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J. Biol. Chem., Vol. 282, Issue 40, 29248-29255, October 5, 2007

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Identification of Residues Important for Agonist Recognition and Activation in GPR40^*

Chi Shing Sum, Irina G. Tikhonova, Susanne Neumann, Stanislav Engel, Bruce M. Raaka, Stefano Costanzi, and Marvin C. Gershengorn¹

From the Clinical Endocrinology Branch and the Laboratory of Biological Modeling, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

GPR40 was formerly an orphan G protein-coupled receptor whose endogenous ligands have recently been identified as free fatty acids (FFAs). The receptor, now named FFA receptor 1, has been implicated in the pathophysiology of type 2 diabetes and is a drug target because of its role in FFA-mediated enhancement of glucose-stimulated insulin release. Guided by molecular modeling, we investigated the molecular determinants contributing to binding of linoleic acid, a C18 polyunsaturated FFA, and GW9508, a synthetic small molecule agonist. Twelve residues within the putative GPR40-binding pocket including hydrophilic/positively charged, aromatic, and hydrophobic residues were identified and were subjected to site-directed mutagenesis. Our results suggest that linoleic acid and GW9508 are anchored on their carboxylate groups by Arg¹⁸³, Asn²⁴⁴, and Arg²⁵⁸. Moreover, His⁸⁶, Tyr⁹¹, and His¹³⁷ may contribute to aromatic and/or hydrophobic interactions with GW9508 that are not present, or relatively weak, with linoleic acid. The anchor residues, as well as the residues Tyr¹², Tyr⁹¹, His¹³⁷, and Leu¹⁸⁶, appear to be important for receptor activation also. Interestingly, His¹³⁷ and particularly His⁸⁶ may interact with GW9508 in a manner dependent on its protonation status. The greater number of putative interactions between GPR40 and GW9508 compared with linoleic acid may explain the higher potency of GW9508.

Received for publication, June 20, 2007 , and in revised form, August 14, 2007.

^* This work was supported by the Intramural Research Program of the NIDDK, National Institutes of Health. 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 To whom correspondence should be addressed: 50 South Dr., Rm. 4134, Bethesda, MD 20892. Tel.: 301-496-4128; Fax: 301-496-9943; E-mail: marving{at}intra.niddk.nih.gov.

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