Kershaw Tom, Wavre-Shapton Silène T, Signoret Nathalie, Marsh Mark
Cell Biology Unit, MRC Laboratory for Molecular Cell Biology, and Department of Cell and Developmental Biology, University College London, London, United Kingdom.
Methods Enzymol. 2009;460:357-77. doi: 10.1016/S0076-6879(09)05218-5.
Chemokine receptors are G protein-coupled receptors (GPCRs) that, through their ability to regulate chemotaxis by responding to small chemoattractant peptides termed chemokines, are involved in the development, maintenance, and functional activities of the immune system. In addition, members of the chemokine receptor family have been implicated in a number of other physiological and pathological processes, including human immunodeficiency virus infection and malaria. These activities are dependent on receptor expression at the cell surface and cellular events that reduce the cell-surface expression of chemokine receptors can abrogate these activities. Moreover, internalization of chemokine receptors by endocytosis is necessary for both receptor degradation and recycling, key regulatory processes that determine cell-surface expression levels. Here we provide detailed methods for the quantitative analysis of CCR5 endocytosis and recycling by flow cytometry, as well as fluorescence and electron microscopic procedures to analyze the endocytosis and intracellular trafficking of CCR5 by immunolabeling of cells or cryosections. In principle, the same approaches can be used for analyzing other chemokine receptors and other GPCR or non-GPCR cell-surface proteins.
趋化因子受体是G蛋白偶联受体（GPCRs），通过对称为趋化因子的小趋化吸引肽作出反应来调节趋化作用，参与免疫系统的发育、维持和功能活动。此外，趋化因子受体家族成员还涉及许多其他生理和病理过程，包括人类免疫缺陷病毒感染和疟疾。这些活动依赖于细胞表面的受体表达，而降低趋化因子受体细胞表面表达的细胞事件可消除这些活动。此外，通过内吞作用使趋化因子受体内化对于受体降解和再循环都是必要的，这是决定细胞表面表达水平的关键调节过程。在这里，我们提供了通过流式细胞术定量分析CCR5内吞作用和再循环的详细方法，以及通过细胞或冷冻切片免疫标记来分析CCR5内吞作用和细胞内运输的荧光和电子显微镜程序。原则上，相同的方法可用于分析其他趋化因子受体以及其他GPCR或非GPCR细胞表面蛋白。
