Cb1-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits

Espen Stang, Frøydis D. Blystad, Maja Kazazic, Vibeke Bertelsen, Tonje Brodahl, Camilla Raiborg, Harald Stenmark, Inger Helene Madshus

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    123 Scopus citations

    Abstract

    Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cb1 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 Cb1 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 Cb1-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 Cb1-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.

    Original languageEnglish (US)
    Pages (from-to)3591-3604
    Number of pages14
    JournalMolecular Biology of the Cell
    Volume15
    Issue number8
    DOIs
    StatePublished - Aug 1 2004

    ASJC Scopus subject areas

    • Molecular Biology
    • Cell Biology

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  • Cite this

    Stang, E., Blystad, F. D., Kazazic, M., Bertelsen, V., Brodahl, T., Raiborg, C., Stenmark, H., & Madshus, I. H. (2004). Cb1-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits. Molecular Biology of the Cell, 15(8), 3591-3604. https://doi.org/10.1091/mbc.E04-01-0041