ChemiSCREEN™ Membrane Preparation Recombinant Human EP₂ Prostanoid Receptor

ChemiSCREEN™ Membrane Preparation Recombinant Human EP₂ Prostanoid Receptor

Chemicon (Millipore)

Full-length human PTGER2 cDNA encoding EP₂

Prostanoids bind to a family of 8 GPCRs to exert their biological effects (Narumiya and FitzGerald, 2001). The protanoid PGE₂ causes pain, vasodilation, immunosuppression of T cells, bone resorption 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. EP₂ couples primarily to Gs to increase intracellular cAMP levels. Mice deficient in EP₂ receptor showed impaired ovulation and fertilization, salt-sensitive hypertension (Kennedy et al., 1999). It has been shown that EP₂ receptors are also involved in cancer associated immunodeficiency. Thus, genetic knockout of the EP₂ receptor reduced tumor growth and prolonged survival in mice that had undergone isograft injection of MC26 or Lewis lung carcinoma cells (Yang et al., 2003). Millipore's EP₂ 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 agonists and antagonists of EP₂. The membrane preparations exhibit a Kd of 14.52 nM for [³H]-PGE₂. With 7.5 nM [³H]-PGE₂, 10µg/well EP₂ Membrane Prep typically yields greater than 3-fold signal-to-background ratio.

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.

GPCR

Chem-1, an adherent mammalian cell line without any endogenous EP₂ expression.

Chem-1

• PTGER2
• EP2

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 1 mg/mL in packaging buffer, and dispensed at 1 mL/vial. Vials were rapidly frozen, and stored at -80°C.

Table 1. Signal:background and specific binding values obtained in a competition binding assay with varying amounts of EP₂ Receptor membrane prep.

	10 µg/well
Signal:Background	3.60
Specific Binding (cpm)	1146

1 unit = 10 µg
Bmax for [³H]-PGE₂ binding: 4.38 pmol/mg protein
Kd for [³H]-PGE₂ binding: ~14.52 nM

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, 500mM NaCl. 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, filtered and stored at 4°C.
Radioligand: [³H]-PGE₂ (Perkin Elmer#: NET-428)
Wash Buffer: 50 mM Hepes, pH 7.4, 500mM NaCl, filtered and stored at 4°C.

GPCR Membrane Preparations

Store at –70°C. Product is stable for at least 6 months from the date of receipt when stored as directed. Do not freeze and thaw.

Human