| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 2, 2002 |
| PATENT TITLE |
Methods of identifying modulators of human IP prostaglandin receptor |
| PATENT ABSTRACT |
A novel prostaglandin receptor has been identified and DNA encoding the receptor has been isolated, purified, sequenced and expressed in host cells. This DNA encoding the novel prostaglandin receptor and host cells expressing the receptor are used to identify modulators of the prostaglandin receptor. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | March 16, 1998 |
| PATENT REFERENCES CITED |
Masu et al. "cDNA cloning of bovine substance-K receptor through oocyte expression system", Nature, vol. 329, p 836 (1987). Adie, E.J. et al. Biochem. J. vol. 258, pp 529-536 (1992). Tsai et al., "Solubilization of Prostacyclin Membrane Receptors from Human Platelets", J. Biol. Chem. 264, 61-67. 1989. R. Coleman, et al., Characterisation Of The Prostanoid Receptors Mediating Contraction of Guinea-Pig Isolated Trachea, (1985), Prostaglandins, 29, pp. 363-375. P. Davies, et. al., Prostaglandins and Inflammation, (1992), Inflammation: Basic Principles And Clinical Correlates, Gallin, Goldstein, Snyderman, eds., 2nd Ed., pp. 123-138. E. Horton, et al., Uterine Luteolytic Hormone: A Physiological Role for Prostaglandin F2a, (1976), Physiol. Rev., 56, pp. 595-651. D. DeWitt, Prostaglandin endoperoxide synthase: regulation of enzyme expression, (1991), Biochim. Biophys, Acta, 1083, pp. 121-134. J. Stjernschantz,et al., Phenyl substituted prostaglandin analogs for glaucoma treatment, (1992), Drugs Future, 17, pp. 691-704. P. Racz, et al., Maintained Intraocular Pressure Reduction With Once-a-Day Application of a New Prostaglandin F2a Analogue (PhXA41), (1993), Arch. Opthalmol., 111, pp. 657-661. J. Senior, et al., In vitro characterization of prostanoid FP-, DP-, IP- and TP-receptors on the non-pregnant human myometrium, (1992), Brit. J. Pharmacol., 107, pp. 215-221. J. Senior, et al., In vitro characterization of prostanoid receptors on human myometrium at term pregnancy, (1993), Brit. J. Pharmacol., 108, pp. 501-506. J. Csepli, et al., The Effect Of The Prostaglandin F2a Analogue ICI 81008 On Uterine Small Arteries And On Blood Pressure, (1975), Prostaglandins, 10, pp. 689-697. R. Coleman, Methods in prostanoid receptor classification, (1987), Prostaglandins And Related Substances--A Practical Approach, IRL Press, 1st Ed., pp. 267-303. R. Coleman, et al., A study of the prostanoid receptors mediating bronchocorstriction in the anaesthetized guinea-pig and dog, (1981), Brit. J. Pharmacol., 74, p. 913. J. Barnard, et al., Evaluation of prostaglandin F2a and prostacyclin interactions in the isolated perfused rat lung, (1992), J. Appl. Physiol., 72, pp. 2469-2474. J. Davis, et al., Prostaglandin F2a stimulates phosphatidylinositol 4,5-bisphosphate hydrolysis and mobilizes intracellular Ca2+ in bovine luteal cells, (1987), Proc. Natl. Acad. Sci. U.S.A., 84, pp. 3728-3732. J. Kitanaka, et al., Astrocytes Possess Prostaglandin F2a Receptors Coupled To Phospholipase C, (1991), Biochem. Biophys. Res. Comm., 178, pp. 946-952. F. Black, et al., Activation of inositol phospholipid breakdown by prostaglandin F2a without any stimulation of proliferation in quiescent NIH-3T3 fibroblasts, (1990), Biochem. Journal, 266, pp. 661-667. A. Nakao, et al., Characterization of Prostaglandin F2a Receptor of Mouse 3T3 Fibroblasts and Its Functional Expression in Xenopus Laevis Oocytes, (1993), J. Cell Physiol., 155, pp. 257-264. W. Powell, et al., Prostaglandin F2a Receptor in Ovine corpora lutea, (1974), Eur. J. Biochem., 41, pp. 103-107. W. Powell, et al., Occurrence and Properties of a Prostaglandin F2a Receptor in Bovine Corpora Lutea, (1975), Eur. J. Biochem., 56, pp. 73-77. W. Powell, et al., Localization of a Prostaglandin F2a Receptor in Bovine Corpus luteum Plasma Membranes, (1976), Eur. J. Biochem., 61, pp. 605-611. M. Molnar, et al., PGF2a and PGE2 binding to rat myometrium during gestation, parturition, and postpartum, (1990), Am. J. Physiol., 258, pp. E740-E747. Th. Bauknecht, et al., Distribution of prostaglandin E2 and prostaglandin F2a receptors in human myometrium, (1981), Acta Endocrinol., 98, pp. 446-450. F. Neuschafer-Rube, et al., Characterization of prostaglandin-F2a-binding sites on rat hepatocyte plasma membranes, (1993), Eur. J. Biochem., 211, pp. 163-169. M. Hirata, et al., Cloning and expression of cDNA for a human thromboxane A2 receptor, (1991), Nature, 349, pp. 617-620. A. Honda, et al., Cloning and Expression of a cDNA for Mouse Prostaglandin E Receptor EP2 Subtype*, (1993), J. Biol. Chem., 268, pp. 7759-7762. Y. Sugimoto, et al., Two Isoforms of the EP3 Receptor with Different Carboxyl-terminal Domains, (1993), J. Biol. Chem., 268, pp. 2712-2718. Y. Sugimoto, et al., Cloning and