Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1996-08-05
1998-06-02
Walsh, Stephen
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 691, 435325, 530350, 536 235, C12Q 100, C07K 14705, C12N 1509
Patent
active
057597898
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The physiological actions of prostaglandin (PG)E.sub.2 are mediated through interaction with the prostaglandin E receptor(s). There are three subtypes of the EP receptor, EP.sub.1, EP2 and EP.sub.3 (for review see Coleman et al., 1989). These three subtypes all show high affinity for PGE2 but show differences in their affinities for various agonists and antagonists and exert their actions through different secondary transduction mechanisms. Thus activation of the EP1 receptor is associated with a rise in IP3 and intracellular calcium, activation of the EP2 receptor results in a rise in intracellular cyclic AMP and activation of the EP3 receptor a fall in intracellular cyclic AMP. To date the only members of this family to be cloned are the mouse EP2 (Honda et al., 1993) and the mouse EP.sub.3.alpha. and EP.sub.3.beta. (Sugimoto et al., 1992; Sugimoto et al., 1993) subtypes. EP2 receptors are normally found on a wide variety of cells including the small intestine, kidney, stomach, muscle, eye, uterus, thymus and trachea, in humans and other animals. Binding of prostaglandin E.sub.2 to the EP2 receptor protein elicits an increase in intracellular cAMP levels. This signal causes the tissues to respond, for example, by smooth muscle relaxation.
Functional activities of the EP2 receptor have been studied using tissue preparations such as guinea-pig ileum circular muscle, cat trachea, guinea-pig trachea and cell preparations, such as lymphocytes and osteoclasts. The above methods for studying EP2 receptor activities have several disadvantages in that they require preparations containing several different but related receptor populations, with different ligand binding properties making measurements of absolute potency and selectivity very difficult. In addition, tissues contain very low levels of EP2 receptor and since tissue samples are required, compounds cannot satisfactorily be tested as effectors of the human EP2 receptor.
SUMMARY OF THE INVENTION
A novel prostaglandin receptor protein termed EP2 has been isolated and purified from human cells. A DNA molecule encoding the full length EP2 protein has been isolated and purified, and the nucleotide sequence has been determined. The EP2 encoding DNA has been cloned into expression vectors and these expression vectors, when introduced into recombinant host cells, cause the recombinant host cells to express a functional EP2 receptor protein. The novel EP2 protein, the EP2-encoding DNA, the expression vectors and recombinant host cells expressing recombinant EP2 are useful in the identification of modulators of EP2 receptor activity.
A method of identifying EP2 receptor modulators is also disclosed which utilizes the recombinant EP2 expressing host cells. Modulators of EP2 activity are useful for the treatment of prostaglandin-related diseases and for modulating the effects of prostaglandins on the EP2 receptor.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-C--The complete DNA sequence encoding the EP2 receptor protein is shown above the complete deduced amino acid sequence of the EP2 receptor protein.
FIG. 2--Expression of the prostaglandin E2 receptor in EP2 cDNA-injected Xenopus oocytes is shown by an inward cAMP-dependent Cl.sup.- current (shown as downward deflection) evoked by bath perfusion of 1 nM PGE2 when the oocyte was injected with 1.6 ng EP2 cDNA plus 2.5 ng CFTR cDNA and voltage-clamped at -60 mV.
FIG. 3--IBMX-induced, CFTR-mediated Cl.sup.- current in (CFTR+anti-sense hEP2) cDNA injected oocytes is shown, noting the lack of response to 1 .mu.M and 3 .mu.M PGE2. binding assays performed in the presence of: Panel A) 10 pM-10 mM PGE2 (l), PGE1 (o), 17-pheyyl-trinor PGE2 (n), iloprost (q), PGF2.alpha. (u), PGD.sub.2 (.diamond.), and U46619 (s) and Panel B, 100 pM-100 uM MB28767 (l), misoprostol (o), butaprost (n), AH6809 (q) and SC19220 (u).
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to cDNA encoding a novel prostaglandin receptor, termed EP2. The present invention is also related to recombinant
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Abramovitz Mark
Adam Mohammed
Bastien Lison
Grygorczyk Richard
Metters Kathleen
Coppola Joseph A.
Merck Frosst Canada Inc.
Teng Sally P.
Tribble Jack L.
Walsh Stephen
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