Monitoring Ligand-Activated Protein–Protein Interactions Using Bioluminescent Resonance Energy Transfer (BRET) Assay

  • Carlos Coriano
  • Emily Powell
  • Wei XuEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1473)


The bioluminescent resonance energy transfer (BRET) assay has been extensively used in cell-based and in vivo imaging systems for detecting protein–protein interactions in the native environment of living cells. These protein–protein interactions are essential for the functional response of many signaling pathways to environmental chemicals. BRET has been used as a toxicological tool for identifying chemicals that either induce or inhibit these protein–protein interactions. This chapter focuses on describing the toxicological applications of BRET and its optimization as a high-throughput detection system in live cells. Here we review the construction of BRET fusion proteins, describe the BRET methodology, and outline strategies to overcome obstacles that may arise. Furthermore, we describe the advantage of BRET over other resonance energy transfer methods for monitoring protein–protein interactions.

Key words

Bioluminescent Resonance Energy Transfer (BRET) Protein–protein interactions Screening assay Imaging assay 


  1. 1.
    Phizicky EM, Fields S (1995) Protein-protein interactions: methods for detection and analysis. Microbiol Rev 59(1):94–123PubMedPubMedCentralGoogle Scholar
  2. 2.
    Xu Y, Piston DW, Johnson CH (1999) A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci U S A 96(1):151–6CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Pfleger KD, Eidne KA (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 3(3):165–74CrossRefPubMedGoogle Scholar
  4. 4.
    Subramanian C et al (2006) A suite of tools and application notes for in vivo protein interaction assays using bioluminescence resonance energy transfer (BRET). Plant J 48(1):138–52CrossRefPubMedGoogle Scholar
  5. 5.
    Xu X et al (2007) Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues. Proc Natl Acad Sci U S A 104(24):10264–9CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Angers S et al (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(7):3684–9PubMedPubMedCentralGoogle Scholar
  7. 7.
    De A et al (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(8):2702–9CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Bacart J et al (2008) The BRET technology and its application to screening assays. Biotechnol J 3(3):311–24CrossRefPubMedGoogle Scholar
  9. 9.
    Powell E et al (2012) Identification of estrogen receptor dimer selective ligands reveals growth-inhibitory effects on cells that co-express ERα and ERβ. PLoS One 7(2):e30993CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Powell E, Xu W (2008) Intermolecular interactions identify ligand-selective activity of estrogen receptor alpha/beta dimers. Proc Natl Acad Sci U S A 105(48):19012–7CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Stoddart LA et al (2015) Application of BRET to monitor ligand binding to GPCRs. Nat Methods 12(7):661–3CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Boute N, Jockers R, Issad T (2002) The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol Sci 23(8):351–4CrossRefPubMedGoogle Scholar
  13. 13.
    Szidonya L, Cserzo M, Hunyady L (2008) Dimerization and oligomerization of G-protein-coupled receptors: debated structures with established and emerging functions. J Endocrinol 196(3):435–53CrossRefPubMedGoogle Scholar
  14. 14.
    Salahpour A et al (2012) BRET biosensors to study GPCR biology, pharmacology, and signal transduction. Front Endocrinol 3:105CrossRefGoogle Scholar
  15. 15.
    Powell E et al (2010) Identification and characterization of a novel estrogenic ligand actinopolymorphol A. Biochem Pharmacol 80(8):1221–9CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Hamdan FF et al (2005) High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay. J Biomol Screen 10(5):463–75CrossRefPubMedGoogle Scholar
  17. 17.
    Couturier C, Deprez B (2012) Setting up a bioluminescence resonance energy transfer high throughput screening assay to search for protein/protein interaction inhibitors in mammalian cells. Front Endocrinol 3:100CrossRefGoogle Scholar
  18. 18.
    Pfleger KD, Seeber RM, Eidne KA (2006) Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions. Nat Protoc 1(1):337–45CrossRefPubMedGoogle Scholar
  19. 19.
    Borroto-Escuela DO et al (2013) Bioluminescence resonance energy transfer methods to study G protein-coupled receptor-receptor tyrosine kinase heteroreceptor complexes. Methods Cell Biol 117:141–64CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Bertrand L et al (2002) The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS). J Recept Signal Transduct Res 22(1-4):533–41CrossRefPubMedGoogle Scholar
  21. 21.
    Kocan M et al (2010) Enhanced BRET technology for the monitoring of agonist-induced and agonist-independent interactions between GPCRs and β-arrestins. Front Endocrinol 1:12Google Scholar
  22. 22.
    Machleidt T et al (2015) NanoBRET-A novel BRET platform for the analysis of protein-protein interactions. ACS Chem Biol 10(8):1797–804CrossRefPubMedGoogle Scholar
  23. 23.
    Pfleger KD et al (2006) Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells. Cell Signal 18(10):1664–70CrossRefPubMedGoogle Scholar
  24. 24.
    Loening AM, Wu AM, Gambhir SS (2007) Red-shifted Renilla reniformis luciferase variants for imaging in living subjects. Nat Methods 4(8):641–3CrossRefPubMedGoogle Scholar
  25. 25.
    Giuliani G et al (2012) New red-shifted coelenterazine analogues with an extended electronic conjugation. Tetrahedron Lett 53(38):5114–5118CrossRefGoogle Scholar
  26. 26.
    Levi J et al (2007) Bisdeoxycoelenterazine derivatives for improvement of bioluminescence resonance energy transfer assays. J Am Chem Soc 129(39):11900–1CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Michelini E et al (2004) Development of a bioluminescence resonance energy-transfer assay for estrogen-like compound in vivo monitoring. Anal Chem 76(23):7069–76CrossRefPubMedGoogle Scholar
  28. 28.
    Koterba KL, Rowan BG (2006) Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET). Nucl Recept Signal 4:e021PubMedPubMedCentralGoogle Scholar
  29. 29.
    Lupien M et al (2007) Raloxifene and ICI182,780 increase estrogen receptor-alpha association with a nuclear compartment via overlapping sets of hydrophobic amino acids in activation function 2 helix 12. Mol Endocrinol 21(4):797–816CrossRefPubMedGoogle Scholar
  30. 30.
    Sievers CK et al (2013) Differential action of monohydroxylated polycyclic aromatic hydrocarbons with estrogen receptors α and β. Toxicol Sci 132(2):359–67CrossRefPubMedGoogle Scholar
  31. 31.
    Pfleger KD, Eidne KA (2003) New technologies: bioluminescence resonance energy transfer (BRET) for the detection of real time interactions involving G-protein coupled receptors. Pituitary 6(3):141–51CrossRefPubMedGoogle Scholar
  32. 32.
    Deriziotis P et al (2014) Investigating protein-protein interactions in live cells using bioluminescence resonance energy transfer. J Vis Exp 87Google Scholar
  33. 33.
    Borroto-Escuela DO et al (2010) Characterization of the A2AR-D2R interface: focus on the role of the C-terminal tail and the transmembrane helices. Biochem Biophys Res Commun 402(4):801–7CrossRefPubMedGoogle Scholar
  34. 34.
    De A et al (2013) Evolution of BRET biosensors from live cell to tissue-scale in vivo imaging. Front Endocrinol 4:131CrossRefGoogle Scholar
  35. 35.
    De A, Loening AM, Gambhir SS (2007) An improved bioluminescence resonance energy transfer strategy for imaging intracellular events in single cells and living subjects. Cancer Res 67(15):7175–83CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of OncologyUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Department of Experimental Radiation OncologyThe University of Texas MD Anderson Cancer CenterHoustonUSA

Personalised recommendations