Epidermal growth factor receptor efficiently activates mitogen-activated protein kinase in HeLa cells and Hep2 cells conditionally defective in clathrin-dependent endocytosis

Epidermal growth factor (EGF)-induced signaling was investigated in cells c onditionally defective in clathrin-dependent endocytosis by overexpression of K44A dynamin in HeLa cells and potassium depletion in Hep2 cells, Overex pression of mutant dynamin disrupts high-affinity EGF-EGF receptor (EGFR) i nteraction (T. Ringerike, E. Stang, L. E. Johannessen, D. Sandnes, F. O. Le vy, and I. H. Madshus, 1998, J. Biol. Chem. 273, 16639-16642), However, the EGFR substrates Shc and c-CbI were as efficiently tyrosine phosphorylated in endocytosis-deficient HeLa cells exhibiting only low-affinity EGFRs as i n HeLa cells with intact endocytosis and with both high- and low-affinity E GFRs. Both Raf and mitogen-activated protein kinase (MAPK) were activated t o the same extent and with the same kinetics. HeLa cells distributed equall y in the cell cycle regardless of EGFR internalization. Upon potassium depl etion of Hep2 cells, EGF-induced EGFR endocytosis was inhibited. However, t he EGFR and MAPK were efficiently activated by EGF in both the absence and the presence of clathrin-dependent endocytosis. The EGFR was weakly tyrosin e phosphorylated by potassium depletion even in the absente of EGF, and thi s activation resulted in detectable activation of MAPK. Our results demonst rate that internalization of EG;FR by clathrin-dependent endocytosis is not required for activation of MAPK (C) 2000 Academic Press.