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Study of G Protein-Coupled Receptor/β-arrestin Interactions Within Endosomes Using FRAP

  • Benjamin Aguila
  • May Simaan
  • Stéphane A. LaporteEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 756)

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.

Key words

Beta-arrestin G protein-coupled receptor Fluorescence recovery after photobleaching Confocal microscopy Yellow fluorescent protein 

Notes

Acknowledgments

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.

References

  1. 1.
    Overington, J. P., Al-Lazikani, B., and Hopkins A. L. (2006) How many drug targets are there? Nat Rev Drug Discov 5, 993–6.PubMedCrossRefGoogle Scholar
  2. 2.
    Lefkowitz, R. J. (1998) G protein-coupled receptors. iii. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization. J Biol Chem 273, 18677–80.PubMedCrossRefGoogle Scholar
  3. 3.
    Claing, A., Laporte, S. A., Caron, M. G., and Lefkowitz, R. J. (2002) Endocytosis of G protein-coupled receptors: Roles of G protein-coupled receptor kinases and beta-arrestin proteins. Prog Neurobiol 66, 61–79.PubMedCrossRefGoogle Scholar
  4. 4.
    Barak, L. S., Ferguson, S. S., Zhang, J., and Caron, M. G. (1997) A beta-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation. J Biol Chem 272, 27497–500.PubMedCrossRefGoogle Scholar
  5. 5.
    Zhang, J., Barak, L. S., Anborgh, P. H., Laporte, S. A., Caron, M. G., and Ferguson, S. S. (1999) Cellular trafficking of g protein-coupled receptor/beta-arrestin endocytic complexes. J Biol Chem 274, 10999–1006.PubMedCrossRefGoogle Scholar
  6. 6.
    Oakley, R. H., Laporte, S. A., Holt, J. A., Caron, M. G., and Barak, L. S. (2000) Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. J Biol Chem 275, 17201–10.PubMedCrossRefGoogle Scholar
  7. 7.
    Simaan, M., Bedard-Goulet, S., Fessart, D., Gratton, J. P., and Laporte, S. A. (2005) Dissociation of beta-arrestin from internalized bradykinin B2 receptor is necessary for receptor recycling and resensitization. Cell Signal 17, 1074–83.PubMedCrossRefGoogle Scholar
  8. 8.
    Gousseva, V., Simaan, M., Laporte, S. A., and Swain, P. S. (2008) Inferring the lifetime of endosomal protein complexes by fluorescence recovery after photobleaching. Biophys J 94, 679–87.PubMedCrossRefGoogle Scholar
  9. 9.
    Snapp, E. L., Altan, N., and Lippincott-Schwartz, J. (2003) Measuring protein mobility by photobleaching gfp chimeras in living cells. Curr Protoc Cell Biol Chapter 21: Unit 21.1.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Benjamin Aguila
    • 1
  • May Simaan
    • 1
  • Stéphane A. Laporte
    • 2
    Email author
  1. 1.Hormones and Cancer Research Unit, Department of Medicine, McGill University Health Center Research InstituteMcGill UniversityMontréalCanada
  2. 2.Hormones and Cancer Research Unit, Departments of Medicine and Pharmacology and Therapeutics, McGill University Health Center Research InstituteMcGill UniversityMontréalCanada

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