Identification of residues important for agonist recognition and activation in GPR40

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, Tyr ⁹¹, 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 Tyr ¹², Tyr⁹¹, His¹³⁷, and Leu¹⁸⁶, appear to be important for receptor activation also. Interestingly, His ¹³⁷ 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.