Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Nov 21;8(1):17179.
doi: 10.1038/s41598-018-35615-w.

A split luciferase-based probe for quantitative proximal determination of Gαq signalling in live cells

Timo Littmann  1 , Takeaki Ozawa  2 , Carsten Hoffmann  3 , Armin Buschauer  4 , Günther Bernhardt  5
Affiliations

A split luciferase-based probe for quantitative proximal determination of Gαq signalling in live cells

Timo Littmann et al. Sci Rep. .

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 β-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 Gαq with its effector phospholipase C-β3. The resulting probe yielded an excellent Z' value of 0.7 and offers a broad and easy applicability to various Gαq-coupling GPCRs (hH1R, hM1,3,5R, hNTS1R), 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 hM3R. 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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic illustration of the sensor principle and the fusion protein library used to determine the best combination of proteins. The activation of the Gαq pathway was probed by fusing complementary luciferase fragments to Gαq and PLC-β3 (A). A fusion protein library was generated by fusing CBRC to Gαq terminally and in three loop regions (numbers in parentheses denote amino acid positions) and by fusing CBRN either N-, or C-terminally to PLC-β3 (B). The different combinations of Gαq and PLC-β3 fusion proteins were expressed in HEK293T cells, co-expressing the hH1R. The relative increase in luminescence of cells stimulated with 10 µM histamine compared to unstimulated cells is shown for each combination (C). Data are presented as means ± SEM from three independent experiments, each performed in triplicate.
Figure 2
Figure 2
Characterization of the Gαq activation sensor. All experiments were performed with HEK293T cells, expressing the Gαq sensor and the hH1R, except for C were the sensor was not present. Increasing concentrations of histamine (addition indicated by arrow) lead to proportionally increasing luminescence emitted from the cells, which can be converted to a CRC (A). The opposite (a gradual decrease in luminescence) became obvious, when stimulated cells (300 nM histamine, first arrow) were subsequently treated with the selective hH1R agonist mepyramine (second arrow) (B). In case of the highest concentration, luminescence decreased to basal levels, indicating full reversibility of the sensor interaction. Since the observed activation kinetics in A was slower than expected for G protein activation, a kinetic Fura-2 assay for the quantification of [Ca2+]i was performed, to guarantee that the sensor does not negatively influence downstream signalling. In cells, in which the sensor was present, the kinetics was the same (C) when compared to cells devoid of the sensor (D). The concentration-dependent response to histamine (addition indicated by arrow) was similar (hH1R alone: pEC50: 7.1 ± 0.1; Gαq sensor present: pEC50: 6.8 ± 0.1), too. Furthermore, we were able to show that the modified Gαq as part of the sensor was still prone to inhibition by FR900359 (E). The sensor shows an exceptionally good Z’ of 0.7 (F). Data in A-D are representative of at least two independent experiments. Data in E are presented as mean along with their SEM from five independent experiments performed in triplicate. Data in F was obtained from an entire 96-well plate.
Figure 3
Figure 3
Characterization of standard ligands at the hH1R, the hM1,3,5R and the hNTS1R. Live HEK293T cells, stably expressing the developed sensor and the indicated receptor, were analysed regarding their response to standard agonists (A) and antagonists (B) for the respective receptors. The substrate D-luciferin was added directly to the cells, and the experiment was carried out at 37 °C. Agonist data was normalized to a reference full agonist for each receptor, maximal stimulation of which was defined as 100% (hH1R: histamine, hM1,5R: carbachol, hM3R: oxotremorine). pEC50, Emax and pKb values are listed in Table 1 and were in good accordance with data described in literature. Data are presented as means ± SEM of at least three independent experiments, each performed in triplicate. s.c.: solvent control.
Figure 4
Figure 4
Live cell luminescence imaging of Gαq sensor-expressing HEK293T cells stimulated via the hM3R. Shown are the results of one experiment, performed in the agonist (N = 3) and the antagonist (N = 2) mode, respectively. D-Luciferin was added to the cells before they were transferred to the bioluminescence microscope with its stage warmed to 37 °C. The first frame always shows cells before stimulation. All images were taken with an exposure time of 5 min and are presented as arbitrary light units in false colour. Upon stimulation with 100 nM oxotremorine (approx. EC60), a constant saturable increase in luminescence was observed leading to a plateau after approx. 45 min (cf. Fig. S6). No increase was detectable, when the cells were pre-incubated with atropine (100 nM). The supplementary videos clips S1 and S2 show all acquired frames of which only selected are shown here.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Katritch V, Cherezov V, Stevens RC. Structure-function of the G protein-coupled receptor superfamily. Annu Rev Pharmacol Toxicol. 2013;53:531–556. doi: 10.1146/annurev-pharmtox-032112-135923. - DOI - PMC - PubMed
    1. Hauser AS, Attwood MM, Rask-Andersen M, Schiöth HB, Gloriam DE. Trends in GPCR drug discovery: new agents, targets and indications. Nat Rev Drug Discov. 2017;16:829–842. doi: 10.1038/nrd.2017.178. - DOI - PMC - PubMed
    1. Lieb S, et al. Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor. Pharmacol Res. 2016;114:13–26. doi: 10.1016/j.phrs.2016.10.010. - DOI - PubMed
    1. Benjamin ER, et al. A miniaturized column chromatography method for measuring receptor-mediated inositol phosphate accumulation. J Biomol Screen. 2004;9:343–353. doi: 10.1177/1087057103262841. - DOI - PubMed
    1. Trinquet E, et al. D-myo-inositol 1-phosphate as a surrogate of D-myo-inositol 1,4,5-tris phosphate to monitor G protein-coupled receptor activation. Anal Biochem. 2006;358:126–135. doi: 10.1016/j.ab.2006.08.002. - DOI - PubMed

Publication types