Transfluor Cell-Based GPCR Assay
A powerful functional assay to detect compound activity against known and/or orphan GPCR targets in a high content screening environment.
Leveraging the principle that virtually all GPCRs rapidly undergo deactivation or "desensitization" by a common pathway, 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 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 of 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.
|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.|
This patented technology, licensed from Duke University, uses cell lines genetically engineered to express both the labeled beta-arrestin and the GPCR of interest and can be used to screen for natural or synthetic ligands, including agonists or antagonists. The Transfluor Assay requires no prior knowledge of the interacting G-protein, making it ideally suited for screening orphan GPCRs (oGPCR)
|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.|
- 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.