ErbB3 interacts with Hrs and is sorted to lysosomes for degradation.

The ErbB family of receptor tyrosine kinases mediates activation of a wide network of signaling pathways. ErbB3 has weak kinase activity, but its six docking sites for the p85 subunit of phosphoinositide 3-kinase make it an important contributor to proliferative signaling. ErbB3 has a relatively short half-life but the exact mechanisms controlling its turnover are unclear as contradictory reports exist. ErbB-mediated signaling is, however, negatively regulated by endocytosis of the receptors, followed by either recycling or degradation. Our previous studies showed that ErbB3 can be endocytosed and degraded in the absence of its ligand heregulin. However, binding of heregulin increased the degradation rate. In the current study we have investigated in more detail the trafficking and degradation of ErbB3 in the presence or absence of heregulin. We report that ErbB3 is internalized by clathrin-mediated endocytosis both in the presence and absence of heregulin. Moreover, we show that both proteasomal and lysosomal activity regulate ErbB3 degradation. Although steady-state expression of ErbB3 is regulated by proteasomal activity to a large extent, probably linked to a previously identified ER-localized quantity control, the results indicate that internalization, both constitutive and ligand-induced, causes lysosomal degradation of ErbB3. Furthermore, we show that ErbB3 interacts with the ESCRT-0 subunit Hrs both in the presence and absence of heregulin. This indicates an ESCRT-mediated sorting of ErbB3 to late endosomes and lysosomes, and in line with this we show that impaired ESCRT function leads to an endosomal accumulation of ErbB3.

Interaction with epsin 1 regulates the constitutive clathrin-dependent internalization of ErbB3.

BACKGROUND: In contrast to other members of the EGF receptor family, ErbB3 is constitutively internalized in a clathrin-dependent manner. Previous studies have shown that ErbB3 does not interact with the coated pit localized adaptor complex 2 (AP-2), and that ErbB3 lacks two AP-2 interacting internalization signals identified in the EGF receptor. Several other clathrin-associated sorting proteins which may recruit cargo into coated pits have, however, been identified, and the study was performed to identify adaptors needed for constitutive internalization of ErbB3. METHODS: A high-throughput siRNA screen was used to identify adaptor proteins needed for internalization of ErbB3. Upon knock-down of candidate proteins internalization of ErbB3 was identified using an antibody-based internalization assay combined with automatic fluorescence microscopy. RESULTS: Among 29 candidates only knock-down of epsin 1 turned out to inhibit ErbB3. Epsin 1 has ubiquitin interacting motifs (UIMs) and we show that ErbB3 interacts with an epsin 1 deletion mutant containing these UIMs. In support of an ErbB3-epsin 1 UIM dependent interaction, we show that ErbB3 is constitutively ubiquitinated, but that both ubiquitination and the ErbB3-epsin 1 interaction increase upon ligand binding. CONCLUSION: Altogether the results are consistent with a model whereby both constitutive and ligand-induced internalization of ErbB3 are regulated through interaction with epsin 1. GENERAL SIGNIFICANCE: Internalization is an important regulator of growth factor receptor mediated signaling and the current study identify mechanisms regulating plasma membrane turnover of ErbB3.