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• W1997758712 abstract "To identify the binding site of the human V1a vasopressin receptor for the selective nonpeptide antagonist SR49059, we have developed a site-directed irreversible labeling strategy that combines mutagenesis of the receptor and use of sulfydryl-reactive ligands. Based on a three-dimensional model of the antagonist docked into the receptor, hypothetical ligand-receptor interactions were investigated by replacing the residues potentially involved in the binding of the antagonist into cysteines and designing analogues of SR49059 derivatized with isothiocyanate or α-chloroacetamide moieties. The F225C, F308C, and K128C mutants of the V1a receptor were expressed in COS-7 or Chinese hamster ovary cells, and their pharmacological properties toward SR49059 and its sulfydryl-reactive analogues were analyzed. We demonstrated that treatment of the F225C mutant with the isothiocyanate-derivative compound led to dose-dependent inhibition of the residual binding of the radio-labeled antagonist [125I]HO-LVA. This inhibition is probably the consequence of a covalent irreversible chemical modification, which is only possible when close contacts and optimal orientations exist between reactive groups created both on the ligand and the receptor. This result validated the three-dimensional model hypothesis. Thus, we propose that residue Phe225, located in transmembrane domain V, directly participates in the binding of the V1a-selective nonpeptide antagonist SR49059. This conclusion is in complete agreement with all our previous data on the definition of the agonist/antagonist binding to members of the oxytocin/vasopressin receptor family. To identify the binding site of the human V1a vasopressin receptor for the selective nonpeptide antagonist SR49059, we have developed a site-directed irreversible labeling strategy that combines mutagenesis of the receptor and use of sulfydryl-reactive ligands. Based on a three-dimensional model of the antagonist docked into the receptor, hypothetical ligand-receptor interactions were investigated by replacing the residues potentially involved in the binding of the antagonist into cysteines and designing analogues of SR49059 derivatized with isothiocyanate or α-chloroacetamide moieties. The F225C, F308C, and K128C mutants of the V1a receptor were expressed in COS-7 or Chinese hamster ovary cells, and their pharmacological properties toward SR49059 and its sulfydryl-reactive analogues were analyzed. We demonstrated that treatment of the F225C mutant with the isothiocyanate-derivative compound led to dose-dependent inhibition of the residual binding of the radio-labeled antagonist [125I]HO-LVA. This inhibition is probably the consequence of a covalent irreversible chemical modification, which is only possible when close contacts and optimal orientations exist between reactive groups created both on the ligand and the receptor. This result validated the three-dimensional model hypothesis. Thus, we propose that residue Phe225, located in transmembrane domain V, directly participates in the binding of the V1a-selective nonpeptide antagonist SR49059. This conclusion is in complete agreement with all our previous data on the definition of the agonist/antagonist binding to members of the oxytocin/vasopressin receptor family. The neurohypophysial antidiuretic hormone arginine vasopressin (AVP) 1The abbreviations used are: AVP, arginine vasopressin; V1aR, vascular V1a vasopressin receptor; V2R, renal V2 vasopressin receptor; V1bR, pituitary V1b vasopressin receptor; OT, oxytocin; OTR, OT receptor; HO-LVA, HO-phenylacetyl1-d-Tyr(Me)2-Phe3-Gln4-Asn5-Arg6-Pro7-Arg8NH2; TM, transmembrane segment; E, extracellular loop; GA, genetic algorithm; GPCR, G protein-coupled receptor.1The abbreviations used are: AVP, arginine vasopressin; V1aR, vascular V1a vasopressin receptor; V2R, renal V2 vasopressin receptor; V1bR, pituitary V1b vasopressin receptor; OT, oxytocin; OTR, OT receptor; HO-LVA, HO-phenylacetyl1-d-Tyr(Me)2-Phe3-Gln4-Asn5-Arg6-Pro7-Arg8NH2; TM, transmembrane segment; E, extracellular loop; GA, genetic algorithm; GPCR, G protein-coupled