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A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation
摘要： Inherent instability of the P23H mutant opsin accounts for approximately 10% of autosomal dominant retinitis pigmentosa cases. Our purpose was to develop an overall set of reliable screening strategies to assess if either stabilization or enhanced degradation of mutant rhodopsin could rescue rod photoreceptors expressing this mutant protein. These strategies promise to reveal active compounds and clarify molecular mechanisms of biologically important processes, such as inhibition of target degradation or enhanced target folding. METHODS. Cell-based bioluminescence reporter assays were developed and validated for high-throughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and image-based analyses. RESULTS. Two stabilization assays of P23H mutant opsin were developed and validated, one based on b-galactosidase complementarity and a second assay involving bioluminescence resonance energy transfer (BRET) technology. Moreover, two additional assays evaluating mutant protein degradation also were employed, one based on the disappearance of luminescence and another employing the ALPHA immunoassay. Imaging of cells revealed the cellular localization of mutant rhodopsin, whereas immunoblots identi?ed changes in the aggregation and glycosylation of P23H mutant opsin. CONCLUSIONS. Our ?ndings indicate that these initial HTS and following assays can identify active therapeutic compounds, even for dif?cult targets such as mutant rhodopsin. The assays are readily scalable and their function has been proven with model compounds. High-throughput screening, supported by automated imaging and classic immunoassays, can further characterize multiple steps and pathways in the biosynthesis and degradation of this essential visual system protein.
关键词： ocular pharmacology,retinal degeneration,GPCR,phototransduction,rod photoreceptors,rhodopsin,misfolded protein
更新于2025-11-21 11:20:48
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Live cell optical assay for precise characterization of receptors coupling to Gα12
摘要： Heterotrimeric G proteins are essential mediators of G Protein-Coupled Receptors (GPCRs) signalling to intracellular effectors. There is a considerable diversity of G protein subunits that channel signals initiated by GPCRs into specific outcomes. In particular, mammalian genomes contain 16 conserved genes encoding G protein α subunits with unique properties. Of four Gα subfamilies (Gi/o, Gq, Gs, and G12/13), members of the G12/13 group have received considerable attention for their roles in carcinogenesis. However, our ability to study activation of G12/13 by GPCRs with the power to distinguish between the two subunits is limited. Here, we present an adaptation of the Bioluminescent Resonance Energy Transfer (BRET)-based assay to specifically monitor activity of Gα12 in living cells. In this kinetic assay, agonist-induced release of Venus-tagged Gβγ subunits from Gα12 is followed in real-time using Nano-luciferase tagged BRET donor. Using this assay, we characterized bradykinin B2 receptor (BDKRB2) and found that the receptor couples to Gα12 in addition to Gαo, and Gαq, but not to Gαs. We demonstrated the utility of this assay to quantify rates of G protein activation and inactivation as well as performing dose-response studies while rank-ordering signalling via individual Gα subunits. We further showed the utility of this assay to other GPCRs by demonstrating Gα12 coupling of cholecystokinin A receptor (CCKAR). Introduction of the Gα12-coupling BRET assay is expected to accelerate characterization of GPCR actions on this understudied G protein.
关键词： GNA12,kinetics,GPCR,bradykinin B2 receptor,BRET
更新于2025-11-21 11:08:12
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Chromenopyrazole-based High Affinity, Selective Fluorescent Ligands for Cannabinoid Type 2 Receptor
摘要： Cannabinoid type 2 receptor (CB2R) is an attractive target for the treatment of pain and inflammatory disorders. Availability of a selective CB2R fluorescent ligand to study CB2R expression and localization in healthy and disease conditions would greatly contribute to improving our understanding of this receptor. Herein, we report a series of chromenopyrazole-based CB2R fluorescent ligands. The highest affinity fluorescent ligand was Cy5-containing 24 (hCB2R pKi = 7.38 ± 0.05), which had 131-fold selectivity over CB1R. In a cAMP BRET assay, 24 behaved as a potent CB2R inverse agonist. Widefield imaging experiments showed that 24 binds to CB2R in live cells with good selectivity and low levels of non-specific fluorescence. The high affinity, selectivity and suitable imaging properties of fluorescent ligand 24 make it a valuable tool for studying CB2R.
