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PathHunter® β-Arrestin Assays

Largest Portfolio of β-Arrestin Cell-based Assays and Reagents for Drug Discovery
 

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. For GPCRs, ligand-induced β-arrestin recruitment activates signaling cascades independent of G-protein signaling to provide a non-amplified signal which is ideal for antagonist mode screening, studying ligand pharmacologies, and deorphanizing GPCRs. With decades of experience and hundreds of customer publications, DiscoverX provides a complete set of tools to analyze β-arrestin biology. The PathHunter β-arrestin assays and reagents offer a powerful and universal screening and profiling platform that can be used for virtually any Gi-, Gs-, or Gq-coupled receptor.

 

Advantages of β-Arrestin Assays and Reagents

  • Largest Portfolio – 400+ cell-based assays covering over 90% of class A & B GPCR targets
  • Accurate Pharmacology – Easily distinguish ligand pharmacological differences
  • Multiplexing Capabilities – Run second messenger assays using the same β-arrestin cell line
  • Universal System – Independent of G-protein coupling, ideal for antagonist screening and GPCR deorphanization

Ultimate Flexibility – Create Your Own GPCR B-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.

Easy-to-Use – Simple, One-Step Add and Read Protocol



Simple no wash, add, mix, and read protocols with a chemiluminescent output that can be read on any benchtop luminometer. Using the β-arrestin assays, you can eliminate the need to load target cells prior to every experiment, eliminate the use of radioactivity, and reduce the number of workflow steps, increasing the efficiency of the lab.

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To view all of our available Arrestin products and services, please check out the links below.

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PathHunter β-Arrestin Principle for GPCR Applications




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 the 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.

Select Applications

  • Perform multiple pathway analysis in the same cell line
  • Uncover unique ligand pharmacologies
  • Correctly rank order ligands
  • Deorphanize GPCRs
  • Evaluate difficult GPCRs
  • Compare ligand responses in different species receptors (orthologs)
  • Study mutant or isoform differences
  • Investigate tissue specific variations using different cell types

Perform Multiple Pathway Analysis in the Same Cell Line


The β-arrestin recruitment assay followed by the second messenger (cAMP) assay were performed on the same cell line expressing human cholinergic muscarinic 2 (CHRM2) fused with 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. Only using the β-arrestin recruitment assay (left) revealed the possible true partial agonist pharmacology for oxo-M due to the non-amplified signal nature of the β-arrestin pathway compared to the amplified G-protein dependent pathway (right).

Uncover Unique Ligand Pharmacologies

Uncover unique pharmacologies for multiple classes of ligands. Cells expressing human β2 adrenergic receptor (ADRB2) were retrovirally infected with PathHunter β-Arrestin2 Retroparticles and assayed for β-arrestin recruitment. Dose curves identified a full agonist (isoproterenol), partial agonist (clenbuterol) and antagonist (propranolol) for ADRB2.

Correctly Rank Order 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 Difficult GPCRs


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 β-arrestin recruitment upon ligand stimulation and acceptable signal:background of 2.6.


For additional β-arrestin application data, visit discoverx.com/arrestin-retroparticles.