Abstract

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⁺ mobilization mediated specifically by each subtype. To reduce endogenous background levels while enhancing recombinant receptor-specific signals, the aequorin luminescence method was used to quantify cytoplasmic Ca²⁺ levels. In TAg-Jurkat T cells transiently co-transfected with apoaequorin and human Edg2 or Edg4 cDNA, LPA dose-dependently increased light emission triggered by increased Ca²⁺ bound to aequorin.N-Palmitoyl-l-serine-phosphoric acid andN-palmitoyl-l-tyrosine-phosphoric acid, which had been previously shown to be antagonists for Xenopus laevis LPA receptors, did not antagonize the Ca²⁺-mobilizing effects of Edg2 and Edg4. Surprisingly, they acted as agonists or partial agonists for Edg2 and Edg4. The Ca²⁺ mobilization by Edg2 and Edg4 was further characterized in stable transfectants of rat HTC4 hepatoma cells. By using the fura-2 fluorescence method, a difference in the kinetics of Ca²⁺ flux with Edg2 and Edg4 was observed. With Edg2, but not Edg4, the initial increase in the Ca²⁺ concentration was followed by a sustained influx of extracellular Ca²⁺. The coincident production of inositol phosphates and the inhibition of Ca²⁺ mobilization by the phospholipase C inhibitor U73122 strongly suggested that Edg2 and Edg4 mobilize Ca²⁺ through inositol trisphosphate generated by phospholipase C activation. Pertussis toxin almost completely blocked LPA-induced Ca²⁺mobilization by Edg2 but only partially blocked that by Edg4, which suggests that Edg2 transduces Ca²⁺ mobilization largely through pertussis toxin-sensitive G_i proteins, whereas Edg4 requires both G_i and G_q.