关键词： chemical tool,Cannabinoid type 2 receptor,GPCR,fluorescent ligand
更新于2025-09-19 17:15:36
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The luminescent HiBiT peptide enables selective quantitation of G proteina??coupled receptor ligand engagement and internalization in living cells
摘要： 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 (cid:2)-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.
关键词： LgBiT,receptor internalization,BRET,ligand engagement,NanoLuc,HiBiT,GPCR,bioluminescent resonance energy transfer
更新于2025-09-19 17:13:59
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Ligand Binding-Induced Structural Changes in the M <sub/>2</sub> Muscarinic Acetylcholine Receptor Revealed by Vibrational Spectroscopy
摘要： M2 muscarinic acetylcholine receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that responds to acetylcholine and mediates various cellular responses in the nervous system. Here, we used Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy analyses on M2R reconstituted in lipid membrane to understand the molecular mechanism behind the ligand binding-induced conformational changes. Upon agonist binding, M2R shows large spectral change of amide-I band corresponding to backbone C=O stretch, which likely connects with the receptor activation in lipid environment. These results pave the way to probe effects of different ligand binding on GPCRs using vibrational spectroscopy.
关键词： ATR-FTIR spectroscopy,GPCR,M2R,Ligand,acetylcholine
更新于2025-09-12 10:27:22
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Small-Angle Neutron Scattering Reveals Energy Landscape for Rhodopsin Photoactivation
摘要： Knowledge of the activation principles for G-protein-coupled receptors (GPCRs) is critical to development of new pharmaceuticals. Rhodopsin is the archetype for the largest GPCR family, yet the changes in protein dynamics that trigger signaling are not fully understood. Here we show that rhodopsin can be investigated by small-angle neutron scattering (SANS) in fully protiated detergent micelles under contrast matching to resolve light-induced changes in the protein structure. In SANS studies of membrane proteins, the zwitterionic detergent [(Cholamidopropyl)dimethylammonio]-propanesulfonate (CHAPS) is advantageous because of the low contrast difference between the hydrophobic core and hydrophilic head groups as compared to alkyl glycoside detergents. Combining SANS results with quasielastic neutron scattering (QENS) reveals how changes in volumetric protein shape are coupled (slaved) to the aqueous solvent. Upon light exposure rhodopsin is swollen by penetration of water into the protein core, allowing interactions with effector proteins in the visual signaling mechanism.
关键词： Detergent,Neutron Scattering,Hydration,GPCR,Protein Dynamics,Vision,Membrane Proteins,Energy landscape,Slaving,Rhodopsin
更新于2025-09-10 09:29:36
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Hot spots for GPCR signaling: lessons from single-molecule microscopy
摘要： G protein-coupled receptors (GPCRs) are among the best-studied membrane receptors, mainly due to their central role in human physiology, involvement in disease and relevance as drug targets. Although biochemical and pharmacological studies have characterized the main steps in GPCR signaling, how GPCRs produce highly specific responses in our cells remains insufficiently understood. New developments in single-molecule microscopy have made it possible to study the protein–protein interactions at the basis of GPCR signaling in previously inconceivable detail. Using this approach, it was recently possible to follow individual receptors and G proteins as they diffuse, interact and signal on the surface of living cells. This has revealed hot spots on the plasma membrane, where receptors and G proteins undergo transient interactions to produce rapid and local signals. Overall, these recent findings reveal a high degree of dynamicity and complexity in signaling by GPCRs, which provides a new basis to understand how these important receptors produce specific effects and might pave the way to innovative pharmacological approaches.
关键词： single-molecule microscopy,plasma membrane,GPCR signaling,protein–protein interactions,pharmacological approaches
更新于2025-09-04 15:30:14