Expression of a cDNA for Mouse Prostaglandin E Receptor EP3 Subtype*, (1992), J. Biol. Chem., 267, pp. 6463-6466. K. Bunce, et al., Differential Effects Of Prostaglandins On Unidirectional Absorption And Secretion In Rat Ileum, (1987), Gastroenterology, 92, p. 1332. Y. Dong, et al., Prostaglandin E receptor subtypes in smooth muscle: agonist activities of stable prostacyclin analogues, (1986), Br. J. Pharmacol., 87, pp. 97-107. B. Hedqvist, et al., Prostaglandin-Induced Neurotransmission Failure In The Field-Stimulated, Isolated Vas Deferens, (1972), Neuropharmacology, 11, pp. 177-187. M. McKenniff, et. al., Characterization of receptors mediating the contractile effects of prostanoids in guinea-pig and human airways, (1988), Eur. J. Pharmacol., 153, pp. 149-159. R. Eglen, et al., The action of prostanoid receptor agonists and antagonists on smooth muscle and platelets, (1988), Br. J. Pharmacol., 94, pp. 591-601. J. Louttit, et al., Prostanoid EP-Receptors In Pig Saphenous Vein, (7/26-31/92), 8th International Conf. on Prostaglandins, Abstract 258. R. Lawrence, et al., Investigation of the prostaglandin E (EP-) receptor subtype mediating relaxation of the rabbit jugular vein, (1992), Br. J. Pharmacol., 105, pp. 817-824. R. Coleman, et al., Prostanoids and their Receptors, (1989), Comprehensive Medicinal Chemistry, 3, pp. 643-714. W. Campbell, et al., Lipid-Derived Autacoids: Eicosanoids And Platelet-Activating Factor, (1990), The Pharmacological Basis of Therapeutics, 8th Edition, pp. 600-617. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method of determining whether a test compound modulates a signal transduction activity of a prostaglandin receptor IP, comprising: a) culturing IP-receptor-expressing host cells under conditions that would allow expression of a recombinant prostaglandin receptor, said host cells being transfected with a nucleic acid molecule encoding said recombinant prostaglandin IP-receptor comprising the amino acid sequence as set forth in SEQ ID NO:3; b) exposing the IP receptor-expressing host cells of step a) to the test compound; c) exposing control host cells to the test compound of step b), wherein said control host cells do not express recombinant prostaglandin IP receptor protein; d) measuring the modulating affect of the test compound which interacts with the recombinant IP receptor from the host cells of step a) and control host cells of step c); and, e) comparing the modulating affect of the test compound on the host cells and control host cells. 2. The method of claim 1 wherein said host cells are Xenopus oocytes. 3. The method of claim 1 wherein the effect is measured by an electrophysiological assay. 4. The method of claim 3 wherein said host cells are Xenopus oocytes. 5. The method of claim 4 wherein the nucleic acid molecule is mRNA. 6. The method of claim 1 wherein the nucleic acid molecule is DNA, wherein said IP receptor-expressing host cells are transiently or stably transformed by said DNA molecule. 7. A method of determining whether a test compound modulates a signal transduction activity of a prostaglandin receptor IP, comprising: a) culturing IP-receptor-expressing host cells under conditions that would allow expression of a recombinant prostaglandin receptor, said host cells being transfected with a nucleic acid molecule encoding said recombinant prostaglandin IP-receptor comprising the amino acid sequence as set forth in SEQ ID NO:3; b) exposing a first population of IP receptor-expressing cells of step a) to a known prostaglandin IP receptor ligand; c) exposing a second population of IP receptor-expressing cells of step a) to the test compound and the known prostaglandin IP-receptor ligand of step b); d) exposing control host cells to the test compound of step c), wherein said control host cells do not express recombinant prostaglandin IP receptor protein; e) measuring the modulating affect of the test compound, which interacts with recombinant IP protein, in the presence and absence of the known IP receptor ligand, from the cells of step b), step c) and step d); and, f) comparing the modulating affect of the test compound as determined from step b), step c) and step d. 8. The method of claim 7 wherein said host cells are Xenopus oocytes. 9. The method of claim 7 wherein the effect is measured by an electrophysiological assay. 10. The method of claim 9 wherein said known prostaglandin IP receptor ligand is a ligand which shows 50% maximum specific binding to the prostaglandin IP receptor protein at a concentration of up to 5 .mu.M when competing with labeled iloprost for binding to the prostaglandin IP receptor protein. 11. The method of claim 9 wherein said known, labeled prostaglandin IP receptor ligand is selected from the group consisting of iloprost, carbacyclin and PGE.sub.2. 