Ferguson Stephen S G, Caron Marc G
The John P. Robarts Institute and Department of Physiology, University of Western Ontario, London, Ontario, Canada.
Methods Mol Biol. 2004;237:121-6. doi: 10.1385/1-59259-430-1:121.
The G protein-coupled receptor (GPCR) superfamily is the largest family of integral membrane proteins. GPCRs respond to a wide variety of sensory and chemical stimuli and contribute directly to the regulation of all major organ systems. As such, GPCRs represent primary drug targets for therapeutic intervention. Although GPCRs respond to a diverse range of ligands and signal through multiple heterotrimeric G proteins, the inactivation of GPCR signaling is mediated by a limited set of proteins. In particular, the desensitization of the majority of GPCRs is mediated by the binding of two arrestin isoforms, beta-arrestin1 and beta-arrestin2, that exhibit overlapping substrate specificity. In response to GPCR activation and phosphorylation by GPCR kinases, beta-arrestins redistribute from the cytosol to the plasma membrane to bind GPCRs and subsequently target the receptors for internalization via clathrin-coated vesicles. This property of beta-arrestins has allowed the development of a green fluorescent protein (GFP)-based assay for detecting GPCR activation by confocal microscopy. This beta-arrestin-GFP translocation methodology is described in detail in this chapter.
G蛋白偶联受体（GPCR）超家族是最大的整合膜蛋白家族。GPCR对多种感官和化学刺激作出反应，并直接参与所有主要器官系统的调节。因此，GPCR是治疗干预的主要药物靶点。尽管GPCR对多种配体作出反应并通过多种异源三聚体G蛋白进行信号传导，但GPCR信号的失活是由一组有限的蛋白质介导的。特别是，大多数GPCR的脱敏是由两种抑制蛋白亚型β抑制蛋白1和β抑制蛋白2的结合介导的，它们表现出重叠的底物特异性。响应GPCR激酶介导的GPCR激活和磷酸化，β抑制蛋白从胞质溶胶重新分布到质膜，以结合GPCR，随后通过网格蛋白包被的囊泡将受体靶向内化。β抑制蛋白的这一特性使得能够开发一种基于绿色荧光蛋白（GFP）的检测方法，通过共聚焦显微镜检测GPCR的激活。本章将详细描述这种β抑制蛋白-GFP转位方法。
