Regulation by intracellular Ca 2+ and cyclic AMP of the growth factor-induced ruffling membrane formation and stimulation of fluid-phase endocytosis and exocytosis

Abstract

Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) induce formation of ruffling membranes [T. Kadowaki et al. (1986) J. Biol. Chem. 261, 16, 141–16, 147] and stimulate the fluid-phase endocytosis and exocytosis [Y. Miyata et al. (1988) Exp. Cell Res. 178, 73–83] in human epidermoid carcinoma KB cells. An increase in intracellular Ca 2+ concentration by treatment with A23187, a calcium ionophore, or an increase in intracellular cAMP level by treatment with dibutyryl cAMP or forskolin almost completely inhibited the insulin-, IGF-I-, or EGF-induced formation of ruffling membranes. Increases in Ca 2+ or cAMP concentration also inhibited almost completely the stimulation of fluid-phase endocytosis and exocytosis elicited by these growth factors. These results suggest that the growth factor-induced ruffling membrane formation and the stimulation of fluid-phase endocytosis and exocytosis have a common regulatory mechanism involving intracellular concentrations of Ca 2+ and cAMP. 125I-EGF binding assays and immunoprecipitation experiments with anti-phosphotyrosine antibody revealed that treatment of KB cells with A23187, dibutyryl cAMP, or forskolin did not inhibit the EGF binding to the cells nor subsequent tyrosine autophosphorylation of its receptors. These results indicate that Ca 2+- and /or cAMP-sensitive intracellular reactions exist downstream from the receptor kinase activation in the process of these early cellular responses.