Grätz, Lukas and Laasfeld, Tönis and Allikalt, Anni and Gruber, Corinna G. and Pegoli, Andrea and Tahk, Maris-Johanna and Tsernant, Mari-Liis and Keller, Max and Rinken, Ago (2021) BRET- and fluorescence anisotropy-based assays for real-time monitoring of ligand binding to M-2 muscarinic acetylcholine receptors. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, 1868 (3): 118930. ISSN 0167-4889, 1879-2596
Full text not available from this repository. (Request a copy)Abstract
BRET and fluorescence anisotropy (FA) are two fluorescence-based techniques used for the characterization of ligand binding to G protein-coupled receptors (GPCRs) and both allow monitoring of ligand binding in real time. In this study, we present the first direct comparison of BRET-based and FA-based binding assays using the human M-2 muscarinic acetylcholine receptor (M2R) and two TAMRA (5-carboxytetramethylrhodamine)-labeled fluorescent ligands as a model system. The determined fluorescent ligand affinities from both assays were in good agreement with results obtained from radioligand competition binding experiments. The assays yielded real-time kinetic binding data revealing differences in the mechanism of binding for the investigated fluorescent probes. Furthermore, the investigation of various unlabeled M2R ligands yielded pharmacological profiles in accordance with earlier reported data. Taken together, this study showed that BRET- and FA-based binding assays represent valuable alternatives to radioactivity-based methods for screening purposes and for a precise characterization of binding kinetics supporting the exploration of binding mechanisms.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | PROTEIN-COUPLED RECEPTORS; CONSTITUTIVE ACTIVITY; BUDDED BACULOVIRUSES; FUNCTIONAL-ACTIVITY; ALLOSTERIC SITE; SMOOTH-MUSCLE; POLARIZATION; ANTAGONISTS; AFFINITY; DIMERIZATION; BRET; Fluorescence anisotropy; Ligand binding; G protein-coupled receptor; M-2 muscarinic acetylcholine receptor; Fluorescent ligands |
| Subjects: | 600 Technology > 610 Medical sciences Medicine 600 Technology > 615 Pharmacy |
| Divisions: | Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical/Medicinal Chemistry I (Prof. Elz) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 26 Aug 2022 05:02 |
| Last Modified: | 26 Aug 2022 05:02 |
| URI: | https://pred.uni-regensburg.de/id/eprint/46974 |
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