PathHunter® GPCR Internalization Assays

A Simple, Primary 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 are functional, cell-based assays that measure GPCR endocytosis. These non-imaging, non-antibody-based, chemiluminescent assays provide a direct and quantitative measurement of internalized GPCRs localized to intracellular endosomes. This allows the fate of activated GPCRs to be monitored in live cells without requiring expensive microscopy.

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Learn more about the updated Cell Culture and Handling Procedure in the new Technical Bulletin.
 
GPCR Internalization
PathHunter GPCR internalization assays are based on the proprietary Enzyme Fragment Complementation (EFC) technology and provide a tool for primary 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.
 

PathHunter Total GPCR Internalization Assay Format



PathHunter total GPCR internalization cell lines are engineered to co-express the ProLink™ (PK; EFC enzyme donor) tagged GPCR, and an EFC enzyme acceptor (EA) tag localized to the endosomes. An alternate format reverses the localization of the enzyme fragments; the GPCR is tagged with the EA and the PK is localized to the endosomes. Ligand-induced activation of GPCR-PK (or EA) leads to internalization of the receptor in EA-tagged (or PK-tagged) endosomes, forcing complementation of the two β-galactosidase (β-gal) enzyme fragments (EA and PK).  The resulting functional enzyme hydrolyzes substrate to generate a chemiluminescent signal.
 

PathHunter Activated GPCR Internalization Assay Format



PathHunter activated GPCR internalization cell lines are engineered to co-express an untagged GPCR, an EA tagged β-arrestin, and a PK tag localized to the endosomes.  Activation of the untagged GPCR induces β-arrestin recruitment, followed by internalization of the GPCR-β-arrestin-EA complex in PK-tagged endosomes.  Similar to the total assay format, this internalization forces complementation of the two β-gal enzyme fragments, forming functional enzyme that hydrolyzes substrate to generate a chemiluminescent signal.

 


DiscoverX offers a variety of products and services for analyzing GPCR internalization.

Products

Cell Lines
Assay Ready Kits
Detection Reagents


Services

GPCR Profiling and Screening
Custom Assay Development


Related Products

Pharmacochaperone Trafficking Assays - also available for ion channels and transporters
Translocation Assays for monitoring cellular movement of proteins to multiple membrane compartments
Parental Cell Lines and Cloning Vectors for creating your own cell-based internalization assays

 

PathHunter GPCR Internalization Assay Highlights

  • ~100 cell-based assays using a simple one-step protocol applicable to multiple target classes
  • Direct functional readout allows for quantitative analysis of GPCR desensitization and recycling as well as uncovering novel classes of compound pharmacologies
  • Chemiluminescent, gain-of-signal assays with no need for antibodies, radioactivity, or imaging
  • Standard instrumentation with no complex data analysis, normalization, or pit/vesicle spot detection required
 

Uncover Unique Pharmacologies

Reveal novel classes of compounds like super-agonists and functional agonists, and evaluate their efficacies and potencies.

Chemokine Receptor 1Cholinergic Receptor, Muscarinic 2

Cells expressing C-C chemokine receptor type 1 (CCR1) (left graph) and Cholinergic Muscarinic M2 receptor (CHRM2) (right graph) were treated with increasing concentrations of indicated compounds and assayed using PathHunter detection reagents. The universal, no wash, single addition protocol results in a high throughput friendly approach that allows you to easily distinguish potency (EC50s) and efficacy (signal-to-background) differences between compounds that induce receptor internalization. The CCR1 internalization assay uncover the super-agonist CCL23 compared to the reference agonist CCL3, which the CHRM2 internalization assay oxotremorine M as 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.

Sphingosine-1-Phosphate Receptor

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 showed agonist ligand pharmacologies as well as depicted accurate potency-based rank order of the ligands.