Polyubiquitination of the epidermal growth factor receptor occurs at the plasma membrane upon ligand-induced activation

We have previously shown that, although overexpression of mutant dynamin in hibits clathrin-dependent endocytosis and disrupts high affinity binding of epidermal growth factor (EGF) to the EGF receptor (EGFR), it does not inhi bit ligand-induced translocation of the EGFR into clathrin-coated pits. In the present study, we demonstrate that, upon ligand binding and incubation at 37 degrees C, the EGFR was polyubiquitinated regardless of overexpressio n of mutant dynamin, In cells not overexpressing mutant dynamin, the EGFR w as rapidly internalized and deubiquitinated, In cells being endocytosis-def icient, due to overexpression of mutant dynamin, however, the EGFR was upon prolonged chase first found in deeply invaginated coated pits, and then ev entually moved out of the coated pits and back onto the smooth plasma membr ane, Polyubiquitination occurred equally efficiently in cells with or witho ut intact clathrin-dependent endocytosis, while the kinetics of ubiquitinat ion and deubiquitination was somewhat different. We further found that the EGF-induced ubiquitination of Eps15 occurred both in the absence and presen ce of endocytosis with the same kinetics as polyubiquitination of the EGFR, but that the EGF-induced monoubiquitination of Eps15 was somewhat reduced upon overexpression of mutant dynamin, Our data show that EGF-induced polyu biquitination of the EGFR occurs at the plasma membrane.