MXPA00009834A - U4, a member of the hematopoietin receptor superfamily - Google Patents

Abstract

Polynucleotides encoding the U4 hematopoietin receptor superfamily chain and fragments thereof are disclosed. U4 proteins and methods for their production are also disclosed.

Description

U4, A MEMBER OF THE HEMATOPOYETINE RECEPTOR SUPERFAMILY Field of the Invention The present invention relates to new members of the mammalian hematopoietin protein superfamily (including, without limitation, human and murine receptor proteins), fragments thereof and recombinant polynucleotides and cells useful for the expression of such proteins. proteins Background of the Invention This application is a continuation-in-part of the application serial number 08 / 784,863, filed on January 16, 1997. A variety of regulatory molecules, known as hematopoietin, have been identified as being involved in the development and proliferation of several populations of hematopoietic or blood cells. Most hematopoietins exhibit certain biological activities by interacting with a receptor on the target cell surface. Cytokine receptors are commonly composed of one, two or three chains. Many cytokine receptors and some cytokines, such as IL-12 p40, are members of the hematopoietin receptor protein superfamily. The identification of new members of the superfamily of hematopoietin receptors can be useful in the regulation of hematopoiesis, in the regulation of immune responses and in the identification of other members of the hematopoietin superfamily, including cytokines and receptors. It would be desirable to identify and determine the? DN and the protein sequence for the hitherto unknown members of the hematopoietin superfamily of receptors.

SUMMARY OF THE INVENTION In accordance with the present invention, polynucleotides encoding the hemopoietin U4 receptor superfamily chain are described, including, without limitation, those from murine and human sources. In certain embodiments, the invention provides an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 4, from nucleotide 242 to nucleotide 1396; (b) the nucleotide sequence of SEQ ID NO: 6, from nucleotide 71 to nucleotide 1225; (c) the nucleotide sequence of SEQ ID NO: 9, from nucleotide 136 to nucleotide 1401; (d) a nucleotide sequence that varies from the sequence of the nucleotide sequence specified in (a), (b) or (c) as a result of the degeneracy of the genetic code; (e) a nucleotide sequence capable of hybridizing under conditions astringent to the nucleotide specified in (a) or (b); (f) a nucleotide sequence encoding a homologous species of the sequence specified in (a), (b) or (c); and (g) an allelic variant of the nucleotide sequence specified in (a), (b) or (c). Preferably, the nucleotide sequence codes for a protein having a biological activity of the hematopoietin U4 receptor superfamily chain. The nucleotide sequence may be operably linked to an expression control sequence. In preferred embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4 from nucleotide 242 to nucleotide 1396; the nucleotide sequence of SEQ ID NO: 4 from nucleotide 122 to nucleotide 1396; the nucleotide sequence of SEQ ID NO: 6 from nucleotide 71 to nucleotide 1225; or the nucleotide sequence of SEQ ID NO: 6 from nucleotide 11 to nucleotide 12256; or the nucleotide sequence of SEQ ID NO: 9 from nucleotide 136 to nucleotide 1401; or the nucleotide sequence of SEQ ID NO: 9 from nucleotide 247 to nucleotide 1401. The invention also provides isolated polynucleotides comprising a nucleotide sequence encoding a peptide or protein comprising an amino acid sequence selected from the group consists of: (a) the amino acid sequence of SEQ ID NO: 5; (b) the amino acid sequence of SEQ ID NO: 5, from amino acid 41 to amino acid 425; (c) the amino acid sequence of SEQ ID NO: 7; (d) the amino acid sequence of SEQ ID NO: 7, from amino acid 24 to amino acid 408; (e) the amino acid sequence of SEQ ID NO: 10; (f) the amino acid sequence of SEQ ID NO: 10, from amino acid 38 to amino acid 421; and (g) fragments of (a) - (f) having biological activity of the hematopoietin receptor superfamily chain TJ4. Other preferred embodiments encode the amino acid sequence of SEQ ID NO: 5; the amino acid sequence of SEQ ID NO: 5 from amino acid 41 to amino acid 425; the amino acid sequence of SEQ ID NO: 7 from amino acid 24 to amino acid 408; the amino acid sequence of SEQ ID NO: 10; and the amino acid sequence of SEQ ID NO: 10 from amino acid 38 to amino acid 421. Host cells, preferably mammalian cells, transformed with the polynucleotides are also provided. In other embodiments, the invention provides a process for the production of a U4 protein. The process comprises: (a) growing a culture of the host cell of the present invention in a suitable culture medium; and (b) purifying the human U4 protein from the culture medium. The proteins produced according to such methods is also provided by the present invention. The present invention also provides an isolated U4 protein comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO: 5; (b) the amino acid sequence of SEQ ID NO: 5, from amino acid 41 to amino acid 425; (c) the amino acid sequence of SEQ ID NO: 7; (d) the amino acid sequence of SEQ ID NO: 7, from amino acid 24 to amino acid 408; and (e) fragments of (a) - (d) having a biological activity of the hemopoietin U4 receptor superfamily chain. Preferably, the protein comprises the amino acid sequence of SEQ ID NO: 5; the amino acid sequence of SEQ ID NO: 5 from amino acid 41 to amino acid 425; the amino acid sequence of SEQ ID NO: 7; or the amino acid sequence of SEQ ID NO: 7 from amino acid 24 to amino acid 408. In other preferred embodiments, the specified amino acid sequence is part of a fission protein (with an additional amino acid sequence not derived from U4) . Preferred fusion proteins comprise an antibody fragment, such as an Fc fragment. Also provided are pharmaceutical compositions comprising a protein of the present invention and a pharmaceutically acceptable carrier. The present invention also provides compositions comprising an antibody that specifically reacts with a protein of the present invention.

Detailed Description of the Preferred Modalities. The inventors of the present application have identified and provided for the first time polynucleotides encoding the hematopoietin receptor superfamily chain U4 (hereinafter referred to as "U4" or "U4 protein"), including without limitation polynucleotides which code for the U4 of murine and human. A region of 79 amino acids of the human IL-5 receptor (LMTNAFISIIDDLSKYDVQVRAAVSSMCREAGLWSEWSQPIYVGNDEHKPLREWFVI VIMATICFILLIL, SEQ ID NO: l) was used to investigate in the EST GenBank database using the TBLASTN algorithm. EST W66776 was identified with homology for this region, suggesting that it could code for a new hematopoietin receptor. Translation of the reverse complement of this EST using the GCG mapping program revealed a protein sequence in the second reading frame that contained the conserved WSXWS motif that was found in hematopoietin receptors. However, a stop codon was also present in this reading frame at nucleotide 227, indicating that this EST was not a new hematopoietin receptor, or that the DNA sequence in this EST was incorrect. To determine if this EST sequence could be related to a hematopoietin receptor, we screened a murine embryo library with an oligonucleotide probe of the sequence CTTGGCTTGG AAGAGGAAAT CCTTGAGAGC (SEQ ID NO: 2). A full length U6-3 (1A) clone was identified and the complete sequence was obtained. The DNA sequence and predicted amino acid sequence for the murine protein were reported as SEQ ID NO: 4 and SEQ ID NO: 5, respectively. The murine protein encodes a new member of the family of hematopoietin receptors. It has a leader sequence, and the conserved cysteine pairs, PP, and the WSXWS motifs characteristic of this family. This clone does not have transmembrane or cytoplasmic domains. The alignment of this clone with the EST in GenBank revealed that the EST had a frame change mutation. SEQ ID NO: 4 provides a nucleotide sequence of a cDNA encoding murine U4. SEQ ID NO: 5 provides the predicted amino acid sequence of the receptor chain, including a putative signal sequence from amino acids 1-40. It is believed that mature murine U4 has amino acid sequence 41-383 of SEQ ID NO: 5. To identify additional related sequences in GenBank, the sequence of W66776 was used to investigate in GenBank using the BLASTN algorithm. A closely related EST, HI4009, derived from human genomic DNA was identified. Subsequently an oligonucleotide derived from this EST CTGAGCGTGC GCTGGGTGTC GCCAC (SEQ ID NO: 3) was used to isolate a cDNA clone from a human cDNA library. A cDNA clone (HU4-3B) coding for a full-length mature protein homolog was completely sequenced. This clone does not have a complete signal sequence, but it codes for all the predicted full-length mature protein. The human clone is 85% homologous at the DNA level with the mouse clone. The predicted amino acid sequences have 95% identity between human and mouse. The nucleotide and amino acid sequence for human U4 are reported as SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

