NK1 Receptor Activation Redistribution Assay

• Designed to assay compounds for their ability to activate the NK1 receptor
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

Neurokinin-1 (NK1) receptor belongs to the group of tachykinin receptors and is stimulated by substance P. NK1 receptor activation has been associated with a number of neurological diseases such as depression. In this assay, the NK1 receptor has been transfected into the GPCR Reporter Assay for Gq-coupled Receptors, where receptor activation leads to release of cytoplasmic Ca²⁺, which in turn induces NFATc1 (nuclear factor of activated T-cells) translocation. Binding of an agonist to the extracellular parts of NK1 receptor causes a conformational change in the receptor. This leads to conformational changes in heterotrimeric G proteins at the intracellular face of the receptor, exchange of GDP for GTP on the alpha subunit (Gαq) and subsequent release of Gαq from the β-γ subunit. GTP-bound Gαq then activates phospholipase C, which catalyzes the formation of DAG and IP3 from PIP2. Free IP3 diffuses into the cytoplasm, and activates IP3 receptors on the endoplasmic reticulum (ER) resulting in Ca²⁺ release from the ER into the cytoplasm. NFATc1 is a transcription factor involved in T-cell signaling and tissue development, and its activity is controlled by the Ca²⁺ /Calmodulin-dependent phosphatase, calcineurin. Inactive NFATc1 resides in the cytosol, but in response to elevated calcium levels, NFATc1 is dephosphorylated by calcineurin, followed by rapid translocation to the nucleus.

Figure 1. Nuclear translocation of EGFP-NFATc1. Cells expressing the NK1 receptor were treated with 10 nM substance P for 30 min (right panel) or untreated (DMSO control, left panel). Activation of the receptor leads to nuclear translocation of EGFP-NFATc1, which can be detected by the image analysis algorithm.

Figure 2. Substance P concentration response curve in the NK1:NFATc1 assay. The EC₅₀ of substance P is ~400 pM. Concentration response was measured in 9 point half log dilution series (n=8). Cells were treated with substance P for 30 min. Cells were then fixed and nuclear translocation was measured using the Cellomics ArrayScan V^TI Reader and the Redistribution V3 BioApplication. % activity was calculated relative to the positive (10 nM substance P) and negative control (0.25% DMSO).