S1P3 Receptor Internalization Redistribution Assay

• Designed to assay compounds for their ability to modulate internalization of S1P₃
• 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

Sphingosine-1-phosphate (S1P) is a pleiotropic platelet-derived lysophospholipid involved in the regulation of cell growth and differentiation, thereby important for angiogenesis, embryogenesis, and atherosclerosis. S1P acts on five subtypes of G-protein-coupled receptors (S1P_1-5). S1P₃, formerly named endothelial differentiation gene-3 (EDG3), is expressed in many cell types, including endothelial and vascular smooth muscle cells. Binding of S1P to the S1P₃ receptor activates G_i, G_q,and G_12/13 subsets of the G-protein families. It has recently been suggested that S1P-induced inflammatory responses are mediated by both S1P₁ and S1P₃ suggesting that these receptors contribute to the regulation of angiogenesis and vascular endothelial cell function.

Figure 1. Internalization of S1P₃-EGFP stimulated with S1P. Cells were treated with 10 μM S1P for 1 hr (right panel) or untreated (DMSO control, left panel). The cytoplasmic spots are detected by the image analysis algorithm.

Figure 2. Concentration response curve in the S1P₃ agonist assay. S1P concentration response curve in the S1P₃ agonist Redistribution assay (n=16). The EC₅₀ value of S1P is 300 nM. Concentration response was measured in 9 point half log dilution series. Cells were incubated with S1P for 60 min. Cells were then fixed and internalization was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (10 μM S1P) and negative control (0.25% DMSO).