SEQ ID NO: 6 provides the nucleotide sequence of a cDNA encoding human U4. SEQ ID NO: 7 provides the predicted amino acid sequence of the receptor chain, including a putative signal sequence from amino acids 1-23. The mature human U4 is believed to have the amino acid sequence 24-380 of SEQ ID NO: 7. The murine and human clones were deposited with the American Type Culture collection on January 15, 1997, with the access numbers ATCC 98305 and ATCC 98306, respectively. The human U4 protein can be expressed by replacing the human leader sequence with the murine leader sequence, or by extending the human leader sequence with amino acids 1-14 of the murine sequence (MPAGRPGPVA QSAR, SEQ ID NO: 8). Additionally, a larger cDNA or genomic clone and coding for the actual leader sequence can be isolated using the sequences described herein as probes. The sequence of the human U4 cDNA described above was used to isolate a larger cDNA encoding human U4 protein. The nucleotide sequence of this larger clone is reported in SEQ ID NO: 9. The predicted amino acid sequence encoded in this manner is reported in SEQ ID NO: 10. The largest cDNA clone was deposited with the American Type Culture collection on March 10, 1998, under accession number ATCC 98688. Any forms of U4 proteins of less than full length are encompassed by the present invention and are collectively referred to herein with full-length and mature forms as "U4" or "U4 proteins." U4 proteins of less than full length can be produced by expressing a corresponding fragment of the polynucleotides encoding the full length U4 protein (SEQ ID NO: 4 or SEQ ID NO: 6). These corresponding polynucleotide fragments are also part of the present invention. Modified polynucleotides as described above can be made by standard techniques of molecular biology, including the construction of desired, desired deletion mutants, site-directed mutagenesis methods or by polymerase chain reaction using appropriate oligonucleotide primers.

For the purposes of the present invention, a protein has "a biological activity of the hematopoietin U4 receptor superfamily chain" if it possesses one or more of the biological activities of the corresponding mature U4 protein. U4 or active fragments thereof (U4 proteins) can be fused to carrier molecules such as immunoglobulins. For example, soluble forms of U4 can be fused via "linker" sequences to the Fc portion of an immunoglobulin.

Other fusion proteins can also be used, such as those with GST, Lex-A or MBP. This invention also encompasses allelic variants of the nucleotide sequences as set forth in SEQ ID NO: 4 or SEQ ID NO: 6, ie, alternative forms that occur naturally, of the isolated polynucleotide of SEQ ID NO: 4 or SEQ ID NO: 6 which also code for U4 proteins, preferably those proteins having a biological activity of U4. Also included in the invention are isolated polynucleotides that hybridize to the nucleotide sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 6 under highly stringent conditions (eg, OlxSSC at 65 ° C). Also included within the present invention are isolated polynucleotides which encode U4 proteins but which differ from the nucleotide sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 6 by virtue of the degeneracy of the genetic code. Also included within the present invention are variations in the nucleotide sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 6 and which are caused by point mutations or by induced modifications. The present invention also provides polynucleotides that encode murine and human U4 homologs from other animal species, particularly other mammal species. The homologous species can be identified and isolated by making probes or primaries from the murine or human sequences described herein and screening a library of an appropriate species, such as for example libraries constructed from PBMCs, thymus or testes of the relevant species . The isolated polynucleotides of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors described in Kaufinan et al., Nucleic Acids Res. 19, 4485-4490 (1991), for the purpose of recombinantly produce the U4 protein. Many suitable expression control sequences are known in the art. General methods for expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein, "operably linked" means enzymatically or chemically linked to form a covalent bond between the isolated polynucleotide of the invention and the expression control sequence, in such a way that the U4 protein is expressed by a host cell that has been transformed (transfected) with the linked polynucleotide sequence / expression control. A number of cell types can act as host cells suitable for the expression of the U4 protein. Any type of cell capable of expressing functional U4 protein can be used. Suitable mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary cells (CHO), human kidney cells 293, A431 cells of human epidermis, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK cells, HL-60, U937, HaK, Rat2, BaF3, 32D, FDCP-1, PC12, Mix or C2C12. The U4 protein can also be produced by operably linking the isolated polynucleotide of the invention to appropriate control sequences in one or more insect expression vectors and employing an insect expression system. Materials and methods for expression systems in báculovirus / insect cells are commercially available as a kit in, for example, Invitrogen, San Diego, California, E.U.A. (the MaxBac® kit) and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), which is incorporated herein by reference. Soluble forms of the U4 protein can also be produced in insect cells using appropriate isolated polynucleotides as described above. Alternatively, the U4 protein can be produced in loeukaryotes such as yeast or in prokaryotes such as bacteria. Suitable strains and yeast potentials include strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces, Candida, or any strain of yeast capable of expressing heterologous proteins. Suitable strains and bacterial potentials include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. Expression in bacteria can result in the formation of inclusion bodies that incorporate the recombinant protein. Thus, the replication of the recombinant protein may be required in order to produce active or more active material. Various methods are known in the art for obtaining correctly refolded heterologous proteins from bacterial inclusion bodies. These methods generally involve the solubilization of the protein from the inclusion bodies, subsequently denaturing the protein completely using a chaotropic agent. When cysteine residues are present in the primary amino acid sequence of the protein, it is often necessary to carry out the refolding in an environment that allows the correct formation of disulfide bonds (a redox system). General methods of refolding are described in Kohno, Meth. Enzvm. 185: 187-195 (1990). European patent EP 0433225 and copending application USSN 08 / 163,877 describe suitable methods The U4 protein of the invention can also be expressed as a product of transgenic animals, for example, as a component of the milk of cows, goats, pigs or sheep transgenic, which are characterized by germ or somatic cells containing a nucleotide sequence encoding the U4 protein The U4 protein of the invention can be prepared by growing a culture of transformed host cells under culture conditions necessary to express the desired protein. The resulting expressed protein can then be purified from the culture medium or cell extracts. Soluble forms of the U4 protein of the invention can be purified from conditioned media. Membrane binding forms of the U4 protein of the invention can be purified by preparing a total membrane fraction from the expression cell and extracting the membranes with a nonionic surfactant such as Triton X-100. The U4 protein can be purified using methods known to those trained in the art. For example, the U4 protein of the invention can be concentrated using a commercially available protein concentration filter, for example, a Millipore Pellicon or Amicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an ion exchange resin, for example, a matrix or a substrate having diethylaminoethyl (DEAE) or polyethylene imine (PEI) groups may be employed. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly used in protein purification. Alternatively, a cation exchange step may be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred (e.g., S-Sepharose® columns). Purification of the U4 protein from the culture supernatant can also include one or more column steps on affinity resins such as concavalin A-agarose, heparin-toyopearl®, or Cibacrom blue 3GA Sepharose®, or by hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether or propyl ether; or by immunoaffinity chromatography. Finally, one or more steps of high resolution reverse phase liquid chromatography (RP-HPLC) employing hydrophobic RP-HPLC media, for example, silica gel having suspended methyl or other aliphatic groups, can be used to further purify the U4 protein Affinity columns including antibodies to the U4 protein can also be used in the purification according to known methods. Some or all of the preceding purification steps, in various combinations or with other known methods, may also be employed to provide an isolated and substantially purified recombinant protein. Preferably, the isolated U4 protein is purified so that it is substantially free of other mammalian proteins. The U4 proteins of the invention can also be used to screen agents that are capable of binding to U4. Binding assays using a desired binding protein, immobilized or not, are well known in the art and can be used for this purpose using the U4 protein of the invention. Screening assays based on purified cells or proteins (cell-free) can be used to identify such agents. For example, the U4 protein can be immobilized in purified form on a carrier and the binding or potential ligands for purified U4 protein can be measured. U4 proteins, purified from cells or recombinantly produced, can be used as a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Said composition may contain, in addition to the U4 or inhibitor and carrier, various diluents, fillers, salts, regulators, stabilizers, solubilizers, and other materials that are well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient (s). The characteristics of the carrier will depend on the administration route. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6. , IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-14, IL-15, G-CSF, pluripotent cell factor and erythropoietin. The pharmaceutical composition may also include anti-cytokine antibodies. The pharmaceutical composition may contain thrombolytic or anti-thrombotic factors such as plasminogen activator and Factor VIII. The pharmaceutical composition may also contain other anti-inflammatory agents. Such additional factors and / or agents can be included in the pharmaceutical composition to produce a synergistic effect with the isolated U4 protein, or to minimize side effects caused by the isolated U4 protein. Conversely, the isolated U4 protein can be included in formulations of the particular cytokine, lymphokine, another hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, another hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent. The pharmaceutical composition of the invention may be in the form of a liposome in which the isolated U4 protein is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids that exist in aggregate form such as miscella, insoluble monolayers, liquid crystals. , or laminar layers that are in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, and the like. The preparation of such liposomal formulations is within the skill of the art, as described, for example, in U.S. Pat. No. 4,235,871; Patent of the U.S.A. No. 4,501,728; Patent of the U.S.A. No. 4,837,028 and U.S. Pat. No. 4,737, 323; all of which is incorporated herein by reference. As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a significant benefit to the patient, eg, improvement of symptoms, cure or increase in curing speed of said conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. In the practice of the method of treatment or use of the present invention, a therapeutically effective amount of the isolated U4 protein is administered to a mammal. The isolated U4 protein can be administered according to the method of the invention, either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, the U4 protein can be administered either simultaneously with cytokine (s), lymphokine (s), other hematopoietic factor (s), thrombolytic or antithrombotic factors, or sequentially. If administered sequentially, the treating physician will decide on the appropriate sequence of administration of the U4 protein in combination with cytokine (s), lymphokine (s), other hematopoietic factor (s), thrombolytic or antithrombotic factors . The administration of the U4 protein used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional forms, such as oral ingestion, inhalation, cutaneous injection, subcutaneous or intravenous. Intravenous administration to the patient is the most preferred. When a therapeutically effective amount of the U4 protein is administered orally, the U4 protein will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as gelatin or an adjuvant. The tablet, capsule and powder contains from about 5 to 95% of the U4 protein, and preferably from about 25 to 90% of the U4 protein. When administered in liquid form, a liquid carrier such as water, petroleum, animal or vegetable oils such as peanut oil, mineral oil, soybean oil, sesame oil, or synthetic oils can be added. The liquid form of the pharmaceutical composition may further contain physiological saline, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of the U4 protein, and preferably from about 1 to 50% of the U4 protein. When a therapeutically effective amount of the U4 protein is administered by intravenous, cutaneous or subcutaneous injection, the U4 protein will be in the form of a parenterally acceptable and pyrogen-free aqueous solution. It is within the skill of the art to prepare such parenterally acceptable protein solutions, with due regard to pH, isotonicity, stability and the like. A pharmaceutically preferred composition for intravenous, cutaneous or subcutaneous injection must contain, in addition to the U4 protein, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose Injection and Sodium Chloride., Ringer's Injection with Lactate, or other vehicle as is known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, regulators, antioxidants, or other additives known to those skilled in the art. The amount of the U4 protein in the pharmaceutical composition of the present invention will depend on the nature and severity of the condition being treated, and on the nature of previous treatments the patient has had. Finally, the attending physician will decide the amount of the U4 protein with which he will treat each individual patient. Initially, the attending physician will administer low doses of the U4 protein and observe the patient's response. Higher doses of the U4 protein can be administered until the optimal therapeutic effect is obtained for the patient, and at that point generally the dose will not increase further. It is contemplated that the various pharmaceutical compositions used in the practice of the method of the present invention should contain about 0.1 μg to about 100 mg of the U4 protein per kilogram of body weight.

The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary depending on the severity of the disease being treated and the potential idiosyncratic condition and response of each individual patient. It is contemplated that the duration of each application of the U4 protein will be in the range of 12 to 24 hours of continuous intravenous administration. Finally the attending physician will decide about the appropriate duration of the intravenous therapy using the pharmaceutical composition of the present invention. It is expected that the polynucleotide and proteins of the present invention will exhibit one or more of the biological uses or activities (including those associated with assays cited herein) that are identified below. The uses or activities described for the proteins of the present invention can be provided by the administration or use of such proteins or by the administration or use of polynucleotides that encode such proteins (such as, for example, in appropriate gene or vector therapies for the introduction of DNA).

Cytokine and Proliferation Activity / Cell Differentiation A protein of the present invention can exhibit cytokine activity, cell proliferation (either induction or inhibition) or cell differentiation (either induction or inhibition), or it can induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by one of a number of routine factor-dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, UNCLE, B9, B9 / 11, BaF3 , MC9 / G, M + (preB M +), 2E8, RB5, DA1, 123, TI 165, HT2, CTLL2, TF-1, Mo7e and CMK. The activity of the protein of the invention may, among other means, be measured by the following methods: Assays for proliferation of thymocytes or T cells include, without limitation, those described in Current Protocols in Immunology, Ed. By J.E. Coligan, A.M. Kruisbeek, D.H. Marguiles, E.M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley Interscience (Chapter 3, In Vitro Assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic Studies in Humans); Takai et al., J. Immunol. 137: 3494-3500, 1986; Bertagnolli et al., J. Immunol. 145: 1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133: 327-341, 1991; Bertagnolli, et al., J. Immunol. 149: 3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994. Assays for determining cytokine production and / or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol. 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto, 1994; and Measurements of mouse and human Interferon ?, Schreiber, R.D. In Current Protocols in Immunology, J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto, 1994. Trials to determine the proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, LS and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173: 1205-1211, 1991; Moreau et al., Nature 336: 690-692, 1988; Greenberger et al., Proc. Nati Acad. Sci. U.S.A. 80: 2931-2938, 1983; Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Nati Acad. Sci. U.S.A. 83: 1857-1861, 1986; Measurement of human Interleukin 11 - Bennett, F. Giannotti, J. Clark, S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a Coligan eds. Vol 1 pp. 6.15.1, John Wiley and Sons, Toronto. 1991. Measurement of mouse and human Interleukin 9 - Ciarletta, A. Giannotti, J., Clark, S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991. Trials to determine the responses of T cell clones to antigens (which will identify, among others, proteins that affect the interactions of APC-T cells as well as the effects of direct T cells by measuring proliferation and cytokine production) include , without limitation, those described in: Current Protocols in Immunology, Ed by JE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro Assays for Mouse Lymphocyte Function, Chapter 6, Cytokines and their cellular receptors, Chapter 7, Immunological studies in Humans); Weinberger et al., Proc. Nati Acad. Sci. U.S.A. 77: 6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11: 405-411, 1981; Takai et al., J. Immunol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140: 508-512, 1988.

