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

Biochim Biophys Acta. 2016 Jun;1863(6 Pt A):1179-88. doi: 10.1016/j.bbamcr.2016.03.011. Epub 2016 Mar 11.

Abstract

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.

Keywords: Clathrin; Endocytosis; Epsin; ErbB3; Ubiquitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Vesicular Transport / genetics
  • Adaptor Proteins, Vesicular Transport / metabolism*
  • Clathrin / genetics
  • Clathrin / metabolism*
  • Endocytosis*
  • HeLa Cells
  • Humans
  • Immunoblotting
  • MCF-7 Cells
  • Microscopy, Confocal
  • Protein Binding
  • RNA Interference
  • Receptor, ErbB-3 / genetics
  • Receptor, ErbB-3 / metabolism*
  • Ubiquitination

Substances

  • Adaptor Proteins, Vesicular Transport
  • Clathrin
  • epsin
  • ERBB3 protein, human
  • Receptor, ErbB-3