Littmann, Timo and Buschauer, Armin and Bernhardt, Gunther (2019) Split luciferase-based assay for simultaneous analyses of the ligand concentration- and time-dependent recruitment of beta-arrestin2. ANALYTICAL BIOCHEMISTRY, 573. pp. 8-16. ISSN 0003-2697, 1096-0309
Full text not available from this repository. (Request a copy)Abstract
Functional selectivity of agonists has gained increasing interest in G protein-coupled receptor (GPCR) research, e.g. due to expectations of drugs with reduced adverse effects. Different agonist-dependent GPCR conformations are conceived to selectively activate a balanced or imbalanced intracellular signalling response, involving e.g. different G alpha subtypes, G beta gamma-subunits and beta-arrestins. To discriminate between the different signalling pathways (bias), sensitive techniques are needed that do not interfere with signalling. We applied split luciferase complementation to the GPCR/beta-arrestin2 interaction and thoroughly analysed the influence of its implementation on intracellular signalling. This led to an assay enabling the functional characterization of ligands at the hH(1)R, and the hM(1,5)R and the hNTS(1)R in live HEK293T cells. As demonstrated at the hM(1,5)R, the assay was sensitive enough to identify iperoxo as a superagonist. Time-dependent analyses of the recruitment beta-arrestin2 became possible, allowing the identification of class A and class B GPCRs, due to the differential duration of their interaction with beta-arrestin2 and their recycling to the cell membrane. The developed beta-arrestin2 recruitment assay, which provides concentration- and time-dependent information on the interaction between GPCRs and beta-arrestin2 upon stimulation of the receptor, should be broadly applicable and of high value for the analysis of agonist bias.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | PROTEIN-COUPLED RECEPTORS; BETA-ARRESTIN; FUNCTIONAL SELECTIVITY; ALLOSTERIC MODULATION; MOLECULAR-MECHANISM; CELLULAR-ASSAYS; DRUG DISCOVERY; BIASED AGONISM; COMPLEMENTATION; ACTIVATION; GPCR; beta-Arrestin recruitment; Split luciferase complementation; Bioluminescence; Ligand characterization; Kinetic measurements |
| Subjects: | 500 Science > 540 Chemistry & allied sciences |
| Divisions: | Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical/Medicinal Chemistry II (Prof. Buschauer) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 14 Apr 2020 11:44 |
| Last Modified: | 14 Apr 2020 11:44 |
| URI: | https://pred.uni-regensburg.de/id/eprint/27010 |
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