Proximity Labeling to Identify β-Arrestin1 Binding Partners Downstream of Ligand-Activated G Protein-Coupled Receptors. Int J Mol Sci 2023 Feb 07;24(4)
Date
02/26/2023Pubmed ID
36834700Pubmed Central ID
PMC9967311DOI
10.3390/ijms24043285Scopus ID
2-s2.0-85148853084 (requires institutional sign-in at Scopus site) 2 CitationsAbstract
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.
Author List
Zhuo Y, Robleto VL, Marchese AAuthor
Adriano Marchese PhD Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
ArrestinsLigands
Receptors, G-Protein-Coupled
beta-Arrestin 1
beta-Arrestin 2
beta-Arrestins
