Littmann, Timo and Ozawa, Takeaki and Hoffmann, Carsten and Buschauer, Armin and Bernhardt, Guenther (2018) A split luciferase-based probe for quantitative proximal determination of G alpha(q) signalling in live cells. SCIENTIFIC REPORTS, 8: 17179. ISSN 2045-2322,
Full text not available from this repository. (Request a copy)Abstract
The earlier an activation of a G protein-dependent signalling cascade at a G protein-coupled receptor (GPCR) is probed, the less amplificatory effects contribute to the measured signal. This is especially useful in case of a precise quantification of agonist efficacies, and is of paramount importance, when determining agonist bias in relation to the beta-arrestin pathway. As most canonical assays with medium to high throughput rely on the quantification of second messengers, and assays affording more proximal readouts are often limited in throughput, we developed a technique with a proximal readout and sufficiently high throughput that can be used in live cells. Split luciferase complementation (SLC) was applied to assess the interaction of G alpha(q), with its effector phospholipase C-beta 3. The resulting probe yielded an excellent Z' value of 0.7 and offers a broad and easy applicability to various G alpha(q)-coupling GPCRs (hH(1)R, hM(1,3,5)R, hNTS(1)R), expressed in HEK293T cells, allowing the functional characterisation of agonists and antagonists. Furthermore, the developed sensor enabled imaging of live cells by luminescence microscopy, as demonstrated for the hM(3)R. The versatile SLC-based probe is broadly applicable e.g. to the screening and the pharmacological characterisation of GPCR ligands as well as to molecular imaging.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | HUMAN HISTAMINE H-1; G-PROTEIN; BINDING-PROPERTIES; HIGH-SENSITIVITY; NEUROPEPTIDE-Y; RECEPTOR; COMPLEMENTATION; PHARMACOLOGY; ASSAYS; VISUALIZATION; |
| Subjects: | 600 Technology > 615 Pharmacy |
| Divisions: | Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical/Medicinal Chemistry II (Prof. Buschauer) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 09 Oct 2019 06:26 |
| Last Modified: | 09 Oct 2019 06:26 |
| URI: | https://pred.uni-regensburg.de/id/eprint/13527 |
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