Kocan Martina, Pfleger Kevin D G
Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Melbourne, Royal Parade, Parkvile, Victoria, Australia.
Methods Mol Biol. 2011;746:357-71. doi: 10.1007/978-1-61779-126-0_20.
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.
生物发光共振能量转移（BRET）已成为实时研究活细胞中蛋白质-蛋白质相互作用的一项极具价值的技术。该技术非常适合用于监测G蛋白偶联受体（GPCR）与蛋白质的相互作用，特别是涉及支架蛋白、调节蛋白和信号蛋白，如β-抑制蛋白，现在已知它们除了在受体脱敏中发挥重要作用外，还具有其他重要功能。BRET实验利用融合蛋白的异源共表达，将一种感兴趣的蛋白质（如GPCR）与一种生物发光供体酶（海肾荧光素酶的变体）相连，另一种感兴趣的蛋白质（如β-抑制蛋白）与一种受体荧光团相连。如果两者靠得很近，供体对细胞可渗透的腔肠素底物快速氧化产生的能量将转移到受体，受体进而会在更长的特征波长处发出荧光。因此，这种能量转移的发生意味着与供体和受体融合的感兴趣的蛋白质直接相互作用或作为复合物的一部分相互作用。BRET检测可以使用扫描光谱法或双滤光片发光测定法进行。BRET方法的最新改进使得活细胞药物筛选以及监测以前无法检测到的蛋白质-蛋白质复合物成为可能，包括组成型GPCR/β-抑制蛋白相互作用。因此，在未来几年，BRET可能在GPCR研究和药物发现中发挥越来越重要的作用。
