Molecular mechanisms controlling endocytic downregulation of EGFR and ErbB3
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AbstractErbB proteins are a family of receptor tyrosine kinases that includes epidermal growth factor receptor (EGFR), ErbB2, ErbB3 and ErbB4. ErbB proteins govern many physiological processes during development and in an adult organism and their deregulated expression or signaling has been linked to many types of cancer. Downregulation of receptors by internalization and subsequent degradation is one of the mechanisms that regulate their activity. EGFR endocytosis is clathrin-dependent and controlled by ubiquitin ligase Cbl. ErbB protein activity can also be modulated therapeutically by tyrosine kinase inhibitors, or antibodies like for instance pertuzumab (PerjetaTM) that targets ErbB2 and inhibits its dimerization. This thesis aims at exploring molecular mechanisms regulating endocytic downregulation of ErbB proteins, how they influence each other with respect to signaling and downregulation, and how that can be experimentally modulated. Using molecular cloning, confocal microscopy and standard biochemical methods we studied endocytosis of EGFR and ErbB3 with respect to clathrin-dependence, signals for endocytosis and interactions with other ErbB family members in mammalian cells. We demonstrated that Cbl-mediated ubiquitination is important for the early steps of EGFR internalization and that ubiquitination can per se be a signal for EGFR endocytosis. We further observed that ErbB3 was endocytosed in a clathrin-dependent manner, but as opposed to EGFR, in the absence of added ligand and its kinase activity. Our data also demonstrated that overexpression of ErbB2 inhibits degradation of ErbB3 and this could be counteracted by pertuzumab that prevents ErbB2-ErbB3 dimerization. We also postulate that ErbB3, having marginal kinase activity, can form homodimers, but depends on heterodimerization for efficient signaling. Better understanding of those cellular processes provides foundation for advancement in cancer screening, diagnostics and treatment.
List of papers. The papers are removed due to copyright restrictions.
Paper I Vibeke Bertelsen, Malgorzata Magdalena Sak, Kamilla Breen, Marianne S. Rødland, Lene E. Johannessen, Linton M. Traub, Espen Stang, and Inger Helene Madshus A chimeric pre-ubiquitinated EGF Receptor is constitutively endocytosed in a clathrin-dependent, but kinase-independent manner. Traffic, Vol. 12(4), 507-520, April 2011. doi:10.1111/j.1600-0854.2011.01162.x
Paper II Malgorzata Magdalena Sak*, Kamilla Breen*, Sissel Beate Rønning, Nina Marie Pedersen, Vibeke Bertelsen, Espen Stang, and Inger Helene Madshus The oncoprotein ErbB3 is endocytosed in the absence of added ligand in a clathrin-dependent manner. Carcinogenesis, Vol. 33(5), 1031-1039, May 2012. doi:10.1093/carcin/bgs128
Paper III Malgorzata Magdalena Sak, Monika Szymanska, Vibeke Bertelsen, Max Hasmann, Espen Stang, and Inger Helene Madshus Pertuzumab counteracts the inhibitory effect of ErbB2 on degradation of ErbB3. Submitted (Carcinogenesis)