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J. Biol. Chem., Vol. 276, Issue 29, 26931-26941, July 20, 2001

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Direct Identification of Human Oxytocin Receptor-binding Domains Using a Photoactivatable Cyclic Peptide Antagonist

COMPARISON WITH THE HUMAN V_1a VASOPRESSIN RECEPTOR^*

Christophe Breton^§, Hichem Chellil, Majida Kabbaj-Benmansour, Eric Carnazzi^¶, René Seyer^¶, Sylvie Phalipou, Denis Morin, Thierry Durroux, Hans Zingg^**, Claude Barberis, and Bernard Mouillac

From the  U469 INSERM and the ^¶ UPR 9023 CNRS, 141 rue de la Cardonille, 34094 Montpellier cedex 5, France and the ^** Laboratory of Molecular Endocrinology, McGill University Health Centre, Montreal, Quebec H3A 1A1, Canada

Received for publication, March 7, 2001, and in revised form, May 2, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Understanding of the molecular determinants responsible for antagonist binding to the oxytocin receptor should provide important insights that facilitate rational design of potential therapeutic agents for the treatment of preterm labor. To study ligand/receptor interactions, we used a novel photosensitive radioiodinated antagonist of the human oxytocin receptor, d(CH₂)₅ [Tyr(Me)²,Thr⁴,Orn⁸,Phe(3¹²⁵I,4N₃)-NH₂⁹]vasotocin. This ligand had an equivalent high affinity for human oxytocin and V_1a vasopressin receptors expressed in Chinese hamster ovary cells. Taking advantage of this dual specificity, we conducted photoaffinity labeling experiments on both receptors. Photolabeled oxytocin and V_1a receptors appeared as a unique protein band at 70-75 kDa and two labeled protein bands at 85-90 and 46 kDa, respectively. To identify contact sites between the antagonist and the receptors, the labeled 70-75- and the 46-kDa proteins were cleaved with CNBr and digested with Lys-C and Arg-C endoproteinases. The fragmentation patterns allowed the identification of a covalently labeled region in the oxytocin receptor transmembrane domain III consisting of the residues Leu¹¹⁴-Val¹¹⁵-Lys¹¹⁶. Analysis of contact sites in the V_1a receptor led to the identification of the homologous region consisting of the residues Val¹²⁶-Val¹²⁷-Lys¹²⁸. Binding domains were confirmed by mutation of several CNBr cleavage sites in the oxytocin receptor and of one Lys-C cleavage site in the V_1a receptor. The results are in agreement with previous experimental data and three-dimensional models of agonist and antagonist binding to members of the oxytocin/vasopressin receptor family.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Oxytocin (OT),¹ a neurohypophyseal nonapeptide, is among the strongest uterotonic agents known to date (1, 2). This hormone acts on the myometrium through specific OT receptors (OTRs) that show a dramatic increase in their expression pattern immediately before parturition (3). In mammals at term, OT-induced contractility of myometrial smooth muscle cells is triggered through an increase in the intracellular calcium level (4). This calcium signaling pathway is dependent on coupling the OTR to both the G_q/11 and G_i proteins (5). The OTR cDNA of several mammalian species has been cloned (6-9), and the deduced amino acid sequence confirmed that the receptor belongs to the large family of seven-helix transmembrane G protein-coupled receptors. OT is widely used in obstetrical practice to promote labor and delivery (10). On the other hand, blockade of the OTR may provide a unique approach for treatment of preterm labor by prolonging uterine quiescence (11-13). Therefore, much effort has been focused on the design and development of OT antagonists as potential tocolytic drugs. The utility of such OT antagonists has been demonstrated clinically by using intravenously administered 1-deamino[D-Tyr(Et)²,Thr⁴,Orn⁸]vasotocin (Atosiban). This peptide antagonist has been shown to inhibit uterine contractions in women with threatened and established preterm labor (14, 15). However, the structure/function relationships of the functional domains of the OTR and the topography of the ligand-binding sites are still poorly investigated. Such knowledge should provide valuable information on the structural requirements for antagonist binding and should be helpful for the rational design of potential therapeutic agents and for a better understanding of the molecular mechanisms leading to receptor inactivation. Agonist-binding sites of the OTR have been investigated previously by site-directed mutagenesis and three-dimensional (3D) molecular modeling (16-18). In parallel, a major contribution to peptide antagonist binding affinity by the upper part of transmembrane domain (TMD) VII has been demonstrated (18).

The photoaffinity labeling technique is an essential complement to modeling and mutagenesis approaches and allows unambiguous direct determination of the ligand/receptor contact regions. So far, radiolabeled OT analogues containing a photoactivatable group at the side chain of residue 8 have been used to photolabel the OTR naturally expressed in the rat mammary gland, the guinea pig uterus, and the rabbit amnion or transfected in COS cells (recombinant porcine receptor). These studies allowed the identification of the receptor as a glycoprotein with an apparent molecular mass between 65 and 80 kDa (18-22). However, the precise localization of the covalent attachment of these ligands to the receptor has not been investigated.

The present study has been performed to localize accurately peptie antagonist-binding domains of the human OTR using a photoaffinity labeling approach. Based on the structure of the previously reported potent and high affinity cyclic peptide antagonist d(CH₂)₅[Tyr(Me)²,Thr⁴,Orn⁸,Tyr(3¹²⁵I)-NH₂⁹]vasotocin (¹²⁵I-OTA) (23), we have designed, synthesized, and characterized a new radiolabeled photosensitive antagonist containing an azido group on the amino acid at position 9, termed ¹²⁵I-ZOTA². Because this novel radioligand displays an equivalent high affinity for the human OTR and the structurally related human V_1a arginine-vasopressin (AVP) receptor, we decided to conduct a comparative photolabeling study on both receptors. Because we already accumulated a large amount of data on linear and cyclic peptide antagonist-binding sites of the V_1a receptor from direct receptor photoaffinity labeling experiments (24-26) as well as from site-directed mutagenesis and 3D molecular modeling studies (27-29; for review, see Ref. 30), we therefore hypothesized that this dual comparative investigation should further increase our understanding of OTR/antagonist interactions. We describe here the photolabeling of the human OTR and the human V_1a AVP receptor with ¹²⁵I-ZOTA and their proteolytic fragmentation using CNBr and Lys-C and Arg-C endoproteinases. Compilation of all the data shows that covalent attachment is restricted to three residues, Leu¹¹⁴-Val¹¹⁵-Lys¹¹⁶, in the upper part of the OTR TMD III. Interestingly, the photolabeled region is equivalent in the V_1a receptor.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Radioiodination and Azidation of the Antagonist Probe-- The detailed procedure of radioiodination and azidation leading to ¹²⁵I-ZOTA has been described elsewhere.² Briefly, a strategy consisting of the solid-phase synthesis of the precursor nonapeptide d(CH₂)₅[Tyr(Me)², Thr⁴,Orn⁸,Phe(4NH₂)-NH₂⁹]vasotocin and its subsequent radioiodination and complete azidation was designed. The HPLC-purified precursor (10³ M) was first iodinated with Na¹²⁵I (1 mCi, Amersham Pharmacia Biotech) in a phosphate buffer (pH 6.8) using Iodo-Gen (Pierce) as oxidant. The monoiodinated peptide d(CH₂)₅[Tyr(Me)², Thr⁴,Orn⁸,Phe(3¹²⁵I,4NH₂)-NH₂⁹]vasotocin was purified by HPLC (Waters C₁₈ µBondapak column). Then diazotization and azidation of this compound was done with an excess of NaNO₂ (10³ M) in HCl (12 M) at 0 °C in dim light for 1 h before addition of NaN₃ (10³ M, progressively allowed to reach room temperature), yielding the final product d(CH₂)₅[Tyr(Me)²,Thr⁴,Orn⁸,Phe(3¹²⁵I,4N₃)-NH₂⁹]vasotocin (¹²⁵I-ZOTA). The ¹²⁵I-ZOTA was purified by HPLC, and its specific activity was 2200 Ci/mmol. The corresponding nonradiolabeled iodinated peptide, termed I-ZOTA, was also synthesized and purified.

