ChemiScreen™ EP1 Calcium-Optimized FLIPR Cell Line

ChemiScreen™ EP1 Calcium-Optimized FLIPR Cell Line

• ChemiScreen
• Chemicon (Millipore)

2 vials

TRANSFECTION: Full-length human PTGER1 cDNA encoding EP1

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 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; EP1 couples primarily to Gq to mobilize intracellular calcium. EP1 appears to mediate the effects of PGE2 in promoting formation of precancerous lesions in animal models of colon cancer (Watanabe et al., 1999). In addition, EP1 has an inhibitory effect on stress-induced aggressive and risk-taking behaviors in mice (Matsuoka et al., 2005). Chemicon's cloned human EP1-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant EP1 expression on the cell surface and contains high levels of the promiscuous G protein Galpha15 to enhance coupling of the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between EP1 and its ligands.

Calcium flux assay, ligand binding assays

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The protein encoded by this gene is a member of the G protein-coupled receptor family. This protein is one of four receptors identified for prostaglandin E2 (PGE2). Through a phosphatidylinositol-calcium second messenger system, G-Q proteins mediate this receptor's activity. Knockout studies in mice suggested a role of this receptor in mediating algesia and in regulation of blood pressure. Studies in mice also suggested that this gene may mediate adrenocorticotropic hormone response to bacterial endotoxin.

FUNCTION: SwissProt: P34995 # Receptor for prostaglandin E2 (PGE2). The activity of this receptor is mediated by G(q) proteins which activate a phosphatidylinositol-calcium second messenger system. May play a role as an important modulator of renal function. Implicated the smooth muscle contractile response to PGE2 in various tissues.
SIZE: 402 amino acids; 41801 Da
SUBCELLULAR LOCATION: Cell membrane; Multi-pass membrane protein.
TISSUE SPECIFICITY: Abundant in kidney. Lower level expression in lung, skeletal muscle and spleen, lowest expression in testis and not detected in liver brain and heart.
PTM: Phosphorylated (Potential).
SIMILARITY: SwissProt: P34995 ## Belongs to the G-protein coupled receptor 1 family.

Human

SPECIFICATIONS:
EC50 for calcium mobilization by PGE2: ~3 nM
IC50 for AH 6809: ~ 860 nM
Signal/noise at agonist E_max: 622

Chem-1

GPCR Cell Lines

Chemicon

Chem-1

Cells are frozen at 2 x 10e6 cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.

EP1

Cell Line

P34995

non-peptide

Prostanoid

Place cells in liquid nitrogen immediately upon receipt. Maintain frozen in liquid nitrogen for up to 5 years. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37ºC water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing 20 mL growth media, and place in a humidified 37°C incubator with 5% CO2.. After 8-24 h, cells will adhere to the plate, at which time the media should be replaced to remove residual DMSO. Cells are passaged by washing with Ca++ and Mg++-free HBSS (10 mL/T75), incubating with 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) for 5-10 minutes at 37°C, and rapping the side of the flask to dislodge the cells. Neutralize the trypsin by addition of 4 volumes growth media. Cells are typically passaged 1:10 with every 3-4 days, and should be passaged at least once after thawing prior to use in calcium flux assays.

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A

1 mL per vial

• PTGER1
• EP1

GPCRs

ChemiScreen™ EP1 Calcium-Optimized FLIPR Cell Line

NM_000955.2

GROWTH MEDIA: DMEM containing 4.5 g/L glucose/10% heat inactivated fetal bovine serum/1x nonessential amino acids/10 mM HEPES/0.25 mg/ml Geneticin (G418)/100 U/ml each penicillin and streptomycin.

2 x 10⁶ cells/mL