Study of G Protein-Coupled Receptor/β-arrestin Interactions Within Endosomes Using FRAP
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.
Key wordsBeta-arrestin G protein-coupled receptor Fluorescence recovery after photobleaching Confocal microscopy Yellow fluorescent protein
We are thankful to A-M. Fay and B. Zimmerman for their helpful comments and for critical reading of the manuscript. This work was supported by a Canadian Institutes of Health Research (CIHR) Operating Grant and a CIHR Confocal Maintenance Grant to S.A.L (MOP-74603 and PRG-82673, respectively). B.A. holds a Fellowship award from the McGill University Health Center Research Institute (MUHC-RI), which is a recognized Fonds de la Recherche en Santé du Québec (FRSQ) supported Institute. S.A.L. holds a Canada Research Chair in Molecular Endocrinology.
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