Cell Culture, Receptor Expression, and Membrane Preparation-- The human V_1a and OTR cDNA clones were generous gifts of Drs. Thibonnier (31) and Kimura (6), respectively. The procedure of CHO cell transfection and stable cell line establishment have been described previously (25, 26). CHO cells stably expressing either the human OT, V_1a, V₂, or V_1b receptor were cultured 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 each, and 0.25 µg/ml amphotericin B in an environment containing 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 (32, 33). As already published, this treatment does not modify the pharmacological properties of the receptors (17, 25, 26). Membranes were prepared as already described (27). In short, cells were washed twice in phosphate-buffered saline 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 800 × 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 the protein content was determined by the Bradford method (Bio-Rad) using bovine serum albumin (BSA) as the standard. Aliquots of membranes were used immediately for binding assays and photolabeling experiments or stored at 80 °C.

Radioligand Binding Assays-- Binding assays were performed at 30 °C using ¹²⁵I-OTA, ¹²⁵I-ZOTA, or [³H]AVP as the radioligands and 1-3 µg (for assays with ¹²⁵I-OTA and ¹²⁵I-ZOTA) or 10-15 µg (for assays with [³H]AVP) of membrane proteins in standard radioligand saturation and competition binding assays as described previously (25-29). Briefly, membranes were incubated in Buffer A supplemented with 1 mg/ml BSA and with radiolabeled and displacing peptides for 30 min (with [³H]AVP) or 60 min (with ¹²⁵I-OTA and ¹²⁵I-ZOTA). Affinities (K_d) for ¹²⁵I-OTA and ¹²⁵I-ZOTA (concentrations from 20 to 2000 pM) as well as for [³H]AVP (concentrations from 0.1 to 20 nM) were directly determined in saturation experiments. Affinities (K_i) for the unlabeled ligand I-ZOTA were determined by competition experiments using [³H]AVP (1-2 nM) as the radioligand. The concentrations of the unlabeled ligands varied from 1 pM to 10 µM. In saturation and competition experiments, depending on the radiolabeled peptide, nonspecific binding was determined by adding unlabeled AVP (10 µM), I-OTA (400 nM), or I-ZOTA (400 nM). Bound and free radioactivity was separated by filtration over Whatman GF/C filters presoaked in a 10 mg/ml BSA solution for 3-4 h. The ligand binding data were analyzed by nonlinear least squares regression using the computer program Ligand (34).

Inositol Phosphate Assays-- The antagonist activity of I-ZOTA was determined by measuring inhibition of OT-stimulated inositol phosphate (IP) accumulation as described previously (25, 26). Briefly, CHO cells expressing the human OT receptor were plated and grown in six-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, PerkinElmer Life Sciences) at a final concentration of 1 µCi/ml in a serum-free, inositol-free medium (Life Technologies, Inc.). Cells were washed twice with phosphate-buffered saline medium, equilibrated at 37 °C in phosphate-buffered saline for 1 h, and then incubated for 10 min in phosphate-buffered saline supplemented with 10 mM LiCl in the presence or absence of increasing concentrations of I-ZOTA (from 10¹² to 10⁶ M). CHO cells were stimulated for 15 min with 10⁸ M OT (a concentration close to the K_act value determined in CHO cells). The reaction was stopped by adding ice-cold perchloric acid. After neutralizing the samples, total IPs were extracted and purified by anion-exchange chromatography (Dowex AG 1-X8 column, 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 + [OT]/K_act) in which IC₅₀ is the concentration of antagonist leading to 50% inhibition, [OT] was kept at 10 nM, and K_act is the concentration of OT inducing half-maximum accumulation of IPs (K_act = 12.9 nM in CHO cells expressing the wild-type human OT receptor (17)).

Photoaffinity Labeling of the Receptors with ¹²⁵I-ZOTA-- Photoaffinity labeling experiments were carried out as described previously with the V_1a receptor photoactivatable antagonists (25, 26). In short, membranes expressing OTR or V_1a (500 µg) were resuspended in 4 ml of binding Buffer A containing BSA (0.5 mg/ml) in Corex glass tubes. Then ¹²⁵I-ZOTA (1-2 nM) was added to the membranes, and samples were incubated at 30 °C for 1 or 3 h in the dark with or without OT or AVP (10 µM) to determine specific labeling. Membranes were separated from unbound ligand by washing with Buffer A without BSA and two subsequent centrifugation steps (20 min, 44,000 × g, 4 °C). The final pellet was resuspended in 1 ml of Buffer A and photolyzed with ultraviolet light (254 nm) for 1-5 min on ice. After photolysis, membranes were washed twice (1 ml of Buffer A) and finally resuspended in Laemmli buffer (35). Samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) using 1-mm-thick 12% acrylamide gels. Gels were fixed in glacial acetic acid:methanol:Me₂SO:water (16:40:2:42), dried, and exposed to Kodak XAR-5 films at 80 °C. The assessment of covalent binding yield was performed after gel drying. Gels were cut into slices, and the radioactive content in each slice was determined. Covalent binding was determined by calculating the amount (in picomoles) of labeled receptors as a percentage of the total number of receptors expressed in the membrane preparation.

Electroelution of the Photolabeled OT and V_1a Receptors-- Photolabeled membranes were first subjected to SDS-PAGE using 12% acrylamide gels as described above. After electrophoresis, the labeled bands were excised from the preparative gel, and the human OTR and V_1a were electroeluted using an electroeluter (model 422, Bio-Rad) in Tris-glycine running buffer (25 mM Tris; 182 mM glycine, pH 8.3; 0.1% SDS). Samples containing electroeluted OTR and V_1a were washed and concentrated using Microcon-50 or Microcon-30, respectively (Amicon). Fragmentation with CNBr and Lys-C/Arg-C endoprotease digestions were performed using these concentrated samples.

Deglycosylation of the Photolabeled OT Receptors with N-Glycosidase F-- The photolabeled CHO membranes (100 µg) or electroeluted OTR was resuspended in deglycosylation buffer (100 mM Na₂HPO₄/NaH₂PO₄, 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% gels.

