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Study of GPCR–Protein Interactions by BRET

  • Martina Kocan
  • Kevin D. G. PflegerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 746)

Abstract

Bioluminescence resonance energy transfer (BRET) has become an extremely valuable technology for the real-time study of protein–protein interactions in live cells. This technique is highly amenable to the monitoring of G protein-coupled receptor (GPCR)–protein interactions, especially involving scaffolding, regulatory and signaling proteins, such as β-arrestins, which are now known to have significant roles in addition to receptor desensitization. The BRET procedure utilizes heterologous coexpression of fusion proteins linking one protein of interest (e.g. a GPCR) to a bioluminescent donor enzyme, a variant of Renilla luciferase, and a second protein of interest (e.g. β-arrestin) to an acceptor fluorophore. If in close proximity, energy resulting from the rapid oxidation of a cell-permeable coelenterazine substrate by the donor will transfer to the acceptor, which in turn fluoresces at a longer characteristic wavelength. Therefore, the occurrence of such energy transfer implies that the proteins of interest fused to the donor and acceptor interact directly or as part of a complex. BRET detection can be carried out using scanning spectrometry or dual-filter luminometry. The latest improvements in BRET methodology have enabled live cell drug screening as well as monitoring of previously undetectable protein-protein complexes, including constitutive GPCR/β-arrestin interactions. Therefore, BRET is likely to play an increasingly important role in GPCR research and drug discovery over the coming years.

Key words

Bioluminescence resonance energy transfer G protein-coupled receptor Arrestin Renilla luciferase8 Rluc8 Fluorophore Venus 

Notes

Acknowledgments

KDGP’s work using the BRET methodology is funded by the National Health and Medical Research Council (NHMRC) of Australia (Project Grant #566736). KDGP is an Australian Research Council (ARC) Future Fellow (FT100100271).

