ChemiSCREEN Membrane Preparation Recombinant Human S1P₃ Lysophospholipid Receptor

Full-length human EDG3 cDNA encoding S1P₃

Sphingosine 1-phosphate (S1P) is a bioactive lipid that binds to and activates a family of GPCRs, S1P_1-5 (also known as EDG receptors). Interactions between S1P and its receptors mediate cytoskeletal rearrangement and cell migration, with functional consequences in angiogenesis, lymphocyte trafficking, and smooth muscle development (Anliker and Chun, 2004). S1P₁ (Edg-1) signals exclusively through G_i, whereas S1P₂ (Edg-5) and S1P₃ (Edg-3) activate G_i, G_q and G_12/13 (Windh et al., 1999). Although S1P₁ and S1P₃ promote cell migration, S1P₂ inhibits cell migration in several cell types; these opposing functions appear to result from differences in the ability of each receptor to activate G_i (Sugimoto et al., 2003). Studies with knockout mice indicate that S1P₂ and S1P₃ have redundant functions in maintaining vascular integrity during embryonic development (Kono et al., 2004). In addition, S1P₃ regulates immune responses by contributing to endothelial barrier function in splenic marginal zones (Girkontaite et al., 2004). Millipore′s S1P₃ membrane preparations are crude membrane preparations made from our proprietary stable recombinant cell lines to ensure high-level of GPCR surface expression; thus, they are ideal HTS tools for screening of S1P₃ interactions with its ligands. The membrane preparation exhibits EC50 of 1.7nM for S1P in a GTPγS binding assay.

Protein Target: S1P3 / EDG3

Inucbation Conditions
ASSAY CONDITIONS: Membranes are permeabilized by addition of saponin to an equal concentration by mass, then mixed with [³⁵S]-GTPγS (final concentration of 0.1 nM) in 20 mM HEPES, pH 7.4/100 mM NaCl/10 mM MgCl₂/0.5 µM GDP in a nonbinding 96-well plate. Unlabeled S1P is added to the final concentration indicated in Figure 1 (final volume 100 µL), and incubated for 30 min at 30°C. The binding reaction is transferred to a GF/B filter plate (Millipore MAHF B1H) previously prewetted with water, and washed 3 times (1 mL per well per wash) with cold 10 mM sodium phosphate, pH 7.4. The plate is dried and counted.

One vial contains enough membranes for at least 200 assays (units), where one unit is the amount of membrane that will yield greater than 1000 cpm specific S1P -stimulated [³⁵S]-GTPγS binding.
The S1P₃ membrane preparation is expected to be functional in a radioligand binding assay; however, the end user will need to determine the optimal radiolabeled ligand for use with this product.

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol with no preservatives.
Packaging method: Membrane protein was adjusted to 1 mg/ml in packaging buffer, rapidly frozen, and stored at -80°C

EC50 in GTPγS binding assay by S1P: ~ 1.7nM

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