Identification and Characterization of UBE2O as a New β‐arrestin Interactor

Abstract

β‐arrestin plays a major role in GPCR regulation and can act as a scaffolding protein for activation of non‐canonical signaling pathways. Thus, it is important to improve our knowledge of β‐arrestin interactors and the role of these protein complexes in GPCR regulation and signaling. The goal of this project was to identify new β‐arrestin interaction partners using the BioID proteomic approach. The BioID technique consists in the fusion of the biotin ligase protein BirA to β‐arrestin and exploits BirA capacity to biotinylate proteins in its proximity. The biotinylated proteins can then be isolated using streptavidin beads and identified by mass spectrometry.We identified 37 potential interactors for β‐arrestin 1 and 86 potential interactors for β‐arrestin 2 localized in different cellular compartments and involved in many different cellular functions and signaling pathways. One of the potential interactors identified is the atypical ubiquitin ligase UBE2O, which caught our attention due to the importance of ubiquitination in β‐arrestin and GPCR regulation. We confirmed the interaction between β‐arrestin 2 and UBE2O by co‐immunoprecipitation. UBE2O is known for its role in the monoubiquitination of BAP1, a tumor suppressor protein that interacts with chromatin‐associated proteins and regulates cell proliferation, resulting in its cytoplasmic sequestration. However, UBE2O function in β‐arrestin and GPCR signaling remains unknown. Considering that β‐arrestin shuttles between the cytoplasm and nucleus, UBE2O could play a role in the regulation of its cellular localization. Using different proteomic and biochemical approaches, we explored the function of UBE2O in GPCR and β‐arrestin ubiquitination and its potential effects on GPCR signaling and trafficking. We were able to identify by mass spectrometry UBE2O substrates involved in GPCR signaling and we showed that UBE2O affects β‐arrestin and GPCR trafficking by ebBRET (Enhanced bystander bioluminescence resonance energy transfer). Considering GPCRs physiological and therapeutic importance, this project will allow improvement of our understanding and knowledge on ubiquitination regulation of GPCR signaling and could contribute to revealing new signaling modalities that might have therapeutic relevance.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.