Monika Szymanska1, Anne Marthe Fosdahl1, Camilla Raiborg2, Markus Dietrich1, Knut Liestøl3, Espen Stang4, Vibeke Bertelsen5. 1. Department of Pathology, Oslo University Hospital, Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Oslo, Norway. 2. Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway; Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway. 3. Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway; Department of Informatics, University of Oslo, Oslo, Norway. 4. Department of Pathology, Oslo University Hospital, Oslo, Norway. Electronic address: espsta@rr-research.no. 5. Department of Pathology, Oslo University Hospital, Oslo, Norway.
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.
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.
Authors: Madhura Bhave; Rosa E Mino; Xinxin Wang; Jeon Lee; Heather M Grossman; Ashley M Lakoduk; Gaudenz Danuser; Sandra L Schmid; Marcel Mettlen Journal: Proc Natl Acad Sci U S A Date: 2020-11-30 Impact factor: 11.205
Authors: Natalya Pashkova; Lokesh Gakhar; Liping Yu; Nicholas J Schnicker; Annabel Y Minard; Stanley Winistorfer; Ivan E Johnson; Robert C Piper Journal: Elife Date: 2021-11-25 Impact factor: 8.140