Chemical and Enzymatic Cleavage of the Photolabeled OT and V_1a Receptors-- The electroeluted receptors were first subjected to single digestion with CNBr or Lys-C/Arg-C endoproteinase. CNBr, Lys-C, and Arg-C cleave proteins specifically at the carboxyl terminus of methionine, lysine, and arginine residues, respectively. Double digestions were also performed (first digestion with Arg-C followed by a second digestion with CNBr). CNBr cleavage of the electroeluted OTR and V_1a was carried out in 100 µl of 70% (v/v) formic acid containing a few crystals of CNBr. The mixture was incubated in the dark for 24 h at room temperature under argon and was then stopped by adding 500 µl of water. The sample volume was reduced under vacuum, and formic acid was removed by solvent exchange with water. Endoproteinase Lys-C (sequencing grade from Lysobacter enzymogenes, Roche Molecular Biochemicals) was used at 0.2 µg/assay in a final volume of 50 µl. 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 Arg-C (sequencing grade from Clostridium histolyticum, Roche Molecular Biochemicals) was used at 0.5 µg/assay for 24 h at 37 °C in a final volume of 50 µl of 0.1 M Tris-HCl, pH 7.6; 10 mM CaCl₂. To perform double fragmentations, the Arg-C digestion reaction was dried under vacuum. The pellet was resuspended in 100 µl of 70% (v/v) formic acid, and CNBr cleavage was performed as indicated above. Results of cleavage were analyzed by a Tricine discontinuous SDS-PAGE system (10-16.5% acrylamide gels) applied to the separation of small molecular mass species (36). Gels were fixed in glacial acetic acid:methanol:Me₂SO:water (10:50:2:38), dried, and exposed to Kodak XAR-5 films at 80 °C. The molecular masses of radiolabeled receptor fragments were determined using a range of low molecular mass markers (Rainbow^TM colored protein, Amersham Pharmacia Biotech).

Site-directed Mutagenesis of the Human OT and V_1a Receptors-- Point mutation M78A, M123A, M296A, M315A, or M330A was introduced in the human OTR cDNA sequence using the QuickChange site-directed mutagenesis kit (Stratagene). The replacement of methionine residues corresponding to potential CNBr cleavage sites by alanine residues was directly performed on the eukaryotic expression vector pCMV (37) and verified by direct dideoxynucleotide sequencing (T7 Sequencing^TM kit, Amersham Pharmacia Biotech). The method used for the construction of the human V_1a receptor mutant K128A has been reported elsewhere (26, 29). All the mutant receptors were transiently expressed in CHO cells by electroporation as published previously (27). For all mutated receptors, cell membrane preparations, receptor photolabeling, and fragmentation experiments were conducted as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Binding and Antagonistic Properties of the New Photoactivatable Ligand-- Pharmacological and functional properties of the wild-type human OTR and AVP V_1a receptor stably expressed in CHO cells have been published previously (17, 26, 27). The affinity of ¹²⁵I-ZOTA, which differs from the OT antagonist ¹²⁵I-OTA (23) only by replacement of the hydroxyl group on Tyr⁹ with a photosensitive azido group (see Fig. 1), was first directly determined for the human OTR by saturation binding experiments. As reported in Table I, the radioiodinated peptide exhibited a high affinity for OTR in the range of that measured for ¹²⁵I-OTA (K_d = 0.25 nM versus 0.18 nM). Like ¹²⁵I-OTA (see Table I), the photoactivatable peptide is indeed nonselective for the OTR, displaying a very similar K_d binding value for the human V_1a AVP receptor (0.45 nM). By doing competition binding experiments using [³H]AVP as the radioligand, the affinity (K_i) of the I-ZOTA was also calculated for the four human AVP/OT receptor subtypes expressed in CHO cells. The results obtained are reported in Table I and confirmed lack of selectivity between the OTR and the V_1a. In CHO cells expressing the human OTR, the photoactivatable peptide I-ZOTA inhibited the IP accumulation induced by 10 nM OT in a concentration-dependent manner (data not shown). The K_inact calculated from experimental IC₅₀ values was 0.15 ± 0.02 nM (n = 4), a value in agreement with the K_d determined in binding experiments. Moreover, no partial agonistic activity of I-ZOTA was detected. In conclusion, the photosensitive cyclic peptide ¹²⁵I-ZOTA is a potent antagonist for the human OTR, displays equivalent high affinity for human OT and V_1a receptors, and thus constitutes a valuable tool to map peptide-binding domains of both receptors.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Structure of the photoreactive antagonist 125I-ZOTA. The structure of 125I-ZOTA is compared with that of the natural hormone OT and that of the antagonist 125I-OTA. The photosensitive compound only differs from its parent peptide antagonist by substitution of an azido group (N3) for a hydroxyl group (OH) on Tyr9. Like OT, the two antagonists are cyclic nonapeptide ligands. Both antagonists can be radioiodinated at the phenyl moiety of Tyr9.

Photoaffinity Labeling of the Human OT and V_1a Receptors-- Photoaffinity labeling of the human wild-type OT and V_1a receptors was directly performed on CHO cell membrane preparations. First, after incubating the photoactivatable ligand ¹²⁵I-ZOTA with the membranes, different UV irradiation times were compared to determine an optimized covalent binding yield. As shown in Fig. 2A, a 254 nm irradiation of the sample for 1 min (lane 2) led to a strong labeling of the receptor and a covalent binding yield, measured as the fraction of specifically bound radioactivity recovered in the labeled bands, reaching 7% (multiple determinations). For longer irradiation times (lanes 3-5), the covalent binding yield decreased. In all cases, ¹²⁵I-ZOTA covalently labeled a major protein band migrating at ~70-75 kDa (Fig. 2, A and C, lane 3). An additional faint band at around 42 kDa that might represent an immature or a nonglycosylated receptor form was sometimes observable. No photolabeling was detected with nonirradiated membranes (lane 1) or in the presence of a saturating 10 µM OT concentration (Fig. 2, A, lane 6, and C, lane 4), indicating that photolabeling of the protein bands was specific for the OTR.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Photoaffinity labeling of the human OTR with 125I-ZOTA: comparison with the human V1a receptor. A, photolabeling of the human OTR. Membrane proteins (500 µg) from CHO cells expressing the OTR were incubated for 1 h with 125I-ZOTA and irradiated at 254 nm for 1-4 min (lanes 2-5). To define nonspecific photolabeling of the membranes, no irradiation was performed (lane 1), or OT was added at a saturating concentration (105 M) during the incubation step (lane 6). Irradiated and washed membranes were separated on a 12% SDS-PAGE system. Equivalent amounts of proteins (25 µg) were loaded onto each well. B, deglycosylation of the photolabeled OTR. CHO cell membranes (100 µg) expressing the receptor were photolabeled and solubilized in deglycosylation buffer in the presence (lane 2) or absence (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% gels. C, comparison of human OTR and V1a receptor photolabeling with 125I-ZOTA. CHO cell membranes (500 µg) expressing the OTR (lane 3) or the V1a receptor (lane 1) were incubated for 1 h with the photoactivatable antagonist and irradiated for 1 min. Equivalent amounts of photolabeled membranes (25 µg) were loaded in each lane before separation by SDS-PAGE. Nonspecific photolabeling of membranes was assessed by adding an excess of AVP (105 M) (lane 2) or OT (105 M) (lane 4) during incubation with 125I-ZOTA. For each experiment described in this figure, the gels were dried and exposed overnight at 80 °C to Kodak XAR-5 films, and autoradiograms are shown. Molecular mass markers are indicated (kDa) on the left. Each panel in this figure is representative of three distinct experiments.

