Study of GPCR-protein interactions by BRET

Martina Kocan, Kevin Pfleger

Research output: Contribution to journalArticleResearchpeer-review

23 Citations (Scopus)


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 I?-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. I?-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/I?-arrestin interactions. Therefore, BRET is likely to play an increasingly important role in GPCR research and drug discovery over the coming years.
Original languageEnglish
Pages (from-to)357 - 371
Number of pages15
JournalMethods in Molecular Biology
Publication statusPublished - 2011
Externally publishedYes

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