References

  1. 1.
    Pfleger, K. D. G. and Eidne, K. A. (2006) Illuminating insights into protein–protein interactions using bioluminescence resonance energy transfer (BRET). Nat. Methods 3, 165–174.PubMedCrossRefGoogle Scholar
  2. 2.
    Pfleger, K. D. G., Seeber, R. M., and Eidne, K. A. (2006) Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein–protein interactions. Nat. Protoc. 1, 337–345.PubMedCrossRefGoogle Scholar
  3. 3.
    Kocan M., See H. B., Seeber R. M., Eidne K. A., and Pfleger K. D. G. (2008) Demonstration of improvements to the bioluminescence resonance energy transfer (BRET) technology for the monitoring of G protein-coupled receptors in live cells. J. Biomol. Screen. 13, 888–898.PubMedCrossRefGoogle Scholar
  4. 4.
    Milligan, G. and Bouvier, M. (2005) Methods to monitor the quaternary structure of G-protein-coupled receptors. FEBS J. 272, 2914–2925.PubMedCrossRefGoogle Scholar
  5. 5.
    Kocan M., See H. B., Sampaio N. G., Eidne K. A., Feldman B. J., and Pfleger K. D. G. (2009) Agonist-independent interactions between β-arrestins and mutant vasopressin type II receptors associated with nephrogenic syndrome of inappropriate antidiuresis. Mol. Endocrinol. 23, 559–571.PubMedCrossRefGoogle Scholar
  6. 6.
    Pfleger, K. D. G., Dromey, J. R., Dalrymple, M. B., Lim, E. M. L., Thomas, W. G., and Eidne, K. A. (2006) Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein–protein interactions in live cells. Cell. Signal. 18, 1664–1670.PubMedCrossRefGoogle Scholar
  7. 7.
    De, A., Loening, A. M., and Gambhir, S. S. (2007) An improved bioluminescence resonance energy transfer strategy for imaging intracellular events in single cells and living subjects. Cancer Res. 67, 7175–7183.PubMedCrossRefGoogle Scholar
  8. 8.
    De A., Ray P., Loening A. M., and Gambhir S. S. (2009) BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein–protein interactions from single live cells and living ­animals. FASEB J. 23, 2702–2709.PubMedCrossRefGoogle Scholar
  9. 9.
    Guo, W., Urizar, E., Kralikova, M., Mobarec, J. C., Shi, L., Filizola, M., and Javitch, J. A. (2008) Dopamine D2 receptors form higher order oligomers at physiological expression levels. EMBO J. 27, 2293–2304.PubMedCrossRefGoogle Scholar
  10. 10.
    Kamal, M., Marquez, M., Vauthier, V., Leloire, A., Froguel, P., Jockers, R., and Couturier, C. (2009) Improved donor/acceptor BRET couples for monitoring β-arrestin recruitment to G protein-coupled receptors. Biotechnol. J. 4, 1337–1344.PubMedCrossRefGoogle Scholar
  11. 11.
    Pfleger, K. D. G. and Eidne, K. A. (2003) New technologies: bioluminescence resonance energy transfer (BRET) for the detection of real time interactions involving G-protein ­coupled receptors. Pituitary 6, 141–151.PubMedCrossRefGoogle Scholar
  12. 12.
    Pfleger, K. D. G. and Eidne, K. A. (2005) Monitoring the formation of dynamic G ­protein-coupled receptor-protein complexes in living cells. Biochem. J. 385, 625–637.PubMedCrossRefGoogle Scholar
  13. 13.
    Hamdan, F. F., Audet, M., Garneau, P., Pelletier, J., and Bouvier, M. (2005) High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based β-arrestin2 recruitment assay. J. Biomol. Screen. 10, 463–475.PubMedCrossRefGoogle Scholar
  14. 14.
    Pfleger, K. D. G., Dalrymple, M. B., Dromey, J. R., and Eidne, K. A. (2007) Monitoring interactions between G-protein-coupled receptors and β-arrestins. Biochem. Soc. Trans. 35, 764–766.PubMedCrossRefGoogle Scholar
  15. 15.
    DeWire, S. M., Ahn, S., Lefkowitz, R. J., and Shenoy, S. K. (2007) β-arrestins and cell ­signaling. Annu. Rev. Physiol. 69, 483–510.PubMedCrossRefGoogle Scholar
  16. 16.
    Dromey, J. R. and Pfleger, K. D. G. (2008) G protein-coupled receptors as drug targets: The role of β-arrestins. Endocr. Metab. Immune Disord. Drug Targets 8, 51–61.PubMedCrossRefGoogle Scholar
  17. 17.
    Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba K., and Miyawaki, A. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90.PubMedCrossRefGoogle Scholar
  18. 18.
    Mercier, J. F., Salahpour, A., Angers, S., Breit, A., and Bouvier, M. (2002) Quantitative assessment of β1- and β2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J. Biol. Chem. 277, 44925–44931.PubMedCrossRefGoogle Scholar
  19. 19.
    Shaner, N. C., Campbell, R.E., Steinbach, P. A., Giepmans, B. N. G., Palmer, A. E., and Tsien, R. Y. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572.PubMedCrossRefGoogle Scholar
  20. 20.
    McVey, M., Ramsay, D., Kellett, E., Rees, S., Wilson, S., Pope, A. J., and Milligan, G. (2001) Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. J. Biol. Chem. 276, 14092–14099.PubMedGoogle Scholar
  21. 21.
    Zhang, J.-H., Chung, T. D. Y., and Oldenburg, K. R. (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73.PubMedCrossRefGoogle Scholar
  22. 22.
    Wu, P. and Brand, L. (1994) Resonance energy transfer: methods and applications. Anal. Biochem. 218, 1–13.PubMedCrossRefGoogle Scholar
  23. 23.
    Jaeger, W. C., Pfleger, K. D. G., and Eidne, K. A. Monitoring GPCR-protein complexes using bioluminescence resonance energy transfer, in G Protein Coupled Receptors: Essential Methods (Poyner, D. and Wheatley, M., ed.), John Wiley & Sons, Hoboken, NJ, 111–132.Google Scholar
  24. 24.
    Levi, J., De, A., Cheng, Z., and Gambhir, S. S. (2007) Bisdeoxycoelenterazine derivatives for improvement of bioluminescence resonance energy transfer assays. J. Am. Chem. Soc. 129, 11900–11901.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Laboratory for Molecular Endocrinology – GPCRs, Western Australian Institute for Medical Research (WAIMR) and Centre for Medical ResearchUniversity of Western AustraliaCrawleyAustralia

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