Immunological Stimulation or Suppression Activity A protein of the present invention may also exhibit immunological stimulation or immune suppression activity, including without limitation the activities for which assays have been described herein. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), for example, in the regulation (up or down) of the growth and proliferation of T lymphocytes and / or B lymphocytes. , as well as carrying out the cytolytic activity of NK cells and other cell populations. These immunological deficiencies can be genetic or viral (eg, HIV) as well as bacterial or fungal infections, or they can result from autoimmune disorders. More specifically, infectious diseases caused by viruses, bacteria, fungi or other infection that can be treated using a protein of the present invention, including HIV infections, hepatitis viruses, herpes viruses, mycobacteria, lesmania spp, malaria spp and various infections by fungi such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where it is generally indicated to strengthen the immune system, for example, in the treatment of cancer. Disorders that can be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, diabetes insulin-dependent mellitus, myasthenia gravis, graft versus host disease and eye inflammation disease by autoimmunity. Said protein of the present invention may also be useful in the treatment of reactions and allergic conditions, such as asthma (particularly allergic asthma) and other respiratory problems. Other conditions, in which immunological suppression is desired (including, for example, organ transplantation) can also be treated using a protein of the present invention. The use of the proteins of the invention may also be possible to regulate immune responses, in a number of ways. The hyporegulation may be in the form of inhibition or blocking of an immune response already in progress, or may involve the prevention of the induction of an immune response. The functions of activated T cells can be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. The immunosuppression of T cell responses is generally an active, not antigen-specific process, which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves the induction of non-response or anergy in T cells, is susceptible to distinction from immunosuppression because it is generally antigen-specific and persists after the exposure to the tolerance-creating agent has ended. Operationally, tolerance can be demonstrated by the lack of a T cell response under re-exposure to a specific antigen in the absence of the creator agent of tolerance. The hyporegulation or prevention of one or more antigenic functions (including without limitation functions of B lymphocyte antigen (such as, for example, B7)), for example, preventing the synthesis of high level lymphokine by activated T cells, will be useful in situations of tissue, skin and organ transplants and in graft versus host disease (GVHD). For example, blocking T cell function should result in reduced destruction of tissue in tissue transplants. Typically, in tissue transplants, rejection of the transplant is initiated through recognition as foreign by the T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule that inhibits or blocks the interaction of a B7 lymphocyte antigen with its natural ligand (s) (s) in immune cells (such as a soluble monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody), before transplantation can lead to the binding of the molecule to the natural ligand (s) in the immune cells without the transmission of the corresponding costimulatory signal . Blocking the antigen function of the B lymphocyte in this case prevents the synthesis of cytokine by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of co-stimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. The induction of long-term tolerance by B lymphocyte antigen blocking reagents can avoid the need for repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens. The effectiveness of a particular blocking reagent in the prevention of a rejection of organ transplantation or GVHD can be evaluated using animal models that are capable of predicting efficacy in humans. Examples of suitable systems that may be used include allogeneic cardiac grafts in rats and islet cell grafts, xenogenic pancreatic grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fibrotic proteins in vivo as described in Lenschow et al. al, Science 257: 789-792 (1992) and Turka et al, Proc. Nati Acad. Sci. USA, 89: 11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the blocking effect of the B lymphocyte antigen function in vivo in the development of The blocking of the antigen function may also be therapeutically useful for the treatment of autoimmune diseases.Many autoimmune disorders are the result of the inappropriate activation of T cells that are reactive against the same tissue and which promotes the production of cytokines. auto-antibodies involved in the pathology of diseases.The prevention of the activation of autoreactive T cells can reduce or eliminate symptoms of the disease.The administration of reagents that block the co-stimulation of T cells breaking the receptor.ligating interactions of the antigens of B lymphocytes, can be used to inhibit the activation of T cells and thus prevent production of auto-antibodies or cytokines that are derived from T cells that may be involved in the unhealthy process. Additionally, blocking reagents can induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief of the disease. The effectiveness of blocking reagents in the prevention or amelioration of autoimmune disorders can be determined by the use of a number of well-characterized animal models of human autoimmune diseases. Examples include experimental murine autoimmune encephalitis, systemic lupus erythematosis in MSUlprllpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856). The upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of upregulating immune responses, may also be useful in therapies. Overregulation of immune responses may be in the form of improving an existing immune response or eliciting an initial immune response. For example, improving an immune response through the stimulation of the B-cell antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold and encephalitis could be alleviated by the systemic administration of stimulant forms of B lymphocyte antigens. Alternatively, anti-viral immune responses can be improved in an infected patient by removing T cells from the patient, costimulating T cells in vitro with APCs pulsed with viral antigen either by expressing a peptide of the present invention or in conjunction with a stimulant form of a soluble peptide of the present invention and reintroducing the T cells activated in vitro within the patient. Another method for improving anti-viral immune responses would be to isolate infected cells from a patient, transfecting them with a nucleic acid encoding a protein of the present invention as described herein, so that the cells express all or a portion of the protein on its surface, and reintroduce the transfected cells into the patient. The infected cells would now be able to deliver a co-stimulatory signal to, and thus activate, the T cells in vivo. In another application, upregulation or amelioration of the antigen function (preferably B lymphocyte antigen function) could be useful in the induction of tumor immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome the specific tolerance to the tumor in the subject. If desired, the tumor cells can be transfected to express a combination of peptides. For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector that directs the expression of a peptide having similar activity to B7-2, alone or in combination with a peptide having activity similar to B7. -1 and / or activity similar to B7-3. The transfected tumor cells are returned to the patient to result in the expression of the peptides on the surface of the transfected cells. Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo. The presence of the peptide of the present invention, which has the activity of a B lymphocyte antigen (s) on the surface of the tumor cells, provides the co-stimulation signal necessary for the T cells to induce a immune response mediated by T cells against transfected tumor cells. In addition, tumor cells lacking MHC class I or MHC class II molecules, or that do not reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a truncated portion of the cytoplasmic domain) of an MHC class I a chain protein and β2 microglobulin protein or an MHC class II a chain protein and a MHC class II β chain protein to express this forms MHC class I or MHC class II proteins on the surface of the cell. Expression of the appropriate MHC class I or classes II, in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a cell-mediated immune response T against the transfected tumor cell. Optionally, a gene encoding an antisense construct that blocks the expression of an associated MHC class II protein, such as the constant chain, can also be cotransfected with a DNA encoding a peptide having the activity of B lymphocyte antigen to promote the presentation of antigens associated with tumor and induce tumor-specific immunity. Thus, the induction of an immune response mediated by T cells in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.

The activity of a protein of the invention can also, among other means, be measured by the following methods: Suitable assays for determining cytotoxicity of splenocytes or thymocytes include, without limitation, those described in Current Protocols in Immunology, Edited by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro Assays for Mouse Lymphocyte Function 3.1.-3.19; Chapter 7, Immunologic Studies in Humans); Herrmann et al., Proc. Nati Acad. Sci. USA 78: 2488-2492, 1981; Herrmann et al., J. Immunol. 128: 1968-1974, 1982; Handa et al., J. Immunol. 135-1564-1572, 1985; Takai et al., J. Immunol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140: 508-512. 1988; Hermann et al., Proc. Nati Acad. Sci. USA 78: 2488-2492, 1981; Herrmann et al., J. Immunol. 128: 1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-1572, 1985; Takai et al., J. Immunol. 137: 3494-3500, 1986; Bowman et al., J. Virology 61: 1992-1998; Takai et al., J. Immunol. 140: 508-512, 1988; Bertagnolli et al., Cellular Immunology 133: 327-341, 1991; Brown et al., J. Immunol. 153: 3079-3092, 1994. Trials to determine T cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T cell-dependent antibody responses and that affect Thl / Th2 profiles. ) include, without limitation, those described in: Maliszewski, J. Immunol. 144: 3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. in Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994. Mixed lymphocyte reaction (MLR) tests (which will identify, among others, proteins that predominantly generate Thl and CTL responses) include, without limitation, those described in Current Protocols in Immunology, Edited by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro Assays for Mouse Lymphocyte Function 3.1.-3.19; Chapter 7, Immunologic Studies in Humans); Takai et al., J. Immunol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140: 508-512, 1988; Bertagnolli et al., J. Immunol. 149: 3778-3783, 1992.

Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate natural T cells) include, without limitation, those described in: Guery et al., J. Immunol. 134: 536-544, 1995; Inaba et al., Journal of Experimental Medicine 173: 549-559, 1991; Macatonia et al., Journal of Immunology 154: 5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182: 255-260, 1995; Nair et al., Journal of Virology 67: 4062-4069, 1993; Huang et al., Science 264: 961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169: 1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94: 797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172: 631-640, 1990. Tests to determine the survival / apoptosis of lymphocytes (which will identify, among others, the proteins that prevent apoptosis after superantigen induction and proteins). regulating lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13: 795-808, 1992; Gorczyca et al., Leukemia 7: 659-670, 1993 Gorczyca et al, Cancer Research 53: 1945-1951, 1993; Itoh et al., Cell 66: 233-243, 1991; Zacharchuk, Journal of Lmmunology 145: 4037-4045, 1990; Zamai et al., Cytometry 14: 891-897, 1993; Gorczyca et al., International Journal of Oncology 1: 639-648, 1992. Tests for proteins that influence early stages of T cell development and confinement include, without limitation, those described in Antica et al., Blood 84; 111 -117, 1994; Fine et al., Cellular Immunology 155: 111-122, 1994; Galy et al., Blood 85: 2770-2778, 1995; Toki et al., Proc. Nat. Acad. Sci. USA 88: 7548-7551, 1991.

