Tango GPCR Assay System
The Tango GPCR assay system provides a readout that is:
- Specific to the target receptor—ensuring a selective readout
- Proximal to the actual site of receptor activation—minimizing false positives
- Independent of the G protein the receptor signals through— enabling study of any GPCR (including orphans)
The Tango GPCR Assay System (Figure 1) is a proprietary platform based upon ligand binding to a specific G protein-coupled receptor (GPCR) that triggers desensitization, a process mediated by the recruitment of protease-tagged arrestin proteins to the activated receptor. The protease-tagged arrestin cleaves a protease site fused to the C-terminus of the GPCR, releasing a non-native transcription factor.
This transcription factor immediately enters the nucleus, thus bypassing additional signaling intermediates. There, the transcription factor directly regulates transcription of a beta-lactamase reporter construct, which is measured upon addition of the live-cell substrate (Figure 2). The blue to green fluorescence ratio obtained directly correlates to receptor modulation by agonists or antagonists. Activation of the reporter gene provides a quantifiable measurement of the degree of interaction between the target receptor and the protease-tagged arrestin partner.
Figure 1. Schematic overview of the Tango Assay system for GPCRs. Ligand binding to target receptor stimulates recruitment of arrestin protease, which triggers release of a tethered transcription factor. Free transcription factor enters the nucleus and stimulates reporter gene activity. This cell-based assay affords a direct and selective measurement of target GPCR activity and is applicable to virtually all classes of GPCRs.
Validation with a diverse array of GPCRs
Tango GPCR assays have been validated for a diverse array of GPCRs, including receptors related to each of the major G protein pathways and activated by a variety of ligand types:
- Gq-, Gs-, and Gi/o-coupled receptors
- Orphan GPCRs
- Hormone receptors
- Neurotransmitter receptors
- Peptide receptors
- Chemokine receptors
Figure 2. Fluorescent detection of þ-lactamase reporter gene response using GeneBLAzer technology. After substrate loading, in the absence of þ-lactamase expression, cells generate green fluorescence. In the presence of B-lactamase expression, the substrate is cleaved and cells generate blue fluorescence.
Agonist and Antagonist Assays
Quantitative measurements of the GPCR activation and arrestin recruitment can be accurately determined with the Tango GPCR Assay System. This method is ideal for high throughput screening (HTS) applications and is highly amenable to miniaturization. Tango GPCR cell lines can be used to monitor agonist-mediated receptor activation, to assess the relative efficacies of different compounds, and to measure receptor inactivation by specific antagonists (Figure 4). The readout is also specific to the target receptor (Figure 3).
Figure 3. Target selectivity. Tango GPCR cell lines and profiling service enable selective interrogation of your target receptor of interest. The vasopressin V2 receptor is expressed in HEK 293 cells and is assayed for activity using a calcium flux assay and a Tango assay. Normally, treatment of cells with vasopressin (a ligand for the target receptor), as well as carbachol and ATP (lgands for endogenous nontarget receptore in HEK 293 cells), results in indistinguishable signals in a calcium flux assay. In contrast, the Tango GPCR cell lines generate a signal only upon stimulation using the target receptor's cognate ligand.
Figure 4. Agonist and antagonist dose response curves with Tango assays. Agonist dose response (A and C) and antagonist dose inhibition (B and D) curves generated with Tango GPCR cell lines EDNRA and NPY2R.
Tango™ technology is available on a custom basis for client-directed targets. Engineered cell lines are provided as a validated master cell line stock. Cells can also be provided on-demand in either 1-10 plate/vial configurations in either a dividing or division arrested format.