ChemiSCREEN Membrane Preparation Recombinant Human S1P₅ Lysophospholipid Receptor

Full-length human EDG8 cDNA encoding S1P₅

Sphingosine 1-phosphate (S1P) is a biologically active lysophospholipid that transmits signals through a family of five G-protein-coupled receptors to regulate cell proliferation, migration, cytoskeletal organization, and differentiation (Spiegel and Milstien , 2003).
S1P₅ can couple with Gi/o and G12/13, and it mediates S1P induced adenylate cyclase inhibition and Ca²⁺ mobilization like the other S1P receptors. However, unlike the other S1P receptors, it mediates inhibition of MAPK activation and cell proliferation (Im et al., 2000). S1P5 is predominantly expressed in the white matter tracts and oligodendrocytes and is particularly abundant in the anterior commissure, corpus collosum, and optic tract (Terai et al., 2003). S1P induces process retraction in pre-oligodendrocytes and supports cell survival in mature oligodendrocytes by activating S1P₅, which indicates a role for S1P₅ in maturation and myelination of oligodendrocytes (Jaillard et al., 2005). Millipore′s S1P₅ 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 S1P₅ interactions with its ligands. The membrane preparations exhibit EC50s of 2.4 nM for S1P in a GTPγS binding assay.

Protein Target: S1P5 / EDG8

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 0.3 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 was 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. The plate is washed 3 times (1 mL per well per wash) with cold 10 mM sodium phosphate, pH 7.4, then 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 S1P₅ 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.

SPECIFICATIONS: 1 unit = 5 µg membrane preparation

EC50 in GTPγS binding assay by S1P: ~ 2.4 nM

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