Proximity Labeling to Identify β-Arrestin1 Binding Partners Downstream of Ligand-Activated G Protein-Coupled Receptors

beta-arrestins are multifaceted adaptor proteins that regulate various aspects of G protein-coupled receptor (GPCR) signaling. beta-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, beta-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 beta-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 beta-arrestin1 (beta arr1-APEX), which we show does not impact its ability to support agonist-stimulated internalization of GPCRs. By using coimmunoprecipitation, we show that beta arr1-APEX interacts with known interacting proteins. Furthermore, following agonist stimulation beta arr1-APEX labeled known beta arr1-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 beta-arrestin1, which we predict are novel ligand-stimulated beta arr1 interacting partners. Our study highlights that beta arr1-APEX-based proximity labeling represents a valuable approach to identifying novel players involved in GPCR signaling.