Department of Biochemistry, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
Department of Pharmacy, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing University, Nanjing, China.
Methods Mol Biol. 2024;2816:161-174. doi: 10.1007/978-1-0716-3902-3_16.
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.
G 蛋白偶联受体（GPCRs）是七螺旋跨膜蛋白，它们通过与其特定配体（包括许多脂质介质）结合来介导各种细胞内信号事件。尽管分析 GPCR 分子相互作用对于理解各种细胞内信号事件至关重要，但由于 GPCR 的疏水性及其动态分子相互作用，使用传统的共免疫沉淀方法来亲和纯化相互作用蛋白具有挑战性。TurboID 系统催化的邻近标记技术是在活细胞中定义靶蛋白分子相互作用的有力技术。TurboID 和 miniTurbo（TurboID 的一种改良版本）是工程化的生物素连接酶，以非特异性方式生物素化邻近的蛋白质。当与靶蛋白融合并在活细胞中表达时，TurboID 或 miniTurbo 介导与靶蛋白近距离的蛋白质的生物素标记，从而能够有效地纯化生物素标记的蛋白质，随后进行鸟枪法蛋白质组学分析。在本章中，我们描述了使用 TurboID 或 miniTurbo 标记 GPCR 邻近蛋白、纯化生物素标记蛋白以及随后进行蛋白质组学分析的样品制备的逐步方案。我们使用 S1PR1 作为模型 GPCR，它是生物活性脂质分子鞘氨醇 1-磷酸（S1P）的受体，在生理和病理条件下发挥各种作用。该分析流程可用于绘制活细胞中脂质 GPCR 的相互作用蛋白图谱。
