Detection of heteromerization of more than two proteins by sequential BRET-FRET
Detection of heteromerization of more than two proteins by sequential BRET-FRET
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DOI：
10.1038/nmeth.1229
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发表时间：
2008-08-01
期刊：
影响因子：
48
通讯作者：
Franco, Rafael
中科院分区：
文献类型：
--
作者：
Carriba, Paulina;Navarro, Gemma;Franco, Rafael
鉴定质膜上的高阶低聚物对于解码控制细胞间通讯的分子网络的特性至关重要。我们将生物发光共振能量转移（BRET）和荧光共振能量转移（FRET）结合在一种称为序列BRET-FRET （SRET）的技术中，该技术允许鉴定由三种不同蛋白质形成的异构体。在SRET中，Renilla荧光素酶（Rluc）底物被Rluc融合蛋白氧化，通过BRET触发第二种融合蛋白的受体激发，随后FRET触发第三种融合蛋白。我们描述了SRET的两种变体，它们使用不同的Rluc底物和适当配对的受体荧光蛋白。利用SRET，我们在活细胞中鉴定了大麻素CB(1)、多巴胺D(2)和腺苷A（2A）受体的复合物。SRET是一种宝贵的技术，可以识别两种以上神经递质受体的异质复合物，这将使我们更好地理解信号是如何在分子水平上整合的。
Identification of higher-order oligomers in the plasma membrane is essential to decode the properties of molecular networks controlling intercellular communication. We combined bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential BRET-FRET (SRET) that permits identification of heteromers formed by three different proteins. In SRET, the oxidation of a Renilla luciferase (Rluc) substrate by an Rluc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. We describe two variations of SRET that use different Rluc substrates with appropriately paired acceptor fluorescent proteins. Using SRET, we identified complexes of cannabinoid CB(1), dopamine D(2) and adenosine A(2A) receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level.