Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

Abstract

Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitininteracting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.

Clathrin-independent endocytosis of ubiquitinated cargos

“…These latter findings also indirectly stress the importance of Cbl-executed ubiquitination of other downstream endocytic proteins for proper clathrin endocytosis. A recent report by Stang et al (25), which shows that Cbl-dependent ubiquitination is required for progression of the EGFR into coated pits, further reinforces this notion. Taken together with these results, our findings suggest a mechanism through which ubiquitination of endocytic machinery, but not cargo ubiquitination, is required for internalization into coated pits.…”

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

“…We have found that ubiquitination of the EGFR is necessary for its recruitment into coated pits (Stang et al, 2004). It has been found that ubiquitination of the EGFR by Cbl can occur both via the direct interaction between the EGFR and Cbl and via indirect interactions through Grb2 (Waterman et al, 2002).…”

“…Epsin is localized to clathrin-coated pits (Stang et al, 2004;Hawryluk et al, 2006) and has been found to be involved in clathrin-mediated endocytosis Wendland et al, 1999). Whereas some authors have proposed that epsin functions as an adaptor in clathrin-mediated endocytosis (reviewed in Wendland, 2002), others have shown that epsin is involved in the formation of membrane curvature within clathrincoated pits.…”

“…Inhibition of EGFR ubiquitination by overexpression of the Cbl-binding protein Sprouty, known to inhibit Cbl's interactions with E2 enzymes (Fong et al, 2003), was found to block the progression of the EGFR into clathrin-coated pits and to block endocytosis of the EGFR (Stang et al, 2004). However, others have found that Cbl-mediated ubiquitination is required for EGFR degradation, but not for internalization (Duan et al, 2003).…”

Ubiquitin-Dependent Sorting in Endocytosis

“…de Melker et al 2004;Stang et al 2004;Sigismund et al 2005 membrane -cytosol interface (Jahnke et al 2012;Kajikawa et al 2012). Another major class of E3 ligases is the neural precursor-cell-expressed, developmentally down-regulated 4 (Nedd4) family, which contains a catalytic HECT (homologous E6-AP carboxy-terminal) domain (Rotin and Kumar 2009).…”