Abstract：
G-protein-coupled receptors (GPCRs) are hepta-helical transmembrane proteins that mediate various intracellular signaling events in response to their specific ligands including many lipid mediators. Although analyses of GPCR molecular interactions are pivotal to understanding diverse intracellular signaling events, affinity purification of interacting proteins by a conventional co-immunoprecipitation method is challenging due to the hydrophobic nature of GPCRs and their dynamic molecular interactions. Proximity labeling catalyzed by a TurboID system is a powerful technique for defining the molecular interactions of target proteins in living cells. TurboID and miniTurbo (a modified version of TurboID) are engineered biotin ligases that biotinylate neighboring proteins in a promiscuous manner. When fused with a target protein and expressed in living cells, TurboID or miniTurbo mediates the biotin labeling of the proteins with close proximity to the target protein, allowing efficient purification of the biotinylated proteins followed by a shot-gun proteomic analysis. In this chapter, we describe a step-by-step protocol for the labeling of GPCR neighboring proteins by TurboID or miniTurbo, purification of the biotin-labeled proteins, and subsequent sample preparation for proteomic analysis. We utilized S1PR1 as a model GPCR, a receptor for a bioactive lipid molecule sphingosine 1-phosphate (S1P) that plays various roles in physiological and pathological conditions. This analysis pipeline enables the mapping of interacting proteins of lipid GPCRs in living cells.
摘要：
G蛋白偶联受体(GPCRs)是七螺旋跨膜蛋白,其响应于其特异性配体(包括许多脂质介质)而介导各种细胞内信号传导事件。尽管GPCR分子相互作用的分析对于理解不同的细胞内信号事件至关重要，由于GPCRs的疏水性和它们的动态分子相互作用,通过常规共免疫沉淀方法亲和纯化相互作用蛋白具有挑战性。由TurboID系统催化的邻近标记是用于定义活细胞中靶蛋白的分子相互作用的强大技术。TurboID和miniTurbo（TurboID的修改版本）是工程化的生物素连接酶，以混杂的方式生物素化相邻的蛋白质。当与靶蛋白融合并在活细胞中表达时，TurboID或miniTurbo介导蛋白质的生物素标记与靶蛋白非常接近，允许有效纯化生物素化的蛋白质，然后进行弹枪蛋白质组学分析。在这一章中,我们描述了通过TurboID或miniTurbo标记GPCR邻近蛋白的分步方案，纯化生物素标记的蛋白质，和随后的样品制备用于蛋白质组学分析。我们利用S1PR1作为GPCR模型，生物活性脂质分子1-磷酸鞘氨醇（S1P）的受体，在生理和病理条件下发挥各种作用。该分析流程能够绘制活细胞中脂质GPCRs的相互作用蛋白。
