Skip to main content
SpringerLink
Log in

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

  • Protocol
  • First Online:
Book cover High-Throughput Screening Assays in Toxicology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1473))

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Phizicky EM, Fields S (1995) Protein-protein interactions: methods for detection and analysis. Microbiol Rev 59(1):94–123

    CAS  PubMed  PubMed Central  Google Scholar 

  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):151–6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Pfleger KD, Eidne KA (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 3(3):165–74

    Article  CAS  PubMed  Google Scholar 

  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):138–52

    Article  CAS  PubMed  Google Scholar 

  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):10264–9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):3684–9

    CAS  PubMed  PubMed Central  Google Scholar 

  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):2702–9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bacart J et al (2008) The BRET technology and its application to screening assays. Biotechnol J 3(3):311–24

    Article  CAS  PubMed  Google Scholar 

  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):e30993

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):19012–7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Stoddart LA et al (2015) Application of BRET to monitor ligand binding to GPCRs. Nat Methods 12(7):661–3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):351–4

    Article  CAS  PubMed  Google Scholar 

  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):435–53

    Article  CAS  PubMed  Google Scholar 

  14. Salahpour A et al (2012) BRET biosensors to study GPCR biology, pharmacology, and signal transduction. Front Endocrinol 3:105

    Article  Google Scholar 

  15. Powell E et al (2010) Identification and characterization of a novel estrogenic ligand actinopolymorphol A. Biochem Pharmacol 80(8):1221–9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):463–75

    Article  CAS  PubMed  Google Scholar 

  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 3:100

    Article  Google Scholar 

  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):337–45

    Article  CAS  PubMed  Google Scholar 

  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 117:141–64

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):533–41

    Article  CAS  PubMed  Google Scholar 

  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 1:12

    Google Scholar 

  22. Machleidt T et al (2015) NanoBRET-A novel BRET platform for the analysis of protein-protein interactions. ACS Chem Biol 10(8):1797–804

    Article  CAS  PubMed  Google Scholar 

  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):1664–70

    Article  CAS  PubMed  Google Scholar 

  24. Loening AM, Wu AM, Gambhir SS (2007) Red-shifted Renilla reniformis luciferase variants for imaging in living subjects. Nat Methods 4(8):641–3

    Article  CAS  PubMed  Google Scholar 

  25. Giuliani G et al (2012) New red-shifted coelenterazine analogues with an extended electronic conjugation. Tetrahedron Lett 53(38):5114–5118

    Article  CAS  Google Scholar 

  26. Levi J et al (2007) Bisdeoxycoelenterazine derivatives for improvement of bioluminescence resonance energy transfer assays. J Am Chem Soc 129(39):11900–1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):7069–76

    Article  CAS  PubMed  Google Scholar 

  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:e021

    PubMed  PubMed Central  Google Scholar 

  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):797–816

    Article  CAS  PubMed  Google Scholar 

  30. Sievers CK et al (2013) Differential action of monohydroxylated polycyclic aromatic hydrocarbons with estrogen receptors α and β. Toxicol Sci 132(2):359–67

    Article  CAS  PubMed  Google Scholar 

  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):141–51

    Article  CAS  PubMed  Google Scholar 

  32. Deriziotis P et al (2014) Investigating protein-protein interactions in live cells using bioluminescence resonance energy transfer. J Vis Exp 87

    Google Scholar 

  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):801–7

    Article  CAS  PubMed  Google Scholar 

  34. De A et al (2013) Evolution of BRET biosensors from live cell to tissue-scale in vivo imaging. Front Endocrinol 4:131

    Article  Google Scholar 

  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):7175–83

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Oncology, University of Wisconsin-Madison, 7459 WIMR II, 1111 Highland Avenue, Madison, WI, 53705‐2275, USA

    Carlos Coriano & Wei Xu

  2. Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA

    Emily Powell

Authors
  1. Carlos Coriano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Emily Powell
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wei Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wei Xu .

Editor information

Editors and Affiliations

  1. Department of Chemistry Rutgers Center for Computational and Integrative Biology, Rutgers University, Camden, New Jersey, USA

    Hao Zhu

  2. National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland, USA

    Menghang Xia

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Coriano, C., Powell, E., Xu, W. (2016). Monitoring Ligand-Activated Protein–Protein Interactions Using Bioluminescent Resonance Energy Transfer (BRET) Assay. In: Zhu, H., Xia, M. (eds) High-Throughput Screening Assays in Toxicology. Methods in Molecular Biology, vol 1473. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6346-1_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6346-1_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6344-7

  • Online ISBN: 978-1-4939-6346-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation