopressin receptor were then proposed and verified experimentally. The two photoactivatable ligands are linear molecules. They are both very flexible and can adopt a quasi-infinite number of conformations within an acceptable energy window. Therefore, looking for the minimum energy conformers would not provide any clue regarding the receptor-bound conformations. An alternative way consists in examining the physicochemical features of both the ligands and the binding cleft and looking for complementarity. However, it is interesting to observe that [¹²⁵I][Lys(3N₃-Phpa)⁸]HO-LVA and [¹²⁵I]3N₃-Phpa-LVA present a striking homology with AVP whose binding mode had already been probed (7). Most of the receptor residues putatively involved in the binding of these two linear photoactivatable peptide antagonists are those already demonstrated to interact with AVP. These two peptides differ from AVP in positions 1, 2, 6, and 8 and at the C terminus but retain the same residues in positions 3, 4, 5, and 7. In the models, the side chains of the 4 conserved residues bind in a similar way compared with homologous features in AVP. Interestingly, the docking procedures lead these antagonists to adopt a pseudocyclic conformation similar to that of the cyclic AVP. Based on these models, the linear peptide antagonists could enter the transmembrane-binding pocket like their agonist counterparts and establish their own network of molecular interactions. This is consistent with the marked hydrophobic nature of these ligands and that of the bottom of the receptor-binding cleft. Interestingly, as confirmed from the mutagenesis results, aromatic/aromatic contacts represent the most important interactions for antagonists, whereas hydrogen bonds with conserved hydrophilic receptor residues seem to represent the most crucial interactions for agonists like AVP (7).

It is now commonly accepted that peptide ligands, agonists or antagonists, partly bind to transmembrane domains of their receptors (for review see Ref. 6). It has been demonstrated with different approaches, such as site-directed mutagenesis, photoaffinity labeling, or spectrofluorimetric methods. However, very few studies have led to the identification of binding sites for peptide antagonists. In the NK-2 neurokinin receptor systems, the N-termini of agonists and of antagonists of similar molecular size have distinct binding sites (39). In the angiotensin AT₁ and the endothelin ET_A receptors, minimal overlapping binding sites between peptide agonists and antagonists have been demonstrated, respectively (40, 41). Very recently, it has been observed that structurally related peptide agonist, partial agonist, and antagonist occupy a similar binding pocket within the rat cholecystokinin CCK-A receptor (42). Similarly, we describe in the present study major transmembrane overlapping binding sites in the V_1a vasopressin receptor for both peptide agonist and two linear peptide antagonists.

	ACKNOWLEDGEMENTS

We are grateful to Dr. T. Durroux for critical reading of the manuscript. Many thanks to M. Passama and L. Charvet for help in the illustrations.

	FOOTNOTES

^* This work was supported by grants from INSERM, PCV97-123 Physique-Chimie du Vivant program of CNRS, and Fondation pour le Recherche Medicale (to S. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 33-4-67-14-29-22; Fax: 33-4-67-54-24-32; E-mail: mouillac@u469.montp.inserm.fr.

² Thr710 and Ala711 positions 10 and 11 in TMR VII. In the three-digit numbers that appear with the three-letter residue codes throughout, the first digit corresponds to the helical TMR, and the next two digits indicate the rank of the residue in the considered helix.

³ N. Cotte, M. N. Balestre, A. Aumelas, E. Mahé, S. Phalipou, D. Morin, C. Barberis, M. Hibert, and B. Mouillac, manuscript in preparation.