AT1 Receptor Activation Redistribution Assay

• Designed to assay compounds for their ability to modulate activation of the AT1R 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

Angiotensin activates a wide spectrum of signaling responses via the Angiotensin II Type-1 receptor (AT1R) having immediate physiological effects on vasoconstriction and blood pressure regulation as well being implicated in inflammation, endothelial dysfunction, atherosclerosis, hypertension, and congestive heart failure. AT1R is a member of the superfamily of G protein–coupled receptors (GPCRs) and couples to Gαq. In this assay, the AT1R 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 translocation. Binding of an agonist to the extracellular parts of AT1R causes a conformational change in the receptor. This leads to activation of heterotrimeric Gq proteins, subsequent release of Gαq from the beta-gamma subunit and activation of 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. Elevated calcium levels leads to dephosphorylation of NFATc1 and rapid translocation from the cytoplasm to the nucleus.

Figure 1. Nuclear translocation of EGFP-NFATc1. Cells expressing the AT1 receptor were treated with 300 nM angiotensin for 30 min (right panel) or untreated (DMSO control, left panel). Activation of the receptor leads to nuclear translocation of EGFP-NFATc1 (arrows), which can be detected by the image analysis algorithm.

Figure 2. Angiotensin concentration response in the AT1R:NFATc1 assay. The EC₅₀ of angiotensin is ~10 nM. Concentration response was measured in 9 point half log dilution series (n=16). Cells were treated with angiotensin 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 (300 nM angiotensin) and negative control (0.25% DMSO).