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 (UlMs) 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. (C) 2016 Elsevier B.V. All rights reserved.