In the present study, isolated pancreatic acinar membranes were used to investigate the mechanism of epidermal growth factor (EGF)-induced activation of phospholipase C (PLC). The data show that EGF caused a rapid and strong increase in tyrosine phosphorylation of the EGF receptor, with a maximum 5-15 s after the beginning of the incubation followed by a decline. With use of [H-3]phosphatidylinositol 4,5-bisphosphate as an exogenous substrate, PLC activity increased fourfold on exposure of the membranes to EGF (85 nM). In contrast, EGF-induced tyrosine phosphorylation of PLC-gamma(1) was rather small, indicating that tyrosine phosphorylation of PLC-gamma(1) is not proportional to changes in PLC activity. EGF-induced activation of PLC was strongly inhibited by pretreatment of the membranes with pertussis toxin, by an antibody raised against a COOH-terminal sequence shared by alpha-subunits of the inhibitory G proteins G(i1) and G(i2), and by an anti-PLC-gamma(1) antibody, whereas anti-G(i) alpha(3), anti-G(q/11)alpha, and anti-PLC-beta(1) antibodies had no effect. In contrast, pertussis toxin or the anti-G(i) alpha(1-2) antibody had no effect on EGF-induced tyrosine phosphorylation of PLC-gamma(1). EGF promoted association of G(i) proteins with both the EGF receptor and PLC-gamma(1) with similar kinetics as EGF-receptor autophosphorylation. All EGF-induced responses were abolished by the specific tyrosine kinase inhibitor pp60(v-src) (137-157), suggesting that EGF-receptor tyrosine kinase activity is essential for G(i1-2)-mediated activation of PLC-gamma(1). However, there was no evidence of tyrosine phosphorylation of G(i) alpha(1-2). Taken together, these data show that EGF causes activation of PLC-gamma(1) by a mechanism requiring activation of G(i1-2) and only a small increase in tyrosine phosphorylation of PLC-gamma(1).
