Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits.
Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitin-interacting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.
Stang
Espen
E
Institute of Pathology, University of Oslo, 0027 Oslo, Norway.
Blystad
Frøydis D
FD
Kazazic
Maja
M
Bertelsen
Vibeke
V
Brodahl
Tonje
T
Raiborg
Camilla
C
Stenmark
Harald
H
Madshus
Inger Helene
IH
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D034281:Dynamins / Q000032:analysis; D004705:Endocytosis / Q000235:genetics / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000032:analysis / Q000502:physiology; D066246:ErbB Receptors / Q000032:analysis / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009154:Mutation / Q000235:genetics; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011990:Receptors, Transferrin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000032:analysis; D020703:ras GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011990:Receptors, Transferrin; C485561:SPRY2 protein, human; D014452:Ubiquitins; D033921:Vesicular Transport Proteins; C114154:epsin; D020703:ras GTPase-Activating Proteins; D004815:Epidermal Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; D034281:Dynamins; C067419:CBL protein, human