RECOMBINANT HUMAN G-PROTEIN-COUPLED LYSOPHOSPHATIDIC ACID RECEPTORS MEDIATE INTRACELLULAR CALCIUM MOBILIZATION

Mobilization of intracellular Ca2+ is a critical cellular response to lysophosphatidic acid (LPA) in many cell types. Recent identification of endothelial differentiation gene (Edg) 2 and Edg4 as subtypes of G protein-coupled receptors for LPA allowed examination of the Ca2+ mobi lization mediated specifically by each subtype. To reduce endogenous b ackground levels while enhancing recombinant receptor-specific signals , the aequorin luminescence method was used to quantify cytoplasmic Ca 2+ levels. In TAg-Jurkat T cells transiently co-transfected with apoae quorin and human Edg2 or Edg4 cDNA, LPA dose-dependently increased lig ht emission triggered by increased Ca2+ bound to aequorin. N-Palmitoyl -L-serine-phosphoric acid and N-palmitoyl-L-tyrosine-phosphoric acid, which had been previously shown to be antagonists for Xenopus laevis L PA receptors, did not antagonize the Ca2+-mobilizing effects of Edg2 a nd Edg4. Surprisingly, they acted as agonists or partial agonists for Edg2 and Edg4. The Ca2+ mobilization by Edg2 and Edg4 was further char acterized in stable transfectants of rat HTC4 hepatoma cells. By using the fura-2 fluorescence method, a difference in the kinetics of Ca2flux with Edg2 and Edg4 was observed. With Edg2, but not Edg4, the ini tial increase in the Ca2+ concentration was followed by a sustained in flux of extracellular Ca2+, The coincident production of inositol phos phates and the inhibition of Ca2+ mobilization by the phospholipase C inhibitor U73122 strongly suggested that Edg2 and Edg4 mobilize Ca2+ t hrough inositol trisphosphate generated by phospholipase C activation. Pertussis toxin almost completely blocked LPA-induced Ca2+ mobilizati on by Edg2 but only partially blocked that by Edg4, which suggests tha t Edg2 transduces Ca2+ mobilization largely through pertussis toxin-se nsitive G(i) proteins, whereas Edg4 requires both G(i) and G(q).