receptor. is involved in the regulation of body fluid osmolality, blood volume, and blood pressure via the stimulation of specific receptors currently classified into V1a vascular (V1aR) and V2 renal (V2R) receptors. In addition, AVP modulates the adrenocorticotropic hormone secretion through V1b pituitary (V1bR) receptors. These different receptor subtypes along with the oxytocin receptor (OTR), which is classified in the same subfamily, possess distinct pharmacological profiles and intracellular second messengers (1Jard S. Elands J. Schmidt A. Barberis C. Imura H. Shizume K. Progress in Endocrinology. Elsevier Science Publishers B.V., Amsterdam1988: 1183-1188Google Scholar, 2Thibonnier M. Berti-Mattera L.N. Dulin N. Conarty D.M. Mattera R. Prog. Brain Res. 1998; 119: 143-158Google Scholar). Moreover, AVP belongs to the family of vasoactive and mitogenic peptides involved in physiological and pathological cell growth and differentiation (3Van Biesen T. Luttrell L.M. Hawes B.E. Leftkowitz R.J. Endocr. Rev. 1996; 17: 698-714Crossref PubMed Scopus (390) Google Scholar). AVP has been shown to be one of the most powerful in vitro vasoconstrictor substances, and its vasoconstrictor and mitogenic actions may contribute to the pathogenesis of arterial hypertension, heart failure, and atherosclerosis (4Johnston C.I. J. Hypertens. 1985; 3: 557-569Crossref PubMed Scopus (94) Google Scholar, 5Goldsmith S.R. Am. J. Med. 1987; 82: 1213-1219Abstract Full Text PDF PubMed Scopus (74) Google Scholar). AVP plays a role in the maintenance of blood pressure in several conditions, including upright posture, dehydration, hemorrhage, adrenal insufficiency, cardiac failure, and during surgery (6Cowley Jr., A.W. Int. Rev. Physiol. 1982; 26: 189-242PubMed Google Scholar, 7Share L. Physiol. Rev. 1988; 68: 1248-1284Crossref PubMed Scopus (211) Google Scholar). An abnormal vascular reactivity specific for AVP has been noted in models of genetic and experimental hypertension, and AVP is instrumental in the genesis and maintenance of several models of experimental hypertension (4Johnston C.I. J. Hypertens. 1985; 3: 557-569Crossref PubMed Scopus (94) Google Scholar, 5Goldsmith S.R. Am. J. Med. 1987; 82: 1213-1219Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 7Share L. Physiol. Rev. 1988; 68: 1248-1284Crossref PubMed Scopus (211) Google Scholar). AVP implication in the development or maintenance of hypertension, or both, is based on measurements of plasma and urinary AVP levels and responses to specific AVP antisera or peptide or nonpeptide antagonists (8Sladek C. Blair M.L. Sterling C. Mangiapane M.L. Hypertension. 1988; 12: 506-512Crossref PubMed Scopus (37) Google Scholar, 9Burrell L.M. Phillipps P.A. Stephenson J.M. Risvanis J. Rolls K.A. Johnston C.I. Hypertension. 1994; 23: 737-743Crossref PubMed Google Scholar, 10Okada H. Suzuki H. Kanno Y. Yamamura Y. Saruta T. Am. J. Physiol. 1994; 267: R1467-R1471Crossref PubMed Google Scholar). The first potent and selective V1aR antagonist to be synthesized is a cyclic peptide, d(CH2)5[Tyr(Me)2]AVP (11Kruszynski M. Lammek B. Manning M. Seto J. Haldar J. Sawyer W.H. J. Med. Chem. 1980; 23: 364-368Crossref PubMed Scopus (440) Google Scholar). In addition to cyclic peptide antagonists, linear peptide antagonists have been developed, such as HO-phenylacetyl1-d-Tyr(Me)2-Phe3-Gln4-Asn5-Arg6-Pro7-Arg-8NH2 (HO-LVA) (12Barberis C. Balestre M.N. Jard S. Tribollet E. Arsenijevic Y. Dreifuss J.J. Bankowski K. Manning M. Chan W.Y. Schlosser S.S. Holsboer F. Elands J. J. Neuroendocrinology. 1995; 62: 135-146Crossref Scopus (92) Google Scholar). However, the lack of oral bioavailability and short half-life of these peptide compounds have limited their use in clinical medicine. During the last decade, nonpeptide AVP antagonists were discovered through random screening of chemical libraries (13Yamamura Y. Ogawa H. Chihara T. Kondo K. Ongawa T. Nakamura S. Mori T. Tominaga M. Yabuuchi Y. Science. 1991; 252: 572-574Crossref PubMed Scopus (279) Google Scholar, 14Serradeil-LeGal C. Wagnon J. Guillon G. Garcia C. Lacour C. Cantau B. Guiraudou P. Christophe B. Barberis C. Villanova G. Nisato D. Maffrand J.P. Jard S. LeFur G. J. Clin. Invest. 