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HTS193M

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ChemiSCREEN Membrane Preparation Recombinant Human S1P5 Lysophospholipid Receptor

Human S1P5 / EDG8 GPCR membrane preparation for Radioligand binding Assays & GTPγS binding.

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About This Item

UNSPSC Code:
41106514
eCl@ss:
32161000
NACRES:
NA.41

biological source

human

Quality Level

recombinant

expressed in Chem-5 cells

manufacturer/tradename

ChemiScreen
Chemicon®

technique(s)

ligand binding assay: suitable (GTPγS)
radioligand binding assay (RLBA): suitable

NCBI accession no.

UniProt accession no.

shipped in

dry ice

General description

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.

Biochem/physiol Actions

Protein Target: S1P5 / EDG8

Quality


SPECIFICATIONS: 1 unit = 5 µg membrane preparation

EC50 in GTPγS binding assay by S1P: ~ 2.4 nM

Specifications

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.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Storage Class Code

12 - Non Combustible Liquids

WGK

WGK 2

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable


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C Jaillard et al.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 25(6), 1459-1469 (2005-02-11)
Endothelial differentiation gene (Edg) proteins are G-protein-coupled receptors activated by lysophospholipid mediators: sphingosine-1-phosphate (S1P) or lysophosphatidic acid. We show that in the CNS, expression of Edg8/S1P5, a high-affinity S1P receptor, is restricted to oligodendrocytes and expressed throughout development from the
D S Im et al.
The Journal of biological chemistry, 275(19), 14281-14286 (2000-05-09)
Three G protein-coupled receptors (Edg-1, Edg-3, and Edg-5) for the lysolipid phosphoric acid mediator sphingosine 1-phosphate have been described by molecular cloning. Using a similar sequence that we found in the expressed sequence tag data base, we cloned and characterized
Sarah Spiegel et al.
Nature reviews. Molecular cell biology, 4(5), 397-407 (2003-05-03)
The evolutionarily conserved actions of the sphingolipid metabolite, sphingosine-1-phosphate (S1P), in yeast, plants and mammals have shown that it has important functions. In higher eukaryotes, S1P is the ligand for a family of five G-protein-coupled receptors. These S1P receptors are

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