Identification of residues responsible for ligand recognition and regioisomeric selectivity of lysophosphatidic acid receptors expressed in mammalian cells
Abstract
The endothelial differentiation gene family encodes three highly homologous G protein-coupled receptors for lysophosphatidic acid (LPA). Based on baculoviral overexpression studies, differences have been proposed in the structure-activity relationship (SAR) of these receptors. We have compared the SAR of the individual receptors either overexpressed transiently at high or at lower levels following stable transfection in LPA-nonresponsive RH7777 cells. The SAR in transfected RH7777 cells was markedly different from that described in insect cells. The LPA3 receptor has been pro posed to be selectiveiy activated by unsaturated LPA species and shows a strong preference for sn-2 versus the sn-1 acyl-LPA regioisomer. Because of the short half-life of sn-2 LPA due to acyl migration under some conditions, we have synthesized acyl migration-resistant analogs using an acetyl group in place of the free hydroxyl group in order to evaluate LPA receptor SAR. Only LPA1 and LPA 2 showed regioisomeric preference and only for the 18:2 fatty acyl-stabilized LPA Sn-1 regioisomer. To identify residues involved in ligand recognition of LPA3, we developed and validated computational models of LPA3 complexes with the analogs studied. The models revealed that Arg-3.28 and Gln-3.29 conserved within the LPA-selective endothelial differentiation gene receptors and the more variable Lys-7.35 and Arg-5.38 Of LPA3 form critical interactions with the polar head-group of LPA. The models identified Leu-2.60 and Val-7.39of LPA3 underlying the regioisomer-selective interaction with the acetyl group of the stabilized regioisomers. Mutation of Leu-2.60 to alanine selectively increased the EC 50 Of the sn-2 acetyl-LPA regioisomers, whereas alanine replacement of Val-7.39 profoundly affected both regioisomers. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Publication Title
Journal of Biological Chemistry
Recommended Citation
Fujiwara, Y., Sardar, V., Tokumura, A., Baker, D., Murakami-Murofushi, K., Parrill, A., & Tigyi, G. (2005). Identification of residues responsible for ligand recognition and regioisomeric selectivity of lysophosphatidic acid receptors expressed in mammalian cells. Journal of Biological Chemistry, 280 (41), 35038-35050. https://doi.org/10.1074/jbc.M504351200
