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 inhibits clathrin-dependent endocytosis and disrupts high affinity binding of epidermal growth factor (EGF) to the EGF receptor (EGFR), it does not inhibit 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 overexpression of mutant dynamin. In cells not overexpressing mutant dynamin, the EGFR was rapidly internalized and deubiquitinated. In cells being endocytosis-deficient, due to overexpression of mutant dynamin, however, the EGFR was upon prolonged chase first found in deeply invaginated coated pits, and then eventually moved out of the coated pits and back onto the smooth plasma membrane. Polyubiquitination occurred equally efficiently in cells with or without intact clathrin-dependent endocytosis, while the kinetics of ubiquitination and deubiquitination was somewhat different. We further found that the EGF-induced ubiquitination of Eps15 occurred both in the absence and presence 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 polyubiquitination of the EGFR occurs at the plasma membrane.
Stang
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Institute of Pathology, University of Oslo, National Hospital, 0027 Oslo, Norway. espenst@ulrik.uio.no
Johannessen
L E
LE
Knardal
S L
SL
Madshus
I H
IH
D002462:Cell Membrane / Q000378:metabolism*; D034281:Dynamins; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D009154:Mutation; D015398:Signal Transduction; D014452:Ubiquitins
D008024:Ligands; D014452:Ubiquitins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020558:GTP Phosphohydrolases; D034281:Dynamins