12. The method of claim 9 wherein said host cells are Xenopus oocytes. 13. The method of claim 12 wherein the nucleic acid molecule is mRNA. 14. The method of claim 7 wherein the nucleic acid molecule is DNA, wherein said IP receptor-expressing host cells are transiently or stably transformed by said DNA molecule. 15. A method of determining whether a test compound competes with a known prostaglandin IP receptor ligand for binding to a prostaglandin IP receptor protein, comprising: a) culturing IP-receptor-expressing host cells under conditions that would allow expression of a recombinant prostaglandin receptor, said host cells being transfected with a nucleic acid molecule encoding said recombinant prostaglandin IP-receptor comprising the amino acid sequence as set forth in SEQ ID NO:3; b) isolating a substantially purified cell membrane preparation from the IP receptor-expressing cells of step a); c) exposing a first population of the purified cell membrane preparation of step b) to a known, labeled prostaglandin IP receptor ligand; d) exposing a second population of the purified cell membrane preparation of step b) to the test compound and the known, labeled prostaglandin IP receptor ligand; e) determining the binding of the known, labeled prostaglandin IP receptor ligand to the membrane preparations of step c) and step d); and, f) comparing the binding of the known, labeled prostaglandin IP receptor ligand protein to the membrane preparations of step c) and step d). 16. The method of claim 15 wherein said known prostaglandin IP receptor ligand is a ligand which shows 50% maximum specific binding to the prostaglandin IP receptor protein at a concentration of up to 5 .mu.M when competing with labeled iloprost for binding to the prostaglandin IP receptor protein. 17. The method of claim 15 wherein said known, labeled prostaglandin IP receptor ligand is selected from the group consisting of iloprost, carbacyclin and PGE.sub.2. 18. The method of claim 15 wherein the nucleic acid molecule is DNA, wherein said IP receptor-expressing host cells are transiently or stably transformed by said DNA molecule. 19. A method of determining whether a test compound competes with a known prostaglandin IP receptor ligand for binding to a prostaglandin IP receptor protein, comprising: a) culturing IP-receptor-expressing host cells under conditions that would allow expression of a recombinant prostaglandin receptor, said host cells being transfected with a nucleic acid molecule encoding said recombinant prostaglandin IP-receptor comprising the amino acid sequence as set forth in SEQ ID NO:3; b) lysing the IP receptor expressing host cells of step a); c) exposing a first population of the lysed cells of step b) to a known, labeled prostaglandin IP receptor ligand; d) exposing a second population of the lysed cells of step b) to the test compound and the known, labeled prostaglandin IP receptor ligand; e) determining the binding of the known, labeled prostaglandin IP receptor ligand to the IP receptor from the lysed cells of step c) and step d); and, f) comparing the binding of the known, labeled prostaglandin IP receptor ligand protein to the IP receptor from the lysed cells of step c) and step d). 20. The method of claim 19 wherein said known prostaglandin IP receptor ligand is a ligand which shows 50% maximum specific binding to the prostaglandin IP receptor protein at a concentration of up to 5 .mu.M when competing with labeled iloprost for binding to the prostaglandin IP receptor protein. 21. The method of claim 19 wherein said known, labeled prostaglandin IP receptor ligand is selected from the group consisting of iloprost, carbacyclin and PGE.sub.2. 22. The method of claim 19 wherein the nucleic acid molecule is DNA, wherein said IP receptor-expressing host cells are transiently or stably transformed by said DNA molecule. 23. A method of determining whether a test compound competes with a known prostaglandin IP receptor ligand for binding to a prostaglandin IP receptor protein, comprising: a) culturing IP-receptor-expressing host cells under conditions that would allow expression of a recombinant prostaglandin receptor, said host cells being transfected with a nucleic acid molecule encoding said recombinant prostaglandin IP-receptor comprising the amino acid sequence as set forth in SEQ ID NO:3; b) exposing a first population of IP receptor-expressing cells of step a) to a known, labeled prostaglandin IP receptor ligand; c) exposing a second population of IP receptor-expressing cells of step a) to the compound and the known, labeled prostaglandin IP-receptor ligand of step b); d) determining the binding of the known, labeled prostaglandin IP receptor ligand to the membrane preparations of step b) and step c); and, e) comparing the binding of the known, labeled prostaglandin IP receptor ligand protein to the membrane preparations of step b) and step c). 