Bagher Amina M, Kelly Melanie E M, Denovan-Wright Eileen M
Department of Pharmacology, Dalhousie University, Halifax, NS, Canada.
Department of Pharmacology and Toxicology, King AbdulAziz University, Jeddah, Saudi Arabia.
Methods Mol Biol. 2019;1947:199-215. doi: 10.1007/978-1-4939-9121-1_11.
G protein-coupled receptors (GPCRs) are the target for many drugs. Evidence continues to accumulate demonstrating that multiple receptors form homo- and heteromeric complexes, which in turn dynamically couple with G proteins, and other interacting proteins. Here, we describe a method to simultaneously determine the identity of up to four distinct constituents of GPCR complexes using a combination of sequential bioluminescence resonance energy transfer 2-fluorescence resonance energy transfer (SRET) with bimolecular fluorescence complementation (BiFC). The method is amenable to moderate throughput screening of changes in response to ligands and time-course analysis of protein-protein oligomerization.
G蛋白偶联受体（GPCRs）是许多药物的作用靶点。越来越多的证据表明，多种受体可形成同聚体和异聚体复合物，这些复合物又能与G蛋白及其他相互作用蛋白动态偶联。在此，我们描述了一种方法，该方法结合了顺序生物发光共振能量转移-2-荧光共振能量转移（SRET）与双分子荧光互补（BiFC），可同时确定GPCR复合物中多达四种不同成分的身份。该方法适用于对配体反应变化的中等通量筛选以及蛋白质-蛋白质寡聚化的时间进程分析。
