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Proximity Labeling to Identify β-Arrestin1 Binding Partners Downstream of Ligand-Activated G Protein-Coupled Receptors.
- Source :
-
International Journal of Molecular Sciences . Feb2023, Vol. 24 Issue 4, p3285. 18p. - Publication Year :
- 2023
-
Abstract
- β-arrestins are multifaceted adaptor proteins that regulate various aspects of G protein-coupled receptor (GPCR) signaling. β-arrestins are recruited to agonist-activated and phosphorylated GPCRs at the plasma membrane, thereby preventing G protein coupling, while also targeting GPCRs for internalization via clathrin-coated pits. In addition, β-arrestins can activate various effector molecules to prosecute their role in GPCR signaling; however, the full extent of their interacting partners remains unknown. To discover potentially novel β-arrestin interacting partners, we used APEX-based proximity labeling coupled with affinity purification and quantitative mass spectrometry. We appended APEX in-frame to the C-terminus of β-arrestin1 (βarr1-APEX), which we show does not impact its ability to support agonist-stimulated internalization of GPCRs. By using coimmunoprecipitation, we show that βarr1-APEX interacts with known interacting proteins. Furthermore, following agonist stimulation βarr1-APEX labeled known βarr1-interacting partners as assessed by streptavidin affinity purification and immunoblotting. Aliquots were prepared in a similar manner and analyzed by tandem mass tag labeling and high-content quantitative mass spectrometry. Several proteins were found to be increased in abundance following GPCR stimulation. Biochemical experiments confirmed two novel proteins that interact with β-arrestin1, which we predict are novel ligand-stimulated βarr1 interacting partners. Our study highlights that βarr1-APEX-based proximity labeling represents a valuable approach to identifying novel players involved in GPCR signaling. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 16616596
- Volume :
- 24
- Issue :
- 4
- Database :
- Academic Search Index
- Journal :
- International Journal of Molecular Sciences
- Publication Type :
- Academic Journal
- Accession number :
- 162140869
- Full Text :
- https://doi.org/10.3390/ijms24043285