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J. Biol. Chem., Vol. 282, Issue 2, 1216-1224, January 12, 2007

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The Molecular Basis for Ligand Specificity in a Mouse Olfactory Receptor

A NETWORK OF FUNCTIONALLY IMPORTANT RESIDUES^*

Tatjana Abaffy¹, Arun Malhotra, and Charles W. Luetje

From the Departments of Molecular and Cellular Pharmacology and Biochemistry and Molecular Biology, University of Miami, Miami, Florida 33101

Sequence differences between members of the mouse olfac-tory receptor MOR42 subfamily (MOR42-3 and MOR42-1) are likely to be the basis for variation in ligand binding preference among these receptors. We investigated the specificity of MOR42-3 for a variety of dicarboxylic acids. We used site-directed mutagenesis, guided by homology modeling and ligand docking studies, to locate functionally important residues. Receptors were expressed in Xenopus oocytes and assayed using high throughput electrophysiology. The importance of the Val-113 residue, located deep within the receptor, was analyzed in the context of interhelical interactions. We also screened additional residues predicted to be involved in ligand binding site, based on comparison of ortholog/paralog pairs from the mouse and human olfactory receptor genomes (Man, O., Gilad, Y., and Lancet, D. (2004) Protein Sci. 13, 240–254). A network of 8 residues in transmembrane domains III, V, and VI was identified. These residues form part of the ligand binding pocket of MOR42-3. C12 dicarboxylic acid did not activate the receptor in our functional assay, yet our docking simulations predicted its binding site in MOR42-3. Binding without activation implied that C12 dicarboxylic acid might act as an antagonist. In our functional assay, C12 dicarboxylic acid did indeed act as an antagonist of MOR42-3, in agreement with molecular docking studies. Our results demonstrate a powerful approach based on the synergy between computational predictions and physiological assays.

Received for publication, October 3, 2006 , and in revised form, November 8, 2006.

^* This work was supported by National Institutes of Health Grants MH66038 (to C. W. L.) and GM69972 (to A. M.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4 and Table S1.

¹ To whom correspondence should be addressed: Dept. of Molecular and Cellular Pharmacology, University of Miami, P. O. Box 016189, Miami, FL 33101. Tel.: 305-243-4468; Fax: 305-243-4555; E-mail: tabaffy{at}med.miami.edu.

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