The luminescent HiBiT peptide enables selective quantitation of G protein–coupled receptor ligand engagement and internalization in living cells

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.

Strategies for Site‐Specific Labeling of Receptor Proteins on the Surfaces of Living Cells by Using Genetically Encoded Peptide Tags

“…[81] Applied to the monitoring of internalization of the adenosine A1 receptor (A 1 AR) and β 2 adrenergic receptor (β 2 AR) in live cells, a decrease of the luminescent signal is observed when internalized receptors are excluded from interactions with the cell-impermeable LgBiT. [81,117] Recently, surface-specific labeling by the HiBiT-LgBiT system was used to study the potential of the relaxin family receptor-1 (RXFP 1 ) to undergo ligand-induced homodimerization. [118] The assay was based on the distancedependent bioluminescence resonance energy transfer (BRET) between a luciferase-and mCitrine-tagged RXFP 1 .…”

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

“…First of all, the high quantum yield of NanoLuc and the possibility to combine BRET assay with NanoLuc-derived split luciferase reporter system (NanoBiT, described above) ensures the development of BRET assay with increased sensitivity. It allows the monitoring of protein–protein interactions at lower and more physiologically-relevant levels [ 182 ] and analysis of interactions with low selectivity or affinity which have previously been challenging [ 183 , 184 ]. The NanoLuc bioluminescence spectrum (λ max = ~460 nm) is about 20% narrower compared with that of RLuc, enabling improved spectral separation of donor and acceptor emissions and the use of various fluorophores as acceptors.…”

A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding

“…Taking advantage of the brightness of NLuc ( Hall et al., 2012 ), we and others have used CRISPR/Cas9-mediated genome engineering to tag and then study genes and proteins expressed under endogenous promotion using either full-length or split NLuc. This has allowed changes in gene expression or protein levels to be measured and quantified ( Lackner et al., 2015 ; Oh-Hashi et al., 2016 ; Schwinn et al., 2018 ), as well as ligand binding and internalization ( Boursier et al., 2020 ; White et al., 2020 ), protein-protein interactions ( White et al, 2017 , 2020 ), post-translational modifications ( Schwinn et al., 2018 ), and protein degradation ( Riching et al., 2018 ) to be monitored in real-time live cell or lysed cell assays by NanoBRET or changes in luminescence for NLuc complementation. Although these studies using CRISPR/Cas9 genome editing and luciferase tags have principally investigated membrane bound and intracellular proteins, vascular endothelial growth factor (VEGFA), a secreted growth factor, was previously tagged with the small NLuc fragment (HiBiT) ( Schwinn et al., 2018 ) and expression measured in a lytic cell assay at a single time point, indicating the potential for use of this approach to monitor secreted chemokines.…”