24. The method of claim 23 wherein said known prostaglandin IP receptor ligand is a ligand which shows 50% maximum specific binding to the prostaglandin IP receptor protein at a concentration of up to 5 .mu.M when competing with labeled iloprost for binding to the prostaglandin IP receptor protein. 25. The method of claim 23 wherein said known, labeled prostaglandin IP receptor ligand is selected from the group consisting of iloprost, carbacyclin and PGE.sub.2. 26. The method of claim 23 wherein the nucleic acid molecule is DNA, wherein said IP receptor-expressing host cells are transiently or stably transformed by said DNA molecule. |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION PGI.sub.2 causes relaxation of arterial smooth muscle and inhibition of platelet aggregation, degranulation and shape change and is, therefore, thought to be important in maintaining vascular homeostasis. Other potential roles for PGI.sub.2 are not well established but include regulation of renal blood flow, renin release and glomerular filtration rate in the kidney cortex, modulation of neurotransmitter release in the heart and stimulation of secretion in the stomach and large intestine. In common with the other prostaglandins, PGI.sub.2 in also involved in the inflammatory response elicting hyperaemia, edema, hyperanalgesia and pyrexia primarily through its role as a vasodilator. The physiological actions of prostaglandin (PG)I.sub.2 are mediated through interaction with the prostaglandin IP receptor. The known distribution of IP receptors is reflective of the physiological actions of PGI.sub.2. They have been extensively characterized by radioligand binding studies in platelets from many species including human and identified in pharmacological studies as present in coronary, pulmonary, renal and several other arterial preparations as well as the heart. IP receptors may also be present in myometrium, penile erectile tissue and the iris sphincter muscle and have been reported in the NCB-20 and NG 108-15 neuronal hybrid cell lines and the mouse mastocytoma P-815 cell line. Functional activities of the IP receptor have been studied using tissue preparations such as arterial smooth muscle and cell based assays using platelets. The above methods for studying IP 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 varying levels of IP receptor and since tissue samples are required, compounds cannot satisfactorily be tested as effectors of the human IP receptor. SUMMARY OF THE INVENTION A novel prostaglandin receptor protein termed IP has been identified from human cells. A DNA molecule encoding the full length IP protein has been isolated and purified, and the nucleotide sequence has been determined. The IP 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 IP receptor protein. The novel IP protein, the IP-encoding DNA, the expression vectors and recombinant host cells expressing recombinant IP are useful in the identification of modulators of IP receptor activity. A method of identifying IP receptor modulators is also disclosed which utilizes the recombinant IP expressing host cells. Modulators of IP activity are useful for the treatment of prostaglandin-related diseases and for modulating the effects of prostaglandins on the IP receptor. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1--The DNA sequence of human IP cDNA clone hLXR3-6 is shown. FIG. 2--The DNA sequence of human IP cDNA clone hLXR3-11 is shown. FIG. 3--The DNA sequence human IP cDNA construct 11/6hLXR3 encoding the IP receptor protein is shown. FIG. 4--The complete deduced amino acid sequence of the IP receptor protein encoded by 11/6hLXR3 is shown. FIG. 5A and 5B --Expression of the prostaglandin 12 receptor in IP cDNA-injected Xenopus oocytes is shown. Panel A shows iloprost induced CFR-mediated Cl.sup.- current evoked by bath perfusion of 1-100 nM iloprost and not by PGE.sub.2 or PGD.sub.2 in an oocyte that was co-injected with 2.6 ng pcDNAIamp-CFTR and 1 ng pcDNAIamp-11/6hLXR3 (hIP cDNA). Panel B shows the absence of endogenous IP receptors in oocytes. Oocytes were injected with CFTR cDNA and challenged with 100 nM iloprost, PGE.sub.2, PGD.sub.2 and 3 mM IBMX. The broken line represents the zero current level. The voltage-clamp experiments shown here were performed 48 hr after nuclear injection and are representative of 6 separate experiments with oocytes from 3 different frogs. FIG. 6--Competition for [.sup.3 H]-iloprost specific binding to pcDNAIamp-hIP transfected COS-M6 membranes. [.sup.3 H]Iloprost binding assays were performed as described in Example 7. The percentage maximum [.sup.3 H]iloprost specific binding at each competing ligand concentration was determined for iloprost (.box-solid.), the stable IP receptor agonist carbacyclin (.quadrature.), PGE.sub.2 (.tangle-solidup.), PGF.sub.2.alpha. (.circle-solid.), PGD.sub.2 (.DELTA.) and the TP-receptor agonist U46619 (.smallcircle.), over a concentration range up to 100 mM. