S1P1 Receptor Activation Redistribution Assay

• Designed to assay compounds for their ability to inhibit a cAMP response through the S1P₁ receptor
• 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 and 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. In this assay, the S1P₁ receptor is stably transfected into a GPCR Reporter Assay for Gs/Gi-coupled Receptors.

This assay system measures translocation of protein kinase A (PKA) in response to changes in the cytoplasmic cAMP concentration. Binding of an agonist to the extracellular parts of the S1P₁ receptor causes activation of the Gi complex at the intracellular face of the receptor. This leads to inhibition of adenylate cyclase, which catalyzes the formation of cAMP from ATP. In cells stimulated with forskolin that induces high levels of cAMP, PKAcat-GFP is found in a diffuse localization in the cytoplasm. S1P₁ activation leads to inhibition of adenylate cyclase and thereby less cAMP. This, in turn, leads to aggregation of PKAcat-GFP in cytoplasmic foci.

Figure 1. Cytoplasmic foci formation of PKAcat-GFP. Cells expressing the S1P₁ receptor were treated with 40 μM forskolin for 5 min in the presence (right panel) or absence (left panel) of 3 nM S1P. Activation of the receptor inhibits the forskolin-induced dispersion of PKAcat-GFP aggregates. Arrows indicated the cytoplasmic foci detected by the image analysis algorithm (right panel).

Figure 2. S1P concentration response in the S1P₁:PKA assay. Concentration response was measured in 9 point half log dilution series (n=32). The EC₅₀ of S1P is ~150 pM. Cells were treated with S1P in the presence of forskolin for 5 min. Cells were then fixed and cytoplasmic spot formation was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetectorV3 BioApplication. % activity was calculated relative to the positive (3 nM S1P) and negative control (0.25% DMSO).