title: Identification of transmembrane domain 1 & 2 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor.
abstract: The vasoactive urotensin-II (UII), a cyclic undecapeptide widely distributed in cardiovascular, renal and endocrine systems, specifically binds the UII receptor (UT receptor), a G protein-coupled receptor (GPCR). The involvement of this receptor in numerous pathophysiological conditions including atherosclerosis, heart failure, hypertension, renal impairment and diabetes potentially makes it an interesting therapeutic target. To elucidate how UII binds the UT receptor through the identification of specific residues in transmembrane domains (TM) one (TM1) and two (TM2) that are involved in the formation of the receptor's binding pocket, we used the substituted-cysteine accessibility method (SCAM). Each residue of TM1 (V49((1.30)) to M76((1.57))) and TM2 (V88((2.41)) to H117((2.70))) was mutated, one by one, to a cysteine. The resulting mutants were then expressed in COS-7 cells and subsequently treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA treatment resulted in a significant binding inhibition of (125)I-UII to mutant I54C((1.35)) in TM1 and mutants Y100C((2.53)), S103C((2.56)), F106C((2.59)), I107C((2.60)), T110C((2.63)) and Y111C((2.64)) in TM2. These results identify key structural residues in TM1 and TM2 that participate in the formation of the UT receptor binding pocket. Together with previous SCAM analysis of TM3, TM4, TM5, TM6 and TM7, these results have led us to identify residues within all 7 TMs that participate in UT's binding pocket and have enabled us to propose a model of this receptor's orthosteric binding site.
authors.affiliation: Department of Pharmacology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H5N4.
authors.forename: Xavier
authors.initials: X
authors.lastname: Sainsily
authors.affiliation: Department of Pharmacology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H5N4.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Cabana
authors.affiliation: Department of Pharmacology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H5N4.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Holleran
authors.affiliation: Department of Pharmacology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H5N4.
authors.forename: Emanuel
authors.initials: E
authors.lastname: Escher
authors.affiliation: Department of Pharmacology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H5N4.
authors.forename: Pierre
authors.initials: P
authors.lastname: Lavigne
authors.affiliation: Department of Pharmacology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H5N4. Electronic address: richard.leduc@usherbrooke.ca.
authors.forename: Richard
authors.initials: R
authors.lastname: Leduc
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D019556:COS Cells; D002522:Chlorocebus aethiops; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D043562:Receptors, G-Protein-Coupled; C473142:Uts2r protein, rat
keywords: G protein-coupled receptor; Molecular model; Substituted-cysteine accessibility method; UT receptor; Urotensin-II