Stimulation of high-affinity GTPase activity and cholera toxin-catalysed [32P]ADP-ribosylation of G(i) by lysophosphatidic acid (LPA) in wild-type and α2C10 adrenoceptor-transfected Rat 1 fibroblasts

Abstract

Lysophosphatidic acid (LPA) stimulated high-affinity GTPase activity in membranes of Rat 1 fibroblasts. This effect was dose-dependent, with maximal effects at 10 μM 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 α2C10 adrenoceptor. The α2 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 [32P]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 [32P]ADP-ribosylation of G(i) in membranes of the α2C10 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 α2C10 adrenoceptor are produced directly by the activation of G(i).