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J. Biol. Chem., Vol. 280, Issue 41, 35038-35050, October 14, 2005

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Identification of Residues Responsible for Ligand Recognition and Regioisomeric Selectivity of Lysophosphatidic Acid Receptors Expressed in Mammalian Cells^*

Yuko Fujiwara, Vineet Sardar¶1, Akira Tokumura||, Daniel Baker**¶¶, Kimiko Murakami-Murofushi, Abby Parrill¶, and Gabor Tigyi¶¶2

From the Departments of Physiology and Pharmaceutical Sciences, ^**Medicine, Vascular Biology, and Genomics/Bioinformatics, Centers of Excellence, and the ^¶¶ University of Tennessee Cancer Institute, University of Tennessee Health Science Center, Memphis, Tennessee 38152, the Department of Chemistry and ^¶Computational Research on Materials Institute at the University of Memphis, Memphis, Tennessee 38152, the ^||Faculty of Pharmaceutical Sciences, University of Tokushima, Tokushima, 770-8505, Japan, and the Department of Biology, Ochanomizu University, Bunkyo-ku, Tokyo, 112 Japan

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 proposed to be selectively 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 LPA₂ 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 headgroup of LPA. The models identified Leu-2.60 and Val-7.39 of LPA₃ underlying the regioisomer-selective interaction with the acetyl group of the stabilized regioisomers. Mutation of Leu-2.60 to alanine selectively increased the EC₅₀ of the sn-2 acetyl-LPA regioisomers, whereas alanine replacement of Val-7.39 profoundly affected both regioisomers.

Received for publication, April 20, 2005 , and in revised form, August 17, 2005.

^* This work was supported by United States Public Health Service Grants CA92160 and HL61469 (to G. T.) and American Heart Association Grants 50006N and 355199B (to A. P.). 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. 1-3.

¹ Present address: Pfizer Global Research and Development Groton Laboratories, Groton, CT 06340.

² To whom correspondence should be addressed: Dept. of Physiology, University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163. Tel.: 901-448-4793; Fax: 901-448-7126; E-mail: gtigyi{at}physio1.utmem.edu.

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