Xu Xin, Wu Guangyu
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA.
Bio Protoc. 2020 Sep 20;10(18). doi: 10.21769/bioprotoc.3761.
G protein-coupled receptors (GPCRs) are the most structurally diverse family of signaling proteins and regulate a variety of cell function. For most GPCRs, the cell surface is their functional destination where they are able to respond a wide range of extracellular stimuli, leading to the activation of intracellular signal transduction cascades. Thus, the quantity of receptor expression at the cell surface is a crucial factor regulating the functionality of the receptors. Over the past decades, many methods have been developed to measure the cell surface expression of GPCRs. Here, we describe an intact live-cell radioligand binding assay to quantify the surface expression of GPCRs at the endogenous levels or after overexpression. In this assay, cell cultures will be incubated with specific cell-nonpermeable radioligands which selectively and stoichiometrically bind to individual GPCRs and the receptor numbers at the cell surface are quantified by the radioactivity of receptor-bound ligands. This method is highly specific for measuring the functional GPCRs at the surface of intact live cells and is particularly useful for endogenous, low-abundant GPCRs.
G蛋白偶联受体（GPCRs）是信号蛋白中结构最多样化的家族，可调节多种细胞功能。对于大多数GPCRs来说，细胞表面是它们的功能目的地，在那里它们能够对广泛的细胞外刺激做出反应，从而激活细胞内信号转导级联反应。因此，细胞表面受体表达的数量是调节受体功能的关键因素。在过去几十年里，已经开发出许多方法来测量GPCRs的细胞表面表达。在这里，我们描述一种完整的活细胞放射性配体结合测定法，以定量内源性水平或过表达后GPCRs的表面表达。在该测定法中，细胞培养物将与特定的细胞不可渗透放射性配体一起孵育，这些配体选择性地、化学计量地结合到单个GPCRs上，并且通过受体结合配体的放射性来定量细胞表面的受体数量。该方法对于测量完整活细胞表面的功能性GPCRs具有高度特异性，并且对于内源性、低丰度的GPCRs特别有用。
