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Abstract
G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the α2A-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning β2-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands.
Highlights
G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets
We previously demonstrated that such ligand-induced conformational changes can be visualized in living cells by Förster Resonance Energy Transfer (FRET)
bioluminescence resonance energy transfer (BRET) occurs between proximally situated donor–acceptor pairs (1.6–8.5 nm)[9], but here a light emitting enzyme luciferase is used as a donor, sidestepping many of the deficiencies associated with direct illumination of the sample
Summary
G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The average distance between the third intracellular loop and the C-terminus of receptors (e.g., β2AR 6.2 nm)[2] is within the range addressable by FRET (2.4–7.2 nm)[3] Tagging these conformationally sensitive sites with fluorescent donors and acceptors permits the recording of receptor activation as a change in energy transfer between these chromophores. Following this principle, a series of intramolecular FRET-based GPCR-biosensors have been generated, notably for the α2A-adrenergic receptor (α2AAR), employing the fluorescent proteins CFP and YFP (α2AARCFP/YFP)[4,5] or CFP with the small Fluorescein Arsenical Hairpin Binder (FlAsH) as fluorophores[6]. We propose that this technology will speed the characterization of new pharmacological compounds acting at GPCRs
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