Hematopoiesis Regulation Activity A protein of the present invention can be useful in the regulation of hematopoiesis and, consequently, in the treatment of deficiencies of myeloid or lymphoid cells. Even a marginal biological activity in support of colony-forming cells or factor-dependent cell lines indicates involvement in the regulation of hematopoiesis, that is, in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines. , indicating utility in this way, for example, in the treatment of various anemias or to be used in conjunction with irradiation / chemotherapy to stimulate the production of erythroid precursors and / or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes / macrophages (ie, traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelosuppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets, thereby allowing the prevention or treatment of various platelet disorders such as thrombocytopenia, and in general to be used in place or in a complementary manner to platelet transfusions; and / or in sustaining the growth and proliferation of hematopoietic stem cells, which are capable of maturing each and every one of the aforementioned hematopoietic cells and therefore find therapeutic utility in various pluripotent stem disorders (such as those usually treated with transplants, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria) as well as in the repopulation of the division of pluripotent cells that results after irradiation / chemotherapy, either in vivo or ex vivo (that is, in conjunction with the bone marrow transplant or with the transplantation of peripheral progenitor cells (homologous or heterologous)) as normal cells or genetically engineered by gene therapy. The activity of a protein of the invention can, among other means, be measured by the following methods: Appropriate tests to determine the proliferation and differentiation of several hematopoietic lines have been cited above. Tests for the differentiation of embryonic pluripotent cells (which will identify, among others, proteins that influence the hematopoiesis of embryonic differentiation) include, without limitation, those described in Johansson et al. Cellular Biology 15: 141-151, 1995; Keller et al., Molecular and Cellular Biology 13: 473-486, 1993; McClanahan et al., Blood 81: 2903-2915, 1993. Tests to determine the survival and differentiation of pluripotent cells (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, MG In Culture of Hematopoietic Cells, R.I. Freshney, et al. Vol pp 265-268, Wiley-Liss, Inc., New York, NY 1994; Hirayama et al., Proc Nati. Acad. Sci. USA 89: 5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc. New York, NY 1994; Neben et al., Experimental Hematology 22: 353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney et al., Eds. Vol pp. 1-21, Wiley-Liss, Inc. New York, NY 1994; Long term bone cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Alien, T. In Culture of Hematopoietic Cells. R.I. Freshney et al., Eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY 1994; Long term culture initiating cell assay, Sutherland, H.J. In Culture of Hematopietic Cells, R.I. Freshney et al., Eds. Vol pp. 139-162, Wiley-Liss, Inc. New York, NY. 1994 Uses and Utilities in Research The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is expressed preferentially (either constitutively or in a particular stage of tissue differentiation or development or in diseased states); as molecular weight markers on Southern gels; as markers or flags of chromosomes (when marked) to identify chromosomes or for mapping of related gene positions; for comparison with endogenous DNA sequences in patients to identify potential genetic disorders; as probes for hybridization and thus discover new related DNA sequences; as a source of information to derive primary PCR for genetic screening; as a probe for "subtracting" known sequences in the process of discovering new polynucleotides; for the selection and preparation of oligomers for binding to a "genetic chip" or other support, including for the examination of expression patterns; to generate anti-protein antibodies using DNA immunization techniques; and as an antigen to generate anti-DNA antibodies or to stimulate another immune response. Where the polynucleotide encodes a protein that binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, example, that described in Guris et al., Cell 75: 791-803 (1993)) to identify polynucleotides that encode the other protein with which the binding occurs or to identify inhibitors of the binding interaction. The proteins provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening, to produce antibodies or to elicit another immune response; as a reagent (including the labeling reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferably expressed (either constitutively or in a particular state of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which the binding occurs or to identify inhibitors of the union interaction. The proteins involved in these binding interactions can also be used to screen peptides or inhibitors of small molecules or agonists of the binding interaction. Any of these research utilities are capable of being developed into a reactive grade or format of equipment for commercialization as a research product.

The methods for making the uses of the above list are of general knowledge for those skilled in the art. References describing such methods include, without limitation, "Molecular Cloning: A Laboratory Manual", 2pd ed. Cold Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S.L. walk. Kimmel eds., 1987.

Nutritional Uses The polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include, without limitation, use as a supplement of amino acids or proteins, use as a carbon source, the use as a source of nitrogen and the use as a source of carbohydrates. In such cases the protein of the present invention can be added to the feed of a particular organism or it can be administered as a separate solid or liquid preparation, such as in the form of a powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the present invention can be added to the medium in or on which the microorganism is cultured. The U4 proteins of the invention can also be used to immunize animals to obtain polyclonal and monoclonal antibodies that specifically react with the U4 protein and which can inhibit the binding of ligands to the receptor. Such antibodies can be obtained using either the complete U4 protein as an immunogen, or using U4 fragments. Small U4 fragments can also be used to immunize animals. The peptide immunogens may additionally contain a cysteine residue at the carboxyl terminus, and be conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Additional peptide immunogens can be generated by replacing tyrosine residues with tyrosine sulphated residues. Methods for synthesizing such peptides are known in the art, for example, as in R.P. Merrifield, J. Amer. Chem. Soc. 855 2149-2154 (1963); J.L. Krstenansky, et al. FEBS Lett. 211, 10 (1987). Neutralization or non-neutralization antibodies (preferably monoclonal antibodies) that bind to the U4 protein may also be useful therapeutic agents for certain tumors and also in the treatment of conditions described above. These monoclonal neutralizing antibodies may be capable of blocking the binding of ligands to the U4 receptor chain.