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to cDNA encoding a novel prostaglandin receptor, termed IP. The present invention is also related to recombinant host cells which express the cloned IP-encoding DNA contained in a recombinant expression plasmid. The present invention is also related to methods for the screening of substances which modulate IP receptor activity. The DNA of the present invention is isolated from IP producing cells. IP, as used herein, refers to a G protein-coupled receptor which can specifically bind prostaglandin molecules. Mammalian cells capable of producing IP include, but are not limited to, cells derived from the small and large intestine, kidney, stomach, vascular smooth muscle, eye, placenta, uterus, thymus and platelets. Transformed mammalian cell lines which produce IP include, but are not limited to, mastocytoma P-815 cells. The preferred cells for the present invention include normal human kidney and platelets and the most preferred cells are human lung cells. Other cells and cell lines may also be suitable for use to isolate IP cDNA. Selection of suitable cells may be done by screening for IP on cell surfaces. Methods for detecting IP activity are well known in the art and measure the binding of radiolabelled ligand specific for the receptor. Cells which possess IP activity in this assay may be suitable for the isolation of IP cDNA. Any of a variety of procedures may be used to clone IP cDNA. These methods include, but are not limited to, direct functional expression of the IP cDNA following the construction of an IP-containing cDNA library in an appropriate expression vector system. Another method is to screen an IP-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled oligonucleotide probe designed from the amino acid sequence of the IP protein. The preferred method consists of screening an IP-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the IP protein. This partial cDNA is obtained by the specific PCR amplification of IP DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence known for other G protein-coupled receptors which are related to the prostaglandin IP receptors. It is readily apparent to those skilled in the art that other types of libraries, as well as libraries constructed from other cells or cell types, may be useful for isolating IP-encoding DNA. Other types of libraries include, but are not limited to, cDNA libraries derived from other cells or cell lines and genomic DNA libraries. It is readily apparent to those skilled in the art that suitable cDNA libraries may be prepared from cells or cell lines which have IP activity. The selection of cells or cell lines for use in preparing a cDNA library to isolate IP cDNA may be done by first measuring cell associated IP activity using the known labelled ligand binding assay cited above and used herein. Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found for example, in Maniatis, T., Fritsch, E. F., Sambrook, J., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982). It is also readily apparent to those skilled in the art that DNA encoding IP may also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techniques can be found in Maniatis, T., Fritsch, E. F., Sambrook, J. in Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982). In order to clone the IP gene by one of the preferred methods, the amino acid sequence or DNA sequence of IP or a homologous protein is necessary. To accomplish this, IP protein or a homologous protein may be purified and partial amino acid sequence determined by automated sequenators. It is not necessary to determine the entire amino acid sequence, but the linear sequence of two regions of 6 to 8 amino acids can be determined for the PCR amplification of a partial IP DNA fragment. Once suitable amino acid sequences have been identified, the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the IP sequence but others in the set will be capable of hybridizing to IP DNA even in the presence of DNA oligonucleotides with mismatches. The mismatched DNA oligonucleotides may still sufficiently hybridize to the IP DNA to permit identification and isolation of IP encoding DNA. Using one of the preferred methods, cDNA clones encoding IP are isolated in a two-stage approach employing polymerase chain reaction (PCR) based technology and cDNA library screening. In the first stage, NH.sub.2 -terminal and internal amino acid sequence information from the purified IP or a homologous protein is used to design degenerate oligonucleotide primers for the amplification of IP-specific DNA fragments. In the second stage, these fragments are cloned to serve as probes for the isolation of full length cDNA from cDNA libraries. The sequence for the cDNA encoding IP is shown in Table 1, and was designated clone 11/6hLXR3. The deduced amino acid sequence of IP from the cloned cDNA is shown in Table 2. Inspection of the determined cDNA sequence reveals the presence of a single, large open reading frame that encodes for a 386 amino acid protein. The cloned IP cDNA obtained through the methods described above may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant IP. Techniques for such manipulations can be found described in Maniatis, T, et al., supra, and are well known in the art. Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. A variety of mammalian expression vectors may be used to express recombinant IP in mammalian cells. Commercially available mammalian expression vectors which may be suitable for recombinant IP expression, include but are not limited to, pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), pcDNAI, pcDNAIamp (Invitrogen), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), 1ZD35 (ATCC 37565), and vaccinia virus transfer vector pTM1. DNA encoding IP may also be cloned into an expression vector for expression in a host cell. Host cells may be prokaryotic or eukaryotic, including but not limited to bacteria, yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Sf9 and drosophila derived cell lines. Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171). The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, infection, protoplast fusion, and electroporation. The expression vector-containing cells are individually analyzed to determine whether they produce IP protein. Identification of IP expressing cells may be done by several means, including but not limited to immunological reactivity with anti-IP antibodies, and the presence of host cell-associated IP activity. Expression of IP DNA may also be performed using in vitro produced synthetic mRNA. Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred. To determine the IP cDNA sequence(s) that yields optimal levels of receptor activity and/or IP protein, IP cDNA molecules including but not limited to the following can be constructed: the full-length open reading frame of the IP cDNA and various constructs containing portions of the cDNA encoding only specific domains of the receptor protein or rearranged domains of the protein. All constructs can be designed to contain none, all or portions of the 5' and/or 3' untranslated region of IP cDNA. IP activity and levels of protein expression can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells. Following determination of the IP cDNA cassette yielding optimal expression in transient assays, this IP cDNA construct is transferred to a variety of expression vectors (including recombinant viruses), including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, E. coli, and yeast cells. Mammalian cell transfectants are assayed for both the levels of IP receptor activity and levels of IP protein by the following methods. Assessing IP receptor activity involves the direct introduction of a labelled ligand to the cells and determining the amount of specific binding of the ligand to the IP-expressing cells. Binding assays for receptor activity are known in the art (Frey et al., 1993, Eur. J. Pharmacol., 244, pp 239-250). Levels of IP protein in host cells is quantitated by a variety of techniques including, but not limited to, immunoaffinity and/or ligand affinity techniques. IP-specific affinity beads or IP-specific antibodies are used to isolate .sup.35 S-methionine labelled or unlabelled IP protein. Labelled IP protein is analyzed by SDS-PAGE. Unlabelled IP protein is detected by Western blotting, ELISA or RIA assays employing IP specific antibodies. Following expression of IP in a host cell, IP protein may be recovered to provide IP in active form, capable of binding IP-specific ligands. Several IP purification procedures are available and suitable for use. Recombinant IP may be purified from cell membranes by various combinations of, or individual application of standard separation techniques including but not limited to detergent solubilization, salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography. In addition, recombinant IP can be separated from other cellular proteins by use of an immuno-affinity column made with monoclonal or polyclonal antibodies specific for full length nascent IP, or polypeptide fragments of IP. Monospecific antibodies to IP are purified from mammalian antisera containing antibodies reactive against IP or are prepared as monoclonal antibodies reactive with IP using the technique of Kohler and Milstein, Nature 256: 495-497 (1975). Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for IP. Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the IP, as described above. IP specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with an appropriate concentration of IP or a peptide derived from the sequence of the IP protein either with or without an immune adjuvant. Preimmune serum is collected prior to the first immunization. Each animal receives between about 0.1 .mu.g and about 1000 .mu.g of IP or IP-related peptide associated with an acceptable immune adjuvant. Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA. The initial immunization consisted of the enzyme in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally (IP) or both. Each animal is bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunization. Those animals receiving booster injections are generally given an equal amount of IP or IP-related peptide in Freund's incomplete adjuvant by the same route. Booster injections are given at about three week intervals until maximal titers are obtained. At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about -20.degree. C. Monoclonal antibodies (mAb) reactive with IP or a peptide derived from the sequence of the IP protein are prepared by immunizing inbred mice, preferably Balb/c, with IP or IP-related peptide . The mice are immunized by the IP or SC route with about 1 .mu.g to about 100 .mu.g, preferably about 10 .mu.g, of IP or IP-related peptide in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred. The mice receive an initial immunization on day 0 and are rested for about three to about 30 weeks. Immunized mice are given one or more booster immunizations of about 1 to about 100 .mu.g of IP in a buffer solution such as phosphate buffered saline by the intravenous (IV) route. Lymphocytes, from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas. Fusion partners may include, but are not limited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected from growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using IP or IP-related peptide as the antigen. The culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973. Monoclonal antibodies are produced in vivo by injection of pristine primed Balb/c mice, approximately 0.5 ml per mouse, with about 2.times.10.sup.6 to about 6.times.10.sup.6 hybridoma cells about 4 days after priming. Ascites fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art. In vitro production of anti-IP mAb is carried out by growing the hydridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb. The mAb are purified by techniques known in the art. Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of IP in body fluids or tissue and cell extracts. It is readily apparent to those skilled in the art that the above described methods for producing monospecific antibodies may be utilized to produce antibodies specific for IP polypeptide fragments, or full-length IP polypeptide. IP antibody affinity columns are made by adding the antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HCl (pH 8). The column is washed with water followed by 0.23 M glycine HCl (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) together with appropriate membrane solubilizing such as detergents and the cell culture supernatants or cell extracts containing IP or IP fragments are slowly passed through the column. The column is then washed with phosphate buffered saline together with appropriate membrane solubilizing such as detergents until the optical density (A.sub.280) falls to background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6) together with appropriate membrane solubilizing such as detergents. The purified IP protein is then dialyzed against phosphate buffered saline together with appropriate membrane solubilizing agent, such as detergents. One method suitable for the isolation of DNA encoding the prostaglandin receptor of the present invention involves the utilization of amino acid and/or DNA sequence information obtained from other G-protein-linked receptors. Since other prostaglandin receptors are known to be G-protein linked, certain regions or domains such as the transmembrane and/or cytoplasmic domains, are expected to have some degree of homology sufficient to produce a probe for the isolation of novel receptors. Prostaglandins and leukotrienes are known to transduce their signals via G-protein-linked receptors. Distinct receptors for PGH.sub.2 /thromboxane A.sub.2, PGI.sub.2, PGE.sub.2, PGD.sub.2, PGF2a, LTB.sub.4, and LTD4 present in various tissues have been described. Some of the receptors have been solubilized and partially purified (Dutta-Roy, A. K. et al., (1987) JBC, 262, pp. 12685; Tsai, A. L. et al., (1989), JBC, 264, pp 61; Watanabe, T. et. al., (1990), JBC, 265, pp. 21237) and the human platelet TXA.sub.2 receptor