Improved 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 compl...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-12-01
|
| Series: | Biosensors |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2079-6374/13/1/48 |
| _version_ | 1797445423976153088 |
|---|---|
| author | Yuxin Wu Isabelle V. von Hauff Niels Jensen Moritz J. Rossner Michael C. Wehr |
| author_facet | Yuxin Wu Isabelle V. von Hauff Niels Jensen Moritz J. Rossner Michael C. Wehr |
| author_sort | Yuxin Wu |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T13:25:38Z |
| format | Article |
| id | doaj.art-c219f7dec9b346958d14ca363f9391c4 |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-09T13:25:38Z |
| publishDate | 2022-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-c219f7dec9b346958d14ca363f9391c42023-11-30T21:24:45ZengMDPI AGBiosensors2079-63742022-12-011314810.3390/bios13010048Improved Split TEV GPCR β-arrestin-2 Recruitment Assays via Systematic Analysis of Signal Peptide and β-arrestin Binding Motif VariantsYuxin Wu0Isabelle V. von Hauff1Niels Jensen2Moritz J. Rossner3Michael C. Wehr4Research Group Cell Signalling, Department of Psychiatry and Psychotherapy, University Hospital, Ludwig Maximilian University of Munich, Nussbaumstr. 7, 80336 Munich, GermanyResearch Group Cell Signalling, Department of Psychiatry and Psychotherapy, University Hospital, Ludwig Maximilian University of Munich, Nussbaumstr. 7, 80336 Munich, GermanySection of Molecular Neurobiology, Department of Psychiatry and Psychotherapy, University Hospital, Ludwig Maximilian University of Munich, Nussbaumstr. 7, 80336 Munich, GermanySection of Molecular Neurobiology, Department of Psychiatry and Psychotherapy, University Hospital, Ludwig Maximilian University of Munich, Nussbaumstr. 7, 80336 Munich, GermanyResearch Group Cell Signalling, Department of Psychiatry and Psychotherapy, University Hospital, Ludwig Maximilian University of Munich, Nussbaumstr. 7, 80336 Munich, GermanyG 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.https://www.mdpi.com/2079-6374/13/1/48GPCRdrug screeningdrug discoverycell-based assaysplit TEV technique |
| spellingShingle | Yuxin Wu Isabelle V. von Hauff Niels Jensen Moritz J. Rossner Michael C. Wehr Improved Split TEV GPCR β-arrestin-2 Recruitment Assays via Systematic Analysis of Signal Peptide and β-arrestin Binding Motif Variants Biosensors GPCR drug screening drug discovery cell-based assay split TEV technique |
| title | Improved Split TEV GPCR β-arrestin-2 Recruitment Assays via Systematic Analysis of Signal Peptide and β-arrestin Binding Motif Variants |
| title_full | Improved Split TEV GPCR β-arrestin-2 Recruitment Assays via Systematic Analysis of Signal Peptide and β-arrestin Binding Motif Variants |
| title_fullStr | Improved Split TEV GPCR β-arrestin-2 Recruitment Assays via Systematic Analysis of Signal Peptide and β-arrestin Binding Motif Variants |
| title_full_unstemmed | Improved Split TEV GPCR β-arrestin-2 Recruitment Assays via Systematic Analysis of Signal Peptide and β-arrestin Binding Motif Variants |
| title_short | Improved Split TEV GPCR β-arrestin-2 Recruitment Assays via Systematic Analysis of Signal Peptide and β-arrestin Binding Motif Variants |
| title_sort | improved split tev gpcr β arrestin 2 recruitment assays via systematic analysis of signal peptide and β arrestin binding motif variants |
| topic | GPCR drug screening drug discovery cell-based assay split TEV technique |
| url | https://www.mdpi.com/2079-6374/13/1/48 |
| work_keys_str_mv | AT yuxinwu improvedsplittevgpcrbarrestin2recruitmentassaysviasystematicanalysisofsignalpeptideandbarrestinbindingmotifvariants AT isabellevvonhauff improvedsplittevgpcrbarrestin2recruitmentassaysviasystematicanalysisofsignalpeptideandbarrestinbindingmotifvariants AT nielsjensen improvedsplittevgpcrbarrestin2recruitmentassaysviasystematicanalysisofsignalpeptideandbarrestinbindingmotifvariants AT moritzjrossner improvedsplittevgpcrbarrestin2recruitmentassaysviasystematicanalysisofsignalpeptideandbarrestinbindingmotifvariants AT michaelcwehr improvedsplittevgpcrbarrestin2recruitmentassaysviasystematicanalysisofsignalpeptideandbarrestinbindingmotifvariants |