To determine whether the photolabeled protein at 70-75 kDa could correspond to the native glycosylated state of the receptor expressed in the CHO cell system, the membranes were treated with N-glycosidase F before SDS-PAGE. As seen in Fig. 2B, this enzymatic treatment converted the unique 70-75-kDa protein band (lane 1) into two bands of ~48 and 38 kDa (lane 2), confirming that the receptor contained carbohydrates. As shown in Fig. 3, the human OTR contains in its extracellular NH₂-terminal domain three potential N-glycosylation sites located at Asn⁸, Asn¹⁵, and Asn²⁶ (6, 38). The molecular mass of the smaller band migrating at 38 kDa (including the antagonist) is close to the theoretical mass of the receptor core deduced from the cDNA sequence (42.8 kDa). As reported for the guinea pig uterine OTR (21), the deglycosylation treatment led to a reaction product with a molecular mass ~50% of the mass of the native receptor that likely represents a final digestion product. The nature of the band obtained at 42 kDa upon photolysis remains unknown. It is interesting to note that deglycosylation of the photolabeled guinea pig receptor resulted in a protein band migrating at 38 kDa as well (21). Taking into account insensitivity of the human OTR to the plasma membrane metalloproteinase proteolysis (18), the upper band at 48 kDa likely represents a form of partial receptor deglycosylation rather than a proteolytic receptor fragment. Indeed, when the human OTR was transiently expressed in CHO cells or when the 70-75-kDa band was electroeluted from preparative SDS-polyacrylamide gels, deglycosylation of the photolabeled materials always gave rise to both 38- and 48-kDa species (not shown). Taken together, the N-glycosidase F effect on the receptor protein and the lack of endoglycosidase H action (data not shown) suggest that the mature OT receptor is a glycoprotein that contains a high percentage of asparagine-linked oligosaccharides.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Schematic representation of the human OTR. The primary sequence of the receptor deduced from the cloned cDNA (6) is shown as well as the predicted arrangement of the protein in the cell membrane. The position of the TMD was predicted from G protein-coupled receptor hydrophobicity analysis and primary sequence comparison and alignment. The three potential N-glycosylation sites are shown at Asn8, Asn15, and Asn26. Shaded residues (Phe91-Gln96) indicate a consensus sequence of the cleavage site for a metalloproteinase (41, 42). Predicted cleavage sites for CNBr (black diamonds), Lys-C (black triangles), and Arg-C (black squares) are also indicated. The tripeptide fragment covalently labeled with the 125I-ZOTA antagonist is highlighted (residues Leu114-Lys116) at the top of TMD III.

Using the same experimental conditions (incubation for 1 h at 30 °C followed by 1 min of UV irradiation), CHO cell membranes expressing the human AVP V_1a receptor were photolabeled with ¹²⁵I-ZOTA. Two broad bands at 85-90 and 46 kDa were covalently labeled (Fig. 2C, lane 1). Equivalent results were obtained in earlier photolabeling studies using two V_1a-selective photoactivatable peptide antagonists (25, 26). The photolabeling of both 85-90- and 46-kDa protein bands was receptor-specific because labeling could be completely suppressed by adding 10 µM AVP (Fig. 2C, lane 2). The covalent binding efficiency of the probe to the V_1a receptor, calculated as the specifically bound radioactivity recovered in the two labeled bands, was estimated at 3% (multiple determinations). As demonstrated in two previous studies (25, 26), the 46-kDa photolabeled band originated from a proteolytic cleavage of the entire receptor migrating at an apparent molecular mass of 85-90 kDa. Indeed, the relative abundance of the 46-kDa species (50% in Fig. 2C, lane 1) could be significantly reduced when incubation with ¹²⁵I-ZOTA was done in the presence of ZnCl₂ and protease inhibitors for 1 h at 4 °C (data not shown). As illustrated in Fig. 4, the sequence Phe¹⁰³-Gln¹⁰⁸ in the V_1a receptor corresponds to a potential cleavage site for a metalloproteinase present in the membrane preparation. This enzyme has been demonstrated to digest the bovine renal V₂ receptor between Gln⁹² and Val⁹³ upon ligand binding (18). As demonstrated previously with two radioiodinated photoactivatable linear peptide antagonists (25, 26), we found here that the human V_1a is sensitive to metalloproteinase proteolysis as well during incubation with the cyclic peptide antagonist ¹²⁵I-ZOTA. Interestingly, although the proteolytic cleavage site of the metalloproteinase is conserved in the human OTR (sequence Phe⁹¹-Gln⁹⁶), this receptor is resistant to the proteolysis (even with a longer 3-h incubation time, data not shown), indicating that other residues, domains, or specific conformations are necessary for an efficient enzymatic action.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Schematic representation of the human V1a receptor. The figure represents the primary human V1a receptor sequence deduced from the cloned cDNA (31). The position of the TMD was predicted from G protein-coupled receptor hydrophobicity analysis and primary sequence comparison and alignment. Three potential N-glycosylation sites are shown at Asn14, Asn27, and Asn196. Shaded residues indicate a consensus sequence of the cleavage site for a metalloproteinase (41, 42). Predicted cleavage sites for CNBr and Lys-C and Arg-C endoproteinases are indicated as in the legend to Fig. 3. The tripeptide fragment covalently labeled with the 125I-ZOTA antagonist is highlighted (residues Val126-Lys128) at the top of TMD III.

Fragmentation of the Human OT and V_1a Photolabeled Receptors and Identification of the Covalently Labeled Regions-- To identify antagonist-binding domains covalently bound to the photoactivatable cyclic peptide ¹²⁵I-ZOTA, photolabeled bands were excised from preparative SDS-polyacrylamide gels, electroeluted, and subjected to chemical cleavage with CNBr or enzymatic digestion with Lys-C and Arg-C endoproteinases. Based on our present knowledge of the localization of ligand-binding sites on G protein-coupled receptors (for review, see Ref. 39), we have systematically excluded fragments corresponding to putative intracellular portions of the receptors as potential domains covalently bound to the ligand. For the human OTR, the unique photolabeled 70-75-kDa band was used to perform the different fragmentation reactions (see Fig. 3 for the localization of CNBr, Lys-C, and Arg-C cleavage sites in the OTR sequence). The photoactivatable peptide itself is protected from the different degradations because it does not possess Met, Lys, or Arg residues.