EXAMPLE OF U4 PROTEIN DNA encoding the full-length murine U4 protein was fused to a spacer sequence encoding Gly-Ser-Gly by PCR and ligated in frame with sequences coding for the CH2 CH3 hinge regions. Human IgGl in the expression vector pED.Fc of COS-1. The DNA was transfected into COS cells and expression of the fusion protein was detected by ELISA using antibodies that detected the IgGl portion of the protein. This showed that the protein could be expressed and secreted in COS cells. All patent and literature references cited here are incorporated as references as if they had been fully established.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Donaldson, Debra. Collins, Mary Neben, Tamlyn Whitters, Matt ew (ii) TITLE OF THE INVENTION: CHAIN OF CYCLOIN RECEPTORS. (iii) NUMBER OF SEQUENCES: 10 (iv) ADDRESS FOR CORRESPONDENCE: (A) SENDER: GENETICS INSTITUTE, INC. (B) STREET: 87 CAMBRIDGEPARK DRIVE (C) CITY: CAMBRIDGE (D) STATE: Massachusetts (E) COUNTRY: USA (F) POSTAL CODE: 02140 (v) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIA: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay #IO, Version # 1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (viii) EMPLOYEE / AGENT INFORMATION: (A) NAME: Brown, Scott A. (B) REGISTRATION NUMBER: 32,724 (C) REREFENCI NUMBER / RECORD: GI5287 (ix) INFORMATION FOR TELECOMMUNICATIONS: (A) PHONE: (617) 498-8224 (B) TELEFAX: (617) 876-5851 (2) INFORMATION FOR THE SEQ ID NO.:l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 70 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide ( iii) HYPOTHETICAL: Yes (xi) DESCRIPTION OF SEQUENCE: SEQ ID N0: 1: Leu Met Thr Asn Wing Phe lie Ser lie lie Asp Leu Ser Lys Tyr 1 5 10 15 Asp Val Gln Val Arg Ala Wing Val Ser Ser Met Cys Arg Glu Wing Gly 20 25 30 Leu Trp Ser Glu Trp Ser Gln Pro lie Tyr Val Gly Asn Asp Glu His 35 40 45 Lys Pro Leu Arg Glu Trp Phe Val lie Val lie Met Wing Thr lie Cys 50 55 60 Phe lie Leu Leu lie Leu 65 70 (2) INFORMATION FOR SEQ ID NO.:2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) Description: / desc = "probe" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: CTTGGCTTGG AAGAGGAAAT CCTTGAGAGC 30 (2) INFORMATION FOR SEQ ID N0.:3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) Description: / desc = "probe" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: CTGAGCGTGC GCTGGGTGTC GCCAC 25 (2) INFORMATION FOR SEQ ID NO.:4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1656 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTCGACCTTC GCTGTCCGCG CCCAGTGACG CGCGTGAGGA CCCGAGCCCC AATCTGCACC 60 CCGCAGACTC GCCCCCGCCC CATACCGGCG TTGCAGTCAC CGCCCGTTGC GCGCCACCCC 120 CATGCCCGCG GGTCGCCCGG GCCCCGTCGC CCAATCCGCG CGGCGGCCGC CGCGGCCGCT 180 GTCCTCGCTG TGGTCGCCTC TGTTGCTCTG TGTCCTCGGG GTGCCTCGGG GCGGATCGGG 240 AGCCCACACA GCTGTAATCA GCCCCCAGGA CCCCACCTTT CTCATCGGCT CCTCCCTGCA 300 AGCTACCTGC TCTATACATG GAGACACACC TGGGGCCACC GCTGAGGGGC TCTACTGGAC 360 CCTCAATGGT CGCCGCCTGC CCTCTGAGCT GTCCCGCCTC CTTAACACCT CCACCCTGGC 420 CCTGGCCCTG GCTAACCTTA ATGGGTCCAG GCAGCAGTCA GGAGACAATC TGGTGTGTCA 480 CGCCCGAGAT GGCAGCATTC TGGCTGGCTC CTGCCTCTAT GTTGGCTTGC CCCCTGAGAA 540 GCCTTTTAAC ATCAGCTGCT GGTCCCGGAA CATGAAGGAT CTCACGTGCC GCTGGACACC 600 GGGTGCACAC GGGGAGACAT TCTTACATAC CAACTACTCC CTCAAGTACA AGCTGAGGTG 660 GTACGGTCAG GATAACACAT GTGAGGAGTA CCACACTGTG GGCCCTCACT CATGCCATAT 720 CCCCAAGGAC CTGGCCCTCT TCACTCCCTA TGAGATCTGG GTGGAAGCCA CCAATCGCCT 780 AGGCTCAGCA AGATCTGATG TCCTCACACT GGATGTCCTG GACGTGGTGA CCACGGACCC 840 CCCACCCGAC GTGCACGTGA GCCGCGTTGG GGGCCTGGAG GACCAGCTGA GTGTGCGCTG 900 GGTCTCACCA CCAGCTCTCA AGGATTTCCT CTTCCAAGCC AAGTACCAGA TCCGCTACCG 960 CGTGGAGGAC AGCGTGGACT GGAAGGTGGT GGATGACGTC AGCAACCAGA CCTCCTGCCG 1020 TCTCGCGGGC CTGAAGCCCG GCACCGTTTA CTTCGTCCAA GTGCGTTGTA ACCCATTCGG 1080 GATCTATGGG TCGAAAAAGG CGGGAATCTG GAGCGAGTGG AGCCACCCCA CCGCTGCCTC 1140 CACCCCTCGA AGTGAGCGCC CGGGCCCGGG CGGCGGGGTG TGCGAGCCGC GGGGCGGCGA 1200 GCCCAGCTCG GGCCCGGTGC GGCGCGAGCT CAAGCAGTTC CTCGGCTGGC TCAAGAAGCA 1260 CGCATACTGC TCGAACCTTA GTTTCCGCCT GTACGACCAG TGGCGTGCTT GGATGCAGAA 1320 GTCACACAAG ACCCGAAACC AGGACGAGGG GATCCTGCCC TCGGGCAGAC GGGGTGCGGC 1380 GAGAGGTCCT GCCGGCTAAA CTCTAAGGAT AGGCCATCCT CCTGCTGGGT CAGACCTGGA 1440 GGCTCACCTG A ATTGGAGCC CCTCTGTACC ATCTGGGCAA CAAAGAAACC TACCAGAGGC 1500 TGGGGCACAA TGAGCTCCCA CAACCACAGC TTTGGTCCAC ATGATGGTCA CACTTGGATA 1560 TACCCCAGTG TGGGTAGGGT TGGGGTATTG CAGGGCCTCC CAAGAGTCTC TTTAAATAAA 1620 TAAAGGAGTT GTTCAGGTCC CGAAAAAAAA GTCGAC 1656 (2) INFORMATION FOR SEQ ID NO.:5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 425 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: Met Pro Wing Gly Arg Pro Gly Pro Val Wing Gln Ser Wing Arg Arg Pro 1 5 10 15 Pro Arg Pro Leu Ser Ser Leu Trp Ser Pro Leu Leu Leu Cys Val Leu 20 25 30 Gly Val Pro Arg Gly Gly Ser Gly Ala His Thr Ala Val lie Ser Pro 35 40 45 Gln Asp Pro Thr Phe Leu lie Gly Ser Ser Leu Gln Ala Thr Cys Ser 50 55 60 lie His Gly Asp Thr Pro Gly Ala Thr Ala Glu Gly Leu Tyr Trp Thr 65 70 75 80 Leu Asn Gly Arg Arg Leu Pro Ser Glu Leu Ser Arg Leu Leu Asn Thr 85 90 95 Ser Thr Leu Ala Leu Ala Leu Ala Asn Leu Asn Gly Ser Arg Gln Gln 100 105 110 Ser Gly Asp Asn Leu Val Cys His Ala Arg Asp Gly Ser lie Leu Ala 115 120 125 Gly Ser Cys Leu Tyr Val Gly Leu Pro Pro Glu Lys Pro Phe Asn lie 130 135 140 Ser Cys Trp Ser Arg Asn Met Lys Asp Leu Thr Cys Arg Trp Thr Pro 145"150 155 160 Gly Ala His Gly Glu Thr Phe Leu His Thr Asn Tyr Ser Leu Lys Tyr 165 170 175 Lys Leu Arg Trp Tyr Gly Gln Asp Asn Thr Cys Glu Glu Tyr His Thr 180 185 190 Val Gly Pro His Ser Cys His lie Pro Lys Asp Leu Wing Leu Phe Thr 195 200 205 Pro Tyr Glu lie Trp Val Glu Wing Thr Asn Arg Leu Gly Ser Wing Arg 210 215 220 Ser Asp Val Leu Thr Leu Asp Val Leu Asp Val Val Thr Thr Asp Pro 225 230 235 240 Pro Pro Asp Val His Val Ser Arg Val Gly Gly Leu Glu Asp Gln Leu 245 250 255 Ser Val Arg Trp Val Ser Pro Pro Wing Leu Lys Asp Phe Leu Phe Gln 260 265 270 Wing Lys Tyr Gln lie Arg Tyr Arg Val Glu Asp Ser Val Asp Trp Lys 275 280 285 Val Val Asp Asp Val Ser Asn Gln Thr Ser Cys Arg Leu Ala Gly Leu 290 295 300 Lys Pro Gly Thr Val Tyr Phe Val Gln Val Arg Cys Asn Pro Phe Gly 305 310 315 320 lie Tyr Gly Ser Lys Lys Wing Gly lie Trp Ser Glu Trp Ser His Pro 325 330 335 Thr Ala Ala Ser Thr Pro Arg Ser Glu Arg Pro Gly Pro "'Gly Gly Gly 340 345 350 Val Cys Glu Pro Arg Gly Gly Glu Pro Ser Gly Pro Val Arg Arg 355 360 365 Glu Leu Lys Gln Phe Leu Gly Trp Leu Lys Lys His Wing Tyr Cys Ser 370 375 380 Asn Leu Ser Phe Arg Leu Tyr Asp Gln Trp Arg Wing Trp Met Gln Lys 385 390 395 400 Ser His Lys Thr Arg Asn Gln Asp Glu Gly Lie Leu Pro Ser Gly Arg 405 410 415 Arg Gly Ala Ala Arg Gly Pro Ala Gly 420 425 (2) INFORMATION FOR SEQ ID NO .: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1579 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GCGGCCGCCG CCGTTGCTGC CCCTGCTGCT GCTGCTCTGC GTCCTCGGGG CGCCGCGAGC _ 60 CGGATCAGGA GCCCACACAG CTGTGATCAG TCCCCAGGAT CCCACGCTTC TCATCGGCTC 120 CTCCCTGCTG GCCACCTGCT CAGTGCACGG AGACCCACCA GGAGCCACCG CCGAGGGCCT 180 CTACTGGACC CTCAACGGGC GCCGCCTGCC CCCTGAGCTC TCCCGTGTAC TCAACGCCTC 240 CACCTTGGCT CTGGCCCTGG CCAACCTCAA TGGGTCCAGG CAGCGGTCGG GGGACAACCT 300 CGTGTGCCAC GCCCGTGACG GCAGCATCCT GGCTGGCTCC TGCCTCTATG TTGGCCTGCC 360 CCCAGAGAAA CCCGTCAACA TCAGCTGCTG GTCCAAGAAC ATGAAGGACT TGACCTGCCG 420 CTGGACGCCA GGGGCCCACG GGGAGACCTT CCTCCACACC AACTACTCCC TCAAGTACAA 480 GCTTAGGTGG TATGGCCAGG ACAACACATG TGAGGAGTAC CACACAGTGG GGCCCCACTC 540 CTGCCACATC CCCAAGGACC TGGCTCTCTT TACGCCCTAT GAGATCTGGG TGGAGGCCAC 600 CAACCGCCTG GGCTCTGCCC GCTCC GATGT ACTCACGCTG GATATCCTGG ATGTGGTGAC 660 CACGGACCCC CCGCCCGACG TGCACGTGAG CCGCGTCGGG GGCCTGGAGG ACCAGCTGAG 720 CGTGCGCTGG GTGTCGCCAC CCGCCCTCAA