1993; 92: 224-231Crossref PubMed Scopus (267) Google Scholar). The availability of these orally active compounds now facilitates the assessment to the potential therapeutic applications of AVP receptor blockade in human diseases. One of these compounds, SR49059 ((2S)1-{(2R, 3S)-5-chloro-3-(2-chloro-phenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl}pyrrolidine-2-carboxamide), is presently the most potent and selective orally active V1aR antagonist described so far (14Serradeil-LeGal C. Wagnon J. Guillon G. Garcia C. Lacour C. Cantau B. Guiraudou P. Christophe B. Barberis C. Villanova G. Nisato D. Maffrand J.P. Jard S. LeFur G. J. Clin. Invest. 1993; 92: 224-231Crossref PubMed Scopus (267) Google Scholar). It has a marked affinity, selectivity, and efficacy toward both animal and human V1aR and is devoid of partial agonist activity. In healthy human volunteers, SR49059 inhibits exogenous AVP-induced platelet aggregation and vasoconstriction (15Brouard R. Laporte V. Serradeil-LeGal C. Pignol R. Jang H.F.D. Lockwood G. Fournie D. Dreux F. Zingg H. Bourque C.W. Bichet D.G. Vasopressin and Oxytocin: Molecular, Cellular, and Clinical Advances. Plenum Press, New York1998: 455-465Google Scholar, 16Weber R. Pechère-Bertschi A. Hayoz D. Gerc V. Brouard R. Lahmy J.P. Brunner H.R. Burnier M. Hypertension. 1997; 30: 1121-1127Crossref PubMed Scopus (24) Google Scholar). Up to now, identification of the binding site of the SR49059 into the V1aR has not been investigated at a molecular level. Several studies based on a combination of receptor three-dimensional modeling and site-directed mutagenesis experiments have suggested that an AVP-binding pocket is buried into a 15–20-Å-deep central cavity of the V1a receptor, defined by the transmembrane helices and surrounded by the extracellular loops (17Mouillac B. Chini B. Balestre M.N. Elands J. Trumpp-Kallmeyer S. Hoflack J. Hibert M. Jard S. Barberis C. J. Biol. Chem. 1995; 270: 25771-25777Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 18Hibert M. Hoflack J. Trumpp-Kallmeyer S. Mouillac B. Chini B. Mahé E. Cotte N. Jard S. Manning M. Barberis C. J. Recept. Signal Transduct. Res. 1999; 19: 589-596Crossref PubMed Scopus (30) Google Scholar). Extracellular residues also play a role in the binding of the hormone (19Chini B. Mouillac B. Ala Y. Balestre M.N. Trumpp-Kallmeyer S. Hoflack J. Elands J. Hibert M. Manning M. Jard S. Barberis C. EMBO J. 1995; 14: 2176-2182Crossref PubMed Scopus (162) Google Scholar, 20Hawtin S.R. Wesley V.J. Parslow R.A. Simms J. Miles A. McEwan K. Wheatley M. Mol. Endocrinol. 2002; 16: 600-609Crossref PubMed Scopus (25) Google Scholar). Binding domains for synthetic peptide antagonists overlap those for peptide agonists into the AVP/OT receptors; however, discrimination of agonist versus antagonist ligands is achieved by conserved aromatic residues (Trp304, Phe307, and Phe308) located at the bottom of the binding pocket in the transmembrane helix VI (21Cotte N. Balestre M.N. Aumelas A. Mahé E. Phalipou S. Morin D. Hibert M. Manning M. Durroux T. Barberis C. Mouillac B. Eur. J. Biochem. 2000; 267: 4253-4263Crossref PubMed Scopus (60) Google Scholar, 22Phalipou S. Cotte N. Carnazzi E. Seyer R. Mahé E. Jard S. Barberis C. Mouillac B. J. Biol. Chem. 1997; 272: 26536-26544Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 23Phalipou S. Seyer R. Cotte N. Breton C. Barberis C. Hibert M. Mouillac B. J. Biol. Chem. 1999; 274: 23316-23327Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 24Breton C. Chellil H. Kabbaj-Benmansour M. Carnazzi E. Seyer R. Phalipou S. Morin D. Durroux T. Zingg H.H. Barberis C. Mouillac B. J. Biol. Chem. 2001; 276: 26931-26941Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Although the binding mode of SR49059 into the V1aR is not yet defined, nonconserved residues Thr333 and Ala334 located in transmembrane region VII have been shown to control the V1aR/V2R receptor binding selectivity for SR49059 and for cyclic peptide antagonists as well (21Cotte N. Balestre M.N. Aumelas A. Mahé E. Phalipou S. Morin D. Hibert M. Manning M. Durroux T. Barberis C. Mouillac B. Eur. J. Biochem. 