MOLECULAR CHARACTERIZATION OF CLATHRIN-DEPENDENT EGFR ENDOCYTOSIS UNVEILS DISTINCT INTERNALIZATION MECHANISMS THAT COUPLE WITH DIFFERENT SIGNALING OUTPUTS

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that induces cell differentiation, proliferation and migration upon ligand activation. EGFR endocytosis is critical for the regulation of its signalling. Despite the enormous amount of work on this topic, the endocytic mechanisms that regulate EGFR signalling have not been completely elucidated. The EGFR can be endocytosed through either clathrin-mediated endocytosis (CME) or non-clathrin endocytosis (NCE), depending on ligand concentration. NCE of the EGFR is mainly associated with receptor degradation; CME, on the other hand, primarily leads to EGFR recycling and sustained signaling, although a minority of receptors is also delivered to degradation through this pathway. Recent proteomic and imaging studies have revealed a complex molecular portrait of CME with more than 30 proteins involved in the assembly and maturation of clathrin-coated pits. The involvement of such a wealth of proteins in CME – together with the large variety of endocytic sorting signals – raises the possibility of the existence of distinct types of clathrin-coated pits, specialized in terms of cargo-selection and intracellular fate. To investigate this possibility, we carried out a complete characterization of ligand-induced EGFR internalization and signaling, upon RNA interference of different endocytic adaptor proteins. Molecular genetics and biochemical assays were employed in this study allowing a comprehensive analysis of EGFR endocytosis. Our results indicate the existence of specialized clathrin adaptors involved in EGFR internalization, namely eps15, eps15R and epsin1, which function in parallel with AP2, previously considered to be the exclusive clathrin adaptor. Importantly, our data show that this molecular heterogeneity of EGFR clathrin vesicles ultimately impacts on EGF-dependent signaling and biological response. On the mechanistic level, we found that monoubiquitination of these adaptors is absolutely required for AP2-independent EGFR internalization through CME. Importantly, this is the first demonstration of a positive role of monoubiquitination in early EGFR internalization events.