Glucocorticoid Receptor (GR) Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of GR
• 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

Glucocorticoids are the most potent and effective drugs for preventing and suppressing inflammation caused by mechanical, chemical, infectious, and immunological stimuli. They exert their activity through binding to the glucocorticoid receptor (GR) resulting in either activation or repression of a large set of glucocorticoid responsive genes. GR is a ligand-dependent transcription factor belonging to the superfamily of steroid hormone receptors, which includes the mineralocorticoid, thyroid hormone, sex hormones, retinoic acid, and vitamin D receptors. The GR receptor contains a DNA binding site, a hormone-independent activation function (AF1) domain in the N-terminus, which is associated with transcriptional activity and binding to co-activators and transcription factors, and a hormone-dependent AF2 domain in the C-terminus responsible for hormone binding as well as binding of co-activators.

In the inactive state, the GR is found in the cytoplasm in complex with heat shock proteins. Upon ligand binding, the GR translocates to the nucleus, dimerizes, and acts as a transcription factor to regulate the expression of its target genes. GR agonists such as dexamethasone induce nuclear translocation and allow the receptor to bind co-activators with intrinsic histone acetylase and methylase activities, such as proteins from the SRC-1, p300/CBP, and PRMT families, whereas GR antagonists, which also induce nuclear translocation of GR, result in the recruitment of histone deacetylases through co-repressors of the N-CoR/SMRT family.

Figure 1. Nuclear translocation of EGFP-GR. Cells were treated without (DMSO control, left panel) or with (right panel) 100 nM dexamethasone for 2 hours. Arrows indicate the nuclear translocation of EGFP-GR detected by the image analysis algorithm.

Figure 2. Concentration response curves in the GR assay: A) Dexamethasone concentration response in the GR assay (n=6). The EC ₅₀ is approximately 1 nM. B) Mifepristone concentration response in the GR assay (n=3). The EC₅₀ is approximately 0.5 nM. The concentration response was measured in 9 point half log dilution series. Cells were treated with compound for 2 hrs. Cells were then fixed and imaging was performed using an image algorithm detecting nuclear translocation. % activity was calculated relative to the positive (100 nM dexamethasone) and negative control (0.25% DMSO)