Abstract
G protein-coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the β-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.
Highlights
G protein– coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases
The sensitivity and robustness of a bioluminescent resonance energy transfer (BRET) target engagement assay stems from its capacity to reproducibly and measure ligand binding across a range of expression levels and in different assay formats
In this study we introduced a versatile cellular platform that takes advantage of the HiBiT/LgBiT complementation reporter to provide robust analyses of GPCR ligand engagement and receptor activation measured as internalization
Summary
Assays typically use fluorescently labeled ligands (fluorescent tracers) in a competitive binding format to measure interactions between ligands and their cognate GPCRs that are genetically fused to an energy donor (i.e. a lanthanide-labeled protein tag or a luciferase). Intracellular luminescence could limit the sensitivity and dynamic range of NanoLuc–GPCR internalization assays in which a decrease in total luminescence corresponds to receptor endocytosis [17] To this end, we sought to use GPCRs genetically fused to the newly described HiBiT tag, a small 11–amino acid peptide that can produce bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc [18, 19]. Using 2-AR as an initial model, we generated ligand binding and internalization analyses in transient and endogenous expression setups, which were in general agreement with reported values These results validated and demonstrated the sensitivity and robustness of our approach, for analyses of endogenously tagged receptors. Applied broadly to the study of other GPCR families and to evaluations of new drug candidates
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