Institut für Pharmakologie und Toxikologie, Friedrich-Schiller-Universität Jena, Universitätsklinikum Jena, Jena, Germany.
7TM Antibodies GmbH, Jena, Germany.
Commun Biol. 2022 Nov 9;5(1):1206. doi: 10.1038/s42003-022-04135-9.
Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.
激动剂驱动的 G 蛋白偶联受体（GPCR）磷酸化分析可以提供有价值的信息，了解受体激活状态和配体药理学。然而，迄今为止，使用高通量应用程序评估 GPCR 磷酸化一直具有挑战性。我们已经开发并验证了一种基于珠的免疫测定法，可用于定量评估激动剂诱导的 GPCR 磷酸化，该方法可以完全在多孔细胞培养板中进行。该测定法涉及使用磁性珠对亲和标记的受体进行免疫沉淀，然后使用磷酸化状态特异性和磷酸化状态非特异性抗 GPCR 抗体进行蛋白检测。作为概念验证，用不同的激动剂和拮抗剂处理了五个典型的 GPCR（MOP、C5a1、D1、SST2、CB2），并生成了浓度反应曲线。然后，我们扩展了我们的方法来建立选择性细胞 GPCR 激酶（GRK）抑制剂测定法，这导致了快速鉴定出一种选择性的 GRK5/6 抑制剂（LDC8988）和一种高效的泛 GRK 抑制剂（LDC9728）。总之，这种多功能的 GPCR 磷酸化测定法可广泛用于配体分析和抑制剂筛选。
