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

Yuko Fujiwara,Vineet Sardar,Akira Tokumura,Daniel Baker,Kimiko Murakami-Murofushi,Abby Parrill,Gabor Tigyi

doi:10.1074/jbc.m504351200

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 LPA(3) 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 LPA(1) 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 LPA(3), we developed and validated computational models of LPA(3) 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 LPA(3) form critical interactions with the polar headgroup of LPA. The models identified Leu-2.60 and Val-7.39 of LPA(3) 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.

Highlights

The radyl-lysoglycerophosphates include several naturally occurring analogs of lysophosphatidic acid (LPA)3 that elicit a multitude of cellular
They noted important differences in ligand preferences between the three receptors. These authors reported that LPA3 was unique in that it could only be activated by unsaturated acyl LPA and showed an centration for half population; EDG, endothelial differentiation gene; Emax, maximal effect; G protein-coupled receptors (GPCRs), G protein-coupled receptor; PA, phosphatidic acid; phosphate-buffered saline (PBS); phosphatebuffered saline; RH7777, rat hepatoma cell; SAR; structure-activity relationship; S1P, sphingosine 1-phosphate; TM, transmembrane domain; FACS, fluorescence-activated cell sorting; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate; PPAR␥, peroxisome proliferator-activated receptor ␥
Residues involved in phosphate recognition are conserved between LPA1 and LPA3 receptors, differences in the SAR between the two receptors [14] led us to hypothesize that residues positioned lower in the transmembrane domains might be involved in ligand recognition and selectivity

Summary

Introduction

The radyl-lysoglycerophosphates include several naturally occurring analogs of lysophosphatidic acid (LPA) that elicit a multitude of cellular. Bandoh et al [14] published the most comprehensive analysis to date of the LPA structure-activity relationship (SAR) using recombinant LPA receptors expressed through baculovirus transduction of Sf9 insect cells They noted important differences in ligand preferences between the three receptors. Our groups have previously examined S1P and LPA recognition of the S1P1, S1P4, and LPA1 EDG family receptors using computational modeling and site-directed mutagenesis (18 –21) These studies have identified two conserved positively charged residues, Arg-3.28 and Arg/ Lys-5.38 in the third and fifth transmembrane domain, respectively, that are required for ligand binding and receptor activation. Our results obtained from transient and stable transfection of LPA3 in RH7777 cells expressing each of the three LPA GPCRs in solutions buffered at pH 7.4 revealed substantial differences compared with results obtained in Sf9 cells in solutions buffered at pH 6.2 [4, 14] in that we could not establish an exquisite requirement for unsaturated fatty acid-containing LPA, and we found no regioisomeric preference for stabilized sn-2 over sn-1 analogs

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Conclusion