STIMULATION OF HIGH-AFFINITY GTPASE ACTIVITY AND CHOLERA TOXIN-CATALYZED [P-32] ADP-RIBOSYLATION OF G(I) BY LYSOPHOSPHATIDIC ACID (LPA) IN WILD-TYPE AND ALPHA-2C10 ADRENOCEPTOR-TRANSFECTED RAT-1 FIBROBLASTS

Lysophosphatidic acid (LPA) stimulated high-affinity GTPase activity in membranes of Rat 1 fibroblasts. This effect was dose-dependent, with maximal effects at 10 mu M LPA, and was attenuated by pertussis toxin but not by cholera toxin pretreatment of the cells, indicating that the effect was likely to be produced by a G(i)-like G-protein. LPA stimulation of high-affinity GTPase was also observed in a clone of Rat 1 fibroblasts that had been transfected to express the human alpha 2C10 adrenoceptor. The alpha 2 adrenoceptor agonist UK14304 also stimulated high-affinity GTPase activity in membranes of these cells, but not in parental Rat 1 cells. LPA was also able to promote cholera toxin-catalysed [P-32]ADP-ribosylation of G(i). This effect of LPA was also prevented by pretreatment of the cells with pertussis toxin but not cholera toxin. LPA-stimulated cholera toxin-catalysed [P-32]ADP-ribosylation of G(i) in membranes of the alpha 2C10 adrenoceptor-expressing clone was additive with that produced by UK14304. Dose-response curves for LPA in the two assays of G-protein activation were coincident. The results presented herein demonstrate conclusively that the pertussis toxin-sensitive effects of LPA in Rat 1 fibroblasts and a clone of these cells expressing the alpha 2C10 adrenoceptor are produced directly by the activation of G(i).