S1P1 Receptor Internalization Redistribution Assay

• Designed to assay compounds for their ability to modulate internalization of S1P₁
• 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. There are five known G protein-coupled S1P receptors in mammals. Binding of S1P to the S1P₁ receptor activates Gαi and Gαo resulting in adenylate cyclase inhibition, phospholipase C activation, Ca²⁺ mobilization, Ras-Erk activation and PI3 kinase activation. Activation of S1P₁ also results in GPCR kinase 2 (GRK2)-dependent receptor internalization, followed by recycling of the receptor back to the plasma membrane.

Figure 1. Internalization of S1P₁ -EGFP stimulated with S1P. Cells were untreated (vehicle control, left panel) or treated with 5 μM S1P (right panel). Arrows indicate S1P-induced S1P₁ internalization detected by the image analysis algorithm.

Figure 2. Concentration response curve in the S1P₁agonist assay. S1P concentration response curve in the S1P₁  agonist Redistribution assay (n = 4). The EC₅₀ value of S1P is 26.9 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 (5 μM S1P) and negative control (0.25% DMSO).