Abstract
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 (EC(50)) of LPA-elicited Ca(2+) 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 LPA(1) and LPA(2) more than in LPA(3). The mutation W4.64A had no effect on activation and modeled LPA interaction of LPA(1) and LPA(2) but reduced the activation and modeled interactions of LPA(3). The R5.38A mutant of LPA(2) and R5.38N mutant of LPA(3) showed diminished activation by LPA; however, in LPA(1) the D5.38A mutation did not, and mutation to arginine enhanced receptor activation. In LPA(2), K7.36A decreased the potency of LPA; in LPA(1) this same mutation increased the E(max). In LPA(3), R7.36A had almost no effect on receptor activation; however, the mutation K7.35A increased the EC(50) in response to LPA 10-fold. In LPA(1-3), the mutation Q3.29E caused a modest increase in EC(50) 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 LPA(2) and LPA(3) receptors, indicating that S1P may function as a weak agonist of endothelial differentiation gene family LPA receptors.
Highlights
Lysophosphatidic acid (LPA)2 and sphingosine 1-phosphate (S1P) are structurally related lysophospholipid growth factors that mediate a variety of cellular effects, including regulation of cellular proliferation, survival, migration, and morphology [1,2,3]
Theoretical Models of LPA1, LPA2, and LPA3: Mutation Site Selection—In LPA1–3, we evaluated the effect of alanine mutation of residues in TM3, TM4, TM5, and TM7 that were computationally predicted to impact binding to LPA C18:1
The number of interactions that were predicted to occur over distances of less than 4.5 Å between the polar head group of LPA and charged residues in TM3, TM5, and TM7 varied between the receptors and were two, two, and three for LPA1, LPA2, and LPA3, respectively
Summary
Reagents—All analogs of LPA and S1P were purchased from Avanti Polar Lipids (Alabaster, AL). The complex with the greatest number of cationic interactions with the LPA phosphate group was chosen and subjected to molecular dynamics simulations using a 1-fs time step at 500 ps. RH7777 cells were transfected with FLAG epitope-tagged LPA receptor constructs, replated after 16 h, and cultured for an additional 24 h. Receptor Activation Assays—FLAG-tagged LPA1, LPA2, and LPA3 receptor constructs were transiently expressed in LPAnonresponsive RH7777 cells using Effectene transfection reagent (Qiagen). The responses to LPA by the wild type and mutant receptors were measured and reported in terms of maximal activation (Emax) and efficacy (EC50) Ϯ S.D. The number of interactions that occur over distances of less than 4.5 Å between the polar head group of LPA and nitrogen atoms of charged residues in TM3, TM5, and TM7 was calculated based upon computational models. NA, not applicable; the residue to be mutated does not occur or that mutation was not made in that receptor
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