The experimental OTR fragmentation patterns derived from CNBr, Lys-C, Arg-C, or Arg-C + CNBr digestions are presented in Fig. 5. For a better understanding of the results, a theoretical fragmentation map of the OTR is shown in Fig. 6. CNBr cleavage of the photolabeled 70-75-kDa OTR yielded three small radiolabeled bands migrating at molecular masses of 4.5, 5.5, and 6.5 kDa (Fig. 5A, lane 2). This result restricted the site of covalent attachment to only one receptor domain, Lys⁷⁹-Met¹²³. Digestion of the photolabeled OTR with Lys-C endoproteinase yielded four fragments at molecular masses of 5, 8, 11, and 16 kDa (Fig. 5B, lane 2). Because the smallest fragment migrated at 5 kDa, only two peptide sequences, His⁸⁰-Lys¹¹⁶ and Met²⁷⁶-Lys³⁰⁶ could be covalently bound to ¹²⁵I-ZOTA. Fragment Ala¹⁹⁹-Lys²²⁶ (3.1 kDa) encompassing TMD V was eliminated as a potential candidate because of the absence of CNBr cleavage sites in this region. Digestion of the photolabeled 70-75-kDa OTR with Arg-C endoproteinase produced four labeled bands at molecular masses of 3.4, 4, 7.8, and 10 kDa (Fig. 5C, lane 2). Several fragments could account for this result, such as Met¹-Arg²⁷, Val⁴¹-Arg⁶⁵, Leu¹¹⁴-Arg¹³⁷, or Leu¹⁵⁵-Arg¹⁷⁸. Considering the cleavage results with CNBr or Lys-C endoproteinase, there was only one reasonable candidate of 3.4 kDa, spanning from Lys¹¹⁴ to Arg¹³⁷ and including the entire TMD III. Successive treatment of the 70-75-kDa photolabeled receptor with Arg-C and CNBr (Fig. 5D, lane 3) generated a new fragment with a molecular mass of 3 kDa, which is slightly smaller than the 3.4 kDa obtained with Arg-C alone (Fig. 5D, lane 2) or the 4.5 kDa produced with CNBr alone (Fig. 5A, lane 2). Among the predicted fragments resulting from a digest with Arg-C, there is only one that fulfills the criteria of having a molecular mass of 3 kDa and possessing an internal CNBr cleavage site, namely the fragment Leu¹¹⁴-Met¹²³. Compilation of the data highlighted an overlap of the different fragmentations in the upper part of TMD III (see Fig. 6) and indicated that covalent binding of ¹²⁵I-ZOTA is restricted to the three amino acid residues Leu¹¹⁴-Val¹¹⁵-Lys¹¹⁶ (see Fig. 3).

View larger version (61K): [in this window] [in a new window]   Fig. 5.   Single and double fragmentations of the human OTR with CNBr and Lys-C/Arg-C endoproteinases. CHO cell membranes (500 µg) expressing the OTR were incubated for 1 h with 125I-ZOTA and irradiated for 1 min. Sample proteins were then separated on a preparative 12% gel, and the photolabeled 70-75-kDa species was electroeluted, washed, and concentrated as described under "Experimental Procedures." Equivalent amounts of electroeluted photolabeled OTR were used in each digestion or chemical cleavage assay (15,000-25,000 cpm). The samples were then loaded on discontinuous 10-16.5% Tricine gels. A, CNBr chemical cleavage. The electroeluted 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 70-75-kDa species was treated (lane 2) or not (lane 1) with the protease for 24 h at 37 °C. C, Arg-C protease digestion. The electroeluted receptor was treated (lane 2) or not (lane 1) with the protease for 24 h at 37 °C. D, Arg-C protease/CNBr double fragmentation. The radiolabeled OTR was treated with Arg-C protease alone (lane 2) or in two steps with Arg-C protease followed by CNBr cleavage (lane 3). The figure shows autoradiograms of dried gels exposed to Kodak XAR-5 films at 80 °C for 48-72 h. Molecular mass markers are indicated on the left in each panel. Each assay is representative of at least three distinct experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Theoretical fragmentation maps of the OTR and V1a receptor. The theoretical fragmentation maps of the OTR (A) and V1a receptor (B) using CNBr, Lys-C endoproteinase, or Arg-C endoproteinase are shown. TMDs are represented as solid black lines. Cleavage sites for CNBr and enzymes are marked with black dots, and the sizes of the theoretical fragments are indicated. Only fragments possessing a molecular mass larger than 1 kDa are shown. In each digestion, the best candidate fragment for covalent attachment of 125I-ZOTA, based on experimental digestions, is shown as a solid black line. For the V1a receptor, cleavage of the receptor with a metalloproteinase during incubation with 125I-ZOTA is indicated by an arrow. The resulting radiolabeled 46-kDa protein used in the different fragmentation reactions starts at Val105. For a direct comparison between these theoretical fragmentation maps and the experimental fragmentation results, the molecular mass of the photoactivatable antagonist 125I-ZOTA covalently attached to the receptors has to be added (1.3 kDa).

We next analyzed the proteolytic fragmentation patterns of the human V_1a receptor (see Fig. 7), and the theoretical digestion map of this receptor is shown again in Fig. 6. In two previous studies (25, 26), we demonstrated that the 46-kDa photolabeled band corresponding to a truncated glycosylated V_1a receptor from Val¹⁰⁵ to the carboxyl-terminal end is derived from the entire 85-90-kDa photolabeled receptor and contains all the covalently bound radioactivity. Therefore, the proteolytic fragmentation of the V_1a receptor was only conducted using the material recovered in the 46-kDa protein (see Fig. 4 for the localization of CNBr, Lys-C, and Arg-C cleavage sites in the V_1a sequence). CNBr cleavage of the photoaffinity-labeled 46-kDa species yielded three labeled fragments migrating at molecular masses of 4, 5.5, and at 7.5 kDa (Fig. 7A, lane 2). Apart from the additional 4-kDa band, the CNBr cleavage pattern was similar to the one described previously using the photoactivatable linear peptide antagonist ¹²⁵I-[Lys(3N₃Phpa)⁸]HO-LVA (26). Based on their predicted size, there are several receptor regions that could correspond to the labeled CNBr cleavage fragments observed: Cys¹¹⁰-Met¹³⁵, Ile¹⁷¹-Met¹⁹¹, Thr²⁹³-Met³¹², and Ser³²⁰-Met³⁴⁹. Digestion of the radioiodinated antagonist-bound 46-kDa photolabeled species with Lys-C endoproteinase yielded three labeled fragments of molecular masses of 5, 8, and 14 kDa (Fig. 7B, lane 2), a result equivalent to that determined with ¹²⁵I-[Lys(3N₃Phpa)⁸]HO-LVA (26). Considering that CNBr cleavage and Lys-C protease digestion of the V_1a receptor are quite similar when the photolabeling is performed either with ¹²⁵I-ZOTA or ¹²⁵I-[Lys(3N₃Phpa)⁸]HO-LVA, the Val¹⁰⁵-Lys¹²⁸ sequence likely corresponds to the 5-kDa labeled band produced upon Lys-C fragmentation (the NH₂ terminus of this fragment is defined by the metalloproteinase cleavage site, Val¹⁰⁵). Arg-C endoproteinase digestion of the photolabeled 46-kDa receptor yielded three labeled fragments at molecular masses of  4, 6.5, and 10 kDa (Fig. 7C, lane 2). Only two fragments could account for a labeled band with an electrophoretic migration at 4 kDa: Val¹⁰⁵- Arg¹¹⁶ and Val¹²⁶-Arg¹⁴⁹. Although the Val¹²⁶-Arg¹⁴⁹ fragment is the best candidate, labeling of both regions could be in agreement with results obtained from the CNBr cleavage and the Lys-C digestion. To confirm this localization, successive treatment of the 46-kDa photolabeled receptor with Arg-C and CNBr was done, generating a new fragment at a molecular mass of 3 kDa (Fig. 7D, lane 2), which is slightly smaller than the 4-kDa bands obtained with CNBr or Arg-C alone. The labeled receptor fragment at 3 kDa corresponds most likely to the Val¹²⁶-Met¹³⁵ sequence. The identification of this fragment as the site of covalent binding for ¹²⁵I-ZOTA would be entirely consistent with the results of the CNBr, Lys-C, and Arg-C cleavages described above. Compilation of all the fragmentation patterns (see Fig. 6) highlighted that the covalent attachment of ¹²⁵I-ZOTA to the human V_1a receptor might also be restricted to the three amino acids Val¹²⁶-Val¹²⁷-Lys¹²⁸ in the upper part of the TMD III (see Fig. 4) that are homologous to Leu¹¹⁴-Val¹¹⁵-Lys¹¹⁶ in the OTR.

