Detects compound activity against known and/or orphan GPCR targets in a high content screening environment
The Transfluor® Assay is a cell-based fluorescence assay used to screen for G-protein coupled receptor (GPCR) ligands and other potential drugs that regulate GPCRs by deactivating or desensitizing a common pathway. By attaching a fluorescent label to beta-arrestin, the location of the receptor-arrestin complex may be monitored during receptor activation. Since desensitization only occurs with an activated receptor, monitoring beta-arrestin translocation and subsequent receptor recycling provides a method to detect the activation of any GPCR.
Studying this mechanism enables researchers to identify the impact of GPCR ligands on a variety of physiological processes including behavior, mood, inflammation, nervous system, and olfactory responses. In addition, researchers can gain insight into the complexities of receptor degradation and recycling for a given target.
- Validated with over 100 GPCRs, the Transfluor assay provides the assurance to work across all GPCR classes (Class I, II, III), regardless of interacting G-protein (Gs, Gi/o and Gq/11).
- Single read-out is compatible with all GPCR subtypes, including Gi, Gs, Go, Gq, eliminating the need for multiple GPCR assays
- No GPCR labeling or tagging avoids custom molecular biology that may alter your target’s response.
- Universal format requires no prior knowledge of interacting G-protein, enabling orphan GPCR screening.
- GFP-based technology does not require additional substrates to monitor beta-arrestin translocation.
- MetaXpress® Application Module automates analysis of beta-arrestin movement to the cell membrane (pits) and endocytic vesicles.
- Ligand Independent Translocation (LITe™) Assay is an agonist-independent assay used to verify the translocation of beta-arrestin-GFP in orphan GPCRs.
Orphan GPCR Assay
In contrast to current methods of screening GPCRs, the Transfluor technology is based on the mechanism for termination of GPCR signaling, known as receptor desensitization. This mechanism is shared by virtually all GPCRs and is activated by ligand binding. Transfluor technology requires no prior knowledge of the interacting G-protein. This important feature of the Transfluor technology makes it ideal for screening orphan GPCRs (oGPCR).
A proprietary technique to assist in validating orphan GPCR screens, called LITe™ (Ligand Independent Translocation), is an agonist-independent assay used to verify the translocation of ß arrestin-GFP in orphan GPCRs.
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High-content screening of known G protein-coupled receptors by arrestin translocation.
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Quantitative cell-based high-content screening for vasopressin receptor agonists using transfluor technology.
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The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors.
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Figure 1.ß2AR-expressing cells were stimulated with isoproterenol. Left: control, center: pits, right: vesicles.
Top: Transfluor assay imaged with ImageXpress. Bottom: Transfluor assay imaged with Discovery-1.
Figure 2.Segmentation and analysis run with the GPCR Cycling Application Module for MetaXpress.
Dose response to isoproterenol. Top: pits, bottom: vesicles. Error bars show standard error.
Partial GPCR ListingThe Transfluor technology monitors receptor activity by detecting movement of beta-arrestin-GFP in the cell.
A partial listing of GPCRs that have been shown to translocate beta-arrestin-GFP is shown below.
- 12 Drosophila GPCRs
- Fz4 frizzled receptor
- TßRIII transforming growth factor-ß