Summary
The bioluminescence resonance energy transfer (BRET) methodology allows for the study of protein-protein interactions as well as conformational changes within proteins or molecular complexes. BRET is a highly versatile technique that can be applied to in vitro studies using purified proteins, crude cell membranes, cell fractions obtained by centrifugation on a density gradient, as well as permeabilized cells. Importantly, BRET also allows for monitoring of protein-protein interactions, in real time, in intact living cells that can be submitted to various stimuli. Moreover, quantitative BRET analysis also permits a pharmacological approach of protein-protein interactions, allowing one to determine whether a given stimulus induces a conformational change within preassociated partners or increases the association (recruitment) between two separated partners. Determination of the proportion of the dimeric vs monomeric form of a protein in the cell also is possible. Therefore, the BRET technology can be considered as a new and powerful tool in the field of protein-protein interactions.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Morin J. G., and Hastings J. W. (1971) Energy transfer in a bioluminescent system. J. Cell Physiol. 77, 313–318.
Lorenz W. W., McCann R. O., Longiaru M., and Cormier M. J. (1991) Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Proc. Natl. Acad. Sci. USA 88, 4438–4442.
Tsien R. Y., Bacskai B. J., and Adams S. R. (1993) FRET for studying intracellular signalling. Trends Cell Biol. 3, 243–245.
Wu P. and Brand L. (1994) Resonance energy transfer: methods and applications. Anal. Biochem. 218, 1–13.
Boute N., Jockers R., and Issad T. (2002) The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol. Sci. 23, 351.
Xu Y., Piston D. W., and Johnson C. H. (1999) A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc. Natl. Acad. Sci. USA 96, 151–156.
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. USA 97, 3684–3689.
McVey M., Ramsay D., Kellett E., et al. (2001) Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. The human delta-opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy. J. Biol. Chem. 276, 14092–14099.
Kroeger K. M., Hanyaloglu A. C., Seeber R. M., Miles L. E., and Eidne K. A. (2001) 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, 12,736–12,743.
Cheng Z. J. and Miller L. J. (2001) Agonist-dependent dissociation of oligomeric complexes of G protein-coupled cholecystokinin receptors demonstrated in living cells using bioluminescence resonance energy transfer. J. Biol. Chem. 276, 48,040–48,047.
Ayoub M. A., Couturier C., Lucas-Meunier E., et al. (2002) 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, 21,522–21,528.
Boute N., Pernet K., and Issad T. (2001) Monitoring the activation state of the insulin receptor using bioluminescence resonance energy transfer. Mol. Pharmacol. 60, 640–645.
Couturier C. and Jockers R. (2003) Activation of the leptin receptor by a ligandinduced conformational change of constitutive receptor dimers. J. Biol. Chem. 278, 26,604–26,611.
Hasbi A., Devost D., Laporte S. A., and Zingg H. H. (2004) Real-time detection of interactions between the human oxytocin receptor and G protein-coupled receptor kinase-2. Mol. Endocrinol. 18, 1277–1286.
Boute N., Boubekeur S., Lacasa D., and Issad T. (2003) Dynamics of the interactionb etween the insulin receptor and protein tyrosine-phosphatase 1B in living cells. EMBO Rep. 4, 313–319.
Lacasa D., Boute N., and Issad T. (2005) Interaction of the insulin receptor with the receptor-like protein tyrosine-phosphatases PTPα and PTPβ in living cells. Mol. Pharmacol. 67, 1206–1213.
Milligan G. (2004) Applications of bioluminescence-and fluorescence resonance energy transfer to drug discovery at G protein-coupled receptors. Eur. J. Pharm. Sci. 21, 397–405.
Issad T., Tavaré J., and Denton R. M. (1991) Analysis of insulin receptor phosphorylation sites in intact rat liver cells by two-dimensional phosphopeptides mapping. Predominance of the trisphosphorylated form of the kinase domain after stimulation by insulin. Biochem. J. 275, 15–21.
Mercier J. F., Salahpour A., Angers S., Breit A., and Bouvier M. (2002) Quantitative assessment of beta 1-and beta 2-adrenergic receptor homo-and heterodimerization by bioluminescence resonance energy transfer. J. Biol. Chem. 277, 44,925–44,931.
Ramsay D., Carr I. C., Pediani J., et al. (2004) High-affinity interactions between human alpha1A-adrenoceptor C-terminal splice variants produce homoand heterodimers but do not generate the alpha1L-adrenoceptor. Mol. Pharmacol. 66, 228–239.
Ramsay D., Kellett E., McVey M., Rees S., and Milligan G. (2002) 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.
Ayoub M. A., Levoye A., Delagrange P., and Jockers R. (2004) Preferential formation of MT1/MT2 melatonin receptor heterodimers with distinct ligand interaction properties compared with MT2 homodimers. Mol. Pharmacol. 66, 312–321.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Issad, T., Jockers, R. (2006). Bioluminescence Resonance Energy Transfer to Monitor Protein-Protein Interactions. In: Ali, H., Haribabu, B. (eds) Transmembrane Signaling Protocols. Methods in Molecular Biology™, vol 332. Humana Press. https://doi.org/10.1385/1-59745-048-0:193
Download citation
DOI: https://doi.org/10.1385/1-59745-048-0:193
Publisher Name: Humana Press
Print ISBN: 978-1-58829-546-0
Online ISBN: 978-1-59745-048-5
eBook Packages: Springer Protocols