Ubiquitination in Control of EGF Receptor Endocytosis
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AbstractGrowth factors and growth factor receptors comprise a signaling network regulating cell differentiation and growth. Tightly regulation of this network is important to avoid excessive signaling and cell growth, which may result in cancer. It is therefore important to gain insight into how this network is regulated and into mechanisms of signal termination. Signaling from epidermal growth factor receptor (EGFR) is terminated by downregulating the receptor from the plasma membrane by clathrin-mediated endocytosis and subsequent lysosomal degradation.
The mechanisms involved in EGFR endocytosis are controversial. In this work we have investigated which adaptor proteins are important for EGFR endocytosis and whether the posttranslational modification termed ubiquitination is involved in EGFR endocytosis. We have shown that EGFR is concentrated in clathrin coated pits at the plasma membrane upon activation with EGF, and that the adaptor proteins Grb2 and Cbl are important for this process. Cbl mediates ubiquitination of activated EGFR, and we found that by inhibiting the activity of Cbl in different ways, EGFR was less effectively ubiquitinated and also less effectively concentrated in clathrin coated pits. We also studied whether the ubiquitin binding protein Epsin 1 could function as an adaptor for recruiting ubiquitinated EGFR into clathrin coated pits. We found that depleting cells of Epsin 1 by RNA interference reduced the recruitment of EGFR to clathrin coated pits, and consistently, EGFR endocytosis was inhibited. Using recombinant DNA-technology we covalently fused ubiquitin to EGFR to study whether ubiquitin could drive endocytosis in the absence of EGFR activation. This study indeed demonstrated that ubiquitin per se is sufficient to mediate endocytosis of EGFR from clathrin coated pits.
In conclusion, this work presents evidence supporting ubiquitination as a mechanism controlling clathrin-mediated endocytosis of EGFR.
List of papers
|Paper I Vibeke Bertelsen, Kamilla Breen, Kirsten Sandvig, Espen Stang, and Inger Helene Madshus. The Cbl-binding protein TULA inhibits dynamin-dependent endocytosis. Experimental Cell Research, Vol 313, 1696-1709, May 2007 The paper is not available in DUO. The published version is available at: http://dx.doi.org/10.1016/j.yexcr.2007.02.017|
|Paper II Espen Stang, Frøydis D. Blystad, Maja Kazazic, Vibeke Bertelsen, Tonje Brodahl, Camilla Raiborg, Harald Stenmark, and Inger Helene Madshus. Cbl-dependent ubiquitination is required for progression of EGF Receptors into clathrin-coated pits. Molecular Biology of the Cell, Vol 15, 3591-3604, August 2004. Molecular Biology of the Cell, Vol 15, 3591-3604, August 2004. The paper is not available in DUO. The published version is available at: http://dx.doi.org/10.1091/mbc.E04-01-0041|
|Paper III Maja Kazazic, Vibeke Bertelsen, Ketil Winther Pedersen, Tram Thu Vuong, Michael Vibo Grandal, Marianne Skeie Rødland, Linton M. Traub, Espen Stang, and Inger Helene Madshus. Epsin recruits ubiquitinated EGF receptors into clathrin-coated pits. Revised manuscript submitted (Traffic) The paper is not available in DUO.|
|Paper IV Vibeke Bertelsen, Malgorzata Magdalena Sak, Kamilla Breen, Espen Stang, and Inger Helene Madshus. Cbl and Ubiquitin promote translocation of EGF Receptor into clathrin-coated pits. Manuscript. The paper is not available in DUO.|