Abstract
Bioluminescence resonance energy transfer (BRET) is a straightforward biophysical technique for studying protein-protein interactions. It requires: (1) that proteins of interest and suitable controls be labeled with either a donor or acceptor molecule, (2) placement of these labeled proteins in the desired environment for assessing their potential interaction, and (3) use of suitable detection instrumentation to monitor resultant energy transfer. There are now several possible applications, combinations of donor and acceptor molecules, potential assay environments and detection system perturbations. Therefore, this review aims to demystify and clarify the important aspects of the BRET methodology that should be considered when using this technique.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Wu, P. & Brand, L. Resonance energy transfer: methods and applications. Anal. Biochem. 218, 1–13 (1994).
Eidne, K.A., Kroeger, K.M. & Hanyaloglu, A.C. Applications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells. Trends Endocrinol. Metab. 13, 415–421 (2002).
Pfleger, K.D.G. & Eidne, K.A. Monitoring the formation of dynamic G protein–coupled receptor-protein complexes in living cells. Biochem. J. 385, 625–637 (2005).
Milligan, G. & Bouvier, M. Methods to monitor the quaternary structure of G protein–coupled receptors. FEBS J. 272, 2914–2925 (2005).
Germain-Desprez, D., Bazinet, M., Bouvier, M. & Aubry, M. Oligomerization of transcriptional intermediary factor 1 regulators and interaction with ZNF74 nuclear matrix protein revealed by bioluminescence resonance energy transfer in living cells. J. Biol. Chem. 278, 22367–22373 (2003).
Boute, N., Jockers, R. & Issad, T. The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol. Sci. 23, 351–354 (2002).
Selvin, P.R. The renaissance of fluorescence resonance energy transfer. Nat. Struct. Biol. 7, 730–734 (2000).
Rizzo, M.A. & Piston, D.W. High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy. Biophys. J. 88, L14–L16 (2005).
Xu, Y., Kanauchi, A., von Arnim, A.G., Piston, D.W. & Johnson, C.H. Bioluminescence resonance energy transfer: monitoring protein-protein interactions in living cells. Methods Enzymol. 360, 289–301 (2003).
Maurel, D. et al. Cell surface detection of membrane protein interaction with homogeneous time-resolved fluorescence resonance energy transfer technology. Anal. Biochem. 329, 253–262 (2004).
Terrillon, S. et al. Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis. Mol. Endocrinol. 17, 677–691 (2003).
Ayoub, M.A. et al. Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. J. Biol. Chem. 277, 21522–21528 (2002).
Yamakawa, Y., Ueda, H., Kitayama, A. & Nagamune, T. Rapid homogeneous immunoassay of peptides based on bioluminescence resonance energy transfer from firefly luciferase. J. Biosci. Bioeng. 93, 537–542 (2002).
Xu, Y., Piston, D.W. & Johnson, C.H. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc. Natl. Acad. Sci. USA 96, 151–156 (1999).
Paulmurugan, R. & Gambhir, S.S. Monitoring protein-protein interactions using split synthetic Renilla luciferase protein-fragment-assisted complementation. Anal. Chem. 75, 1584–1589 (2003).
Liu, J. & Escher, A. Improved assay sensitivity of an engineered secreted Renilla luciferase. Gene 237, 153–159 (1999).
Jensen, A.A., Hansen, J.L., Sheikh, S.P. & Brauner-Osborne, H. Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET). Eur. J. Biochem. 269, 5076–5087 (2002).
Ormo, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).
Angers, S. et al. Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. USA 97, 3684–3689 (2000).
Koshimizu, T.A., Tsujimoto, G., Hirasawa, A., Kitagawa, Y. & Tanoue, A. Carvedilol selectively inhibits oscillatory intracellular calcium changes evoked by human alpha1D- and alpha1B-adrenergic receptors. Cardiovasc. Res. 63, 662–672 (2004).
Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).
Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).
Hamdan, F.F., Audet, M., Garneau, P., Pelletier, J. & Bouvier, M. 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, 463–475 (2005).
Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. & Tsien, R.Y. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276, 29188–29194 (2001).
Arai, R., Nakagawa, H., Kitayama, A., Ueda, H. & Nagamune, T. Detection of protein-protein interaction by bioluminescence resonance energy transfer from firefly luciferase to red fluorescent protein. J. Biosci. Bioeng. 94, 362–364 (2002).
Tannous, B.A., Kim, D.E., Fernandez, J.L., Weissleder, R. & Breakefield, X.O. Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol. Ther. 11, 435–443 (2005).
Pfleger, K.D.G. & Eidne, K.A. New technologies: bioluminescence resonance energy transfer (BRET) for the detection of real time interactions involving G-protein coupled receptors. Pituitary 6, 141–151 (2003).
Gales, C. et al. Real-time monitoring of receptor and G protein–interactions in living cells. Nat. Methods 2, 177–184 (2005).
