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. 2018 Jul;65(1):9-14.
doi: 10.2144/btn-2018-0039.

Luciferase complementation based-detection of G-protein-coupled receptor activity

Hideaki Yano  1 , Ning Sheng Cai  1 , Jonathan A Javitch  2   3 , Sergi Ferré  1
Affiliations

Luciferase complementation based-detection of G-protein-coupled receptor activity

Hideaki Yano et al. Biotechniques. 2018 Jul.

Abstract

Protein complementation assays (PCA) are used as pharmacological tools, enabling a wide array of applications, ranging from studies of protein-protein interactions to second messenger effects. Methods to detect activities of G protein-coupled receptors (GPCRs) have particular relevance for drug screening. Recent development of an engineered luciferase NanoLuc created the possibility of generating a novel PCA, which in turn could open a new avenue for developing drug screening assays. Here we identified a novel split position for NanoLuc and demonstrated its use in a series of fusion constructs to detect the activity of GPCRs. The split construct can be applied to a variety of pharmacological screening systems.

Keywords: GPCR; assay development; biosensor; complementation; dopamine receptor; luciferase.

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Figures

Figure 1:
Figure 1:
Drug induced complementation of split luciferase fragments. Throughout, time scale corresponds to substrate (5 μM coelenterazine H) incubation time. A. Kinetic measurement of NanoLuc complementation between FRB-Nluc1 and FKBP-Nluc2 after three hours of incubation with vehicle (black), 100 nM rapamycin (orange), 10 μM FK506 (blue), 100 nM rapamycin + 10 μM FK506 (red), 30 μg/ml cycloheximide + vehicle (black open), 30 μg/ml cycloheximide + 100 nM rapamycin (orange open), 30 μg/ml cycloheximide + 10 μM FK506 (blue open). B. Kinetic measurement of NanoLuc complementation between FRB-Nluc1 and FKBP-Nluc2 with simultaneous addition of vehicle (black), 100 nM rapamycin (orange), 10 μM FK506 (blue), 100 nM rapamycin +10 μM FK506 added after 5 min (red). C. Time dependent drug induced change of NanoLuc activity in cells separately expressing split fragments immediately after addition of vehicle with FRB-Nluc1 fragment (black top hemi circle), 100 nM rapamycin with FRB-Nluc1 fragment (orange top hemi circle), 10 μM FK506 with FRB-Nluc1 fragment (blue top hemi circle), vehicle with FKBP-Nluc2 fragment (black bottom hemi circle), 100 nM rapamycin with FKBP-Nluc2 fragment (orange bottom hemi circle), 10 μM FK506 with FKBP-Nluc2 fragment (blue bottom hemi circle), vehicle with FRB-Nluc1 fragment cells and FKBP-Nluc2 fragment cells combined (black square), and 100 nM rapamycin with FRB-Nluc1 fragment cells and FKBP-Nluc2 fragment cells combined (light blue square). D. NanoLuc complementation between D2R-Nluc1 and D2R-Nluc2 immediately after three hours of incubation with vehicle (black), and 100 nM rapamycin (orange). E-H. Rluc complementation (same experimental scheme and legend for A-D applied).
Figure 2:
Figure 2:
Dopamine (DA) induced G protein engagement, activation, and β-arrestin2 recruitment measured by NanoLuc complementation. A. Specific NanoLuc complementation between D1-Nluc1 (0.5 μg transfected) with non-interacting FRB-Nluc2 (black; transfected amount = y axis) or with cognate G protein Gαs-Nluc2 (orange; transfected amount = y axis). B-E. DA induced complementation change between D1-Nluc1 and Gαs-Nluc2 (B), Gαs-Nluc1 and γ2-Nluc2 (C), D1-Nluc1 and γ2-Nluc2 (D), and D1-Nluc1 and β-arrestin2-Nluc2 (E). pEC50: D1N1 GsN2 – 6.78±0.25, D1 Gαs-Nluc1 γ2-Nluc2 – 6.96±0.11, D1-Nluc1 γ2-Nluc2 – 7.78±0.16, D1-Nluc1 β-arrestin2-Nluc2 – 6.00±0.10.
Figure 3:
Figure 3:
Confirmation of pharmacological characteristics in drug induced G protein engagement. A. D1-Nluc1 Gαs-Nluc2 engagement with dopamine (black), norepinephrine (orange), SKF38393 (blue), A77636 (red), 10 μM DA + SCH23390 (open black). B. D2-Nluc1 Gαi-Nluc2 engagement with dopamine (black), norepinephrine (orange), 10 μM DA + Eticlopride (open black).
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