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.
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 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.