has been purified to apparent homogeneity (Ushikubi, F. et. al., (1989), JBC, 264, pp. 16496). The purified thromboxane receptor exhibited a very broad band on a SDS-polyacrylamide gel centered at appr. 57 kDa. Enough protein was obtained for partial sequence information. An approach to the isolation of other eicosanoid receptor genes by homology screening was taken, with the assumption that these receptors are related in primary structure (Sugimoto, Y. et al., (1992), JBC, 267, pp. 6463). Since these receptors are of the G-protein-coupled receptor superfamily there are areas of homology which are likely to be found in the transmembrane region and in the cytoplasmic domains. Therefore, various known G-protein linked receptors related to the prostaglandin receptors may be utilized to provide DNA probes to regions of the receptor protein-encoding DNA sought, which is likely to have homology, such as the transmembrane region. Using an antisense 16-fold degenerate 26 mer oligonucleotide based upon a stretch of nine amino acids in transmembrane domain VII of the published mouse EP.sub.2 receptor amino acid sequence a human lung library was screened from which human IP cDNA clones were isolated. From two such cDNA clones one was constructed. This 1.417 kb cDNA clone encodes a 386-amino acid protein. This protein was designated as the IP receptor. Like many other G-protein coupled receptors the IP receptor shares several common features. Firstly, there is 1 potential N-linked glycosylation site at Asn7 in the putative extracellular amino terminus. Secondly, conserved cysteine residues are found in extracellular loops 1 and 2. There are serine residues, potential sites of protein kinase phosphorylation, in the C-terminus. The IP receptor possesses a conserved arginine (position 279) found in all known eicosanoid receptors within transmembrane seven. This region is the most highly conserved among the eicosanoid receptors. The novel prostaglandin receptor of the present invention is suitable for use in an assay procedure for the identification of compounds which modulate the receptor activity. Modulating receptor activity, as described herein includes the inhibition or activation of the receptor and also includes directly or indirectly affecting the normal regulation of the receptor activity. Compounds which modulate the receptor activity include agonists, antagonists and compounds which directly or indirectly affect regulation of the receptor activity. The prostaglandin receptor of the present invention may be obtained from both native and recombinant sources for use in an assay procedure to identify receptor modulators. In general, an assay procedure to identify prostaglandin receptor modulators will contain the prostaglandin receptor of the present invention, and a test compound or sample which contains a putative prostaglandin receptor modulator. The test compounds or samples may be tested directly on, for example, purified receptor protein whether native or recombinant, subcellular fractions of receptor-producing cells whether native or recombinant, and/or whole cells expressing the receptor whether native or recombinant. The test compound or sample may be added to the receptor in the presence or absence of a known labelled or unlabelled receptor ligand. The modulating activity of the test compound or sample may be determined by, for example, analyzing the ability of the test compound or sample to bind to the receptor, activate the receptor, inhibit receptor activity, inhibit or enhance the binding of other compounds to the receptor, modify receptor regulation, or modify an intracellular activity. The identification of modulators of IP receptor activity are useful in treating disease states involving the IP receptor activity. Other compounds may be useful for stimulating or inhibiting activity of the receptor. Selective agonists or antagonists of the IP receptor may be of use in the treatment of diseases and disease states including, but not limited to, edema associated with inflammation, pain response and fever, and may have utility in the inhibition of platelet aggregation and hence in the treatment of vascular diseases, prevention of post-injury blood clotting and rejection in organ tranplantation and by-pass surgery, congestive heart failure, pulmonary hypertension, gangrene, Raynauds disease, bone resorption, shock, and gastric acid secretion. Modulators may also be useful as cytoprotective agents. The isolation and purification of an IP-encoding DNA molecule would be useful for establishing the tissue distribution of IP receptors, studying changes in IP receptor expression in disease states, as well as establishing a process for identifying compounds which modulate IP receptor activity. |
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