GPCR Internalization Assays
A Simple, Secondary, and Orthogonal Screening Tool for Identifying Safer Drugs
GPCR internalization is essential to prevent cells from undergoing excessive receptor stimulation or periods of prolonged inactivity. If GPCR internalization is inhibited by a ligand, a protein mutation, or aberrant signaling, undesirable effects may occur, often resulting in drug tolerance, unwanted side effects, and disease.
PathHunter® GPCR internalization assays from Eurofins DiscoverX® are functional, cell-based assays that measure GPCR endocytosis. These non-imaging, non-antibody-based, mechanism of action (MOA)-reflective chemiluminescent cellular assays directly and quantitatively measure internalized GPCRs localized to intracellular endosomes. This allows the fate of activated GPCRs to be monitored in live cells without requiring expensive microscopy.
Product Highlights
- Cell-Based Assays – Over 100 GPCR Internalization chemiluminescent, gain-of-signal cell-based assays negating the need for detection antibodies, radioactivity, or imaging
- Functional Assays – Direct functional readout allows for quantitative analysis of GPCR desensitization and recycling as well as uncovering novel classes of compound pharmacologies
- Simple Platform – High throughput cell-based assay platform utilizes standard instrumentation and no complex data analysis, normalization, or pit/vesicle spot detection requirement
- Flexible Products – Cell lines and assay-ready eXpress kits that can be used to analyze GPCR internalization regardless of coupling status
PathHunter GPCR internalization assays are based on the proprietary Enzyme Fragment Complementation (EFC) technology and provide a tool for secondary or orthogonal screening to identify safer drugs with less undesirable effects like drug tolerance, unwanted side effects, and disease. There are two assay formats for detecting receptor internalization in whole cells. The total internalization assay detects ligand-induced GPCR endocytosis via all endocytosis mechanisms; the activated internalization assay looks at only ligand-induced, β-arrestin-mediated receptor internalization.
Receptor Internalization Assay Principles
PathHunter total GPCR internalization assay principle. Cell lines are engineered to co-express two fragments of the β-galactosidase (β-gal) enzyme – a small EFC enzyme donor (ED) and a larger EFC enzyme acceptor (EA). In A., the ED is tagged to the GPCR, and the EA is localized to the endosome. In B., the ED is localized to the endosome, and the EA tagged to the GPCR. In both formats, small molecule or biologic (e.g. antibody) ligands that bind the GPCR-tagged fusion protein leads to internalization (endocytosis) of the receptor to the endosome, forcing the complementation of the two β-gal enzyme fragments (ED and EA). The resulting functional β-gal enzyme hydrolyzes a substrate to generate a dose-dependent chemiluminescent signal.
PathHunter activated GPCR internalization assay principle. Cell lines are engineered to co-express an untagged GPCR, an EA tagged β-arrestin, and an ED tag localized to the endosomes. Activation of the untagged GPCR induces β-arrestin recruitment, followed by internalization of the GPCR-β-arrestin-EA complex to the ED-tagged endosomes. Similar to the total assay format, this internalization forces the complementation of the two β-gal enzyme fragments, forming a functional enzyme that hydrolyzes substrate to generate a chemiluminescent signal.
Quantify GPCR Desensitization and Recycling
Measure protein internalization of GPCRs from the plasma membrane to the endosome upon ligand stimulation. A GPCR internalization assay was generated using ENDO-EA U2OS parental cells expressing PK1-tagged opioid receptor mu, OPRM1 (Cat. No. 93-0745C3; GPCR internalization total format). The data indicate translocation of the ED-tagged receptor from the plasma membrane to the EA-tagged endosome upon stimulation with agonist [Met5]- enkephalin (EC50 = 589 nM; signal-to-background (S/B) = 4.3).
Uncover Unique Pharmacologies
Reveal novel classes of compounds like super-agonists and functional agonists, and evaluate their efficacies and potencies. Cells expressing C-C chemokine receptor type 1 (CCR1) (A.) and cholinergic muscarinic M2 receptor (CHRM2) (B.) were treated with increasing concentrations of indicated compounds and assayed using PathHunter detection reagents. The universal, no wash, single addition protocol provides a high-throughput friendly approach that allows you to easily distinguish potency (EC50s) and efficacy (signal-to-background) differences between compounds that induce receptor and the CHRM2 internalization assay oxotremorine M reveals a more potent agonist compared to acetylcholine or carbachol.
Correctly Rank Order Ligands
Compare agonists and evaluate their pharmacology to determine the best leads for your drug discovery program or specific application. Cells expressing sphingosine-1-phosphate receptor (S1P1) were treated with increasing concentrations of indicated compounds and assayed for receptor internalization using PathHunter detection reagents. The dose response curves shows agonist ligand pharmacologies as well as depicted accurate potency-based rank order of the ligands.
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