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.
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 alpha-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.
authors.affiliation: Laboratoire de Pharmacochimie de la Communication Cellulaire, UMR 7081 CNRS, 74 Route du Rhin, 67401 Illkirch, France.
authors.forename: Chouaïb
authors.initials: C
authors.lastname: Tahtaoui
authors.forename: Marie-Noëlle
authors.initials: MN
authors.lastname: Balestre
authors.forename: Philippe
authors.initials: P
authors.lastname: Klotz
authors.forename: Didier
authors.initials: D
authors.lastname: Rognan
authors.forename: Claude
authors.initials: C
authors.lastname: Barberis
authors.forename: Bernard
authors.initials: B
authors.lastname: Mouillac
authors.forename: Marcel
authors.initials: M
authors.lastname: Hibert
mesh_terms: D000595:Amino Acid Sequence; D065092:Antidiuretic Hormone Receptor Antagonists; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D006727:Hormone Antagonists / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007211:Indoles / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011759:Pyrrolidines / Q000737:chemistry / Q000378:metabolism*; D017483:Receptors, Vasopressin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D065092:Antidiuretic Hormone Receptor Antagonists; D006727:Hormone Antagonists; D007211:Indoles; D008024:Ligands; D046911:Macromolecular Substances; D011759:Pyrrolidines; D017483:Receptors, Vasopressin; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C082134:relcovaptan; D003545:Cysteine