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 \u2013 together with the large variety of endocytic sorting signals \u2013 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

Threshold-controlled ubiquitination of the EGFR directs receptor fate

How the cell converts graded signals into threshold-activated responses is a question of great biological relevance. Here, we uncover a nonlinear modality of epidermal growth factor receptor (EGFR)-activated signal transduction, by demonstrating that the ubiquitination of the EGFR at the PM is threshold controlled. The ubiquitination threshold is mechanistically determined by the cooperative recruitment of the E3 ligase Cbl, in complex with Grb2, to the EGFR. This, in turn, is dependent on the simultaneous presence of two phosphotyrosines, pY1045 and either one of pY1068 or pY1086, on the same EGFR moiety. The dose-response curve of EGFR ubiquitination correlate precisely with the non-clathrin endocytosis (NCE) mode of EGFR internalization. Finally, EGFR-NCE mechanistically depends on EGFR ubiquitination, as the two events can be simultaneously re-engineered on a phosphorylation/ubiquitination-incompetent EGFR backbone. Since NCE controls the degradation of the EGFR, our findings have implications for how the cell responds to increasing levels of EGFR signalling, by varying the balance of receptor signalling and degradation/attenuation

USP9X Controls EGFR Fate by Deubiquitinating the Endocytic Adaptor Eps15

Following activation by its cognate ligand(s), the epidermal growth factor receptor (EGFR) is rapidly routed to the lysosome for degradation in a ubiquitination-dependent fashion. This pathway represents the major mechanism of long-term attenuation of EGFR signaling, and its deregulation is a significant feature in different types of cancers. Here we demonstrate, through a systematic RNAi-based approach, that several deubiquitinating (DUB) enzymes extend or decrease EGFR half-life upon EGF stimulation. We focus on USP9X, whose depletion severely affects EGFR turnover, interfering with its internalization and trafficking. We identify the endocytic protein Eps15 as one of the critical substrates of USP9X, and we map the Eps15 ubiquitination sites. We found that Eps15 monoubiquitination occurs already at minimal dose of EGF stimulation and is essential for EGFR internalization. Overall, our findings identify USP9X as a novel regulator of EGFR endocytosis and suggest a model whereby cycles of ubiquitination and deubiquitination events on endocytic accessory proteins may regulate the internalization and trafficking of the EGFR toward the lysosomes. Savio et al. undertook a siRNA screen to find 18 DUBs that affect EGFR degradation. They focus on USP9X, whose depletion significantly delayed EGFR internalization and trafficking. USP9X counteracts monoubiquitination of Eps15 that occurs at minimal EGF dose and is critical for EGFR internalization