Research Group Cell Signalling, Department of Psychiatry and Psychotherapy, University Hospital, Ludwig Maximilian University of Munich, Nussbaumstr. 7, 80336 Munich, Germany.
Section of Molecular Neurobiology, Department of Psychiatry and Psychotherapy, University Hospital, Ludwig Maximilian University of Munich, Nussbaumstr. 7, 80336 Munich, Germany.
Biosensors (Basel). 2022 Dec 29;13(1):48. doi: 10.3390/bios13010048.
G protein-coupled receptors (GPCRs) are major disease-relevant drug targets; robust monitoring of their activities upon drug treatment is key to drug discovery. The split TEV cell-based assay technique monitors the interaction of an activated GPCR with β-arrestin-2 through TEV protein fragment complementation using a luminescent signal as the readout. In this work, split TEV GPCR β-arrestin-2 recruitment assays were optimized to monitor the endogenous ligand-induced activities of six GPCRs (DRD1, DRD2, HTR2A, GCGR, AVPR2, and GLP1R). Each GPCR was tested in four forms; i.e., its wildtype form, a variant with a signal peptide (SP) to facilitate receptor expression, a variant containing the C-terminal tail from the V2 vasopressin receptor (V2R tail) to promote β-arrestin-2 recruitment, and a variant containing both the SP and V2R tail. These 24 GPCR variants were systematically tested for assay performance in four cell lines (HEK-293, PC12 Tet-Off, U-2 OS, and HeLa). We found that the assay performance differed significantly for each GPCR variant and was dependent on the cell line. We found that V2R improved the DRD2 split TEV assays and that HEK-293 cells were the preferred cell line across the GPCRs tested. When taking these considerations into account, the defined selection of assay modifications and conditions may improve the performance of drug development campaigns that apply the split TEV technique as a screening tool.
G 蛋白偶联受体（GPCRs）是主要的与疾病相关的药物靶点；在药物治疗时，对其活性进行稳健的监测是药物发现的关键。基于分裂 TEV 细胞的测定技术通过使用荧光信号作为读出物来监测激活的 GPCR 与β-arrestin-2 的相互作用，该方法通过 TEV 蛋白片段互补实现。在这项工作中，优化了分裂 TEV GPCR β-arrestin-2 招募测定法，以监测六种 GPCR（DRD1、DRD2、HTR2A、GCGR、AVPR2 和 GLP1R）的内源性配体诱导的活性。每个 GPCR 以四种形式进行测试；即其野生型形式、带有信号肽（SP）以促进受体表达的变体、含有来自 V2 加压素受体的 C 末端尾部（V2R 尾部）以促进β-arrestin-2 募集的变体，以及含有 SP 和 V2R 尾部的变体。这些 24 种 GPCR 变体在四种细胞系（HEK-293、PC12 Tet-Off、U-2 OS 和 HeLa）中系统地测试了测定性能。我们发现，每种 GPCR 变体的测定性能差异很大，并且取决于细胞系。我们发现 V2R 改善了 DRD2 分裂 TEV 测定法，并且在测试的 GPCR 中，HEK-293 细胞是首选的细胞系。考虑到这些因素，适当选择测定修饰和条件可能会提高应用分裂 TEV 技术作为筛选工具的药物开发活动的性能。
