Study of G protein-coupled receptor/β-arrestin interactions within endosomes using FRAP

Methods Mol Biol. 2011;756:371-80. doi: 10.1007/978-1-61779-160-4_23.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arrestins / analysis
  • Arrestins / genetics
  • Arrestins / metabolism*
  • Cell Line
  • DNA, Complementary / genetics
  • Endosomes / metabolism*
  • Fluorescence Recovery After Photobleaching / methods*
  • Humans
  • Luminescent Proteins / analysis
  • Luminescent Proteins / genetics
  • Microscopy, Confocal / methods
  • Protein Interaction Mapping / methods*
  • Receptors, G-Protein-Coupled / analysis
  • Receptors, G-Protein-Coupled / metabolism*
  • Transfection
  • beta-Arrestin 2
  • beta-Arrestins

Substances

  • ARRB2 protein, human
  • Arrestins
  • DNA, Complementary
  • Luminescent Proteins
  • Receptors, G-Protein-Coupled
  • beta-Arrestin 2
  • beta-Arrestins