2000; 267: 4253-4263Crossref PubMed Scopus (60) Google Scholar). To precisely investigate the SR49059-V1aR binding interactions at a molecular level, we first constructed a three-dimensional model of the antagonist docked into the receptor based on the x-ray crystal structure of bovine rhodopsin (25Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Trong I.L. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5001) Google Scholar). To validate or invalidate the hypotheses of potential ligand-receptor interactions, we developed a site-directed irreversible labeling strategy that combines mutagenesis of the receptor and binding of chemically reactive probes derived from SR49059. This approach generates a chemical bond between the nucleophilic moiety of a cysteine residue incorporated into the receptor and the electrophilic moiety of a sulfydryl-reactive group (isothiocyanate or an α-chloroacetamide) in the SR49059 analogue. Because such a chemical bond formation is only possible when a close contact exists between reactive elements created both on the ligand and on the protein, the covalent link can be taken as a proof of direct interaction versus a long range effect (26Foucaud B. Perret P. Grutter T. Goeldner M. Trends Pharmacol. Sci. 2001; 22: 170-173Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). This strategy combines the advantages of three-dimensional modeling and site-directed mutagenesis techniques with those of a direct unambiguous identification of contact regions between a receptor and a specific ligand, like in photoaffinity labeling studies. This is an elegant complement to receptor photolabeling; however, it is much easier to develop than photolabeling, because it does not require the use of a radiolabeled ligand and the overproduction of the target receptor. However, this strategy necessitates introduction of a mutation in the receptor, which could be by itself detrimental to the overall structure of the protein. In this study, we demonstrated that the use of cysteine mutants of V1aR as chemical sensors for sulfydryl-reactive ligands allows unambiguous localization of the SR49059 nonpeptide-binding site and offers a reliable molecular model of the V1a receptor. Materials—SR49059 and its affinity probe derivatives were synthesized according to a SANOFI Recherche Montpellier patent (27Garcia, G., Nisato, D., Roux, R., Serradeil-LeGal, C., and Valette, G. (February 1, 1995) European Patent 636,608Google Scholar). The synthesis will be published in a paper to come. 2C. Tahtaoui, M. N. Balestre, P. Klotz, D. Rognan, C. Barberis, B. Mouillac, and M. Hibert, manuscript in preparation. [3H]AVP (60–80 Ci·mmol–1) was purchased from PerkinElmer Life Sciences. HO-LVA was kindly provided by Dr. M. Manning (Toledo, OH) and was radioiodinated in our laboratory as previously described (12Barberis C. Balestre M.N. Jard S. Tribollet E. Arsenijevic Y. Dreifuss J.J. Bankowski K. Manning M. Chan W.Y. Schlosser S.S. Holsboer F. Elands J. J. Neuroendocrinology. 1995; 62: 135-146Crossref Scopus (92) Google Scholar). Alignment of Amino Acid Sequences—The amino acid sequences of the three human AVP receptor subtypes and that of the OT receptor were retrieved from the Swiss-Prot data base (accession numbers were as follows: V1a receptor, P37288; V1b receptor, P47901; V2 receptor, P30518; and OTR, P30559) and aligned to the sequence of bovine rhodopsin (accession number P02699) using the ClustalW multiple alignment program (28Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55399) Google Scholar). A slow pairwise alignment using BLOSUM matrix series (29Henikoff S. Henikoff J.