EGF-dependent translocation of green fluorescent protein-tagged PLC-γ1 to the plasma membrane and endosomes

Growth factor-dependent translocation of phospholipase C-gamma1 (PLC-gamma1) was investigated using a green fluorescent protein-tagged PLC-gamma1 (PLC-gamma1-GFP) expressed in human epidermoid carcinoma A-431 cells. In the absence of growth factors, PLC-gamma1-GFP was present throughout the cytoplasm of A-431 cells. Treatment of the cells with epidermal growth factor (EGF) produced a very rapid redistribution of PLC-gamma1-GFP to the plasma membrane in a nonuniform manner. This translocation to the plasma membrane was insensitive to an inhibitor of phosphatidylinositol 3-kinase and was independent of cell adhesion. However, the translocation was disrupted by an agent which depolymerizes the actin cytoskeleton. At later times following the addition of EGF, PLC-gamma1-GFP appeared associated with intracellular vesicles. Stimulation of A-431 cells by Texas red-conjugated EGF for more than 10 min resulted in punctate intracellular PLC-gamma1-GFP distribution that colocalized with Texas red-conjugated EGF. This suggests that PLC-gamma1 is translocated to endosomes after EGF treatment, probably by associating with the internalized and autophosphorylated EGF receptor. Fractionation studies demonstrated that the EGF-induced plasma membrane-localized PLC-gamma1 is concentrated in caveolae microdomains. Disruption of caveolae with methyl-beta -cyclodextrin resulted in the ablation of EGF-induced, but not bradykinin-induced, mobilization of intracellular Ca2+. This treatment, however, only partially decreased PLC-gamma1 membrane translocation. (C) 2001 Academic Press.