Amsterdam Institute for Molecules, Medicines and Systems, Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
Methods Mol Biol. 2021;2268:233-248. doi: 10.1007/978-1-0716-1221-7_16.
Cytosolic β-arrestins are key regulators of G protein-coupled receptors (GPCRs) by sterically uncoupling G protein activation, facilitating receptor internalization, and/or acting as G protein-independent signaling scaffolds. The current awareness that GPCR ligands may display bias toward G protein signaling or β-arrestin recruitment makes β-arrestin recruitment assays important additions to the drug discovery toolbox. This chapter describes two NanoLuc-based methods to monitor β-arrestin2 recruitment to the human histamine H receptor by measuring bioluminescence resonance energy transfer and enzyme-fragment complementation in real-time on living cells with reasonable high throughput. In addition to the detection of agonism, both assay formats can be used to qualitatively evaluate the binding kinetics of antihistamines on the human histamine H receptor.
细胞质 β-arrestins 通过空间上阻断 G 蛋白激活、促进受体内化和/或充当 G 蛋白非依赖性信号支架，成为 G 蛋白偶联受体 (GPCR) 的关键调节剂。目前人们已经意识到，GPCR 配体可能偏向于 G 蛋白信号或 β-arrestin 募集，这使得 β-arrestin 募集测定成为药物发现工具包的重要补充。本章描述了两种基于 NanoLuc 的方法，通过在活细胞上实时测量生物发光共振能量转移和酶片段互补，监测人组氨酸 H 受体的β-arrestin2 募集，具有较高的高通量。除了检测激动剂外，这两种测定方法都可以用于定性评估抗组胺药与人组氨酸 H 受体的结合动力学。
