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PathHunter® β-Arrestin2 Retroparticles

PathHunter® β-Arrestin2 Retroparticles enable the creation of stable β-arrestin2 cell lines in any cell type and the development of a β-arrestin2 assay for analyzing the activation of GPCRs, desensitization of most 7-transmembrane receptors, and regulation of other signaling molecules such as protein kinases. The retroparticles are Moloney murine leukemia retrovirus (MMLV) expressing β-arrestin2 fused to the β-galactosidase (β-gal) reporter fragment called EA (enzyme acceptor). The retroparticles can be used for effective transduction of a wide variety of dividing target cells (e.g. CHO, HEK293, U2OS) resulting in high–level expression of the encoded β-arrestin2-EA fusion protein. This product is intended for use with a plasmid expressing a ProLink™-tagged GPCR (or another β-arrestin binding protein) to create assays that report on β-arrestin binding or ligand-induced β-arrestin2 recruitment to the GPCR or β-arrestin binding protein of interest.

Catalog No. Size Price Quantity
93-1087Retroparticles$3085.00

For account specific pricing, please contact Customer Service at customerservicedrx@eurofins.com.
Contents
0.5 mL, > 5x106 pfu/mL

Product Highlights

  • Ultimate Flexibility – Analyze mutant, ortholog or difficult GPCRs or other β-arrestin binding proteins
  • Simple Solution – Rapid implementation in any lab without the need for costly assay development
  • Broad Applicability – Create β-arrestin cell-based assays in any dividing cell type
  • Easy-to-Use – Simple assay protocol with readout on any standard luminometer
  • [User Manual] PathHunter® β-Arrestin2 Retroparticles

    The PathHunter β-Arrestin2 Retroparticles includes Moloney murine leukemia retrovirus (MMLV) expressing β-arrestin2 fused to the β-galactosidase (β-gal) reporter fragment called EA (enzyme acceptor). The Retroparticles can be used for effective transduction of a wide variety of dividing target cells (e.g. CHO, HEK 293, U2OS) resulting in hig...

    [User Manual] PathHunter® β-Arrestin2 Retroparticles

Assay Principle

The expression-ready PathHunter® β-Arrestin2 Retroparticles (retroviruses) are packaged with single stranded (+) mRNA that encodes a hygromycin resistance selection marker and a large β–galactosidase (β-gal) enzyme fragment called the enzyme acceptor (EA) that is fused to the scaffold protein β-arrestin2. With the addition of a ProLink-tagged β-arrestin binding protein (i.e. GPCR), you can take advantage of DiscoverX's proprietary Enzyme Fragment Complementation technology to ultimately create your own β-Arrestin cell-based assay.

Make Your Own GPCR β-Arrestin Cell-based Assays

For GPCR applications, this product is designed to evaluate ligand-induced arrestin recruitment to any GPCR of interest. The GPCR of interest must first be fused to the enzyme donor, ProLink (PK or ED), a smaller β-gal fragment. When the PK-GPCR fusion plasmid is stably co-expressed with the β-arrestin2-EA cell line, cells stimulated with the appropriate ligand will induce recruitment of β-arrestin2-EA to the C-terminal tail of the PK-tagged GPCR. This brings both fragments (EA and PK) of β-gal into close enough proximity to reconstitute a fully functional active enzyme capable of hydrolyzing a substrate molecule and generating a chemiluminescent signal that can be read on any standard luminometer. NOTE: β-Arrestin2-EA Retroparticles and GPCR-PK can be introduced into the target cells in either order.



Easy-to-Follow Protocol with Readout on Any Standard Luminometer


A. Generate stable PathHunter β-arrestin cell lines that express β-arrestin-EA fusion proteins and B. develop a GPCR β-arrestin assay with your GPCR of interest.

Select Applications

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

Compare Ligand Responses in Different Species (Receptor Orthologs)

Screen drugs in varying animal derived receptors prior to animal studies. M. fascicularis (monkey) (blue curve) and human (red curve) chemokine CXCR1 receptors were analyzed for β-arrestin recruitment with the agonist human interleukin-8 (IL-8). Two stable cell lines expressing monkey or human CXCR1-PK fusion proteins were generated and retrovirally infected with PathHunter β-Arrestin2 Retroparticles. Upon ligand stimulation, the β-arrestin recruitment assay showed a 10-fold less agonist potency (EC50) for the monkey receptor compared to the human receptor indicating species variations between CXCR1 GPCR orthologs.

Study Mutant or Isoform Differences

Isoforms (VGV, VNV, VSV) of the 5HT2C human serotonin receptor were compared to the canonical form (INI) of the receptor for β-arrestin recruitment. Four different 5HT2C-PK cell lines were created and retrovirally infected with PathHunter β-Arrestin2 Retroparticles. β-Arrestin recruitment assays were performed using the ligand 5-HT (serotonin). Results revealed a 10-fold greater ligand potency (EC50) for the canonical receptor compared to the other 3 isoforms indicating slight differences between the serotonin receptors for β-arrestin recruitment.

Investigate Tissue Specific Variations Using Different Cell Types

Tissue specific variations for prostaglandin E receptor 4 (PTGER4) were investigated using the β-arrestin recruitment assay in 2 different parental cell types. HEK 293 (human embryonic kidney 293, blue curve) and U2OS (human bone osteosarcoma epithelial, red curve) cells containing PTGER4-PK were each retrovirally infected with the PathHunter β-Arrestin2 Retroparticles. Cells were then assayed for β-arrestin recruitment in the presence of prostaglandin E2 and revealed a slight tissue type difference with ligand bias toward the U2OS cell line (10-fold greater ligand potency, EC50) compared to the HEK 293 cell line.


Tissue specific variations for cholecystokinin A receptor (CCKAR or CCK1) were investigated using the β-arrestin recruitment assay in 3 different parental cell types. CHO-K1 (Chinese hamster ovary K1, blue curve), HEK 293 (human embryonic kidney 293, red curve) and U2OS (human bone osteosarcoma epithelial, orange curve) cells containing CCKAR-PK were each retrovirally infected with the PathHunter β-Arrestin2 Retroparticles. Cells were then assayed for β-arrestin recruitment in the presence of the ligand CCK8 and revealed slight tissue type differences with ligand bias towards the CHO-K1 cell line (10-fold greater ligand potency, EC50) compared to the HEK 293 and U2OS cell lines