A chimeric pre-ubiquitinated EGF receptor is constitutively endocytosed in a clathrin-dependent, but kinase-independent manner.

The roles of EGF receptor (EGFR) kinase activity and ubiquitination in EGFR endocytosis have been controversial. The adaptor protein and ubiquitin ligase Cbl has reportedly been required. Consistently, we now report that siRNA-mediated knock-down of c-Cbl and Cbl-b significantly slowed clathrin-dependent internalization of activated wild-type (wt) EGFR by inhibiting recruitment of the EGFR to clathrin-coated pits. However, a chimeric protein consisting of wt-EGFR, a C-terminal linker and four linearly connected ubiquitins was found to interact with Eps15 and epsin 1 and to be constitutively endocytosed in a clathrin-dependent manner. Interestingly, endocytosis of this fusion protein did not require binding of EGF. Nor was kinase activity required, and the fusion protein was endocytosed in the presence of an EGFR kinase inhibitor, which efficiently counteracted tyrosine phosphorylation. This demonstrates that ubiquitination over-rides the requirement for kinase activity in recruitment of the EGFR to clathrin-coated pits.

Pertuzumab counteracts the inhibitory effect of ErbB2 on degradation of ErbB3.

Overexpression of ErbB2 and ErbB3 is found in several human cancers, and ErbB2-ErbB3 heterodimers are known as the most potent signaling units among ErbB dimers. While ErbB2 probably undergoes weak endocytosis, ErbB3 is readily internalized even in the absence of added ligand and without requirement for kinase activity. Overexpression of ErbB2 has been demonstrated to inhibit epidermal growth factor-induced internalization and degradation of epidermal growth factor receptor. This happens due to epidermal growth factor receptor-ErbB2 dimerization and can be counteracted by the anti-ErbB2 antibody pertuzumab, which binds the dimerization arm of ErbB2. Pertuzumab does also inhibit ErbB2-ErbB3 dimerization, but to what extent this has effect on constitutive and/or ligand-induced downregulation of ErbB3 is not known. In this study, we demonstrate that expression of ErbB2 as such did not block constitutive internalization of ErbB3, but that heregulin-induced degradation of ErbB3 was significantly slowed in cells expressing high levels of ErbB2. Incubation with pertuzumab did, however, counteract this effect. This indicates that the formation of ErbB2-ErbB3 heterodimers inhibits downregulation of ErbB3 and supports the notion that pertuzumab inhibits ErbB2 dimerization. The inhibitory effect of pertuzumab on ligand-induced ErbB2-ErbB3 heterodimerization was confirmed by the observation that pertuzumab inhibited heregulin-induced phosphorylation of ErbB3 in cells expressing ErbB2 and efficiently reduced heregulin-induced downstream signaling in cells expressing low levels of ErbB2. Altogether the results indicate that pertuzumab can be a valuable therapeutic agent not only in cancers overexpressing ErbB2 but also in cancers co-expressing ErbB2 and ErbB3.

The oncoprotein ErbB3 is endocytosed in the absence of added ligand in a clathrin-dependent manner.

The oncoprotein ErbB3 is overexpressed in several human cancers, for example in pancreatic adenocarcinoma and in ovarian cancers, and ErbB3-containing heterodimers have been demonstrated to be potent signaling units in carcinogenesis. This especially applies to ErbB2-ErbB3 and epidermal growth factor receptor (EGFR)-ErbB3 heterodimers providing anti-apoptotic signaling. Relatively little is understood about the signaling of EGFR-ErbB3 heterodimers and especially about mechanisms involved in downregulation of ErbB3 from the plasma membrane. This is in contrast to EGFR homodimers, for which trafficking has been extensively characterized. In the present study, we have investigated mechanisms involved in endocytosis of ErbB3 in porcine aortic endothelial cells stably expressing either ErbB3 only or stably expressing ErbB3 and EGFR. Our data show that ErbB3 is endocytosed in the absence of added ligand, independently of its tyrosine phosphorylation state and in a clathrin-dependent manner. Functional EGFR-ErbB3 heterodimers were observed to be formed, and dimerization with ErbB3 was observed to negatively affect endocytosis of the EGFR.