Forster, Lisa and Graetz, Lukas and Moennich, Denise and Bernhardt, Guenther and Pockes, Steffen (2020) A Split Luciferase Complementation Assay for the Quantification of beta-Arrestin2 Recruitment to Dopamine D-2-Like Receptors. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 21 (17): 6103. ISSN , 1422-0067
Full text not available from this repository. (Request a copy)Abstract
Investigations on functional selectivity of GPCR ligands have become increasingly important to identify compounds with a potentially more beneficial side effect profile. In order to discriminate between individual signaling pathways, the determination of beta-arrestin2 recruitment, in addition to G-protein activation, is of great value. In this study, we established a sensitive split luciferase-based assay with the ability to quantify beta-arrestin2 recruitment to D-2long and D(3)receptors and measure time-resolved beta-arrestin2 recruitment to the D-2long receptor after agonist stimulation. We were able to characterize several standard (inverse) agonists as well as antagonists at the D2longR and D3R subtypes, whereas for the D4.4R, no beta-arrestin2 recruitment was detected, confirming previous reports. Extensive radioligand binding studies and comparisons with the respective wild-type receptors confirm that the attachment of the Emerald luciferase fragment to the receptors does not affect the integrity of the receptor proteins. Studies on the involvement of GRK2/3 and PKC on the beta-arrestin recruitment to the D-2long R and D3R, as well as at the D1R using different kinase inhibitors, showed that the assay could also contribute to the elucidation of signaling mechanisms. Its broad applicability, which provides concentration-dependent and kinetic information on receptor/beta-arrestin2 interactions, renders this homogeneous assay a valuable method for the identification of biased agonists.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | AGONIST-INDUCED DESENSITIZATION; PROTEIN-COUPLED RECEPTORS; RESTLESS LEGS SYNDROME; BETA-ARRESTIN; D-3 RECEPTORS; HIGH-AFFINITY; FUNCTIONAL-CHARACTERIZATION; MOLECULAR-CLONING; MULTIPLE CLASSES; DRUG-ADDICTION; GPCR; dopamine D-2-like receptors; beta-arrestin; GRK; Emerald luciferase; functional assay |
| Subjects: | 500 Science > 540 Chemistry & allied sciences |
| Divisions: | Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical/Medicinal Chemistry I (Prof. Elz) Chemistry and Pharmacy > Institute of Pharmacy > Alumni or Retired Professors > Pharmaceutical/Medicinal Chemistry II (Prof. Buschauer) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 17 Mar 2021 09:41 |
| Last Modified: | 17 Mar 2021 09:41 |
| URI: | https://pred.uni-regensburg.de/id/eprint/43926 |
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