Identification of transmembrane domain 1 & 2 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor Xavier Sainsily, Je ´ro ˆme Cabana, Brian J. Holleran, Emanuel Escher, Pierre Lavigne, Richard Leduc * Department of Pharmacology, Institut de Pharmacologie de Sherbrooke, Faculty of Medicine and Health Sciences, Universite ´ de Sherbrooke, Sherbrooke, Que ´bec, Canada J1H5N4 1. Introduction The cyclic undecapeptide urotensin-II (UII), initially isolated from the caudal neurosecretory cells of the Teleost goby fish [1], is described as the most potent vasoactive peptide in mammals [2]. UII binds the urotensin-II receptor (UT receptor), a member of family ‘‘A’’ of the G protein-coupled receptor (GPCR) superfamily [2]. Many structural features are associated with this family such as a short N-terminus, a highly conserved residue in each transmembrane domain (TM), a (D/E)R (3.50) Y ‘‘Ionic lock’’ motif [3] at the interface between the TM3 and the second intracellular loop (ICL), a C(W/F)xP (6.50) ‘‘rotamer toggle switch’’ motif [4] in TM6, a NP (7.50) xxY ‘‘tyrosine toggle switch’’ motif in TM7, and potential serine/threonine phosphorylation sites in the cyto- plasmic tail [5,6]. The UT receptor also has two cysteine residues which participate in disulfide bonding between the first and second extracellular loops (ECL) (Fig. 1). The UII/UT receptor system has been detected in the central nervous system, and is widely expressed in human tissues, including heart, liver, kidney and pancreas. This system is considered as a pharmacological target in the pathophysiology of hypertension, atherosclerosis, diabetes, heart failure and renal diseases [7–9]. The molecular mechanisms by which agonists bind to and activate GPCRs through conformational changes are not complete- ly understood. Although for many years the only available structural model of a GPCR was rhodopsin [10], recently the three-dimensional crystal structures of other GPCRs such as the b-adrenergic receptors [11,12], adenosine A2A receptor [13], chemokine receptors [14,15], serotonin receptors [16,17] and opioid receptors [18–22] have been determined. More recently, Biochemical Pharmacology 92 (2014) 280–288 A R T I C L E I N F O Article history: Received 19 June 2014 Accepted 21 August 2014 Available online 29 August 2014 Keywords: Urotensin-II G protein-coupled receptor UT receptor Substituted-cysteine accessibility method Molecular model A B S T R A C T 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. ß 2014 Elsevier Inc. All rights reserved. * Corresponding author. Tel.: +1 819 564 5413; fax: +1 819 564 5400. E-mail address: richard.leduc@usherbrooke.ca (R. Leduc). Contents lists available at ScienceDirect Biochemical Pharmacology jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/bio c hem p har m http://dx.doi.org/10.1016/j.bcp.2014.08.023 0006-2952/ß 2014 Elsevier Inc. All rights reserved.