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HTS097M

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ChemiSCREEN Membrane Preparation Recombinant Human S1P3 Lysophospholipid Receptor

Human S1P3 / EDG3 GPCR membrane preparation for GTPγS Binding & Radioligand Binding Assays.

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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-1 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 EDG3 cDNA encoding S1P3
Sphingosine 1-phosphate (S1P) is a bioactive lipid that binds to and activates a family of GPCRs, S1P1-5 (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 Gi, whereas S1P2 (Edg-5) and S1P3 (Edg-3) activate Gi, Gq and G12/13 (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 Gi (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.

Biochem/physiol Actions

Protein Target: S1P3 / EDG3

Quality

EC50 in GTPγS binding assay by S1P: ~ 1.7nM

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 0.1 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.

Physical form

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

Legal Information

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

Storage Class

12 - Non Combustible Liquids

wgk_germany

WGK 2

flash_point_f

Not applicable

flash_point_c

Not applicable


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Mari Kono et al.
The Journal of biological chemistry, 279(28), 29367-29373 (2004-05-13)
Sphingosine-1-phosphate (S1P) elicits diverse cellular responses through a family of G-protein-coupled receptors. We have shown previously that genetic disruption of the S1P(1) receptor, the most widely expressed of the family, results in embryonic lethality because of its key role within
R T Windh et al.
The Journal of biological chemistry, 274(39), 27351-27358 (1999-09-17)
Sphingosine 1-phosphate (S1P) is one of several bioactive phospholipids that exert profound mitogenic and morphogenic actions. Originally characterized as a second messenger, S1P is now recognized to achieve many of its effects through cell surface, G protein-coupled receptors. We used
Irute Girkontaite et al.
The Journal of experimental medicine, 200(11), 1491-1501 (2004-12-08)
Marginal zones (MZs) are microdomains in the spleen that contain various types of immune cells, including MZ B cells, MOMA1(+) metallophilic macrophages, and mucosal addressin cell adhesion molecule 1 (MAdCAM-1)(+) endothelial cells. MAdCAM-1(+) and MOMA1(+) cells line the sinus, that
Lysophospholipid G protein-coupled receptors.
Anliker, Brigitte and Chun, Jerold
The Journal of Biological Chemistry, 279, 20555-20558 (2004)
Naotoshi Sugimoto et al.
Molecular and cellular biology, 23(5), 1534-1545 (2003-02-18)
The G protein-coupled receptors S1P2/Edg5 and S1P3/Edg3 both mediate sphingosine-1-phosphate (S1P) stimulation of Rho, yet S1P2 but not S1P3 mediates downregulation of Rac activation, membrane ruffling, and cell migration in response to chemoattractants. Specific inhibition of endogenous Galpha12 and Galpha13

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