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10915-10919Crossref PubMed Scopus (4284) Google Scholar) and a gap opening penalty of 15.0 were chosen for aligning the amino acid sequences to the sequence of bovine rhodopsin. Because the disulfide bridge occurring between the third transmembrane segment (TM III) and the second extracellular loop (E II) in the structure of bovine rhodopsin is conserved in all AVP/OT receptors, we manually adjusted the alignment of E II to align the corresponding cysteines. Preparation of Starting Protein Coordinates—The three-dimensional model of the human V1a receptor was constructed by mutating the side chains of the amino acids in rhodopsin. Standard geometries for the mutated side chains were given by the BIOPOLYMER module of SYBYL (SYBYL 6.8, Tripos Inc., St. Louis, MO). Whenever possible, the side chain torsional angles were kept to the values occurring in bovine rhodopsin. Otherwise, a short scanning of side chain angles was performed to remove steric clashes between the mutated side chain and the other amino acids. The third intracellular loop between helices V and VI, which shows a high degree of variability, was not included in any of the three models. This loop is not involved in direct interactions with the ligand (31Strader C.D. Dixon R.A. Cheung A.H. Candelore M.R. Blake A.D. Sigal I.S. J. Biol. Chem. 1987; 262: 16439-16443Abstract Full Text PDF PubMed Google Scholar). We therefore assume that omitting this loop should not influence our docking results. Insertions/deletions occurred only in loops but not in secondary structure elements (α-helix and β-sheet). The insertions/deletions in the loops were achieved through a simple knowledge-based loop search procedure using the LOOPSEARCH module of the SYBYL package. In this procedure, a set of 1478 high resolution x-ray structures was searched for loops of similar length and similar distance between the Cα atoms of the residues delimiting the loop window. The loop showing the highest sequence identity and the lowest root mean square deviation was then selected for insertion in the model. Special caution had to be given with E II, which has been described in bovine rhodopsin to fold back over transmembrane helices (25Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Trong I.L. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5001) Google Scholar) and thereby limit the size of the active site. Hence, amino acids of this loop could be involved in direct interactions with the ligands. A driving force to this peculiar fold of the E II loop might be the presence of a disulfide bridge between cysteines in TM III and E II. Because this covalent link is conserved in all receptors modeled in the current study, the E II loop was modeled using a rhodopsin-like constrained geometry around the E II-TM III disulfide bridge. After the heavy atoms were modeled, all of the hydrogen atoms were added, and the protein coordinates were then minimized with AMBER (32Case D.A. Pearlman D.A. Caldwell J.W. Cheatham T.E. Ross W.S. Simmerling C.L. Darden T.A. Merz J.K.M. Stanton R.V. Cheng A.L. Vincent J.J. Crowley M. Ferguson D.M. Radmer R.J. Seibel G.L. Singh U.C. Weiner P.K. Kollman P.A. AMBER 5.0 computer program. University of California, San Francisco, CA1997Google Scholar) using the AMBER95 force field (33Cornel W.D. Cieplak P. Bayly C.I. Gould I.R. Merz J.K.M. Ferguson D.M. Spellmeyer D.M. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11497) Google Scholar). Topology and charge parameters for the nonpeptide V1a antagonist were computed using a previously described procedure (34Rognan D. Mukhija S. Folkers G. Zerbe O. J. Computer-Aided Mol. Design. 2001; 15: 103-115Crossref PubMed Scopus (7) Google Scholar). The minimizations were carried out by 1,000 steps of steepest descent followed by conjugate gradient minimization until the root mean square gradient of the potential energy was less than 0.05 kcal/mol·Å. A twin cut-off (10.0 and 15.0 Å) was used to calculate nonbonded electrostatic interactions at every minimization step, and the nonbonded pair list was updated every 25 steps. A distance-dependent (ϵ = 4r) dielectric function was used. Modeling the Antagonist-bound V1a Receptor Model—To obtain an “antagonist-bound” model of the V1a receptor, the above-described coordinates were refined by the following procedure. Conivaptan, a known V1a antagonist (35Yatsu T. Tomura Y. Tahara A. Wada K. Tsukada J. Uchida W. Tanaka A. Takenaka T. Eur. J. Pharmacol. 1997; 321: 225-230Crossref PubMed Scopus (77) Google Scholar) was first manually docked into the putative active site (21Cotte N. Balestre M.N. Aumelas A. Mahé E. Phalipou S. Morin D. Hibert M. Manning M. Durroux T. Barberis C. Mouillac B. Eur. J. Biochem. 