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J. Biol. Chem., Vol. 282, Issue 24, 17405-17412, June 15, 2007

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Systematic Analysis of the Entire Second Extracellular Loop of the V_1a Vasopressin Receptor

KEY RESIDUES, CONSERVED THROUGHOUT A G-PROTEIN-COUPLED RECEPTOR FAMILY, IDENTIFIED^*

From the School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom

The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V_1a vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe¹⁸⁹, Trp²⁰⁶, Phe²⁰⁹, and Tyr²¹⁸ are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp²⁰⁶ and Phe²⁰⁹ project into the binding crevice. Indeed, Phe²⁰⁹ is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe¹⁸⁹ and Tyr²¹⁸, located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues.

Received for publication, March 13, 2007

^* This work was supported by grants (to M. W.) from the Wellcome Trust and the Biotechnology and Biological Sciences and Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Novartis Pharma AG, WSJ-386.9.59, CH-4002 Basel, Switzerland.

² Present address: Dept. of Pharmacology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia.

³ Present address: Institute of Life Sciences, University of Wales, Swansea SA2 8PP, United Kingdom.

⁴ To whom correspondence should be addressed. Tel.: 44-121-414-3981; Fax: 44-121-414-5925; E-mail: m.wheatley{at}bham.ac.uk.

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