Epidermal growth factor activates phospholipase C-gamma 1 via G(i)1-2 proteins in isolated pancreatic acinar membranes.
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 [3H]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(i)1 and G(i)2, and by an anti-PLC-gamma 1 antibody, whereas anti-G(i) alpha 3, anti-Gq/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 pp60v-arc (137-157), suggesting that EGF-receptor tyrosine kinase activity is essential for G(i)1-2-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(i)1-2 and only a small increase in tyrosine phosphorylation of PLC-gamma 1.
Piiper
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A
II Medical Department, University of Frankfurt, Germany.
Stryjek-Kaminska
D
D
Zeuzem
S
S
D000818:Animals; D000906:Antibodies / Q000276:immunology; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D008566:Membranes / Q000201:enzymology; D010179:Pancreas / Q000201:enzymology*; D037342:Pertussis Toxin; D010766:Phosphorylation; D051381:Rats; D010738:Type C Phospholipases / Q000276:immunology / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000906:Antibodies; D007527:Isoenzymes; D010566:Virulence Factors, Bordetella; D014443:Tyrosine; D004815:Epidermal Growth Factor; D037342:Pertussis Toxin; D066246:ErbB Receptors; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins