Using BRET to Detect Ligand-Specific Conformational Changes in Preformed Signalling Complexes

  • Nicolas Audet
  • Graciela Piñeyro
Part of the Methods in Molecular Biology book series (MIMB, volume 756)


Bioluminescence energy transfer (BRET) has become a powerful tool to study protein–protein interactions and conformational changes among interacting proteins. In particular, BRET assays performed in living cells have revealed that heptahelical receptors (7TMRs), heterotrimeric G proteins and their proximal effectors form constitutive signalling complexes. BRET technology has also allowed us to demonstrate that these multimeric protein arrays remain intact throughout initial stages of receptor signalling, thus providing a platform for direct transmission of conformational information from activated receptors to downstream signalling partners. A clear example of the latter are the distinct intermolecular re-arrangements undergone by 7TMRs and G protein subunits following activation of the receptor by different ligands. Here we present protocols describing the type of BRET assay that has been used to reveal the existence of constitutive signalling arrays formed by 7TMRs and proximal signalling partners as well as the ability of complex components to undergo ligand-specific conformational changes.

Key words

G protein-coupled receptor Heterotrimeric guanine nucleotide-binding protein Bioluminescence resonance energy transfer Signal transduction Protein complex 



This work was supported by Discovery Grant from The National Sciences and Engineering Council of Canada (NSERC) to GP.


