Elsevier

Biochemical Pharmacology

Volume 86, Issue 11, 1 December 2013, Pages 1584-1593
Biochemical Pharmacology

Identification of transmembrane domain 3, 4 & 5 residues that contribute to the formation of the ligand-binding pocket of the urotensin-II receptor

https://doi.org/10.1016/j.bcp.2013.09.015Get rights and content

Abstract

Urotensin-II (UII), a cyclic undecapeptide, selectively binds the urotensin-II receptor (UT receptor), a G protein-coupled receptor (GPCR) involved in cardiovascular effects and associated with numerous pathophysiological conditions including hypertension, atherosclerosis, heart failure, pulmonary hypertension and others. In order to identify specific residues in transmembrane domains (TM) three (TM3), four (TM4) and five (TM5) that are involved in the formation of the UT receptor binding pocket, we used the substituted-cysteine accessibility method (SCAM). Each residue in the F118(3.20) to S146(3.48) fragment of TM3, the L168(4.44) to G194(4.70) fragment of TM4 and the W203(5.30) to V232(5.59) fragment of TM5, was mutated, individually, to a cysteine. The resulting mutants were then expressed in COS-7 cells and subsequently treated with the positively charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA treatment resulted in a significant reduction in the binding of 125I-UII to TM3 mutants L126C(3.28), F127C(3.29), F131C(3.33) and M134C(3.36) and TM4 mutants M184C(4.60) and I188C(4.64). No loss of binding was detected following treatment by MTSEA for all TM5 mutants tested. In absence of a crystal structure of UT receptor, these results identify key determinants in TM3, TM4 and TM5 that participate in the formation of the UT receptor binding pocket and has led us to propose a homology model of the UT receptor.

Introduction

Urotensin-II (Glu-Thr-Pro-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val, UII) is a cyclic undecapeptide described as the most potent vasoconstrictor identified in mammals, firstly isolated from the caudal neurosecretery system of the Teleost fish [1]. UII signals through the urotensin-II receptor (UT receptor) detected in the central nervous system and widely expressed in human tissues, including the left atrium and ventricle of the heart, smooth muscle cells of the coronary artery and aorta, as well as endothelial cells from several vascular beds [2]. The UII/UT receptor system is considered as a pharmacological target in the pathophysiology of hypertension, heart failure, and cardiac fibrosis and hypertrophy [3], [4]. The UT receptor is a member of family ‘A’ of the larger G protein-coupled receptor (GPCR) superfamily [2]. Many features associated with this family such as a short N-terminus, a highly conserved residue in each transmembrane domain (TM), a D/ERY motif in the second intracellular loop, a CW/FxP ‘toggle switch’ motif [5] in TM6, a NPxxY motif in TM7, and potential serine/threonine phosphorylation sites in the cytoplasmic tail are found in the UT receptor [6].

The molecular mechanisms by which agonists bind to and activate GPCRs through conformational changes are not completely understood. Although for many years, the only available structural model of a GPCR was rhodopsin [7], recently the three-dimensional structures of other GPCRs such as the β adrenergic receptors [8], [9], adenosine A2A receptor [10], chemokine CXCR4 receptor [11], and opioid receptors [12], [13], [14] have been determined. These studies have enabled a better understanding of how diffusible ligands can recognize and interact with residues of the binding pocket of GPCRs.

Despite these major advances, many questions remain regarding the subtle variations found in different GPCR binding pockets. Hence, a variety of biophysical and biochemical approaches are needed to identify those key determinants that make-up the binding cavity. The substituted-cysteine accessibility method (SCAM) [15], [16], [17] is an ingenious approach for systematically identifying TM residues that contribute to the formation of the binding-site pocket of GPCRs. In this approach, consecutive residues within TMs are mutated to cysteine, one at a time and the mutant receptors are expressed in heterologous cells. If ligand binding to a cysteine-substituted mutant is unchanged compared to wild-type receptor, it is assumed that the structure of the mutant receptor, especially around the binding site, is similar to that of the wild-type receptor and that the substituted cysteine lies in an orientation similar to that of the residue of the wild-type receptor. In TMs, the sulfhydryls of cysteines oriented towards the aqueous binding-site pocket should react more quickly with charged sulfhydryl reagents like methanethiosulfonate-ethylammonium (MTSEA) than the sulfhydryls of cysteines that face the interior of the protein or the lipid bilayer. Two criteria are used to determine whether engineered cysteines are positioned at the surface of the binding-site pocket: (i) the reaction with the MTSEA reagent alters binding irreversibly and (ii) the reaction is retarded by the presence of the ligand. This approach has been used by us and others to identify residues that line the surface of GPCR binding-site pockets [18], [19], [20], [21], [22], [23], [24]. Indeed, using SCAM analysis, we have previously identified five MTSEA-sensitive residues in TM6 and TM7 of the rat UT receptor (rUT receptor) [23]. In this study, we report the application of SCAM to probe TM3, TM4 and TM5 of the rUT receptor.

Section snippets

Materials

Bovine serum albumin (BSA) and bacitracin were from Sigma–Aldrich (Oakville, ON, Canada). X-tremeGENE HP transfection reagent was from Roche Applied Science (Indianapolis, IN, USA). The sulfhydryl-specific alkylating reagent MTSEA (CH3SO2single bondSCH2CH2NH3+) was from Toronto Research Chemicals (Toronto, ON, Canada). Dulbecco's modified Eagle's medium (DMEM), Fetal Bovine Serum (FBS), phosphate-buffered saline (PBS), and penicillin/streptomycin were from Wisent Bioproduct (St-Bruno, QC, Canada).

Binding properties of mutant receptors with cysteines in TM3, TM4 and TM5

To identify residues in TM3, TM4 and TM5 that face the binding-site pocket of the rUT receptor, we mutated 28 consecutive residues between F118(3.20) and S146(3.48) of TM3, 27 consecutive residues between L168(4.44) and G194(4.70) of TM4 and 30 consecutive residues between W203(5.30) and V232(5.59) of TM5 to cysteine, one at a time (Fig. 1). Each mutant receptor was transiently expressed in COS-7 cells. To assess the conservation of the overall conformation of these receptors after the

Discussion

The rationale of this study, which relied on SCAM analyses, was to gain an insight into the orientation of TM3, TM4 and TM5 residues within the binding pocket of the rUT receptor. Using this approach, we had previously identified residues in TM6 and TM7 that participated in the formation of the rUT receptor binding pocket [23]. SCAM is based on the reactivity of engineered cysteines to MTSEA, a reagent that reacts 109 times faster with ionized cysteines than with the un-ionized thiols [17] and

Acknowledgement

This work was supported by a grant from the Canadian Institutes for Health Research (CIHR).

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      This approach has been used by us and others to identify residues that line the surface of GPCR binding-site pockets [29–37]. Indeed, using SCAM analysis, we have previously identified eleven MTSEA-sensitive residues in TM3, TM4, TM5, TM6 and TM7 of the rat UT receptor (rUT receptor) [34,36] that make up that receptor's binding pocket. In this study, we report the identification of residues within TM1 and TM2 that forms the UT binding pocket and we propose a complete molecular model of the ligand-binding cavity of the rUT receptor.

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