Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-07-29
2001-03-06
Kunz, Gary L. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S252300, C435S320100, C435S069100
Reexamination Certificate
active
06197933
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the cloning and expression of the human EP
3
prostaglandin receptor. Methods of identifying compounds capable of both binding to and activating the human EP
3
receptor are also disclosed.
BACKGROUND OF THE INVENTION
Prostaglandins are a group of lipid-soluble hormone mediators derived from the metabolism of arachidonic acid via the cyclooxygenase enzymatic pathway. In the prostaglandin biosynthetic pathway, arachidonic acid is first converted to the endoperoxide PGH2 by PGH2 synthases followed by the cell-specific isomerization or reduction of PGH2 to the active prostaglandins: PGD
2
, PGE
2
, PGF
2&agr;
, prostacyclin (PGI
2
) and thromboxane (TxA
2
). Following enzymatic conversion, prostaglandins exert their actions locally on the cells in which they were synthesized (autocrine) and/or on nearby cells (paracrine) through specific G protein-coupled receptors (Smith, (1992)
Am. J. Physiol.,
263: F181-F191) to either stimulate or inhibit the production of second messengers. Prostaglandins elicit a diverse spectrum of often opposing biological effects including muscle contraction and relaxation, platelet aggregation, vasodilation and inflammation.
PGE
2
exhibits a broad range of actions in a number of tissues by binding to at least three EP receptor subtypes. It acts through pharmacologically distinct stimulatory (EP
2
) and inhibitory (EP
3
) PGE receptor subtypes to stimulate and inhibit cAMP formation, respectively (Sonnenburg, and Smith, (1988)
J. Biol. Chem.,
263: 6155-6160). PGE
2
also stimulates calcium release and protein kinase C activity in the rabbit kidney collecting tubule, most likely by binding to the EP
1
receptor subtype that is coupled to stimulation of phospholipase C (Hebert et al., (1990)
Am. J. Physiol.,
259: F318-F325). The EP
3
receptor subtype is involved in inhibition of gastric acid secretion, modulation of neurotransmitter release, inhibition of sodium and water reabsorption in the kidney tubule, potentiation of platelet aggregation at low concentrations (below 1 &mgr;M) and inhibition of platelet aggregation at higher concentrations (Tynan et al., (1984)
Prostaglandins,
27: 683-696; Matthews and Jones, (1993)
British J. Pharmacol.,
108: 363-369).
Development of therapeutic prostaglandins requires selective action at receptor subtypes. The murine EP
2
and EP
3
prostaglandin receptors have been cloned and sequenced (Honda et al., (1993)
J. Biol. Chem.,
268: 7759-7762; Sugimoto et al., (1992)
J. Biol. Chem..,
267: 6463-6466). The deduced protein sequences indicate that both are members of the G protein-linked receptor superfamily, having seven putative membrane-spanning hydrophobic domains. The proteins share significant amino acid sequence similarity with other members of this family including the thromboxane (TP) receptor (Hirata et al., (1991)
Nature
349: 617-620), rhodopsin and the adrenergic receptors. In order to characterize the pharmacology of the murine EP
3
receptor, the gene was transfected into COS-7 cells which lack the EP
3
receptor and competition binding assays using tritiated PGE
2
were performed on the plasma membrane fraction (Sugimoto et al., (1992)
J. Biol. Chem.,
267: 6463-6466).
However, these results only addressed the binding of compounds to the murine receptors. There is still the need to identify compounds which specifically bind to the human EP
3
receptor, since the pharmacology of rodent G-protein coupled receptors does not always match their human homologs (Oksenberg et al., (1992)
Nature,
360: 161-163; Link et al., (1992)
Mol. Pharmacol.,
42: 16-27).
SUMMARY OF THE INVENTION
One embodiment of the present invention is an isolated DNA molecule encoding the human prostaglandin EP
3
receptor. Preferably, this molecule has the nucleotide sequence of SEQ ID NOS: 3, 5, 7 or 16.
Another embodiment of the present invention is an isolated, unique 18 nucleotide DNA sequence contained within SEQ ID NOS: 3, 5, 7 or 16.
