Regulation of Rac and Cdc42 Pathways by Gi during Lysophosphatidic Acid-induced Cell Spreading

Abstract

The pertussis toxin-sensitive G protein, Gi, has been implicated in lysophosphatidic acid-induced cell mitogenesis and migration, but the mechanisms remain to be detailed. In the present study, we found that pertussis toxin blocks lysophosphatidic acid-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. This prevention of cell spreading was eliminated by the expression of constitutively active mutants of Rho family small GTP-binding proteins, Rac and Cdc42, but not by Rho. In addition, activation of the endogenous forms was suppressed by pertussis toxin, indicating that G i-induced cell spreading is mediated through the Rac and Cdc42 pathway. Transfection of constitutively active mutants of Gα1 and Gα11 and Gβγ subunits enhanced spreading of pertussis toxin-treated cells. Gβ1 with Gγ12, a major Gγ form in fibroblasts, was more effective for increasing cell spreading than Gβ1γ2 or Gβ1 plus Gγ1.2S2A, a mutant in which Ser-2, a phosphorylation site for protein kinase C, is replaced with alanine. In addition, a protein kinase C inhibitor diminished Gβ1γ12-induced cell spreading, suggesting a role for phosphorylation of the protein. These findings indicate that both Gαi and Gβγ stimulate Rac and Cdc42 pathways with lysophosphatidic acid-induced cell spreading on fibronectin.