biostudies-literatureWu YChina Scholarship CouncilFaculty of Medicine of the Ludwig-Maximilians-Universität München48https://www.ebi.ac.uk/biostudies/studies/S-EPMC9855867biostudies-literatureUnknown13(1)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 aBiosensorsImproved Split TEV GPCR β-arrestin-2 Recruitment Assays via Systematic Analysis of Signal Peptide and β-arrestin Binding Motif Variants.PMC9855867not applicable201906230338von Hauff IVRossner MJJensen NWehr MCWu YfalseImproved Split TEV GPCR β-arrestin-2 Recruitment Assays via Systematic Analysis of Signal Peptide and β-arrestin Binding Motif Variants.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 a2022-01-01T00:00:00Z2022 Dec2025-04-04T11:33:11.122Z2025-04-04T11:33:11.122ZS-EPMC98558673667188310.3390/bios13010048