EGF receptor is a prototype ErbB receptor tyrosine kinase that plays essential physiological roles in mediating cell proliferation, differentiation and migration in response to external stimuli. Activation of ErbB receptors is linked to tumor progression, and their overactivity or overexpression is directly liked to human cancer. ErbB receptor activation also mediates cytoprotection from chemotherapy and radiation, and their inhibition represents an important strategy for radio- and chamo-sentization of tumors. Understanding the mechanisms that control the signaling potency of ErbB receptors is therefore a major goal in cancer biology. Ligand-induced downregulation, representing a balance between lysosomal degradation and recycling to cell surface, constitutes a major determinant of the signaling potency of ErbB receptors. Recent work by us and others has established the Cbl proto-oncoprotein as a crucial regulator of ErbB receptor downregulation. Cbl, a ubiquitin ligase, targets activated ErbB receptors for ubiquitination, which facilitates their lysosomal sorting. How Cbl-dependent ubiquitination functions as a lysosomal sorting signal for ErbB receptors is unknown. Based on recent studies, we hypothesize that human ESCRT-1 complex, which incorporates the TSG 101 tumor suppressor, mediates the Cbl- and ubiquitin-dependent ErbB sorting to lysosomes. Here, we will test this hypothesis using a well-characterized ErbB receptor, the EGF receptor. We will use Cbl-/- cells, Cbl-insensitive EGFR mutants, conditionally ubiquitin-deficient mutant cells and EGFR-ubiquitin chimeras to establish the role of Cbl-dependent ubiquitination in EGFR down-regulation. We will use two-color confocal immunofluorescence and electron microscopy to identify the endocytic compartment(s) where Cbl-dependent EGFR sorting occurs. We will demonstrate biochemical interaction and co-localization of EGFR and ESCRT-1 complex, and use over-expression and dominant-negative strategies to establish the requirement of ESCRT-1 for lysosomal sorting of EGFR. Finally, functional analyses will assess the role of ESCRT-1 complex in Cbl-mediated EGFR down-regulation. Insights gained from these studies should further our understanding or receptor tyrosine kinase down-regulation, and may identify newer targets to rationally design anti-cancer agents and to potentiate existing chemotherapeutic and radiation-based cancer cell killing, by countering receptor tyrosine kinase-mediated cytoprotection. ? ?
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