Technologies & Platforms / Enzyme Fragment Complementation Technology / PathHunter EFC Cell-Based Assay Platform / Protein Translocation / GPCRs - Internalization
Technologies & Platforms

PathHunter® GPCR Internalization Assay Platform

PathHunter GPCR Internalization assays are whole cell functional 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 and allow the fate of unlabelled, activated GPCRs to be monitored in live cells without the requirement of expensive microscopy.

PathHunter Total GPCR Internalization System

PathHunter® Total GPCR Internalization cell lines are engineered to co-express the ProLink™ (PK) tagged GPCR, and an Enzyme Acceptor (EA) tag localized to the endosomes. An alternate format reverses the localization of the enzyme fragments:  The GPCR is tagged  with EA and PK is localized to the endosomes.  Activation of the GPCR-PK (or EA) induces internalization of the receptor in EA-tagged (or PK-tagged) endosomes, forcing complementation of the two β-galactosidase enzyme fragments (EA and PK).  The resulting functional enzyme hydrolyzes substrate to generate a chemiluminescent signal.
 
Total GPCR Internalization System

PathHunter Activated GPCR Internalization Assays

PathHunter® Activated GPCR Internalization cell lines are engineered to co-express an untagged GPCR, Enzyme Acceptor (EA) tagged β-Arrestin, and a ProLink™ (PK) tag localized to the endosomes.  Activation of the untagged GPCR induces β-Arrestin recruitment, followed by internalization of the Receptor/Arrestin-EA complex in PK-tagged endosomes.  This forces complementation of the two β-galactosidase enzyme fragments (EA and PK), forming functional enzyme that hydrolyzes substrate to generate a chemiluminescent signal.

GPCR Internalization PathHunter EFC Schematic
In both formats, complementation of the two enzyme fragments results in an increase in enzyme activity that can be easily measured using PathHunter® Detection Reagents.
Cells expressing C-C chemokine receptor type 1, CCR1 (left panel) and Cholinergic Muscarinic M2 receptor, CHRM2 (right panel) 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 and efficacy differences between compounds that induce receptor internalization.

Features & Benefits

  • Direct functional readout – uncover novel classes of compounds like superagonists and functional antagonists
  • Simple one step protocol apllicable to multiple target classes.
  • Stable cell lines – results in predictable pharmacology and reproducibility
  • Chemiluminescent, gain-of-signal assays – no antibodies, radioactivity or imaging
  • Standard instrumentation – no complex data analysis, normalization or pit/vesicle spot detection required