Maurel Damien, Comps-Agrar Laëtitia, Brock Carsten, Rives Marie-Laure, Bourrier Emmanuel, Ayoub Mohammed Akli, Bazin Hervé, Tinel Norbert, Durroux Thierry, Prézeau Laurent, Trinquet Eric, Pin Jean-Philippe
Centre National de la Recherche Scientifique (CNRS), UMR 5203, Institut de Génomique Fonctionnelle, 141 Rue de la Cardonille, Montpellier F-34000, France.
Nat Methods. 2008 Jun;5(6):561-7. doi: 10.1038/nmeth.1213. Epub 2008 May 18.
Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput-compatible format. Using this approach, we examined whether G protein-coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers--a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABA(B) receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.
细胞表面蛋白在细胞间通讯中起着重要作用。它们组装成包含不同受体和效应器的异源复合物。对这类蛋白质复合物的阐明和操控提供了新的治疗可能性。我们描述了一种将时间分辨荧光共振能量转移（FRET）与快速标记技术相结合的方法，以高通量兼容的形式定量分析活细胞表面的蛋白质-蛋白质相互作用。使用这种方法，我们研究了G蛋白偶联受体（GPCRs）是单体形式，还是组装成二聚体或更大的寡聚体——这是一个激烈争论的问题。我们获得了A类和C类GPCRs寡聚状态的证据。我们还观察到，针对神经递质谷氨酸和γ-氨基丁酸（GABA）的GPCRs具有不同的四级结构：代谢型谷氨酸受体组装成严格的二聚体，而GABA(B)受体则自发形成异二聚体的二聚体，这为调节G蛋白偶联效率提供了一种方法。这种方法将有助于对活细胞中细胞表面蛋白相互作用进行系统分析。