2000; 267: 4253-4263Crossref PubMed Scopus (60) Google Scholar) so that its shape optimally fit in that of the binding pocket. The resulting protein-ligand complex was then refined by minimization using the above-described AMBER parameters. Removing the ligand atoms from the minimized complex finally yielded one set of coordinates for an antagonist-bound receptor model. Automated Docking of a Selective V1a Antagonist (SR49059)—The Gold 1.2 docking program (36Jones G. Wilett P. Glen R.C. Leach A.R. Taylor R. J. Mol. Biol. 1997; 267: 727-74837Crossref PubMed Scopus (5294) Google Scholar) was used to automatically dock the selective V1a antagonist SR49059. For each of the 10 independent genetic algorithm (GA) runs, a maximum number of 1,000 GA operations was performed on a single population of 50 individuals. Operator weights for cross-over, mutation, and migration were set to 100, 100, and 0, respectively. To allow poor nonbonded contacts at the start of each GA run, the maximum distance between hydrogen donors and fitting points was set to 5 Å, and nonbonded Van der Waals energies were cut off at a value equal to the kij well depth of the van der Waals energy for the atom pair i and j). To further speed up the calculation, the GA docking was stopped when the top three solutions were within 1.5 Å root mean square deviation. If this criterion is met, we can assume that these top solutions represent a reproducible pose for the ligand. Site-directed Mutagenesis of the Human V1aR—Point mutations F225C, Y300C, F308C, and K128C introduced in the cDNA sequence of the human V1aR vasopressin receptor were directly achieved on the eukaryotic expression vector pCMV (37Schall T.J. Lewis M. Koller K.J. Lee A. Rice G.C. Wong G.H.W. Gatanaga T. Granger G.A. Lentz R. Raab H. Kohr W.J. Goeddel D.V. Cell. 1990; 61: 361-370Abstract Full Text PDF PubMed Scopus (844) Google Scholar) using the QuikChange™ site-directed mutagenesis kit (Stratagene). All of the mutations were verified by direct dideoxynucleotide sequencing (T7™ sequencing kit; Amersham Biosciences). Cell Culture, Receptor Expression, and Membrane Preparations— The wild-type human V1aR and its mutants were transiently expressed in COS-7 cells or Chinese hamster ovary cells by electroporation as previously described (17Mouillac B. Chini B. Balestre M.N. Elands J. Trumpp-Kallmeyer S. Hoflack J. Hibert M. Jard S. Barberis C. J. Biol. Chem. 1995; 270: 25771-25777Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 22Phalipou S. Cotte N. Carnazzi E. Seyer R. Mahé E. Jard S. Barberis C. Mouillac B. J. Biol. Chem. 1997; 272: 26536-26544Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, the cells were suspended in electroporation buffer (107 cells/0.3 ml) and incubated with plasmid DNA (20 μg of carrier DNA and 1–4 μg of expression vector containing the mutant cDNA insert) for 10 min at room temperature before being pulsed (280 V, 950 microfarads; GeneZapper system, Kodak Scientific Imaging). After electroporation, the cells were plated in Petri dishes and grown in Dulbecco's modified Eagle's medium (Invitrogen) 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. To increase the level of expression of some mutants, the cells were treated overnight with 5 mm sodium butyrate (38Kassis S. Henneberry R.C. Fishman P.H. J. Biol. Chem. 1984; 259: 4910-4916Abstract Full Text PDF PubMed Google Scholar, 39Park C. Chamberlin M.E. Pan C.J. Chou J.Y. Biochemistry. 1996; 35: 9807-9814Crossref PubMed Scopus (12) Google Scholar). As already published, this treatment does not modify the pharmacological properties of the receptors (22Phalipou S. Cotte N. Carnazzi E. Seyer R. Mahé E. Jard S. Barberis C. Mouillac B. J. Biol. Chem. 1997; 272: 26536-26544Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 23Phalipou S. Seyer R. Cotte N. Breton C. Barberis C. Hibert M. Mouillac B. J. Biol. Chem. 1999; 274: 23316-23327Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Transfected cells were harvested 48–72 h after electroporation, and the membranes were prepared as already described (17Mouillac B. Chini B. Balestre M.N. Elands J. Trumpp-Kallmeyer