GGATTTCCTC TTTCAAGCCA AATACCAGAT 780 CCGCTACCGA GTGGAGGACA GTGTGGACTG GAAGGTGGTG GACGATGTGA GCÁACCAGAC 840 CTCCTGCCGC CTGGCCGGCC TGAAACCCGG CACCGTGTAC TTCGTGCAAG TGCGCTGCAA 900 CCCCTTTGGC ATCTATGGCT CCAAGAAAGC CGGGATCTGG AGTGAGTGGA GCCACCCCAC 960 AGCCGCCTCC ACTCCCCGCA GTGAGCGCCC GGGCCCGGGC GGCGGGGCGT GCGAACCGCG 1020 GGGCGGAGAG CCGAGCTCGG GGCCGGTGCG GCGCGAGCTC AAGCAGTTCC TGGGCTGGCT 1080 CAAGAAGCAC GCGTACTGCT CCAACCTCAG CTTCCGCCTC TACGACCAGT GGCGAGCCTG 1140 GATGCAGAAG TCGCACAAGA CCCGCAACCA GGACGAGGGG ATCCTGCCCT CGGGCAGACG 1200 GGGCACGGCG AGAGGTCCTG CCAGATAAGC TGTAGGGGCT CAGGCCACCC TCCCTGCCAC 1260 GTGGAGACGC AGAGGCCGAA CCCAAACTGG GGCCACCTCT GTACCCTCAC TTCAGGGCAC 1320 CTGAGCCACC CTCAGCAGGA GCTGGGGTGG CCCCTGAGCT CCAACGGCCA TAACAGCTCT 1380 GACTCCCACG TGAGGCCACC TTTGGGTGCA CCCCAGTGGG TGTGTGTGTG TGTGTGAGGG 1440 TTGGTTGAGT TGCCTAGAAC CCCTGCCAGG GCTGGGGGTG AGAAGGGGAG TCATTACTCC 1500 CCATTACCTA GGGCCCCTCC AAAAGAGTCC TTTTAAATAA ATGAGCTATT TAGGTGCTGT 1560 GAAAAAAAAA AAAAAAAAA 1579 (2) INFORMATION FOR SEQ ID NO.:7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 408 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: Arg Pro Pro Pro Leu Leu Pro Leu Leu Leu Leu Leu Cys Val Leu Gly 1 5 10 15 Wing Pro Arg Wing Gly Ser Gly Wing His Thr Wing Val lie Pro Pro Gln 20 25 30 Asp Pro Thr Leu Leu lie Gly Be Ser Leu Leu Wing Thr Cys Ser Val 35 40 45 His Gly Asp Pro Pro Gly Wing Thr Wing Glu Gly Leu Tyr Trp Thr Leu 50 55 60 Asn Gly Arg Arg Leu Pro Pro Glu Leu Ser Arg Val Leu Asn Wing Ser 65 70 75 80 Thr Leu Ala Leu Ala Leu Ala Asn Leu Asn Gly Ser Arg Gln Arg Ser 85 90 95 Gly Asp Asn Leu Val Cys His Wing Arg Asp Gly Ser lie Leu Wing Gly 100 105 110 Ser Cys Leu Tyr Val Gly Leu Pro Pro Glu Lys Pro Val Asn lie Ser 115 120 125 Cys Trp Ser Lys Asn Met Lys Asp Leu Thr Cys Arg Trp Thr Pro Gly 130 135 140 Wing His Gly Glu Thr Phe Leu His Thr Asn Tyr Ser Leu Lys Tyr Lys 145 150 155 160 Leu Arg Trp Tyr Gly Gln Asp Asn Thr Cys Glu Glu Tyr His Thr Val 165 170 175 Gly Pro His Ser Cys His Lie Pro Lys Asp Leu Wing Leu Phe Thr Pro 180 185 190 Tyr Glu lie Trp Val Glu Wing Thr Asn Arg Leu Gly Ser Wing Arg Ser 195 200 205 Asp Val Leu Thr Leu Asp lie Leu Asp Val Val Thr Thr Asp Pro Pro 210 215 220 Pro Asp Val His Val Ser Arg Val Gly Gly Leu Glu Asp Gln Leu Ser 225 230 235 240 Val Arg Trp Val Pro Pro Pro Wing Leu Lys Asp Phe Leu Phe Gln Wing 245 250 255 Lys Tyr Gln lie Arg Tyr Arg Val Glu Asp Ser Val Asp Trp Lys Val 260 265 270 Val Asp Asp Val Ser Asn Gln Thr Ser Cys Arg Leu Wing Gly Leu Lys 275 280 285 Pro Gly Thr Val Tyr Phe Val Gln Val Arg Cys Asn Pro Phe Gly lie 290 295 300 Tyr Gly Ser Lys Lys Wing Gly Lie Trp Ser Glu Trp Ser His Pro Thr 305 310 315 320 Ala Ala Ser Thr Pro Arg Ser Glu Arg Pro Gly Pro Gly Gly Gly Ala 325 330 335 Cys Glu Pro Arg Gly Gly Glu Pro Ser Gly Pro Val Arg Arg Glu 340 345 350 Leu Lys Gln Phe Leu Gly Trp Leu Lys Lys His Wing Tyr Cys Ser Asn 355 360 365 Leu Ser Phe Arg Leu Tyr Asp Gln Trp Arg Wing Trp Met Gln Lys Ser 370 375 380 His Lys Thr Arg Asn Gln Asp Glu Gly Lie Leu Pro Ser Gly Arg Arg 385 390 395 400 Gly Thr Ala Arg Gly Pro Ala Arg 405 (2) INFORMATION FOR SEQ ID NO.:8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: Met Pro Ala Gly Arg Pro Gly Pro Val Ala Asn Ser Ala Arg 1 5 10 (2) INFORMATION FOR SEQ ID NO .: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1787 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GAATTCCGGC GCGTCCGCGC CCAGCGACGT GCGGGCGGCC TGGCCCGCGC CCTCCCGCGC 60 CCGGCCTGCG TCCCGCGCCC TGCGCCACCG CCGCCGAGCC GCAGCCCGCC GCGCGCCCCC 120 GGCAGCGCCG GCCCCATGCC CGCCGGCCGC CGGGGCCCCG CCGCCCAATC CGCGCGGCGG 180 CCGCCGCCGT TGCTGCCCCT GCTGCTGCTG CTCTGCGTCC TCGGGGCGCC GCGAGCCGGA 240 TCAGGAGCCC ACACAGCTGT GATCAGTCCC CAGGATCCCA CGCTTCTCAT CGGCTCCTCC 300 CTGCTGGCCA CCTGCTCAGT GCACGGAGAC CCACCAGGAG CCACCGCCGA GGGCCTCTAC 360 TGGACCCTCA ACGGGCGCCG CCTGCCCCCT GAGCTCTCCC GTGTACTCAA CGCCTCCACC 420 TTGGCTCTGG CCCTGGCCAA CCTCAATGGG TCCAGGCAGC GGTCGGGGGA CAACCTCGTG 480 TGCCACGCCC GTGACGGCAG CATCCTGGCT GGCTCCTGCC TCTATGTTGG CCTGCCCCCA 540 GAGAAACCCG TCAACATCAG CTGCTGGTCC AAGAACATGA AGGACTTGAC CTGCCGCTGG 600 ACGCCAGGGG CCCACGGGGA GACCTTC CTC CACACCAACT ACTCCCTCAA GTACAAGCTT 660 AGGTGGTATG GCCAGGACAA CACATGTGAG GAGTACCACA CAGTGGGGCC CCACTCCTGC 720 CACATCCCCA AGGACCTGGC TCTCTTTACG CCCTATGAGA TCTGGGTGGA GGCCACCAAC 780 CGCCTGGGCT CTGCCCGCTC CGATGTACTC ACGCTGGATA TCCTGGATGT GGTGACCACG 840 GACCCCCCGC CCGACGTGCA CGTGAGCCGC GTCGGGGGCC TGGAGGACCA GCTGAGCGTG 900 CGCTGGGTGT CGCCACCCGC CCTCAAGGAT TTCCTCTTTC AAGCCAAATA CCAGATCCGC 960 TACCGAGTGG AGGACAGTGT GGACTGGAAG GTGGTGGACG ATGTGAGCAA CCAGACCTCC 1020 TGCCGCCTGG CCGGCCTGAA ACCCGGCACC GTGTACTTCG TGCAAGTGCG CTGCAACCCC 1080 TTTGGCATCT ATGGCTCCAA GAAAGCCGGG ATCTGGAGTG AGTGGAGCCA CCCCACAGCC 1140 GCCTCCACTC CCCGCAGTGA GCGCCCGGGC CCGGGCGGCG GGGCGTGCGA ACCGCGGGGC 1200 GGAGAGCCGA GCTCGGGGCC GGTGCGGCGC GAGCTCAAGC AGTTCCTGGG CTGGCTCAAG 1260 AAGCACGCGT ACTGCTCCAA CCTCAGCTTC CGCCTCTACG ACCAGTGGCG AGCCTGGATG 1320 CAGAAGTCGC ACAAGACCCG CAACCAGGAC GAGGGGATCC TGCCCTCGGG CAGACGGGGC 1380 ACGGCGAGAG GTCCTGCCAG ATAAGCTGTA GGGGCTCAGG CCACCCTCCC TGCCACGTGG 1440 AGACGCAGAG GCCGAACCCA AACTGGGGCC ACCTCTGT AC CCTCACTTCA GGGCACCTGA 1500 GCCACCCTCA GCAGGAGCTG GGGTGGCCCC TGAGCTCCAA CGGCCATAAC AGCTCTGACT 1560 CCCACGTGAG GCCACCTTTG GGTGCACCCC AGTGGGTGTG TGTGTGTGTG TGAGGGTTGG 1620 TTGAGTTGCC TAGAACCCCT GCCAGGGCTG GGGGTGAGAA GGGGAGTCAT TACTCCCCAT 1680 TACCTAGGGC CCCTCCAAAA GAGTCCTTTT AAATAAATGA GCTATTTAGG TGCAAAAAAA 1740 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAACC 1787 (2) INFORMATION FOR SEQ ID NO.:10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 422 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10: Met Pro Wing Gly Arg Arg Gly Pro Wing Wing Gln Ser Wing Arg Arg Pro 1 5 10 15 Pro Pro Leu Leu Pro Leu Leu Leu Leu Leu Cys Val Leu Gly Ala Pro 20 25 30 Arg Wing Gly Ser Gly Wing His Thr Wing Val lie Pro Pro Gln Asp Pro 35 40 45 Thr Leu lie Gly Be Ser Leu Leu Wing Thr Cys Ser Val His Gly 50 55 60 Asp Pro Pro Gly Ala Thr Ala Glu Gly Leu Tyr Trp Thr Leu Asn Gly 65 70 75 80 Arg Arg Leu Pro Pro Glu Leu Ser Arg Val Leu Asn Wing Ser Thr Leu 85 90 95 Wing Leu Wing Leu Wing Asn Leu Asn Gly Ser Arg Gln Arg Ser Gly Asp 100 105 110 Asn Leu Val Cys His Wing Arg Asp Gly Ser lie Leu Wing Gly be Cys 115 120 125 Leu Tyr Val Gly Leu Pro Pro Glu Lys Pro Val Asn lie Ser Cys Trp 130 135 140 Ser Lys Asn Met Lys Asp Leu Thr Cys Arg Trp Thr Pro Gly Ala His 145 150 155 160 Gly Glu Thr Phe Leu His Thr Asn Tyr Ser Leu Lys Tyr Lys Leu Arg 165 170 175 Trp Tyr Gly Gln Asp Asn Thr Cys Glu Glu Tyr His Thr Val Gly Pro 180 185 190 His Ser Cys His lie Pro Lys Asp Leu Ala Leu Phe Thr Pro Tyr Glu 195 2 205 lie Trp Val Glu Wing Thr Asn Arg Leu Gly Ser Wing Arg Ser Asp Val 210 215 220 Leu Thr Leu Asp lie Leu Asp Val Val Thr Thr Asp Pro Pro Pro Asp 225 230 235 240 Val His Val Ser Arg Val Gly Gly Leu Glu Asp Gln Leu Ser Val Arg 245 250 255 Trp Val Pro Pro Pro Wing Leu Lys Asp Phe Leu Phe Gln Wing Lys Tyr 260 265 270 Gln lie Arg Tyr Arg Val Glu Asp Ser Val Asp Trp Lys Val Val Asp 275 280 285 Asp Val Ser Asn Gln Thr Ser Cys Arg Leu Ala Gly Leu Lys Pro Gly 290 295 300 Thr Val Tyr Phe Val Gln Val Arg Cys Asn Pro Phe Gly lie Tyr Gly 305 310 315 320 Ser Lys Lys Wing Gly lie Trp Ser Glu Trp Ser His Pro Thr Ala Ala 325 330 335 Be Thr Pro Arg Ser Glu Arg Pro Gly Pro Gly Gly Gly Wing Cys Glu 340 345 350 Pro Arg Gly Gly Glu Pro Ser Gly Pro Val Arg Arg Glu Leu Lys 355 360 365 Gln Phe Leu Gly Trp Leu Lys His Ala Tyr Cys Ser Asn Leu Ser 370 375 380 Phe Arg Leu Tyr Asp Gln Trp Arg Wing Trp Met Gln Lys Ser His Lys 385 390 395 400 Thr Arg Asn Gln Asp Glu Gly lie Leu Pro Gly Arg Arg Gly Thr 405 410 415 Wing Arg Gly Pro Wing Arg 420

