GPCR Beta-arrestin assays -

Technologies & Platforms

GPCR β-Arrestin Product Solutions

Universal GPCR Assays for Ligand Bias, Receptor Deorphanization, and Antagonist Discovery

With β-arrestins cell-based assays, you can easily monitor GPCR activity for antagonist mode screening, study ligand pharmacologies, and deorphanize GPCRs. Implement these assays to measure GPCR β-arrestin recruitment and subsequent internalization upon ligand or therapeutic activation of GPCRs in a dose-dependent manner.

β-Arrestins are ubiquitously expressed and function in the activation of GPCRs, desensitization of most 7-transmembrane receptors, and regulation of other signaling molecules such as protein kinases. β-Arrestin activation is part of the G protein-independent independent pathway that results from the ligand-activated GPCR phosphorylation by specific GPCR kinases referred to as GKRs. This phosphorylation leads to β-arrestin recruitment and binding that blocks G protein-mediated signaling, and results in the internalization (or endocytosis) of the GPCR; 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.

Eurofins DiscoverX^® provides a complete set of analytical tools and brings decades of experience and hundreds of customer publications to β-arrestin biology. PathHunter® β-Arrestin cell lines, assays, kits, and reagents all offer a powerful and universal screening and profiling platform that can be used for virtually any Gi-, Gs-, or Gq-coupled receptor.

Product Highlights

Largest Portfolio – Covering ~90% of class A & B GPCR targets with over 400 cell-based assays, cell lines, kits, and reagents
Accurate Pharmacology – Easily distinguish ligand pharmacological differences with excellent superior performance for screening antagonists, positive allosteric modulators, and partial agonists
Multiplexing Capabilities – Run second messenger cAMP or calcium assays using the same β-arrestin cell line for ligand bias studies
Universal System – Independent of G-protein coupling, ideal for antagonist screening and GPCR deorphanization

Key Resources

Products

Assay-Ready eXpress^™ and Bioassay Kits

β-Arrestin eXpress and Bioassay Kits – Complete assay-ready kits with specific cell line, optimized reagents, and plates for quick Enzyme Fragment Complementation (EFC) based chemiluminescent detection. Bioassays include additional optimization for high lot-to-lot reproducibility. Choose from Known, Orphan, or Orthologs assay-ready kits.

Cell Lines

β-Arrestin Cell Lines – Stable cell lines confirmed for past 10 passages and rigorously and continuously validated for accurate pharmacology through Eurofins Discovery services with billions of data points screened. Choose from Known, Orphan, or Orthologs cell lines.

Custom and Toolbox Products

Custom Capabilities – Custom cell lines, assays, and protein development capabilities optimized to your requirements.
Parental Cell Lines – Engineered cells to generate your own β-arrestin assays and investigate β-arrestin recruitment and GPCR desensitization and internalization of your GPCR target of interest.
Retroviral Particles – Create target-specific, stable β-arrestin cell lines in any divided cell type. The product comes with a complete set of retroparticles and reagents combined with an easy-to-follow protocol to help you generate your own β-arrestin cell-based assays.

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.

Assay Workflow

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.

Select Applications

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.

Deorphanize GPCRs

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.