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J. Biol. Chem., Vol. 283, Issue 18, 12175-12187, May 2, 2008

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Subtype-specific Residues Involved in Ligand Activation of the Endothelial Differentiation Gene Family Lysophosphatidic Acid Receptors^*

William J. Valentine, James I. Fells, Donna H. Perygin, Sana Mujahid, Kazuaki Yokoyama, Yuko Fujiwara, Ryoko Tsukahara, James R. Van Brocklyn^¶, Abby L. Parrill, and Gabor Tigyi¹

From the Department of Physiology, The University of Tennessee Health Science Center, Memphis, Tennessee 38163, the Department of Chemistry and Computational Research on Materials Institute, University of Memphis, Memphis, Tennessee 38152, and the ^¶Department of Pathology, Ohio State University, Columbus, Ohio 43210

Lysophosphatidic acid (LPA) is a ligand for three endothelial differentiation gene family G protein-coupled receptors, LPA_1–3. We performed computational modeling-guided mutagenesis of conserved residues in transmembrane domains 3, 4, 5, and 7 of LPA_1–3 predicted to interact with the glycerophosphate motif of LPA C18:1. The mutants were expressed in RH7777 cells, and the efficacy (E_max) and potency (EC₅₀) of LPA-elicited Ca²⁺ transients were measured. Mutation to alanine of R3.28 universally decreased both the efficacy and potency in LPA_1–3 and eliminated strong ionic interactions in the modeled LPA complexes. The alanine mutation at Q3.29 decreased modeled interactions and activation in LPA₁ and LPA₂ more than in LPA₃. The mutation W4.64A had no effect on activation and modeled LPA interaction of LPA₁ and LPA₂ but reduced the activation and modeled interactions of LPA₃. The R5.38A mutant of LPA₂ and R5.38N mutant of LPA₃ showed diminished activation by LPA; however, in LPA₁ the D5.38A mutation did not, and mutation to arginine enhanced receptor activation. In LPA₂, K7.36A decreased the potency of LPA; in LPA₁ this same mutation increased the E_max. In LPA₃, R7.36A had almost no effect on receptor activation; however, the mutation K7.35A increased the EC₅₀ in response to LPA 10-fold. In LPA_1–3, the mutation Q3.29E caused a modest increase in EC₅₀ in response to LPA but caused the LPA receptors to become more responsive to sphingosine 1-phosphate (S1P). Surprisingly micromolar concentrations of S1P activated the wild type LPA₂ and LPA₃ receptors, indicating that S1P may function as a weak agonist of endothelial differentiation gene family LPA receptors.

Received for publication, October 26, 2007 , and in revised form, February 15, 2008.

^* This work was supported by United States Public Health Service Grants HL61469 (to G. T.), CA921160 (to G. T.), HL084007 (to A. L. P.), and HL07641-19 (to W. J. V.) and by Southeast American Heart Association Postdoctoral Fellowship 0625325 (to Y. F.) and predoctoral fellowship (to J. I. F.). 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 and 2.

¹ To whom correspondence should be addressed: Dept. of Physiology, The University of Tennessee Health Science Center Memphis, 894 Union Ave., Memphis, TN 38163. Tel.: 901-448-4793; E-mail: gtigyi{at}physio1.utmem.edu.

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