Study of GPCR–Protein Interactions by BRET

  • Martina Kocan
  • Kevin D. G. PflegerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 746)


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.

Key words

Bioluminescence resonance energy transfer G protein-coupled receptor Arrestin Renilla luciferase8 Rluc8 Fluorophore Venus 



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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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Laboratory for Molecular Endocrinology – GPCRs, Western Australian Institute for Medical Research (WAIMR) and Centre for Medical ResearchUniversity of Western AustraliaCrawleyAustralia

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