Nooh Mohammed M, Bahouth Suleiman W
Faculty of Pharmacy Cairo University, Cairo, Egypt.
The University of Tennessee Health Sciences Center, Memphis, TN, United States.
Methods Cell Biol. 2017;142:67-78. doi: 10.1016/bs.mcb.2017.07.010. Epub 2017 Sep 19.
G protein-coupled receptors (GPCRs) are recognized as one of the most fruitful group of therapeutic targets, accounting for more than 40% of all approved pharmaceuticals on the market. Therefore, the search for selective agents that affect GPCR function is of major interest to the pharmaceutical industry. This chapter describes methods for measuring agonist-promoted GPCR trafficking, which involves the internalization of the GPCR and its subsequent recycling back to the plasma membrane or retention and eventual degradation. These pathways will be analyzed by confocal cellular imaging, using the β-adrenergic receptor (β-AR) as a primary model. A major problem encountered in studying GPCR trafficking is the unavailability of antibodies that would recognize the native receptor in cells or tissues. Therefore, wild-type, point mutants, and β-AR chimeras are generated as epitope-tagged proteins, which are stably- or transiently expressed in mammalian cells. GPCR are labeled with a fluorophore-conjugated antibody directed against the N-terminal epitope tag. The trafficking of the fluorophore-tagged GPCR between divergent trafficking pathways that result in retention and eventual degradation or recycling and reinsertion into the plasma membrane can be followed by confocal immunofluorescence microscopy techniques outlined in this review.
G蛋白偶联受体（GPCRs）被认为是最具成效的治疗靶点之一，占市场上所有获批药物的40%以上。因此，寻找影响GPCR功能的选择性药物是制药行业的主要兴趣所在。本章介绍了测量激动剂促进的GPCR转运的方法，这涉及GPCR的内化及其随后循环回到质膜或滞留并最终降解。这些途径将通过共聚焦细胞成像进行分析，以β-肾上腺素能受体（β-AR）作为主要模型。研究GPCR转运时遇到的一个主要问题是缺乏能够识别细胞或组织中天然受体的抗体。因此，野生型、点突变体和β-AR嵌合体被构建为带有表位标签的蛋白质，并在哺乳动物细胞中稳定或瞬时表达。GPCR用针对N端表位标签的荧光团偶联抗体进行标记。通过本综述中概述的共聚焦免疫荧光显微镜技术，可以追踪荧光团标记的GPCR在不同转运途径之间的转运情况，这些途径会导致滞留和最终降解，或者循环并重新插入质膜。
