A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes

Autor: Carina Höring, Ulla Seibel, Katharina Tropmann, Lukas Grätz, Denise Mönnich, Sebastian Pitzl, Günther Bernhardt, Steffen Pockes, Andrea Strasser
Jazyk: angličtina
Rok vydání: 2020
Předmět:
Zdroj: International Journal of Molecular Sciences, Vol 21, Iss 22, p 8440 (2020)
Druh dokumentu: article
ISSN: 1422-0067
1661-6596
DOI: 10.3390/ijms21228440
Popis: In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTPγS, [γ-32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z’ factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTPγS binding assay.
Databáze: Directory of Open Access Journals
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