View larger version (63K): [in this window] [in a new window]   Fig. 7.   Single and double fragmentations of the human V1a vasopressin receptor with CNBr and Lys-C/Arg-C endoproteinases. CHO cells membranes (500 µg) expressing the wild-type human V1a vasopressin receptor were incubated for 3 h (a condition that favors the preferential accumulation of the 46-kDa species) with 125I-ZOTA and irradiated for 1 min. 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 V1a receptor were used in each digestion or chemical cleavage assay (15,000-25,000 cpm). A, CNBr chemical cleavage. The electroeluted 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 electroeluted receptor was treated (lane 2) or not (lane 1) with Lys-C protease for 24 h at 37 °C. C, Arg-C protease digestion. The electroeluted receptor was treated (lane 2) or not (lane 1) with Arg-C protease for 24 h at 37 °C. D, Arg-C protease/CNBr fragmentation. The electroeluted V1a receptor was treated (lane 3) or not (lane 1) with Arg-C protease alone or subjected to a double fragmentation with Arg-C protease and CNBr (lane 2). The figure shows autoradiograms of dried gels exposed to Kodak XAR-5 films at 80 °C for 48-72 h. Molecular mass markers are indicated on the left in each panel. Each assay is representative of at least three distinct experiments.

Site-directed Mutagenesis of the Human OT and V_1a Receptors-- To confirm further the localization of the covalent binding of ¹²⁵I-ZOTA in the TMD III of the human OTR, we next carried out site-directed mutagenesis of the potential CNBr cleavage sites in this region. Thus, M78A and M123A mutant receptors were first constructed (see Fig. 3 for localization of these Met residues). Moreover, to eliminate the possibility that photolabeling might have occurred elsewhere in the receptor, other potential CNBr cleavage sites in TMD VI and TMD VII were also mutated (M296A, M315A, M330A; see Fig. 3). All mutant receptors were transiently expressed in CHO cells and photolabeled with ¹²⁵I-ZOTA. CNBr cleavage fragments obtained using radiolabeled mutant receptors were then compared with the fragments obtained with the wild-type OTR. The fragmentation patterns of M296A, M315A, and M330A mutant receptors were equivalent to that of the wild-type OTR (the three labeled fragments at 4.5, 5.5, and 6.5 kDa were recovered, data not shown), confirming that regions including TMD VI and TMD VII were not photolabeled. By contrast, as illustrated in Fig. 8, the CNBr cleavage of both M78A and M123A mutant receptors only produced a unique major 6.5-kDa radiolabeled band (lanes 4 and 6). The two smaller bands at 4.5 and 5.5 kDa visualized in the wild-type OTR fragmentation (lane 2) were not present anymore, confirming that the covalently bound domain of the OTR with ¹²⁵I-ZOTA is delimited by Met⁷⁸ and Met¹²³ residues.

View larger version (63K): [in this window] [in a new window]   Fig. 8.   Chemical CNBr fragmentation of photolabeled M78A and M123A mutant OTR. Photolabeling of CHO cell membranes (1 mg) expressing wild-type, mutant M78A, or mutant M123A OTR as well as preparative 12% SDS-PAGE were performed as described in the legends to Figs. 2 and 5. Radioactive material at 70-75 kDa, corresponding to each receptor, was electroeluted, washed, and concentrated before being subjected to CNBr cleavage. Equivalent amounts of photolabeled receptors were used in each chemical cleavage assay (20,000 cpm). The radiolabeled samples, i.e. wild-type (lanes 1 and 2), mutant M78A (lanes 3 and 4), and mutant M123A (lanes 5 and 6) were treated (lanes 2, 4, and 6) or not (lanes 1, 3, and 5) with CNBr for 24 h in the dark at room temperature. An autoradiogram of a discontinuous 10-16.5% dried gel exposed to Kodak XAR-5 film at 80 °C for 72 h is shown. Molecular mass markers are indicated on the left. This fragmentation pattern is representative of three distinct experiments.

To confirm as well our hypothesis regarding the localization of the covalent attachment site of ¹²⁵I-ZOTA to TMD III in the human V_1a receptor, we used in this study a K128A mutant V_1a receptor (Lys¹²⁸ constitutes a potential cleavage site for Lys-C protease, see Fig. 4), which was constructed and described previously (26, 29). Following expression in CHO cells and photolabeling of the mutated receptor, the corresponding 46-kDa radiolabeled fragment was digested with Lys-C, and the resulting fragmentation was compared with that obtained with the wild-type receptor. As shown in Fig. 9, the 5-kDa band produced upon cleavage of the wild-type receptor with Lys-C endoproteinase (lane 2) was no more visible when the mutant K128A receptor was used instead (lane 4). The Lys-C fragmentation pattern yielded only the 8- and 14-kDa photolabeled fragments, confirming that covalent binding of ¹²⁵I-ZOTA to the human V_1a receptor is located in TMD III. Taken together, these results demonstrate that upon UV irradiation the cyclic peptide antagonist ¹²⁵I-ZOTA attaches covalently to both OTR and V_1a at an equivalent position in the upper part of TMD III.

View larger version (57K): [in this window] [in a new window]   Fig. 9.   Endoproteinase Lys-C fragmentation of the photolabeled K128A mutant V1a receptor. Photolabeling of CHO cell membranes (1 mg) expressing wild-type or mutant K128A V1a receptors was performed as described in the legend to Fig. 7. The corresponding 46-kDa photolabeled species were then electroeluted from a preparative 12% SDS-polyacrylamide gel and subjected to Lys-C endoproteinase digestion. Equivalent amounts of photolabeled receptors were used in each digestion assay (10,000 cpm). The radiolabeled samples, i.e. wild-type (lanes 1 and 2) or mutant K128A (lanes 3 and 4) were treated (lanes 2 and 4) or not (lanes 1 and 3) with Lys-C proteinase for 24 h at 37 °C. An autoradiogram of a discontinuous 10-16.5% dried gel exposed to Kodak XAR-5 film at 80 °C for 72 h is shown. Molecular mass markers are indicated on the left. This fragmentation pattern is representative of three distinct experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It is well established that OTR is a major therapeutic target in the control of labor, thus analysis of the structure/function relationships of its functional domains, particularly the ligand binding pocket, is very important. However, relatively little information on the characterization of OTR antagonist-binding sites is currently available. In one report, the upper part of TMD VII has been demonstrated to participate in the binding of an ¹²⁵I-OTA-derived antagonist (18). To provide further information on structural requirements for antagonist binding to the human OTR, we have undertaken a photoaffinity labeling study with a novel photoactivatable cyclic peptide ligand. The structure of I-ZOTA or that of its radiolabeled counterpart ¹²⁵I-ZOTA is based on that of I-OTA for which properties were described more than 10 years ago (23). We have first pharmacologically and functionally characterized ¹²⁵I-ZOTA² (present study) and demonstrated that this OT antagonist combined high affinity, low nonspecific binding, easiness and efficiency of radioiodination, and appreciable covalent binding yield. Like ¹²⁵I-OTA (40), ¹²⁵I-ZOTA behaved as a nonselective compound displaying equivalent high affinity for both the human OTR and the human AVP V_1a receptor. Taking advantage of this lack of selectivity, we decided to conduct a comparative photoaffinity labeling study on both receptors.