Hanyaloglu, A.C., Seeber, R.M., Kohout, T.A., Lefkowitz, R.J. & Eidne, K.A. Homo- and hetero-oligomerization of thyrotropin-releasing hormone (TRH) receptor subtypes. Differential regulation of beta-arrestins 1 and 2. J. Biol. Chem. 277, 50422–50430 (2002).
Kroeger, K.M., Hanyaloglu, A.C., Seeber, R.M., Miles, L.E. & Eidne, K.A. Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. Detection in living cells using bioluminescence resonance energy transfer. J. Biol. Chem. 276, 12736–12743 (2001).
Bertrand, L. et al. 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, 533–541 (2002).
Mercier, J.F., Salahpour, A., Angers, S., Breit, A. & Bouvier, M. Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J. Biol. Chem. 277, 44925–44931 (2002).
Issafras, H. et al. Constitutive agonist-independent CCR5 oligomerization and antibody-mediated clustering occurring at physiological levels of receptors. J. Biol. Chem. 277, 34666–34673 (2002).
Gomes, I., Filipovska, J., Jordan, B.A. & Devi, L.A. Oligomerization of opioid receptors. Methods 27, 358–365 (2002).
Breit, A., Lagace, M. & Bouvier, M. Hetero-oligomerization between β2- and β3-adrenergic receptors generates a β-adrenergic signaling unit with distinct functional properties. J. Biol. Chem. 279, 28756–28765 (2004).
De, A. & Gambhir, S.S. Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescence resonance energy transfer. FASEB J. 19, 2017–2019 (2005).
Ayoub, M.A., Levoye, A., Delagrange, P. & Jockers, R. Preferential formation of MT1/MT2 melatonin receptor heterodimers with distinct ligand interaction properties compared with MT2 homodimers. Mol. Pharmacol. 66, 312–321 (2004).
Wilson, S., Wilkinson, G. & Milligan, G. The CXCR1 and CXCR2 receptors form constitutive homo- and heterodimers selectively and with equal apparent affinities. J. Biol. Chem. 280, 28663–28674 (2005).
Couturier, C. & Jockers, R. Activation of the leptin receptor by a ligand-induced conformational change of constitutive receptor dimers. J. Biol. Chem. 278, 26604–26611 (2003).
Veatch, W. & Stryer, L. The dimeric nature of the gramicidin A transmembrane channel: conductance and fluorescence energy transfer studies of hybrid channels. J. Mol. Biol. 113, 89–102 (1977).
Devost, D. & Zingg, H.H. Homo- and hetero-dimeric complex formations of the human oxytocin receptor. J. Neuroendocrinol. 16, 372–377 (2004).
Perroy, J., Pontier, S., Charest, P.G., Aubry, M. & Bouvier, M. Real-time monitoring of ubiquitination in living cells by BRET. Nat. Methods 1, 203–208 (2004).
Hu, C.D. & Kerppola, T.K. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat. Biotechnol. 21, 539–545 (2003).
Ramsay, D., Kellett, E., McVey, M., Rees, S. & Milligan, G. Homo- and hetero-oligomeric interactions between G protein–coupled receptors in living cells monitored by two variants of bioluminescence resonance energy transfer (BRET): hetero-oligomers between receptor subtypes form more efficiently than between less closely related sequences. Biochem. J. 365, 429–440 (2002).
Pfleger, K.D.G. et al. Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells. Cell. Signal. (in the press).
Cormack, B.P., Valdivia, R.H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).
Acknowledgements
We acknowledge U. Schmidt for providing spectral data, M. Dalrymple, R. Seeber and G. Pfleger for proofreading the manuscript, and L. Coleman for assistance with manuscript preparation. The authors' work using the BRET methodology is funded by the National Health and Medical Research Council (NHMRC) of Australia (project grants #254646, #303256 and #404087). K.D.G.P. and K.A.E. are supported by NHMRC Peter Doherty (#353709) and Principal Research (#212064) Fellowships, respectively.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
K.A.E. was involved with the development of the Mithras instrument (Berthold Technologies) for BRET detection. K.D.G.P. and K.A.E. have been involved in the testing of the EnduRen substrate (Promega) for use with BRET and, in the past, Promega have contributed to the conference expenses of K.D.G.P. No payments in terms of salaries have been received and neither author receives any commission for the sale of these products.
Rights and permissions
About this article
Cite this article
Pfleger, K., Eidne, K. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 3, 165–174 (2006). https://doi.org/10.1038/nmeth841
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth841
This article is cited by
-
Electron transfer in protein modifications: from detection to imaging
Science China Chemistry (2023)
-
NanoBRET in C. elegans illuminates functional receptor interactions in real time
BMC Molecular and Cell Biology (2022)
-
Oligomerization of the heteromeric γ-aminobutyric acid receptor GABAB in a eukaryotic cell-free system
Scientific Reports (2022)
-
Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer
Scientific Reports (2021)