Carriba Paulina, Navarro Gemma, Ciruela Francisco, Ferré Sergi, Casadó Vicent, Agnati Luigi, Cortés Antoni, Mallol Josefa, Fuxe Kjell, Canela Enric I, Lluís Carmen, Franco Rafael
Institut d'Investigacions Biomèdiques August Pi i Sunyer, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, University of Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain.
Nat Methods. 2008 Aug;5(8):727-33. doi: 10.1038/nmeth.1229. Epub 2008 Jun 29.
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.
识别质膜中的高阶寡聚体对于解读控制细胞间通讯的分子网络特性至关重要。我们将生物发光共振能量转移（BRET）和荧光共振能量转移（FRET）结合在一种称为顺序BRET-FRET（SRET）的技术中，该技术可识别由三种不同蛋白质形成的异源二聚体。在SRET中，Renilla荧光素酶（Rluc）融合蛋白对Rluc底物的氧化通过BRET触发第二个融合蛋白的受体激发，并随后通过FRET激发第三个融合蛋白。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。使用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是一种用于识别两种以上神经递质受体异源复合物的宝贵技术，这将使我们能够更好地理解信号在分子水平上是如何整合的。