Claims (19)

1. Novelty of the Invention 1. An isolated polynucleotide comprising a nucleotide sequence that is selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 4, from nucleotide 242 to nucleotide 1396; (b) the nucleotide sequence of SEQ ID NO: 6, from nucleotide 71 to nucleotide 1225; (c) the nucleotide sequence of SEQ ID NO: 9, from nucleotide 136 to nucleotide 1401; (d) a nucleotide sequence that varies from the sequence of the nucleotide sequence specified in (a), (b) or (c) as a result of the degeneracy of the genetic code; (e) a nucleotide sequence capable of hybridizing under conditions astringent to the nucleotide specified in (a) or (b); (f) a nucleotide sequence encoding a homologous species of the sequence specified in (a), (b) or (c); and (g) an allelic variant of the nucleotide sequence specified in (a), (b) or (c).
2. 2. The polynucleotide of claim 1, wherein said nucleotide sequence codes for a protein having a biological activity of the hemopoietin U4 receptor superfamily chain.
3. 3. The polynucleotide of claim 1, wherein said polynucleotide sequence is operably linked to an expression control sequence.
4. The polynucleotide of claim 1, comprising the nucleotide sequence of SEQ ID NO: 4 from nucleotide 122 to nucleotide 1396.
5. 5. The polynucleotide of claim 1, comprising the nucleotide sequence of SEQ ID NO: 6 from nucleotide 11 to nucleotide 1225.
6. 6. A host cell transformed with the polynucleotide of claim 3.
7. 7. The host cell of claim 6, wherein said cell is a mammalian cell.
8. 8. A process for producing a U4 protein, said process comprising: (a) growing a culture of the host cell of claim 6 in a suitable culture medium; and (b) purifying the U4 protein from the culture medium.
9. 9. An isolated U4 protein comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO: 5; (b) the amino acid sequence of SEQ ID NO: 5, from amino acid 41 to amino acid 425; (c) the amino acid sequence of SEQ ID NO: 7; (d) the amino acid sequence of SEQ ID NO: 7, from amino acid 24 to amino acid 408; (e) the amino acid sequence of SEQ ID NO: 10; (f) the amino acid sequence of SEQ ID NO: 10, from amino acid 38 to amino acid 421; and (g) fragments of (a) - (f) having a biological activity of the hemopoietin U4 receptor superfamily chain.
10. The protein of claim 9, comprising the amino acid sequence of SEQ ID NO: 5.
11. The protein of claim 9, comprising the amino acid sequence of SEQ ID NO: 5 from amino acid 41 to the amino acid 425.
12. The protein of claim 9, comprising the amino acid sequence of SEQ ID NO: 7.
13. 13. The protein of claim 9, comprising the amino acid sequence of SEQ ID NO: 7 from amino acid 24 through amino acid 408.
14. 14. A pharmaceutical composition comprising a protein of claim 9 and a pharmaceutically acceptable carrier.
15. 15. A protein produced according to the process of claim 8.
16. 16. A composition comprising an anti-cue that specifically reacts with a protein of claim 9.
17. 17. An isolated polynucleotide comprising a nucleotide sequence encoding a peptide or protein comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO: 5; (b) the amino acid sequence of SEQ ID NO: 5, from amino acid 41 to amino acid 425; (c) the amino acid sequence of SEQ ID NO: 7; (d) the amino acid sequence of SEQ ID NO: 7, from amino acid 24 to amino acid 408; and (e) fragments of (a) - (d) having a biological activity of the hemopoietin U4 receptor superfamily chain.
18. 18. The protein of claim 9, wherein said amino acid sequence is part of a fission protein.
19. 19. The protein of claim 1, comprising an Fc fragment. Extract of the Description Polynucleotides encoding the U4 hematopoietin receptor superfamily chain and fragments thereof are described. U4 proteins and methods for their production are also described.