PathHunter™ Activated GPCR Internalization Assays
PathHunter™ Activated GPCR Internalization assays
provide a quantitative measurement of
internalized GPCR protein localized to the early endocytic compartment. Unlike other imaging
or antibody-based internalization technologies, PathHunter assays are simple, non-radioactive
chemiluminescent assays that are amenable to highthroughput screening.
•Differentiate between slow and fast recyclers
•Compare pharmacology of selective and non-selective agonists
•Combine with traditional second messenger assays to identify functional antagonists,
novel inhibitors or biased ligands
Figure 3. The small enzyme fragment of β-galactosidase (ProLink™) is localized to the surface of cellular endosomes and the larger, complementing enzyme fragment (termed Enzyme Acceptor or
EA) is fused to β-Arrestin. Stimulation of the receptor results in Arrestin binding to the activated
GPCR, internalization of the receptor and trafficking to cellular endosomes resulting in enzyme complementation and an increase in activity that is easily measured using PathHunter Detection Reagents.
Figure 4. (C) Cells expressing the native, untagged Cholinergic Muscarinic M2 Receptor (CHRM2) were treated with increasing concentrations of Oxotremorine-M and Acetylcholine, two known agonists.
(A, B & D) Agonist dose response curves wer also performed on cells expressing the Neurotensin NTSR1 (B), Chemokine CCR5 (A) and Cholinergic Muscarinic CHRM5 (D) receptors. U2OS cells expressing each receptor were treated with increasing concentrations of control agonist and assayed using the PathHunter Detection Reagents. Together, this data demonstrates that the PathHunter Activated GPCR Internalization Technology is broadly applicable across a large number of GPCR families.