A further embodiment of the present invention are the proteins derived from the aforementioned DNA sequences.
The present invention also embodies a vector containing SEQ ID NOS: 3, 5, 7 or 16 operably linked to a heterologous promoter.
Another aspect of the present invention provides isolated antibodies directed to the human EP
3
receptor protein. Preferably, these antibodies are polyclonal; most preferably, these antibodies are monoclonal.
A further embodiment consists of a method of screening compounds for binding to the human EP
3
receptor by the following steps:
transfecting cells with a DNA molecule encoding a human EP
3
receptor operably linked to a promoter;
culturing the cells to express the human EP
3
receptor;
incubating the cultured cells in the presence of a labeled compound to be tested for binding affinity to the human EP
3
receptor; and
measuring the amount of label bound to the cells.
Preferably, the cell line is COS-7, the human EP
3
receptor is encoded by the polynucleotide of SEQ ID NOS: 3, 5, 7, or 16, and the expression vector is mammalian. Most preferably, the mammalian expression vector is pBC12BI. Additionally, the compound of interest may advantageously be either radiolabeled, calorimetrically labeled or fluorimetrically labeled. In another aspect of this preferred embodiment, prior to the incubation step, cell membranes containing the expressed human EP
3
receptor are isolated.
Still another embodiment of the present invention is a method of determining the ability of a compound to inhibit ligand binding to the human EP
3
receptor by the steps of:
transfecting cells with a DNA sequence encoding the human EP
3
receptor operably linked to a promoter;
culturing the cells to express the human EP
3
receptor;
incubating the cultured cells in the presence of a labeled ligand having binding affinity for the receptor and a test compound; and
determining the level of binding of the ligand to the expressed human EP
3
receptor, wherein a lower level of ligand binding in the presence of the compound indicates that the compound binds to the receptor.
Preferably, the cell line is COS-7. Advantageously, the compound may be either radioactively, colorimetrically or fluorimetrically labeled. Alternatively, the ligand may be labeled. Most preferably, the expression vector is pBC12BI and the ligand is PGE
2
. In another aspect of this preferred embodiment, prior to the incubation step, cell membranes containing the expressed human EP
3
receptor are isolated.
Another aspect of the present invention provides a method for identifying compounds that are receptor agonists by the following steps:
transfecting cells with the human EP
3
receptor gene operably linked to a promoter;
transfecting cells with a DNA segment encoding cyclic AMP-responsive chloramphenicol acetyltransferase (CAT);
incubating the cells in the presence or absence of an activator of adenylate cyclase and in the presence of a compound to be tested; and
assaying the amount of CAT produced, where a change in CAT activity indicates that the compound is an agonist of the receptor.
Preferably, the cell line is mammalian; most preferably, the mammalian cells are JEG-3 choriocarcinoma cells, and the activator of adenylate cyclase is forskolin.
The present invention further provides a cell line in continuous culture expressing the human EP
3
receptor encoded by the DNA of SEQ ID NOS: 3, 5, 7 or 16. Preferably these cells are CHO cells.
REFERENCES:
patent: 0557966 A1 (1993-02-01), None
patent: WO95/00552 (1995-01-01), None
patent: WO95 00551 (1995-01-01), None
patent: WO95 00552 (1995-01-01), None
Adam M., et al. Cloning and expression of three isoforms of the human EP3 prostanoid receptor. FEBS Letters. 338. pp. 170-174, 1994.
Rudinger J., et al. Characteristics of the amino acids as components of a peptide hormone sequence. in Peptide Hormones. pp. 1-7. Edited by Parsons, JA; Mill Hill, London, 1976.
Namba T, et al. Mouse thromboxane A2 receptor: cDNA cloning, expression and northern blot analysis. Biochem Biophys Res Commun. 1992 May 15;184(3):1197-203.
Namb
Gil Daniel W.
Regan John W.
Allergan Inc.
Baran Robert J.
Fisher Carlos A.
Kunz Gary L.
Landsman Robert S.
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