PathHunter β-Arrestin Assay Principle
The β-arrestin G protein-independent pathway and PathHunter GPCR β-Arrestin assay principle. A. Ligand-induced β-arrestin (types 1 or 2) recruitment activates signaling cascades independently of G-protein signaling to provide a stoichiometric, non-amplified signal. The ligand-activated GPCR is phosphorylated by a specific GPCR kinase (GKRs, not shown here), leading to β-arrestin recruitment. This β-arrestin binding blocks G protein-mediated signaling and results in the internalization (or endocytosis) of the GPCR and ending of the attenuation of GPCR signaling (known as desensitization). Subsequently, the GPCR is recycled back to the plasma membrane or degraded in the lysosome. This stoichiometric (1 receptor: 1 ligand), non-amplified system requires full ligand occupancy of the ligand bound to the receptor to give a full signal, which lowers sensitivity to agonists, but improves the ability to detect differences of efficacy between agonists and superior sensitivity for antagonists (compared to second messenger systems). By analyzing the β-arrestin pathway, you can fine tune GPCR biology when screening antagonists as well as deorphanize GPCRs and distinguish between full, super, and partial agonists. B. PathHunter GPCR β-Arrestin assays take advantage of DiscoverX’s proprietary Enzyme Fragment Complementation technology. The GPCR is fused in frame with a small enzyme donor fragment ProLink™ (PK) and co-expressed in cells stably expressing a fusion protein of β-arrestin and a larger, N-terminal deletion mutant of β-galactosidase (called enzyme acceptor or EA). Activation of the GPCR stimulates binding of β-arrestin to the PK-tagged GPCR and forces complementation of the two enzyme fragments, resulting in the formation of an active β-galactosidase enzyme. This interaction leads to an increase in enzyme activity that can be measured using chemiluminescent PathHunter Detection Reagents. Ultimately, β-arrestin recruitment assays offer an easy-to-use alternative to second messenger cAMP and calcium G-protein dependent pathways to enable enhanced profiles of compound pharmacology – a universal assay that expands opportunities for development of novel drugs.
Simple homogeneous protocols with no washing and a chemiluminescent output that can be read on any benchtop luminometer. Eliminate the need to load target cells before every experiment and the use of radioactivity and antibodies. Reduce the number of workflow steps to increase the efficiency of lab experimentation using the β-arrestin assays for your GPCR drug discovery needs.
- Perform multiple pathway analysis using the same cell line
- Uncover unique ligand pharmacologies
- Determine potency-based rank order of ligands
- Deorphanize GPCRs
- Evaluate GPCRs that are difficult to assay
- Compare ligand responses in different species receptors (orthologs)
- Study mutant or isoform differences
- Investigate tissue specific variations using different cell types
- Create your own GPCR β-arrestin cell-based assays in any cell type
Perform Multiple Pathway Analysis using the Same Cell Line and Uncover Unique Ligand Pharmacologies
Conveniently use only one cell line for analyzing multiple GPCR pathways and uncover unique pharmacologies for your ligand that may not have been discovered by analyzing only a single pathway. The β-arrestin recruitment assay (A.) followed by the second messenger (cAMP) assay (B.) were performed on the same cell line expressing human cholinergic muscarinic 2 (CHRM2) fused with the Enzyme Fragment Complementation small enzyme donor PK. Cells expressing CHRM2-PK fusion proteins were infected with PathHunter β-Arrestin2 Retroparticles and tested using 3 ligands: acetylcholine, carbachol and oxotremorine-M (oxo-M). Both pathway assays showed agonist pharmacology for all 3 ligands. The β-arrestin recruitment assay in particular showed the true partial agonist pharmacology for oxo-M was revealed due to the non-amplified signal nature of the β-arrestin pathway compared to the amplified G-protein dependent pathway.
Determine Potency-Based Rank Order of Ligands
Activation and inhibition of prostaglandin D2 (CRTH2) was analyzed with 3 ligands – prostanglandin D2 (PGD2), indomethacin and ramatroban. Cells expressing CRTH2-PK fusion proteins were retrovirally infected with PathHunter β-Arrestin2 Retroparticles and assayed for β-arrestin recruitment. Dose response curves showed agonist and antagonist ligand pharmacologies as well as depicted accurate potency-based rank order of the ligands.
GPR17, a class A orphan receptor that is phylogenetically related to purinergic and CysLT receptors, has emerged as a modulator of CNS myelination. The small molecule MDL29,951 was analyzed for its ability to deorphanize GPR17 and function as a potential treatment for demyelinating CNS diseases like multiple sclerosis. CHO-K1 cells expressing the GPR17-PK fusion protein were retrovirally infected with the PathHunter β-Arrestin2 Retroparticles and assayed for β-arrestin recruitment. Results indicate compound induced concentration-dependent agonist response for MDL29,951 revealing a potential modulator of GPR17 and potential therapeutic for multiple sclerosis patients.
Evaluate GPCRs that are Difficult to Assay
Lipid ligands and lysophospholipid GPCRs tend to be problematic to work with. The difficult to study GPCR lysophospholipid, EDG2 (also called LPA1 or LPAR1), was evaluated for β-arrestin recruitment using the ligand 1-Oleoyl-LPA. A CHO-K1 human EDG2-PK cell line was retrovirally infected with PathHunter β-Arrestin2 Retroparticles and assayed for β-arrestin recruitment. Results revealed successful measurement of β-arrestin recruitment upon ligand stimulation and acceptable signal:background of 2.6.
Create Your Own GPCR β-Arrestin Cell-Based Assays in Any Cell Type
Create your own GPCR β-arrestin cell-based assays to evaluate ligand-induced β-arrestin recruitment to any GPCR in any cell type. Using the Enzyme Fragment Complementation technology, simply infect your target cells with the PathHunter β-Arrestin Retroparticles, transfect the cells with a GPCR plasmid, and perform a PathHunter GPCR β-arrestin assay with your ligand of interest. Note: β-Arrestin2-EA Retroparticles and GPCR-PK can be introduced into the target cells in either order.