Unique ligand selectivity of the GPR92/LPA5 lysophosphatidate receptor indicates role in human platelet activation

Abstract

Lysophosphatidic acid (LPA) is a ligand for LPA1-3 of the endothelial differentiation gene family G-protein-coupled receptors, and LPA4-8 is related to the purinergic family G-protein-coupled receptor. Because the structure-activity relationship (SAR) of GPR92/LPA5 is limited and whether LPA is its preferred endogenous ligand has been questioned in the literature, in this study we applied a combination of computational and experimental site-directed mutagenesis of LPA5 residues predicted to interact with the headgroup of LPA. Four residues involved in ligand recognition in LPA5 were identified as follows: R2.60N mutant abolished receptor activation, whereas H4.64E, R6.62A, and R7.32A greatly reduced receptor activation. We also investigated the SAR of LPA5 using LPA analogs and other non-lysophospholipid ligands. SAR revealed that the rank order of agonists is alkyl glycerol phosphate > LPA > farnesyl phosphates ≫ N-arachidonoylglycine. These results confirm LPA5 to be a bona fide lysophospholipid receptor. We also evaluated several compounds with previously established selectivity for the endothelial differentiation gene receptors and found several that are LPA5 agonists. A pharmacophore model of LPA5 binding requirements was developed for in silico screening, which identified two non-lipid LPA5 antagonists. Because LPA5 transcripts are abundant in human platelets, we tested its antagonists on platelet activation and found that these non-lipid LPA5 antagonists inhibit platelet activation. The present results suggest that selective inhibition of LPA5 may provide a basis for future anti-thrombotic therapies. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.