Pampillo Macarena, Babwah Andy V
The Children's Health Research Institute, Victoria Research Laboratories, The University of Western Ontario, A4-140, 800 Commissioners Road East, London, ON, Canada, N6C 2V5.
Methods Mol Biol. 2015;1272:119-32. doi: 10.1007/978-1-4939-2336-6_9.
GPCR internalization is a critical regulatory step in determining receptor activity. While internalization terminates G protein-coupled signaling, it might be required for G protein-independent signaling. A large number of clinical therapies are based on preventing or promoting GPCR internalization. Thus, for any given GPCR, it is important to characterize its internalization and understand the factors that regulate such internalization. Here we describe different experimental protocols to evaluate the internalization of any GPCR transiently expressed in HEK 293 cells. The protocols describe the use of immunofluorescence and imaging techniques as well as flow cytometry. The techniques described use the FLAG-tagged kisspeptin receptor (KISS1R) as an example but are equally applicable to any other GPCR.
G蛋白偶联受体（GPCR）的内化是决定受体活性的关键调节步骤。内化虽然会终止G蛋白偶联信号传导，但可能是G蛋白非依赖性信号传导所必需的。大量临床治疗方法都基于预防或促进GPCR的内化。因此，对于任何给定的GPCR，表征其内化并了解调节这种内化的因素都很重要。在这里，我们描述了不同的实验方案，以评估在HEK 293细胞中瞬时表达的任何GPCR的内化。这些方案描述了免疫荧光和成像技术以及流式细胞术的使用。所描述的技术以带有FLAG标签的亲吻素受体（KISS1R）为例，但同样适用于任何其他GPCR。
