CRTH2 Receptor Internalization Redistribution Assay

• Designed to assay compounds for their ability to modulate internalization of CRTH2
• 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

Chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes (CRTH2) has attracted interest as a potential therapeutic target in inflammatory and allergic diseases. Prostaglandin D2 has a well-established role in inflammation, and there are two known cellular receptors: DP and CRTH2. They are both members of the superfamily of G protein–coupled receptors (GPCRs). CRTH2 is Gi-coupled, while DP is Gs-coupled. Prostaglandin D2 activation of the CRTH2 receptor causes rapid phosphorylation, desensitization, and endocytosis of CRTH2, with subsequent intracellular processing and recycling to the cell membrane. Evidence for differential regulation of the CRTH2 and D2 receptors has been published, and small molecule antagonists have been identified. For example, Ramatroban (BAY-u3405) is used in the clinic as a treatment for allergic rhinitis.

Figure 1. Internalization of CRTH2-EGFP stimulated with Prostaglandin D2. Cells were treated with 300 nM Prostaglandin D2 for 2 hr (right panel) or untreated (DMSO control, left panel). Arrows indicate the CRTH2-EGFP internalization that is detected by the image analysis algorithm.

Figure 2. Concentration response curves in the CRTH2 assay: A) Prostaglandin D2 concentration response in the CRTH2 agonist assay (n=15). The EC₅₀ is ~10 nM. Concentration response was measured in 9 point half log dilution series. Cells were treated with prostaglandin D2 for 2 hr. 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 (300 nM prostaglandin D2) and negative control (0.25% DMSO). B) BAY-u3405 concentration response in the CRTH2 assay run in antagonist format (n=8). Cells were treated with prostaglandin D2 in the presence of a half log dilution series of BAY-u3405 for 2 hr. Cells were then fixed and analyzed on the Cellomics ArrayScan V^TI Reader. The EC₅₀ of BAY-u3405 is ~0.3 μM.