Abstract
Bioluminescence resonance energy transfer (BRET) has become an extremely valuable technology for the real-time study of protein–protein interactions in live cells. This technique is highly amenable to the monitoring of G protein-coupled receptor (GPCR)–protein interactions, especially involving scaffolding, regulatory and signaling proteins, such as β-arrestins, which are now known to have significant roles in addition to receptor desensitization. The BRET procedure utilizes heterologous coexpression of fusion proteins linking one protein of interest (e.g. a GPCR) to a bioluminescent donor enzyme, a variant of Renilla luciferase, and a second protein of interest (e.g. β-arrestin) to an acceptor fluorophore. If in close proximity, energy resulting from the rapid oxidation of a cell-permeable coelenterazine substrate by the donor will transfer to the acceptor, which in turn fluoresces at a longer characteristic wavelength. Therefore, the occurrence of such energy transfer implies that the proteins of interest fused to the donor and acceptor interact directly or as part of a complex. BRET detection can be carried out using scanning spectrometry or dual-filter luminometry. The latest improvements in BRET methodology have enabled live cell drug screening as well as monitoring of previously undetectable protein-protein complexes, including constitutive GPCR/β-arrestin interactions. Therefore, BRET is likely to play an increasingly important role in GPCR research and drug discovery over the coming years.
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Acknowledgments
KDGP’s work using the BRET methodology is funded by the National Health and Medical Research Council (NHMRC) of Australia (Project Grant #566736). KDGP is an Australian Research Council (ARC) Future Fellow (FT100100271).
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Kocan, M., Pfleger, K.D.G. (2011). Study of GPCR–Protein Interactions by BRET. In: Willars, G., Challiss, R. (eds) Receptor Signal Transduction Protocols. Methods in Molecular Biology, vol 746. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-126-0_20
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DOI: https://doi.org/10.1007/978-1-61779-126-0_20
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