A split luciferase-based probe for quantitative proximal determination of G\u03b1q signalling in live cells

Timo Littmann,Carsten Hoffmann,Günther Bernhardt,Armin Buschauer,Takeaki Ozawa

doi:10.1038/s41598-018-35615-w

Timo Littmann, Carsten Hoffmann + Show 3 more

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https://doi.org/10.1038/s41598-018-35615-w

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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.

Highlights

G protein-coupled receptors (GPCR) consist of seven transmembrane helices and are responsible for transducing stimuli, e.g. by hormones or neurotransmitters, across the cellular membrane
As our aim was to probe the Gαq/phospholipase C (PLC)-β3 interaction (Fig. 1A), and because Gαq is a rather small protein of which it is well known that both termini are of major importance for interactions with βγ-subunits, with a GPCR and the association with the cell membrane[31,32,33,34], we pursued a slightly different strategy: we fused the smaller luciferase fragment (CBRC) to Gαq and incorporated CBRC into three different flexible loop regions, localized within the helical domain of Gαq[40] (Fig. 1B)
To identify the best combination of fusion proteins in terms of luminescence intensity and S/B ratio, we expressed all combinations of Gαq and PLC-β3 fusion proteins shown in Fig. 1B in HEK293T cells co-expressing the human histamine H1 receptor

Summary

Introduction

G protein-coupled receptors (GPCR) consist of seven transmembrane helices and are responsible for transducing stimuli, e.g. by hormones or neurotransmitters, across the cellular membrane. Since there is an increasing interest in the discrimination of biased agonism with respect to G protein-dependent and β-arrestin-dependent pathways[13], techniques are needed, allowing a proximal quantification of signalling events[14] Such methods comprise [35S]GTPγS-incorporation[15] and steady-state [32,33P]-based GTPase assays[16] or FRET17- and bioluminescence resonance energy transfer (BRET)-based techniques[18]. Most of the aforementioned approaches are compromised e.g. by requiring cell lysis, the preparation of membranes, the availability of radiolabelled chemicals or by low throughput To overcome these limitations, we decided to use SLC to quantify the interaction of Gαq with PLC-β3. Advantages of SLC involve a high signal-to-background (S/B) ratio, enabling live cell and in vivo imaging[27] and the availability of luciferases catalysing chemical reactions, accompanied by the emission of bright light of different wavelengths (broad spectral diversity)[28,29,30]

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