ChemiSCREEN™ Human DP Prostanoid Receptor Membrane Preparation

ChemiSCREEN™ Human DP Prostanoid Receptor Membrane Preparation

• ChemiScreen
• Chemicon (Millipore)

200 units

Full-length human PTGDR cDNA encoding DP

Prostanoids are a series of arachidonic acid metabolites produced by the action of cyclooxygenase and subsequently by isomerases and synthases. 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 PGD₂ is produced by mast cells upon activation by allergens, and is present at high levels in allergic diseases. PGD₂ binds to two receptors, DP and CRTH2. DP activates G_s to increase cAMP levels, and lack of DP results in reduced allergic response in animal models of bronchial asthma (Matsuoka et al., 2000). Chemicon's DP 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 DP interactions and its ligands. The membrane preparations exhibit a Kd of 10.39 nM for [³H]-PGD2. With 8 nM [³H]-PGD₂, 5 µg/well and 10 µg/well DP Membrane Prep typically yield greater than 4-fold signal-to-background ratio.

Radioligand binding assay and GTPS binding.

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The protein encoded by this gene is a G-protein-coupled receptor. It has been shown to function as a prostanoid DP receptor. The activity of this receptor is mainly mediated by G-S proteins that stimulate adenylate cyclase resulting in an elevation of intracellular cAMP and Ca2+. Knockout studies in mice suggest that the ligand of this receptor, prostaglandin D2 (PGD2), functions as a mast cell-derived mediator to trigger asthmatic responses.

FUNCTION: SwissProt: Q13258 # Receptor for prostaglandin D2 (PGD2). The activity of this receptor is mainly mediated by G(s) proteins that stimulate adenylate cyclase, resulting in an elevation of intracellular cAMP. A mobilization of calcium is also observed, but without formation of inositol 1,4,5-trisphosphate.
SIZE: 359 amino acids; 40271 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Expressed in retina and small intestine.
DISEASE:SwissProt: Q13258 # Genetic variations in PTGDR are associated with susceptibility to asthma-related traits type 1 (ASRT1) [MIM:607277]. Asthma-related traits include clinical symptoms of asthma, such as coughing, wheezing and dyspnea.
SIMILARITY: SwissProt: Q13258 ## Belongs to the G-protein coupled receptor 1 family.

Human

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

	10 µg/well	5 µg/well
Signal:Background	4.86	4.12
Specific Binding (cpm)	1345	885

Table 2. IC50 values for ligands obtained in a competition binding assay with DP membrane preparation.

	IC50 (nM)
Prostaglanding D2	12.1
13,14-dihydro-15-keto prostaglandin D2	>10000
BW A868C	4.8

SPECIFICATIONS: 1 unit = 10 µg
Bmax for [³H] PGD2 binding: 5.3 pmol/mg protein;
Kd for [³H] PGD2 binding: ~10.39nM

Chem-1

Chemicon

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] PGD2 (Perkin Elmer#:NET-616 )
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 a unit is the amount of membrane that will yield greater than 4-fold signal:background with ³H-labeled PGD₂ at 8 nM

Chem-1

Liquid in packaging buffer: 50 mM Tris pH 7.4, 10% glycerol and 1% BSA no preservatives.
Packaging method: Membranes protein were adjusted to 0.5 mg/ml in 1 ml packaging buffer, rapidly frozen, and stored at -80°C.

DP

Q13258

non-peptide

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

Prostanoid

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A

• PTGDR
• DP
• MGC49004
• AS1
• ASRT1

GPCR Membrane Preparations

ChemiSCREEN™ Human DP Prostanoid Receptor Membrane Preparation

NM_000953.2

GPCR