ChemiSCREEN™ Membrane Preparation Recombinant Human S1P₅ Lysophospholipid Receptor

ChemiSCREEN™ Membrane Preparation Recombinant Human S1P₅ Lysophospholipid Receptor

Chemicon (Millipore)

200 units

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.

Radioligand binding assay and GTPγS binding.

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

The lysosphingolipid sphingosine 1-phosphate (S1P) regulates cell proliferation, apoptosis, motility, and neurite retraction. Its actions may be both intracellular as a second messenger and extracellular as a receptor ligand. S1P and the structurally related lysolipid mediator lysophosphatidic acid (LPA) signal cells through a set of G protein-coupled receptors known as EDG receptors. Some EDG receptors (e.g., EDG1; MIM 601974) are S1P receptors; others (e.g., EDG2; MIM 602282) are LPA receptors.[supplied by OMIM]

FUNCTION: SwissProt: Q9H228 # Receptor for the lysosphingolipid sphingosine 1- phosphate (S1P). S1P is a bioactive lysophospholipid that elicits diverse physiological effect on most types of cells and tissues. Is coupled to both the G(i/0)alpha and G(12) subclass of heteromeric G-proteins (By similarity). May play a regulatory role in the transformation of radial glial cells into astrocytes and may affect proliferative activity of these cells.
SIZE: 398 amino acids; 41775 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Widely expressed in the brain, most prominently in the corpus callosum, which is predominantly white matter. Detected in spleen, peripheral blood leukocytes, placenta, lung, aorta, and fetal spleen. Low-level signal detected in many tissue extracts. Isoform 1 is predominently expressed in peripheral tissues, whereas isoform 2 is expressed in brain, spleen and peripheral blood leucocytes.DEVELOPMENTAL STAGE: At 24 weeks of gestation, fragments of radial glial fibers are positive within the cortical plate and subplate of allocortical areas. These positive fragments often appear enlarged as varicosities and some of them terminate at blood vessels. Between 28 and 30 weeks of gestation, all iso- and allocortical areas contain immunolabelled radial glial fibers revealing curvature next to sulci. After 32 weeks of gestation, radial glial fibers gradually disappear; instead positive transitional stages between radial glia and astrocytes were found.DISEASE:SwissProt: Q9H228 # Overexpressed in leukemic large granular lymphocyte (LGL). LGL is a lymphopropliferative disorder often associated with autoimmune disease.
SIMILARITY: SwissProt: Q9H228 ## Belongs to the G-protein coupled receptor 1 family.

Human

SPECIFICATIONS: 1 unit = 5 µg membrane preparation

EC50 in GTPγS binding assay by S1P: ~ 2.4 nM

Chem-5

Chemicon

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.

Chem-5

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

S1P5 / EDG8

Membranes

Q9H228

non-peptide

Lysophospholipid

Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.

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A

• EDG8
• SPPR-2
• SPPR-1
• Edg-8
• S1P5
• S1PR5

GPCR Membrane Preparations

ChemiSCREEN™ Membrane Preparation Recombinant Human S1P₅ Lysophospholipid Receptor

NM_030760.3

GPCR