The photolabeled OTR migrated on SDS-polyacrylamide gels as a unique glycosylated broad band with an apparent molecular mass of 70-75 kDa. The size of the human OTR is consistent with the molecular mass reported for OTRs isolated from rat mammary gland, guinea pig uterus, or rabbit amnion (19-22). Deglycosylation of the photolabeled OTR converted the 70-75-kDa protein into two bands of molecular masses of 48 and 38 kDa, the latter corresponding roughly to the expected size of the peptidic core of the native receptor as described for the guinea pig OTR (21). By contrast, using the same photolabeling conditions (incubation for 1 h at 30 °C followed by 1 min of irradiation), the human V_1a receptor was degraded during incubation with the ligand, leading to two bands of molecular masses of 85-90 and 46 kDa. As described previously, the 85-90-kDa molecular species corresponds to the glycosylated native state of the receptor, whereas the 46-kDa species represented an NH₂-terminal-truncated receptor. The latter results from a proteolytic cleavage of the 85-90-kDa band by an endogenous metalloproteinase present in the CHO membrane preparation (25, 26). Equivalently, photolabeling of bovine kidney membranes with a tritiated photoactivatable agonist containing an arylazido group at the side chain of Lys⁸ gave rise to two AVP V₂ receptor bands, a glycosylated native receptor at 58 kDa and an NH₂-terminal-truncated form at 30 kDa (41, 42). In that case, the proteolytic cleavage occurred between Gln⁹² and Val⁹³ in the six-amino acid sequence FQVLPQ located in TMD II and conserved in all neurohypophyseal hormone receptors, including the OTR. According to the authors, the proteolytic cleavage of the V₂ receptor would require a receptor conformational change dependent on the agonistic properties of the ligand (42). We have presently shown that the human OTR is fully resistant to the proteolysis after binding of the cyclic photoactivatable antagonist, a result previously shown by others with a different photoactivatable antagonist (18, 42). By contrast, the metalloproteinase cleavage occurred in the human V_1a receptor after incubation with ¹²⁵I-ZOTA and under the same experimental conditions. This finding is equivalent to that obtained when incubating V_1a-expressing membranes with two different photoactivatable linear peptide antagonists (25, 26). Taken together, the present data suggest that cyclic as well as linear peptide antagonists are able to induce a conformational change leading to proteolytic cleavage in the V_1a receptor, whereas the OTR remains resistant.

Using CNBr chemical cleavage and Lys-C/Arg-C protease digestions of the photolabeled human OTR and V_1a, we demonstrated that ¹²⁵I-ZOTA covalently bound to the upper part of TMD III. This result has been confirmed using double fragmentation (CNBr followed by Arg-C protease) experiments and site-directed mutagenesis of potential CNBr or Lys-C cleavage sites in this OTR or V_1a receptor region. The covalently attached region of both receptors has been restricted to three amino acid residues only, Leu¹¹⁴-Val¹¹⁵-Lys¹¹⁶ in the OTR, which corresponds to Val¹²⁶-Val¹²⁷-Lys¹²⁸ in the human V_1a receptor (Figs. 3 and 4). Based on the high resolution x-ray structure of bovine rhodopsin (43), these residues are predicted to be located at the top of TMD III. Interestingly, the Leu¹¹⁴-Val¹¹⁵-Lys¹¹⁶ motif in the OTR and the corresponding Val¹²⁶-Val¹²⁷-Lys¹²⁸ motif in the V_1a are located in a position homologous to that of Glu¹¹³ in rhodopsin known for interacting with the retinal Schiff base. As illustrated in Fig. 10, this photoaffinity labeling study not only constituted the first direct identification of the human OTR antagonist-binding sites determined so far but also allowed us to localize a third photolabeled region in the V_1a receptor (25, 26). Indeed, the first extracellular loop and the upper part of TMD VII of the V_1a receptor were identified as contact regions for two structurally related V_1a-selective photoactivatable linear peptide antagonists, ¹²⁵I-[Lys(3N₃Phpa)⁸]HO-LVA and ¹²⁵I-3N₃Phpa-LVA. The Lys¹²⁸ in the human V_1a, corresponding to Lys¹¹⁶ in the human OTR, is well conserved throughout the AVP/OT receptor family (30). Moreover, this residue has been shown to play a pivotal role in the binding of agonists (27, 29), two different classes of peptide antagonists such as linear peptides (25, 26) or the cyclic peptide d(CH₂)₅[Tyr(Me)²]AVP (29) (see also Fig. 10), and also the nonpeptide compound SR 49059 (29). In the present study, the presence of Lys¹²⁸ residue in the tripeptide sequence covalently attached to the ligand is very interesting. As for V_1a-selective linear and cyclic peptide antagonists as well as for SR 49059, this Lys residue might be responsible for a direct interaction between the receptors and ¹²⁵I-ZOTA. It is very likely that the protonated amine group of Lys¹²⁸ or Lys¹¹⁶, depending on the receptor subtype, could be involved in a classical -cation interaction with the electron cloud of the phenyl ring of Phe⁹ of the ligand. This kind of interaction is often encountered in protein structures (44). Alternatively, the ammonium group of Lys could form a hydrogen bond with a carbonyl function of the peptide backbone. The predicted interaction between Lys¹²⁸ and ¹²⁵I-ZOTA is in good agreement with 3D antagonists/V_1a receptor models that we have constructed previously for V_1a-selective linear and cyclic peptide antagonists (25, 26, 29). Moreover in the present study, we have performed the photolabeling of the K128A mutant V_1a receptor using a high concentration of ¹²⁵I-ZOTA (around 2-3 nM), but we were not able to directly measure the K_d of the radioligand in saturation studies. This suggests that the affinity of the ligand was reduced and also reinforces a putative role for this residue in the binding of the ligand. As a control, we tried as well to directly measure the affinity for [³H]AVP, but as already demonstrated in COS cells (29), mutation of Lys¹²⁸ of the human V_1a receptor into an alanine significantly decreased affinity for this radioligand. The affinity of AVP for the mutant K128A was finally determined in competition studies using the V_1a-selective antagonist ¹²⁵I-HO-LVA (45), and K_i was 43 ± 6.8 nM (n = 3). This value is in agreement with that measured in COS cells (29). Altogether, these data magnify the role of Lys¹²⁸ (Lys¹¹⁶ in OTR) and demonstrate again that this residue contributes to the overlap of agonist- and antagonist-binding sites in the human V_1a receptor binding pocket (29). The role of Lys¹²⁸ could be compared with that of Asp¹¹³ of cationic neurotransmitter receptors localized again in the upper part of TMD III. Substitution of this residue dramatically affects the affinity of both ammonium group-containing agonists and antagonists in the -adrenergic receptor (46).