  1. 1.
    Yao, X., Parnot, C., Deupi, X., Ratnala, V. R., Swaminath, G., Farrens, D., and Kobilka, B. (2006) Coupling ligand structure to specific conformational switches in the beta2-adrenoceptor. Nat Chem Biol 2, 417–22.PubMedCrossRefGoogle Scholar
  2. 2.
    Granier, S., Kim, S., Shafer, A. M., Ratnala, V. R., Fung, J. J., Zare, R. N., and Kobilka, B. (2007) Structure and conformational changes in the C-terminal domain of the beta2-adrenoceptor: insights from fluorescence resonance energy transfer studies. J Biol Chem 282, 13895–905.PubMedCrossRefGoogle Scholar
  3. 3.
    Oldham, W. M., and Hamm, H. E. (2008) Heterotrimeric G protein activation by G-protein-coupled receptors. Nat Rev Mol Cell Biol 9, 60–71.PubMedCrossRefGoogle Scholar
  4. 4.
    Kapoor, N., Menon, S. T., Chauhan, R., Sachdev, P., and Sakmar, T. P. (2009) Structural evidence for a sequential release mechanism for activation of heterotrimeric G proteins. J Mol Biol 393, 882–97.PubMedCrossRefGoogle Scholar
  5. 5.
    Brinkerhoff, C. J., Traynor, J. R., and Linderman, J. J. (2008) Collision coupling, crosstalk, and compartmentalization in G-protein coupled receptor systems: can a single model explain disparate results? J Theor Biol 255, 278–86.PubMedCrossRefGoogle Scholar
  6. 6.
    Hein, P., and Bunemann, M. (2009) Coupling mode of receptors and G proteins. Naunyn Schmiedebergs Arch Pharmacol 379, 435–43.PubMedCrossRefGoogle Scholar
  7. 7.
    Citri, Y., and Schramm, M. (1980) Resolution, reconstitution and kinetics of the primary action of a hormone receptor. Nature 287, 297–300.PubMedCrossRefGoogle Scholar
  8. 8.
    Hebert, T. E., Gales, C., and Rebois, R. V. (2006) Detecting and imaging protein-protein interactions during G protein-mediated signal transduction in vivo and in situ by using fluorescence-based techniques. Cell Biochem Biophys 45, 85–109.PubMedCrossRefGoogle Scholar
  9. 9.
    Lohse, M. J., Nikolaev, V. O., Hein, P., Hoffmann, C., Vilardaga, J. P., and Bünemann, M. (2008) Optical techniques to analyze real-time activation and signaling of G-protein-coupled receptors. Trends Pharmacol Sci 29, 159–65.PubMedCrossRefGoogle Scholar
  10. 10.
    Pineyro, G. (2009) Membrane signalling complexes: implications for development of functionally selective ligands modulating heptahelical receptor signalling. Cell Signal 21, 179–85.PubMedCrossRefGoogle Scholar
  11. 11.
    Galés, C., Rebois, R. V., Hogue, M., Trieu, P., Breit, A., Hébert, T. E., and Bouvier, M. (2005) Real-time monitoring of receptor and G-protein interactions in living cells. Nat Methods 2, 177–84.PubMedCrossRefGoogle Scholar
  12. 12.
    Rebois, R. V., Robitaille, M., Galés, C., Dupré, D. J., Baragli, A., Trieu, P., Ethier, N., Bouvier, M., and Hébert, T. E. (2006) Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells. J Cell Sci 119, 2807–18.PubMedCrossRefGoogle Scholar
  13. 13.
    Audet, N., Galés, C., Archer-Lahlou, E., Vallières, M., Schiller, P. W., Bouvier, M., and Pineyro, G. (2008) Bioluminescence resonance energy transfer assays reveal ligand-specific conformational changes within preformed signaling complexes containing delta-opioid receptors and heterotrimeric G proteins. J Biol Chem 283, 15078–88.PubMedCrossRefGoogle Scholar
  14. 14.
    Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., and Bouvier, M. (2000) Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci U S A 97, 3684–9.PubMedGoogle Scholar
  15. 15.
    Milligan, G., and Bouvier, M. (2005) Methods to monitor the quaternary structure of G protein-coupled receptors. Febs J 272, 2914–25.PubMedCrossRefGoogle Scholar
  16. 16.
    Marullo, S., and Bouvier, M. (2007) Resonance energy transfer approaches in molecular pharmacology and beyond. Trends Pharmacol Sci 28, 362–5.PubMedCrossRefGoogle Scholar
  17. 17.
    Bouvier, M., Heveker, N., Jockers, R., Marullo, S., and Milligan, G. (2007) BRET analysis of GPCR oligomerization: newer does not mean better. Nat Methods 4, 3–4; author reply 4.Google Scholar
  18. 18.
    Salahpour, A., and Masri, B. (2007) Experimental challenge to a ‘rigorous’ BRET analysis of GPCR oligomerization. Nat Methods 4, 599–600; author reply 601.Google Scholar
  19. 19.
    Maehle, A. H. (2005) The quantification and differentiation of the drug receptor theory, c. 1910–1960. Ann Sci 62, 479–500.PubMedCrossRefGoogle Scholar
  20. 20.
    Colquhoun, D. (2006) The quantitative analysis of drug-receptor interactions: a short history. Trends Pharmacol Sci 27, 149–57.PubMedCrossRefGoogle Scholar
  21. 21.
    Roettger, B. F., Ghanekar, D., Rao, R., Toledo, C., Yingling, J., Pinon, D., and Miller, L. J. (1997) Antagonist-stimulated internalization of the G protein-coupled cholecystokinin receptor. Mol Pharmacol 51, 357–62.PubMedGoogle Scholar
  22. 22.
    Willins, D. L., and Meltzer, H. Y. (1998) Serotonin 5-HT2C agonists selectively inhibit morphine-induced dopamine efflux in the nucleus accumbens. Brain Res 781, 291–9.PubMedCrossRefGoogle Scholar
  23. 23.
    Azzi, M., Charest, P. G., Angers, S., Rousseau, G., Kohout, T., Bouvier, M., and Piñeyro, G. (2003) Beta-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors. Proc Natl Acad Sci U S A 100, 11406–11.PubMedCrossRefGoogle Scholar
  24. 24.
    Audet, N., Paquin-Gobeil, M., Landry-Paquet, O., Schiller, P. W., and Pineyro, G. (2005) Internalization and Src activity regulate the time course of ERK activation by delta opioid receptor ligands. J Biol Chem 280, 7808–16.PubMedCrossRefGoogle Scholar
  25. 25.
    Groer, C. E., Tidgewell, K., Moyer, R. A., Harding, W. W., Rothman, R. B., Prisinzano, T. E., and Bohn, L. M. (2007) An opioid agonist that does not induce micro-opioid receptor--arrestin interactions or receptor internalization. Mol Pharmacol 71, 549–57.PubMedCrossRefGoogle Scholar
  26. 26.
    Urban, J. D., Clarke, W. P., von Zastrow, M., Nichols, D. E., Kobilka, B., Weinstein, H., Javitch, J. A., Roth, B. L., Christopoulos, A., Sexton, P. M., Miller, K. J., Spedding, M., and Mailman, R. B. (2007) Functional selectivity and classical concepts of quantitative pharmacology. J Pharmacol Exp Ther 320, 1–13.PubMedCrossRefGoogle Scholar
  27. 27.
    Ehlert, F. J. (2008) On the analysis of ligand-directed signaling at G protein-coupled receptors. Naunyn Schmiedebergs Arch Pharmacol 377, 549–77.PubMedCrossRefGoogle Scholar
  28. 28.
    Kenakin, T. (2005) New concepts in drug discovery: collateral efficacy and permissive antagonism. Nat Rev Drug Discov 4, 919–27.PubMedCrossRefGoogle Scholar
  29. 29.
    Kenakin, T. (2007) Functional selectivity through protean and biased agonism: who steers the ship? Mol Pharmacol 72, 1393–401.PubMedCrossRefGoogle Scholar
  30. 30.
    Pfleger, K. D., 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, 337–45.PubMedCrossRefGoogle Scholar
  31. 31.
    Loening, A. M., Fenn, T. D., Wu, A. M., and Gambhir, S. S. (2006) Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output. Protein Eng Des Sel 19, 391–400.PubMedCrossRefGoogle Scholar
  32. 32.
    Kocan, M., See, H. B., Seeber, R. M., Eidne, K. A., and Pfleger, K. D. (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, 888–98.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Nicolas Audet
    • 1
    • 2
  • Graciela Piñeyro
    • 3
    • 2
  1. 1.Department of Pharmacology, Faculty of MedicineUniversity of MontrealMontrealCanada
  2. 2.Centre de Recherche du CHU Ste-Justine, BureauMontrealCanada
  3. 3.Department of Psychiatry, Faculty of MedicineUniversity of MontrealMontrealCanada

Personalised recommendations