Description
General description
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 (Arikawa et al., 2003; Sugimoto et al., 2003; Goparaju et al., 2005). Studies with knockout mice indicate that S1P₂ and S1P₃ have redundant functions in maintaining vascular integrity during embryonic development (Kono et al., 2004). Chemicon′s S1P₂ 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 S1P₂ interactions with its ligands. The cell line exhibits EC50 of 7.2nM for S1P in a GTPγS binding assay.
human EDG-5 cDNA encoding S1P₂
Application
GTPγS Binding and Radioligand Binding Assay.
Human S1P2 / EDG5 GPCR membrane preparation for GTPγS Binding & Radioligand Binding Assays.
Biochem/physiol Actions
GPCR Class: A
Protein Target: S1P2 / EDG5
Target Sub-Family: Lysophospholipid
Features and Benefits
Inucbation 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 S1P₂ 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.
Preparation Note
Maintain frozen at -70°C for up to 2 years. Do not freeze and thaw.
Analysis Note
EC50 in GTPγS binding assay by S1P: ~ 7.2nM
Legal Information
CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
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.
