EPIDERMAL GROWTH-FACTOR INDUCES RAPID AND TRANSIENT ASSOCIATION OF PHOSPHOLIPASE C-GAMMA-1 WITH EGF-RECEPTOR AND FILAMENTOUS ACTIN AT MEMBRANE RUFFLES OF A431 CELLS

Addition of epidermal growth factor to A431 cells results in dramatic changes in cell morphology, Initially the cells form membrane ruffles accompanied by increased actin polymerization, followed by cell roundi ng, Activation of the tyrosine kinase of the receptor by binding epide rmal growth factor leads also to phosphorylation and activation of pho spholipase C-gamma 1, a key enzyme in the phosphoinositide pathway, In this study we have investigated the localization of phospholipase C-g amma 1 during cell activation by epidermal growth factor. It is shown that addition of the growth factor to A431 cells leads to a translocat ion of phospholipase C-gamma 1 from the cytosol to the membrane fracti on, Interestingly, this relocation is exclusively directed to the memb rane ruffles, Most of the phospholipase C-gamma 1 associates to the me mbrane and a small fraction to the underlying skeleton, Immunocytochem ical studies demonstrated that phospholipase C-gamma 1 co-localizes wi th the epidermal growth factor receptor and also filamentous actin at the membrane ruffles, Moreover, using anti-phosphotyrosine antibodies we found that the membrane ruffles are significantly enriched in phosp hotyrosyl proteins, Between 5 and 10 minutes after stimulation the mem brane ruffles disappear and also the co-localization of phospholipase C-gamma 1 with the epidermal growth factor receptor and filamentous ac tin, These results support the notion that activation of A431 cells by epidermal growth factor leads to the formation of a signalling comple x of its receptor, phospholipase C-gamma 1 and filamentous actin which is primarily localized at membrane ruffles.