View larger version (30K): [in this window] [in a new window]   Fig. 10.   Schematic model of the human OTR and V1a receptor binding pockets. The arrangement of transmembrane domains of OTR and V1a receptors viewed from the extracellular surface is based on the two-dimensional crystal projection map of frog rhodopsin (47). The TMDs are represented as shaded cylinders, and the first extracellular loop is shown as a solid black line between TMD II and TMD III. Receptor regions that have been demonstrated to be covalently attached to photoactivatable peptide antagonists are indicated with black triangles. In the human V1a receptor, the top of TMD VII, the first extracellular loop, and the top of TMD III have been photolabeled with 125I-3N3Phpa-LVA (25), 125I-[Lys(3N3Phpa)8]HO-LVA (26), and 125I-ZOTA (this study), respectively. In the human OTR, only the top of TMD III has been demonstrated so far to be covalently attached to 125I-ZOTA (this study). Residues or regions likely to be involved in peptide antagonist binding affinity, on the basis of site-directed mutagenesis studies, are indicated with black spheres. In the human V1a receptor, Gln108 (TMD II), Lys128 (TMD III), Gln185 (TMD IV), and Phe307 (TMD VI) play a role in the binding of V1a-selective cyclic (29) and linear (25, 26) peptide antagonists. In the human OTR, the top of TMD VII has been proposed to be a major contributor to high affinity binding of an 125I-OTA-related cyclic peptide antagonist (18).

As depicted in Fig. 10, most of the interactions between linear or cyclic peptide antagonist ligands and the V_1a receptor (25-30) are located in a receptor central pocket surrounded by the first extracellular loop and the upper parts of TMD II, TMD III, TMD VI, and TMD VII. The covalent attachment of ¹²⁵I-ZOTA at the top of TMD III and the demonstration that the upper part of TMD VII is involved in the binding of an I-OTA-derived antagonist (18) are in agreement with such a model. Based on photoaffinity labeling results, 3D docking of two linear peptide antagonists into the V_1a receptor was equivalent to that of the natural hormone AVP. When bound to the receptor despite an open chain, the two linear peptide antagonists could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Moreover, based on site-directed mutagenesis results, 3D docking of the cyclic peptide antagonist d(CH₂)₅[Tyr(Me)²]AVP is also equivalent to that of the hormone AVP. According to these models, AVP and its peptide antagonists, at least in the V_1a receptor, could enter this transmembrane central binding pocket and interact with overlapping binding regions. As for AVP and the three peptide antagonists, we hypothesize that ¹²⁵I-ZOTA is also able to enter this central binding pocket and interact with the V_1a receptor in a similar way while establishing its own network of molecular interactions. Because (i) ¹²⁵I-ZOTA displays an equivalent affinity for the human OTR and the human V_1a receptor, (ii) several residues involved in the binding of all classes of antagonists are conserved throughout the AVP/OT receptor family, and (iii) OTR and V_1a share a significant sequence identity in the upper part of TMD II, TMD III, TMD VI, and TMD VII as well as in the first extracellular loop, we propose that binding sites for ¹²⁵I-ZOTA in the OTR and V_1a receptor could be equivalent. However, to allow a more complete definition of the binding sites of the OTR for cyclic peptide antagonists such as ¹²⁵I-ZOTA or Atosiban (14, 15) and to generate meaningful information on receptor/antagonist interactions, it will be necessary to perform other photolabeling studies.

In conclusion, covalent attachment of ¹²⁵I-ZOTA to the upper part of TMD III of both the OTR and V_1a receptor is fully consistent with our previous 3D antagonist/V_1a receptor models. Taken together, these findings suggest once again as demonstrated previously that agonist and peptide antagonist binding pockets might be common to all receptor subtypes of the OT/AVP family and that specific residues can differentiate agonist versus antagonist binding in these receptors (29). The delineation of a three-amino acid "contact domain" is a major step "en route" to a more detailed mapping of the molecular interactions between OT-related ligands and the OTR. The use of other photoactivatable antagonist ligands as well as molecular modeling of the OTR based on the recent 3D crystal structure of bovine rhodopsin (43) should be very helpful in the future for rationalizing the design of OT antagonists based on the receptor structure.

	Aknowledgments

We thank M. Passama and L. Charvet for help with the illustrations. We especially thank Drs. Tuhinadri Sen and Jean-Philippe Pin for critical reading of the manuscript.

	FOOTNOTES

^* This research was supported by grants from INSERM and CNRS.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.

^§ Present address: Laboratoire d'Endocrinologie des Annélides, UPRESA CNRS 8017, SN3, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France.

Present address: Laboratoire des Amino Acides, Peptides et Protéines, UMR 5810 CNRS, Faculté de Pharmacie, 15 avenue Charles Flahaut, 34060 Montpellier, France.

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.

Published, JBC Papers in Press, May 3, 2001, DOI 10.1074/jbc.M102073200

² E. Carnazzi, A. Aumelas, B. Mouillac, C. Breton, L. Guillou, C. Barberis, and R. Seyer, manuscript submitted for publication.

	ABBREVIATIONS

The abbreviations used are: OT, oxytocin; OTR, OT receptor; 3D, three-dimensional; TMD, transmembrane domain; AVP, arginine-vasopressin; I-OTA, d(CH₂)₅[Tyr(Me)²,Thr⁴,Orn⁸,Tyr(3I)-NH₂⁹]vasotocin; ¹²⁵I-OTA, d(CH₂)₅[Tyr(Me)²,Thr⁴,Orn⁸,Tyr(3¹²⁵I)-NH₂⁹]vasotocin; I-ZOTA, d(CH₂)₅[Tyr(Me)²,Thr⁴,Orn⁸,Phe(3I,4N₃)-NH₂⁹]vasotocin; ¹²⁵I-ZOTA, d(CH₂)₅[Tyr(Me)²,Thr⁴,Orn⁸,Phe(3¹²⁵I,4N₃)-NH₂⁹]vasotocin; CHO, Chinese hamster ovary; BSA, bovine serum albumin; IP, inositol phosphate; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis- (hydroxymethyl)ethyl]glycine; [Lys(3N₃Phpa)⁸]HO-LVA, 4-hydroxyphenylpropionyl¹-D-Tyr(Me)²-Phe³-Gln⁴-Asn⁵-Arg⁶-Pro⁷-Lys(3-azidophenylpropionyl)⁸-NH₂; 3N₃Phpa-LVA, 3-azidophenylpropionyl¹-D-Tyr(Me)²-Phe³-Gln⁴-Asn⁵-Arg⁶-Pro⁷-Arg⁸-Tyr⁹-NH₂; HO-LVA, 4-hydroxyphenylacetyl¹-D-Tyr(Me)²-Phe³-Gln⁴-Asn⁵-Arg⁶-Pro⁷-Arg⁸-NH₂.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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