ChemiScreen Membrane Preparation
Recombinant Human EP4 Prostanoid Receptor

Human EP4

Prostanoids are a series of arachidonic acid metabolites produced by the action of cyclooxygenase and further modified by isomerases and syntheses. Cells rapidly secrete prostanoids after synthesis, whereupon the prostanoids bind to a family of 8 GPCRs to exert their biological effects (Narumiya and FitzGerald, 2001). The prostaglandin PGE2 causes pain, vasodilation, immunosuppression of T cells, bone remodeling and promotion of carcinogenesis. Four related GPCRs, EP1, EP2, EP3 and EP4, each bind to PGE2, but the different G protein coupling status of each receptor leads to distinct biological effects. EP4 couples primarily to Gs to increase intracellular cAMP levels. During neonatal development, EP4 participates in closure of the ductus arteriosus, a process required for switching circulation from the placenta to the lungs (Nguyen et al., 1997). In addition, EP4 mediates PGE2-induced bone formation by promoting osteoblastogenesis, and selective EP4 agonists are being evaluated as potential treatments for osteoporosis (Yoshida et al., 2002). Chemicon′s EP4 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 antagonists of EP4 interactions with prostaglandin E2. The membrane preparations exhibit a Kd of 1.27-1.85 nM for [³H]-Prostaglandin E2. With 5 µg/well EP4 Membrane Prep and 1.5 nM [³H]-Prostaglandin E2, a greater than 3-fold signal-to-background ratio was obtained.

Radioligand binding assay, and GTPγS binding.

GPCR Class: A

Protein Target: EP4

Target Sub-Family: Prostanoid

Inucbation Conditions
Membranes are mixed with radioactive ligand and unlabeled competitor (see Figures 1 and 2 for concentrations tested) in binding buffer in a nonbinding 96-well plate, and incubated for 1-2 h. Prior to filtration, a GF/C 96-well filter plate is coated with 0.33% polyethyleneimine for 30 min, then washed with 50mM HEPES, pH 7.4, 0.5% BSA. Binding reaction is transferred to the filter plate, and washed 3 times (1 mL per well per wash) with Wash Buffer. The plate is dried and counted.

Binding buffer: 50 mM Hepes, pH 7.4, 5 mM MgCl₂, 1 mM CaCl₂, 0.2% BSA, filtered and stored at 4°C
Radioligand: [³H] prostaglandin E2 (Perkin Elmer # NET428)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl , 0.1% BSA, filtered and stored at 4°C.

One package contains enough membranes for at least 200 assays (units), where an unit is the amount of membrane that will yield greater than 3-fold signal:background with 3H-labeled prostaglandin E2 at 1.5 nM.

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

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

SPECIFICATIONS:
Signal:background and specific binding values obtained in a competition binding assay with varying amounts of EP4 membrane prep:

	10 µg/well
Signal:Background	3.9
Specific Binding (cpm)	643

1 unit = 10 µg membrane preparation
Bmax: 16.1 pmol/mg
Kd: 1.57 nM

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