AT1 Receptor Internalization Redistribution Assay

• Designed to assay compounds for their ability to modulate internalization of AT1R
• 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

The renin-angiotensin system is a central component of the physiological and pathological responses of the cardiovascular system. Its primary effector hormone, angiotensin, activates a wide spectrum of signaling responses via the AT1 receptor (AT1R) thereby mediating its immediate physiological effects of vasoconstriction and blood pressure regulation. It is also implicated in inflammation, endothelial dysfunction, atherosclerosis, hypertension, and congestive heart failure. The AT1R is a member of the superfamily of G protein–coupled receptors (GPCRs), and angiotensin-induced AT1R activation via Gq/11 stimulates phospholipases A2, C, and D. This leads to activation of inositol trisphosphate/Ca²⁺ signaling, protein kinase C isoforms, MAPKs, and tyrosine kinases as well as scaffold proteins. Angiotensin activation of the AT1R causes rapid phosphorylation, desensitization, and endocytosis of the AT1R with subsequent intracellular processing and recycling to the cell membrane.

In addition to the established therapeutic actions of angiotensin in hypertension, the actions of angiotensin have lead to novel clinical applications of angiotensin-converting enzyme (ACE) inhibitors and AT1R blockers within the fields of cancer, diabetes, and metabolic diseases. Antagonists of AT1R internalization are likely to be relevant in the treatment of hypertension and related diseases.

Figure 1. Internalization of AT1R-EGFP. Cells were treated with 500 nM angiotensin I for 30 min (right panel) or untreated (DMSO control, left panel). Arrows indicate the AT1R internalization detected by the image analysis algorithm.

Figure 2. Concentration response curve in the AT1R assay: The EC₅₀ of angiotensin I is approximately 20 nM (n=32). Cells were treated with angiotensin I for 30 min. Cells were then fixed and receptor internalization was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (500 nM angiotensin I) and negative control (0.25% DMSO).