Stimulation of high-affinity GTPase activity and cholera toxin-catalysed [32P]ADP-ribosylation of Gi 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 microM 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 Gi-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 [32P]ADP-ribosylation of Gi. 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 Gi 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 Gi.
Carr
C
C
Department of Biochemistry, University of Glasgow, U.K.
Grassie
M
M
Milligan
G
G
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008246:Lysophospholipids / Q000494:pharmacology*; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D008246:Lysophospholipids; D011942:Receptors, Adrenergic, alpha; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins