Detection of GPCR/beta-arrestin interactions in live cells using bioluminescence resonance energy transfer technology

Bioluminescence resonance energy transfer (BRET) is a powerful and increasingly popular technique for studying protein-protein interactions in live cells and real time. In particular, there has been considerable interest in the ability to monitor interactions between G protein-coupled receptors (GPCRs) and proteins that serve as key regulators of receptor function, such as beta-arrestin. The BRET methodology involves heterologous co-expression of genetically fused proteins that link one protein of interest (e.g., a GPCR) to a bioluminescent donor enzyme and a second protein of interest (e.g., beta-arrestin) to an acceptor fluorophore. If the fusion proteins are in close proximity, resonance energy will be transferred from the donor to the acceptor molecule and subsequent fluorescence from the acceptor can be detected at a characteristic wavelength. Such fluorescence is therefore indicative of the proteins of interest linked to the donor and the acceptor interacting directly or as part of a complex. In addition to monitoring protein-protein interactions to elucidate cellular function, BRET also has the exciting potential to become an important technique for live cell high-throughput screening for drugs targeting GPCRs, utilizing ligand-induced interactions with beta-arrestins.