Department of Infection and Immunity, Immuno-Pharmacology and Interactomics, Luxembourg Institute of Health (LIH), Esch-sur-Alzette, Luxembourg; Faculty of Science, Technology and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg.
Department of Infection and Immunity, Immuno-Pharmacology and Interactomics, Luxembourg Institute of Health (LIH), Esch-sur-Alzette, Luxembourg; Faculty of Science, Technology and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg; Department of Oncology, Tumor Immunotherapy and Microenvironment, Luxembourg Institute of Health (LIH), Luxembourg City, Luxembourg.
Methods Cell Biol. 2022;169:309-321. doi: 10.1016/bs.mcb.2022.04.001. Epub 2022 May 4.
G protein-coupled receptor kinases (GRKs) are a family of seven soluble receptor-modifying enzymes which are essential regulators of GPCR activity. Following agonist-induced receptor activation and G protein dissociation, GRKs prime the receptor for desensitization through phosphorylation of its C terminus, which subsequently allows arrestins to bind and initiate the receptor internalization process. While GRKs constitute key GPCR-interacting proteins, to date, no method has been put forward to readily and systematically determine the preference of a specific GPCR towards the seven different GRKs (GRK1-7). This chapter describes a simple and standardized approach for systematic profiling of GRK1-7-GPCR interactions relying on the complementation of the split Nanoluciferase (NanoBiT). When applied to a set of GPCRs (MOR, 5-HT, B2AR, CXCR3, AVPR2, CGRPR), including two intrinsically β-arrestin-biased receptors (ACKR2 and ACKR3), this methodology yields highly reproducible results highlighting different GRK recruitment profiles. Using this assay, further characterization of MOR, a crucial target in the development of analgesics, reveals not only its GRK fingerprint but also related kinetics and activity of various ligands for a single GRK.
G 蛋白偶联受体激酶（GRK）是一类七聚体可溶性受体修饰酶，是 GPCR 活性的重要调节因子。在激动剂诱导受体激活和 G 蛋白解离后，GRK 通过磷酸化其 C 端使受体失活，随后允许 arrestin 结合并启动受体内化过程。虽然 GRK 是关键的 GPCR 相互作用蛋白，但迄今为止，尚无方法可轻易且系统地确定特定 GPCR 对七种不同 GRK（GRK1-7）的偏好。本章描述了一种简单而标准化的方法，用于系统分析 GRK1-7-GPCR 相互作用，该方法依赖于 Nanoluciferase（NanoBiT）的分割互补。当应用于一组 GPCR（MOR、5-HT、B2AR、CXCR3、AVPR2、CGRPR），包括两个内在的β-arrestin 偏向性受体（ACKR2 和 ACKR3）时，该方法产生了高度可重复的结果，突出了不同的 GRK 募集谱。使用该测定法，对关键的镇痛药开发靶标 MOR 进行进一步表征，不仅揭示了其 GRK 指纹，还揭示了各种配体与单个 GRK 的相关动力学和活性。
