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Updated: May 29, 2026

Monitoring GPCR-β-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery
Published on: June 28, 2019
Study of G protein-coupled receptor/β-arrestin interactions within endosomes using FRAP
Benjamin Aguila1, May Simaan, Stéphane A Laporte
1Hormones and Cancer Research Unit, Department of Medicine, McGill University Health Center Research Institute, McGill University, Montréal, QC, Canada.
Abstract:
β-arrestins, through their scaffolding functions, are key regulators of G protein-coupled receptor (GPCR) signaling and intracellular trafficking. However, little is known about the dynamics of β-arrestin/receptor interactions and how these complexes, and complexes with other regulatory proteins, are controlled in cells. Here, we use yellow fluorescent protein (YFP)-tagged β-arrestin 2 and a fluorescence recovery after photobleaching (FRAP) imaging approach to probe the real-time interaction of β-arrestin with a GPCR, the bradykinin type 2 receptor (B2R). We provide a detailed protocol to assess the avidity of β-arrestin2-YFP for B2R within endosomes in HEK293 cells. β-arrestin2-YFP associated with internalized receptors is photobleached with intense light, and fluorescence recovery due to the entry of nonbleached β-arrestin2-YFP is monitored over time as a measure of the rate exchange of β-arrestin2-YFP within the endosome. This approach can be extended to other GPCR/β-arrestin complexes and their putative regulators to provide information about the kinetics of similar protein-protein interactions in cells. Moreover, these techniques should provide insight into the role of β-arrestins in the intracellular trafficking and signaling of GPCRs.
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