S. Hoflack J. Hibert M. Jard S. Barberis C. J. Biol. Chem. 1995; 270: 25771-25777Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). In short, the 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. The supernatants were recovered and centrifuged at 44,000 × g for 20 min at 4 °C. The 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. The 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 as the standard. Aliquots of membranes were used immediately for binding assays or stored at –80 °C. Radioligand Binding Assays—The binding assays were performed at 30 °C using [125I]HO-LVA or [3H]AVP as the radioligands and 1–3 μg (for assays with [125I]HO-LVA) or 10–15 μg (for assays with [3H]AVP) of membrane proteins in standard radioligand saturation and competition binding assays as previously described (17Mouillac B. Chini B. Balestre M.N. Elands J. Trumpp-Kallmeyer S. Hoflack J. Hibert M. Jard S. Barberis C. J. Biol. Chem. 1995; 270: 25771-25777Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Briefly, the membranes were incubated in Buffer A supplemented with 1 mg/ml bovine serum albumin (binding buffer) and with radiolabeled and displacing ligands for 30 min (with [3H]AVP) or 60 min (with [125I]HO-LVA). Affinities (Kd) for [125I]HO-LVA (concentrations from 50 to 800 pm) as well as for [3H]AVP (concentrations from 0.1 to 20 nm) were directly determined in saturation experiments. Affinities (Ki) or apparent affinities (apparent Ki) for the unlabeled ligands were determined by competition experiments using [125I]HO-LVA (100–500 pm) or [3H]AVP (1–2 nm) as the radioligands. The concentration 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) or HO-LVA (400 nm). Bound and free radioactivity were separated by filtration over Whatman GF/C filters presoaked in a 10 mg/ml bovine serum albumin solution (with [3H]AVP) or in a 0.5% polyethylenimine solution (with [125I]HO-LVA) for 3–4 h. The ligand binding data were analyzed by nonlinear least squares regression using the computer program Ligand (40Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7771) Google Scholar). All of the assays were performed in triplicate on at least three separate batches of electroporated cells. Chemical Modification of the Mutant Receptors and Measure of Residual Radioligand Binding—To covalently and irreversibly label the cysteine mutants of the human V1aR, membrane preparations of COS-7 cells were first incubated with sulfydryl-modifying antagonist ligands (100 nm-10 μm), their nonreactive analogues (1–10 μm), or vehicle (Me2SO) in binding buffer for 30 min at 37 °C. The protein content was controlled before this treatment. Next, the membranes were pelleted at 21,000 × g for 20 min at 4 °C, washed, and centrifuged four times with binding buffer without the ligands. The protein content of the pelleted and washed membranes was checked again before the radioligand binding assays. Residual binding of V1aR was assessed by incubating the homogenate with different concentrations of [125I]HO-LVA (from 50 to 800 pm) or [3H]AVP (from 0.1 to 20 nm) depending on the mutant (saturation experiments). Nonspecific binding was determined in parallel assays using saturating concentrations of HO-LVA (400 nm) or AVP (10 μm). Protein concentrations used in the receptor irreversible labeling assays are not equivalent to the ones used in classical binding assays (1–3 μg for assays with [125I]HO-LVA and 10–15 μg for assays with [3H]AVP). Indeed, because several washes of the membranes are necessary for eliminating the irreversible ligand after preincubation of the receptors, more than half of the protein content is lost during this protocol. Thus, 25–40 μg of proteins are preincubated with the ligand when using the F225C mutant. Only 5–10 μg of proteins are preincubated with the irreversible ligand when using the wild-type V1a r" @default.
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• W1997758712 title "Identification of the Binding Sites of the SR49059 Nonpeptide Antagonist into the V1a Vasopressin Receptor Using Sulfydryl-reactive Ligands and Cysteine Mutants as Chemical Sensors" @default.
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