GPCR Research for Drug Discovery
G protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. Discover how to measure the response along each step of the GPCR signaling cascade with easy-to-use, bioluminescence-based assays.
GPCR Signaling PathwaysLuciferase-based assays offer convenient, sensitive methods to measure GPCR activity for multiple signaling pathways.
Dr. Stephen Hill describes a novel approach, using NanoBRET™ and NanoLuc® technology, to study the effects of drugs on a GPCR that recognizes and responds to neurotransmitters.
Interactions of activated GPCRs with intracellular proteins, such as G proteins, GPCR kinases and β-arrestins, are critical steps in G protein signaling, making these key areas of research for drug discovery. Sensitive bioluminescent assays enable measurement of these interactions in live cells to assess real-time kinetics in high-throughput compatible formats.
Internalization of ligand activated GPCRs is an important step in regulating receptor signaling. The HiBiT tagging system makes it possible to develop simple, quantitative assays for receptor internalization and subsequent recycling that save time and eliminate the variability associated with antibody-based methods.
HiBiT-Tagged GPCR Internalization
- Assays on live cells for real-time kinetic analysis,
- No fixing, washes or antibodies required
- Total receptor measurement on the same cells
- Compatible with endogenously tagged receptors for measuring native biology
Isoproterenol-dependent internalization monitored with the Nano-Glo® HiBiT Extracellular Detection System. The HiBiT tag was introduced into the β2-AR gene in HEK293 cells using CRISPR gene editing.
Titration of forskolin using the suspension cell line HEK293. In a white, clear-bottom, 384-well plate, 2,000 HEK293 cells were exposed to the indicated concentration of forskolin. The cAMP-Glo™ Max Assay was performed as described in the technical manual. Each point represents eight data points; the error bars represent the standard deviation. Data analysis was performed with GraphPad Prism® software using a sigmoidal dose-response (variable slope) equation.
Determining the IC50 value of SCH23390 in D1 receptor-expressing HEK293 cells. Cells were resuspended in Induction Buffer and 2,000 cells were added to each well of a white solid 384-well plate. Cells were treated with the indicated amount of antagonist, SCH23390, in the presence of 100nM agonist, SKF38393. In the negative control reactions, SCH23390 was replaced with alprenolol. The cAMP-Glo™ Max Assay was performed as described in the technical manual. Data analysis was performed with GraphPad Prism® software using a sigmoidal dose-response (variable slope) equation.
Live-Cell GloSensor™ cAMP Assay
- Real-time detection of cAMP in live cells
- Broad dynamic range, showing up to 500-fold changes in light output
- Extreme sensitivity allowing detection of Gi-coupled receptor activation or inverse agonist activity in the absence of artificial stimulation by compounds such as forskolin
Overview of the GloSensor™ cAMP Assay. Genetically encoded biosensor variants contain cAMP binding domains fused to mutant forms of Photinus pyralis luciferase. Upon binding to cAMP, conformational changes occur that promote large increases in light output.
Real-time Gαs monitoring. HEK293 cells were transiently transfected with pGloSensor™-22F cAMP Plasmid and treated with compounds as shown.
Sensitive enough to detect inverse agonists without forskolin. HEK293 cells expressing Dopamine D1 receptor were transiently transfected with pGloSensor™-22F cAMP Plasmid and treated with 10µM compounds at 28°C.
Sensitive enough to detect Gαi activity without forskolin. HEK293 cells expressing GPR44 were transiently transfected with pGloSensor™-22F cAMP Plasmid and treated with prostaglandin D2 (PGD2). Note that forskolin pretreatment increases the fold response (not shown).
Lighting Up GPCR Research with Bioluminescent Tagging
A summary of a research publication that used the HiBiT assay to measure surface expression of orexin and prokineticin receptors.
A NanoBRET™ Biosensor for GPCR:G protein Interaction with the Kinetics and Temporal Resolution of Patch Clamping
Review of a study to detect signaling through Gα12/13 proteins, which had previously proved intractable, using NanoBRET™ technology.
Measuring cAMP Levels and Cytotoxicity in a Single Plate Well
Multiplexing of a fluorescent real-time cytotoxicity assay with a bioluminescent cAMP assay, to examine cytotoxic effects through coupling of GPCRs with adenylate cyclase activity.