EGF-dependent translocation of green fluorescent protein-tagged PLC-gamma1 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 Ca(2+). This treatment, however, only partially decreased PLC-gamma1 membrane translocation.
Wang
X J
XJ
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, 37232-0146, USA.
Liao
H J
HJ
Chattopadhyay
A
A
Carpenter
G
G
D021941:Caveolae / Q000201:enzymology / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D021962:Membrane Microdomains / Q000201:enzymology / Q000378:metabolism; D051966:Phospholipase C gamma; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D010738:Type C Phospholipases; D051966:Phospholipase C gamma