title: Study of G protein-coupled receptor/β-arrestin interactions within endosomes using FRAP.
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.
authors.affiliation: Hormones and Cancer Research Unit, Department of Medicine, McGill University Health Center Research Institute, McGill University, Montréal, QC, Canada.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Aguila
authors.forename: May
authors.initials: M
authors.lastname: Simaan
authors.forename: Stéphane A
authors.initials: SA
authors.lastname: Laporte
mesh_terms: D000818:Animals; D019390:Arrestins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D011992:Endosomes / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D018613:Microscopy, Confocal / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000378:metabolism*; D014162:Transfection; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605254:ARRB2 protein, human; D019390:Arrestins; D018076:DNA, Complementary; D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins