JP2004201693A - Receptor of denervated muscle kinase (dmk) and tyrosine kinase superfamily - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel tyrosine kinase called denervated muscle kinase "Dmk". <P>SOLUTION: There are provided a gene, designated as dmk, that encodes a novel tyrosine kinase receptor expressed in high levels in denervated muscle; also assay systems that may be used to detect and/or measure ligands that bind the dmk gene product: and further diagnostic and therapeutic methods based on the interaction between Dmk and agents that initiate signal transduction through binding to Dmk. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

(1. Introduction)
The present invention provides novel orphan receptor molecules and their uses, as well as assay systems useful for identifying novel ligands that interact with these receptors.

(2. Background of the Invention)
The ability of a polypeptide ligand to bind to cells and thereby elicit a phenotypic response such as cell growth, survival, or differentiation within these cells is often mediated by transmembrane tyrosine kinases. The extracellular portion of each receptor tyrosine kinase (RTK) is generally the most distinctive portion of the molecule because it provides a protein with ligand-identifying properties. Binding of the ligand to the extracellular domain causes signal transduction via the intracellular tyrosine kinase catalytic domain, which transmits a biological signal to a target protein in the cell.

  This particular arrangement of sequence motifs in the cytoplasmic catalytic domain determines access to potential kinase substrates (Mohammadi et al., 1990, Mol. Cell. Biol., 11: 5068-5078; Fantl et al., 1992, Cell, 69: 413-413).

  All known growth factor RTKs are believed to dimerize following ligand binding (Schlessinger, J., 1988, Trend Biochem. Sci. 13: 443-447; Ullrich and Schlessinger, 1990, Cell, 61: 203-212; Schlessinger and Ullrich, 1992, Neuron 9: 383-391). : Kinase function is activated by molecular interaction between cytoplasmic domains that dimerize. In some cases, such as platelet-derived growth factor (PDGF), the ligand is a dimer that binds two receptor molecules (Hart et al., 1988, Science, 240: 1529-1531; Heldin, 1989, J. Biol. Chem. 264: 8905-8912), but in the case of EGF, for example, the ligand is a monomer (Weber et al., 1984, J, Biol. Chem., 259: 14631-14636).

  The tissue distribution of a particular tyrosine kinase receptor within a higher organism provides relevant data regarding the biological function of the receptor. Tyrosine kinase receptors for growth and differentiation factors, such as fibroblast growth factor (FGF), are widely expressed and are therefore believed to play a general role in tissue growth and maintenance. Members of the Trk RTK family of receptors (Glass and Yancopoulos, 1993, Trends in Cell Biol, in press) are more generally restricted to cells of the nervous system, and the neurotrophic factors that bind to these receptors are expressed in the brain. And promotes differentiation of diverse peripheral neuronal groups (Lindsey, RM, 1993, Neurotrophic Factors, SE, Lowhlin and JH, Fallom, eds., Pages 257-284 (San Diego, CA: Academic Press). )). The localization of one such Trk family receptor, trkB, in tissue has led to some of the potential biological roles of this receptor as well as the ligands (referred to herein as cognate) that bind to this receptor. Insight was provided. Thus, for example, in adult mice, significant levels of trkB mRNA were also observed in lung, muscle, and ovary, but trkB was found to be preferentially expressed in brain tissue. In addition, trkB transcripts were detected in mid- and late-gestation fetuses. By in situ hybridization analysis of 14 and 18 day old mouse fetuses, the trkB transcript was found to be in the central nervous system, including the brain, spinal cord, spinal and cranial ganglia, the spinal trunk of the sympathetic nervous system, and various nerve-sensitive pathways. And localized in the peripheral nervous system. This suggests that the trkB gene product is a receptor involved in neurogenesis and early neural development, and may play a role in the adult nervous system.

  The cellular environment in which the RTK is expressed can affect the biological response exhibited in binding the ligand to the receptor. Thus, for example, if a neuron cell that expresses a Trk receptor is exposed to a neurotrophic factor that binds to that receptor, the neuron will survive and differentiate. If the same receptor is expressed by fibroblasts, exposure to neurotrophic factors results in fibroblast proliferation (Glass et al., 1991, Cell 66: 405-413). Thus, the extracellular domain provides a determinant for ligand specificity, and once signaling is initiated, the cellular environment will determine the consequences of that signaling phenotype.

  Many RTK families have been identified based on the sequence homology of these intracellular domains. For example, two members of the TIE (tyrosine kinase with immunoglobulin and EGF homology domains) family, known as TIE-1 and TIE-2, have 79% sequence homology to the intracellular region (Maisompierre et al., 1993; Oncogene 8: 1631-1637). These receptors share similar motifs with the extracellular domain, but are only 32% identical in sequence. This indicates a potentially different biological role, with both genes being widely expressed in endothelial cells of fetal and offspring tissues, while significant levels of tie-2 transcripts are also present. As well as in other fetal cell populations including the lens epithelium, epicardium and mesenchymal areas.

  Receptors and signaling pathways utilized by NGF include the product of the trk proto-oncogene (Kaplan et al., 1991, Nature 350: 156-160; Klein et al., 1991, Cell 65: 189-197). (1989, EMBO J. 8: 3701-3709) describe a simple form of trkB, which encodes a second member of the thiosine protein kinase family of receptors known to be highly associated with the human trk proto-oncogene. Reported separation. TrkB binds to BDNF, NT-4 and, to a lesser extent, NT-3 and mediates a functional response thereto (Squinto et al., 1991, Cell 65: 885-903: Ip et al., 1992, Proc. Natl.Acad.Sci.USA 89: 3060-3064; Klein et al., 1992, Neuron, 8: 947-956). At the amino acid level, the products of trk and trkB were found to share 57% homology in the extracellular region containing nine of the eleven cysteines present in trk. This homology was found to increase to 88% within the tyrosine kinase catalytic domain. The Trk gene family has now expanded to include the trkC locus as well as NT-3, which has been identified as a preferred ligand for trkC (Lambale et al., 1991, Cell 66: 967-979).

  Two novel human genes, lor1 and rr2, encode proteins with regions homologous to the Trk family tyrosine kinase receptor domain in the cytoplasmic portion, but these proteins differ significantly from the Trk family in the extracellular portion ( Masakowski and Carroll, 1992, J. Biol Chem. 267: 26181-26190).

  Another receptor with a kinase domain associated with the Trk family has been identified in the Torpedo califomica, which may play a role in motor neuron-induced synapses on muscle fibers. Jennings et al., Proc. Natl. Acad. Sci. ScL USA 90: 2895-2899 (1993). This kinase was isolated from electrical organs, a tissue homologous to muscle. Like rr, the tyrosine kinase domain of this protein is related to the Trk family, but the extracellular domain is somewhat different from Trk. This protein was found to be expressed at high levels in Torpedo's skeletal muscle and at very low levels in adult Torpedo's brain, spine, heart, liver and testis.

  Because RTKs are thought to mediate many important functions during development, the identification and isolation of new RTKs can be used as a means to identify new ligands that may play important roles in development. Such new RTKs are often used to search for additional members of the known family of tyrosine kinase receptors, for example, using PCR-based screening that includes regions of known homology between members of the Trk family. (See, eg, Maisonpierre et al., 1993, Oncogene 8: 1631-1637). By isolating such a so-called "orphan" tyrosine kinase receptor, in the absence of an associated known ligand, and then determining the tissue in which such receptor is expressed, the growth, proliferation of cells in the target tissue , And insights into the regulation of regeneration. Further, such receptors can be used to isolate cognate ligands, which in turn can be used to regulate the survival, growth, and regeneration of cells that express the receptor.

It is an object of the present invention to provide a novel tyrosine kinase called denervated muscle kinase "Dmk".

The present invention provides the following sections:
Item 1. (A) the nucleic acid sequence shown in FIG. 1 or (b) a part of the sequence, which encodes the extracellular domain, transmembrane portion or intracellular domain of the amino acid sequence shown in FIG. 1 or (c) A) a substantially purified recombinant nucleic acid molecule that is part of the sequence and comprises a sequence encoding the extracellular domain, transmembrane portion and intracellular domain of the amino acid sequence shown in FIG. 1;
Item 2. 1) comprising the nucleotides 192-1610 of FIG. 1; b) the nucleotides 1611-1697 of FIG. 1; c) the nucleotides 1698-2738 of FIG. 1; The described nucleic acid;
Item 3. a) contains the amino acid sequence shown in FIG. 1; or b) contains a part of the protein of (a) containing an extracellular domain, a transmembrane portion, or an intracellular domain; or;) extracellularly Contains a portion of the protein of (a), which contains the transmembrane and intracellular domains; d) is functionally equivalent to the protein of a), b), or c); or e) a) A substantially purified protein having substantial similarity to the protein of, b) or c);
Item 4. Item 4. The protein according to Item 3, comprising the amino acids of 20 to 492 of FIG. 1;
Item 5. An expression vector comprising an expression control sequence operably linked to a nucleic acid molecule encoding the protein according to item 3 or 4;
Item 6. Item 6. An expression vector according to Item 5, comprising an expression control sequence operatively linked to the nucleic acid molecule according to Item 1 or 2.
Item 7. Item 7. The vector according to Item 5 or 6, which contains an immediate gene promoter. Item 8. The vector according to Item 7, wherein the immediate gene promoter is a fos promoter or a jun promoter;
Item 9. A cell carrying the expression vector defined in any of items 5 to 8;
Item 10. A microorganism having the expression vector according to any one of Items 5 to 8;
Item 11. Item 10. The cell according to Item 9, which is a cell derived from the PC12 cell line or the NIH 3T3 cell line, which carries the vector defined in Item 7 or 8.
Item 12. Item 12. The cell according to Item 11, further comprising a gene encoding a detectable marker that can be expressed under the control of an immediate gene promoter;
Item 13. A fibroblast cell line that is growth factor-dependent in a serum-free medium and contains the expression vector according to any of items 6 to 8;
Item 14. 7. A nucleotide sequence that hybridizes to the nucleic acid molecule of paragraph 1, 5 or 6, wherein said sequence encodes a tyrosine kinase that is expressed at higher levels in denervated muscle than in normal muscle;
Item 15. 5. A process for identifying a ligand that binds to the protein according to item 3 or 4, wherein a) a growth factor-dependent cell that does not survive in a serum-free medium is replaced with a nucleic acid defined in item 1 or any of items 5 to 8. B) culturing such transformed cells in a serum-free medium in the presence of a potential Dmk-binding ligand; and c) transforming said transformant in a serum-free medium. Identifying a ligand that supports the survival of the transformed cell;
Item 16. Item 16. A ligand identified by the method according to Item 15;
Item 17. Item 3 or 4 in a patient sample, where a difference in the level of expression of the Dmk protein in the patient as compared to a healthy person indicates that the patient's disorder may be primarily or secondaryly associated with Dmk metabolism. Comparing the expression level of the Dmk protein defined in (1) with the expression level of the Dmk protein defined in item (3) or (4) in a comparative sample from a healthy subject. Ex vivo diagnostic methods for:
Item 18. 18. The method of claim 17, wherein the disorder is idiopathic ataxia;
Item 19. A protein identified in item 3 or 4, or a peptide fragment or derivative of said protein, for use in a method of treating the body of a human or animal suffering from a muscular disorder or other disorder;
Item 20. A method for cloning at least a portion of a tyrosine kinase receptor gene, comprising: (i) using a collection of cDNA molecules as a template, and a) a degenerate primer based on SEQ ID NO: 3; and b) SEQ ID NO: 5, SEQ ID NO: 7, a combination of oligonucleotide primers encompassing a degenerate primer based on a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13, or the following sequences: c) SEQ ID NO: 4; Nucleic acid encoding tyrosine kinase by polymerase chain reaction using a combination of oligonucleotides comprising primers having a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14 Amplifying the sequence; and; The method comprises the step of cloning the amplified nucleic acid terpolymer, a;
Item 21. 21. A substantially purified nucleic acid cloned by the method of item 20;
Item 22. Item 22. A cell carrying the nucleic acid molecule of Item 21;
Item 23. Item 22. A microorganism having the nucleic acid molecule of Item 21;
Item 24. A microorganism having ATCC Deposit No. 75498.

  The present invention provides a gene called dmk. This gene encodes a novel tyrosine kinase receptor that is expressed at high levels in denervated muscle. The invention also provides an assay system that can be used to detect and / or measure a ligand that binds to a dmk gene product. The present invention also provides diagnostic and therapeutic methods based on the interaction between Dmk and an agent that initiates signal transduction through binding to Dmk.

(3. Summary of the Invention)
The present invention provides a novel tyrosine kinase called denervated muscle kinase "Dmk". It is expressed in normal and denervated muscle and other tissues, including the heart, spleen and retina. This protein is thought to be related to the Trk family of tyrosine kinases.

  The invention further provides an isolated nucleic acid molecule encoding Dmk.

  The invention also provides proteins or peptides comprising the extracellular domain of Dmk, and nucleic acids encoding such extracellular domains.

  The invention further provides a vector comprising an isolated nucleic acid molecule encoding Dmk or its extracellular domain, which can be used to express Dmk in bacteria, yeast, and mammalian cells.

  The present invention further provides the use of a Dmk receptor or an extracellular or intracellular domain thereof in screening for a drug that interacts with Dmk. The novel drugs that bind to the receptors described herein can be used to treat cells that have been engineered to express the receptor, as well as mediate survival and differentiation in cells that naturally express the receptor. Provide survival and growth where possible.

  In certain embodiments, the extracellular domain of Dmk (soluble receptor) is utilized for screening for cognate ligands.

  The present invention also provides a nucleic acid probe capable of hybridizing with a sequence contained within a nucleic acid sequence encoding human Dmk, which is useful for detecting Dmk expressing human and animal tissues.

  The present invention further provides antibodies for Dmk.

  The invention also has diagnostic and therapeutic utility. In certain embodiments of the invention, the methods for detecting abnormal forms of receptor function or expression described herein can be used in the diagnosis of muscle disorders or other disorders. In other embodiments, manipulation of the receptor or agonist that binds to the receptor includes Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease) and idiopathic torsion dystonia. , Neurological diseases, diseases of muscle or neuromuscular unit disorders. In a further embodiment, the extracellular domain of the receptor is utilized as a blocking agent that blocks binding of the receptor to target cells.

  In a further embodiment of the invention, administering to a patient suffering from an excess of Dmk an effective amount of an antisense RNA or antisense oligodeoxyribonucleotide corresponding to the dmk gene coding region, thereby reducing the expression of excess Dmk. Can be treated.

(5. Detailed Description of the Invention)
The present invention provides novel tyrosine kinase molecules related to the trk family of tyrosine kinases. The sequence of this protein is shown in FIG. 1 as SEQ ID NO: 1. The coding region for this mature protein is believed to begin at or around serine-glycine-threonine at or around site 20 of the coding region.

  The novel tyrosine kinases described herein have been found to be induced in denervated skeletal muscle. Therefore, it is called Dmk (Denervated Muscle Kinase). In addition to being found in both normal and denervated muscle, and especially in the heart, Dmk also has been found to be substantially present in the spleen, ovary, and retina, but does not appear to be limited to these . It appears to be early in development, but is also found in adult tissues.

  Dmk may be related to the Torpedo RTK identified by Jennings et al., Supra. However, although Dmk is thought to be induced in denervated muscle, it differs in that no such induction has been reported for Torpedo RTKs. In addition, Torpedo RTK has an extracellular Tallingle domain, whereas Dmk does not. However, these kinases can be members of the same or related families.

  The gene encoding Dmk has been cloned and its DNA sequence has been determined (FIG. 1; SEQ ID NO: 2). The extracellular domain of this mature protein is believed to be encoded by a nucleic acid sequence beginning at or around site 192 and ending at or around site 1610. The transmembrane portion of this protein is believed to be encoded by a nucleic acid sequence beginning at or around site 1611 and ending at or around site 1697. The intracellular domain will be encoded by a nucleic acid sequence beginning at or around site 1698 and ending at or around site 2738. The cDNA clone encoding Dmk was deposited on July 13, 1993 with the American Type Culture Collection and given accession number ATCC 75498.

  The invention also provides proteins or peptides comprising the extracellular domain of Dmk and nucleic acids encoding this extracellular domain. The extracellular domain of the protein is believed to consist of amino acids at or around sites 20-492 of the coding region set forth as SEQ ID NO: 1.

  Mouse Dmk can be used to identify comparable proteins in humans using the materials and methods described herein for searching human libraries, preferably from muscle. Furthermore, the similarity between Dmk and TorpedoRTK suggests the utility of regions of sequence homology within these genes to develop useful primers to search for additional related RTKs.

  Accordingly, the present invention provides nucleic acids or oligonucleotides longer than about 10 bases in length that hybridize to DNA encoding the Dmk described herein and maintain stable binding thereto under stringent conditions. provide. However, such hybridizing nucleic acids are novel and non-obvious compared to any conventional nucleic acids, including nucleic acids encoding other tyrosine kinases or nucleic acids complementary thereto.

Stringent conditions as used herein include (1) using low ionic strength and high temperature for washing, for example, 0.15 M NaCl / 0.015 M sodium citrate / 0.1% NaDodSO ₄ At 50 ° C., or (2) 0.1% bovine pH 6.5 containing 750 mM NaCl, 75 mM sodium citrate at 42 ° C. with denaturing agents such as formamide during hybridization. Using 50% (vol / vol) formamide with serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer.

  When using a nucleotide sequence encoding for some or all of the Dmk of the present invention to isolate Dmk from novel family members or other species, the length of the sequence will be at least as long as the vertebrate's own dmk. It should be large enough to hybridize to the endogenous mRNA from the source. Typically, a sufficient sequence size can be about 15 contiguous bases (DNA or RNA).

  Strategies for identifying new RTKs using denatured oligodeoxyribonucleotide primers corresponding to protein regions surrounding amino acids conserved in tyrosine kinases have been described previously (Wilks et al., 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 1603-1607; Partanen, J. et al., 1990, Proc.Natl.Acad.Sci.U.S.A. 87: 8913-8917; Lai and Lemke, 1991; Neuron 6: 691-704; Masakowski and Carroll, 1992, J. Biol. Chem. 267: 26181-26190). The discovery by Applicants of a relationship between Dmk and TorpedoRTK has identified unknown regions of homology that can be used in screening techniques.

The following primers, based on the amino acid homology domain Asp-Val-Trp-Ala-Tyr-Gly (SEQ ID NO: 3) between Dmk and Torpedo's RTK, were added to another RT corresponding to a known homology region characteristic of RTKs. Can be used in combination with a primer to isolate related tyrosine kinases (eg, members of other families) [All codes used herein, representing amino acids and nucleotides, are 37C. F. R. §1.822 (b)]:
5 'GAATTCGAGCTCCCRWANGCCCANACRTC-3' (SEQ ID NO: 4)
Alternative primers corresponding to known regions of homology characteristic of RTKs include:
5 '
1) Asp-Leu-Ala-Thr-Arg-Asn (SEQ ID NO: 5)
5′-TCTTGACTCGAGAYYTNGCCNMGMGNAA-3 ′ (SEQ ID NO: 6)
2) Asp-Leu-Ala-Ala-Arg-Asn (SEQ ID NO: 7)
5-'TCTGTCACTGAGAYYTNGCNGCNMGNAA-3 '(SEQ ID NO: 8)
3 '
1) Asp-Val-Trp-Ser-Leu-Gly (SEQ ID NO: 9)
3′-CTRCANACCWSNATRCCCTCGAGCTTAAG-5 ′ (SEQ ID NO: 10)
2) Asp-Val-Trp-Ser-Phe-Gly (SEQ ID NO: 11)
3'-CTRCANACCWSNAARCCCTCGAGCTTAAG-5 '(SEQ ID NO: 12)
3) Asp-Val-Trp-Ser-Tyr-Gly (SEQ ID NO: 13)
3'-CTRCANACCWSNRRANCCTCCGAGCTTAAG-5 '(SEQ ID NO: 14)
Alternatively, regions of homology shared by members of the related family, such as the Dmk and Trk families, can be used in strategies designed to isolate new RTKs.

  The present invention further provides a substantially purified protein molecule or a functionally equivalent molecule substantially comprising the amino acid sequence as shown in FIG. 1 for Dmk (SEQ ID NO: 1). Functionally equivalent molecules include those in which a residue in the sequence has been replaced with an amino acid residue at which a silent change occurs. For example, one or more amino acid residues in the sequence may be replaced by another amino acid of similar polarity that acts as a functional equivalent, resulting in a silent change. Substituents for an amino acid in the sequence may be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

  Also included in the scope of the invention are proteins or their fragments or derivatives that are variously modified during or after translation, for example, by glycosylation, proteolytic cleavage, binding to antibody molecules or other cellular ligands, and the like.

  The present invention further contemplates isolation of proteins having substantial similarity to the Dmk proteins described herein. Substantial similarity, as used herein, refers to a protein that is Dmk from a different species, or that is a member of a family within a species that is at least 40% identical in position. Substantial similarities at the protein level include the ability of a subject protein to compete with Dmk for binding to several monoclonal antibodies raised against an epitope of Dmk.

  The Dmk proteins described herein are useful in 1) screening strategies, 2) purification strategies, and 3) diagnostic applications. With respect to screening strategies, expression cloning strategies based on cell viability and proliferation assays provide a way to screen for cognate ligands (Glass et al., (1991) Cell 66: 405-413). Other strategies for identifying such receptors may be more appropriate because ligands that bind Dmk may bind to membranes (Armitage et al., 1992, Nature 357: 80-82; Smith et al., 1993, Cell 73: 1349-1360). In a preferred embodiment, the extracellular domain of Dmk is fused to a marker to create a chimeric protein that allows for the identification and purification of the extracellular domain when bound to a homolog.

  For example, as described in Smith et al., Supra, where the cognate ligand binds to the membrane, the extracellular portion of Dmk can be fused to the heavy chain (Fc) of a truncated immunoglobulin. This fusion product can then be used to identify cells that express a surface ligand that binds to the receptor, for example, by flow cytometry. Alternatively, other tags, such as myc, used to label the extracellular domain of Dmk are also useful for screening and purification of Dmk binding ligands (Davis et al., 1991, Science 253: 59-63; Squinto et al., 1990, Neuron 5 ,: 757-766).

  In another embodiment, the extracellular portion of the RTK that binds to a known ligand is replaced with the extracellular portion of Dmk. A measurable effect, such as a phenotypic change or induction of an early response gene, usually associated with binding of a known ligand to the receptor, can be used to screen for cognate ligands that induce a similar effect.

  For example, a cell line carrying an introduced Dmk receptor or a chimeric protein comprising the extracellular domain of Dmk and another intracellular domain of an RTK fused to a transmembrane domain (Dmk-chimeric receptor), and a receptor. The non-parental cell line can be exposed to any possible source of an agent that can operate through the receptor; with any specific effect on the cell line carrying the receptor or chimera (eg, cell survival or proliferation), Drugs acting on the receptor can be identified and ultimately such drugs can be purified. Once a particular receptor / ligand system has been defined, various additional specific assay systems may be utilized, for example, to test for additional agonists or antagonists of Dmk.

  When the receptor is introduced into cells that do not normally express the Dmk or Dmk-RTK chimeric receptor according to the present invention, the receptor can be used to identify ligands that bind to the receptor based on the discriminating response of the cell. . The present invention contemplates that the type of response elicited will depend on the cell utilized rather than the specific receptor introduced into the cell. Thus, for example, the expression of a Dmk receptor in PC12 pheochromocytoma cells can result in the differentiation of PC12 cells upon exposure to a ligand that binds to this receptor, while in fibroblasts, the same receptor binds to a Dmk-binding ligand. In response, it can mediate both survival and proliferation. By selecting the appropriate cell line, one will be able to obtain the response with the greatest utility for the assay, and discover agents that can act on the tyrosine kinase receptor. "Drug" refers to any molecule, including but not limited to peptide molecules and non-peptide molecules, that work in a system described in a receptor-dependent manner.

  One of the more useful systems to be developed involves the introduction of the desired receptor into a growth factor-dependent fibroblast cell line; such receptors, which do not normally mediate a proliferative response, nonetheless, Following introduction into fibroblasts, they can be assayed by a variety of well-established methods used to quantify the effects of fibroblast growth factors (eg, thymidine incorporation or other types of proliferation assays; van Zoelen, 1990, "Use of Biological Assays for the Detection of Polypeptide Growth Factors" Progress in Factor Research, Vol. 2, pp. 131-152; Zhan and M. Goldfarb, 1966, Mol. Cell. Biol. Vol. 6, pages 3541-3544). These assays have the further advantage that any preparation can be assayed, both for cell lines having the introduced receptor and for parent cell lines lacking the receptor; only specific effects on cell lines having the receptor are It is determined to be mediated through the introduced receptor.

  Cells expressing the orphan receptor described herein can naturally express the receptor or can be genetically engineered to express the receptor. For example, a nucleic acid sequence obtained as described in Section 6 or Section 8 (below) may be transfected, transduced, microinjected, and the like via any transgenic animal using any method known in the art. Can be introduced into cells by electroporation.

  The specific binding of a test agent to an orphan receptor can be measured in a number of ways. For example, the actual binding of a test agent to a cell may be determined by (i) a test agent bound to the surface of an intact cell; (ii) a test agent cross-linked to a receptor protein in a cell lysate; or (iii) a receptor in vitro. Can be detected or measured by detecting or measuring the test agent bound to the. The specific interaction between the test agent and the receptor can be assessed by using a reagent that exhibits the unique properties of that interaction.

  Alternatively, the specific binding of a test agent to the receptor can be attributed to the secondary biological effects of receptor / ligand binding, including but not limited to inducing neurite sprouting, immediate gene expression or receptor phosphorylation. Can be measured by evaluating For example, the ability of a test agent to induce neurite sprouting can be tested in cells lacking the receptor and in comparative cells expressing a chimeric receptor containing, for example, a Dmk extracellular domain and an intracellular domain of a Trk family member; Neurite sprouting, which does not take place in control cells lacking, but occurs in receptor expressing cells, is indicative of a specific test agent / receptor interaction. By detecting immediate genes (eg, fos and jun) in receptor minus and receptor plus cells, or by detecting phosphorylation of the receptor protein using standard phosphorylation assays known in the art. A similar analysis can be performed.

  Similarly, the invention provides for (i) exposing cells expressing a tyrosine kinase receptor as described herein to a test agent, and (ii) detecting specific binding of the test agent to the receptor. Wherein the specific binding to the receptor is positively correlated with the signaling activity, and a method for identifying an agent having a signaling activity is provided. Specific binding may be detected by assaying for either direct binding or a secondary biological effect of binding, as described above. Such methods can be used to identify new neurotrophic factors or other pharmacologically active factors, such as cardioprotective activity, or in the pharmaceutical industry, a number of peptide and non-peptide drugs for such activities (eg, , Peptidomimetics) can be particularly useful.

  In certain non-limiting, preferred embodiments of the invention, a large grid comprising alternating rows of PC12 (or fibroblasts, see below) that are receptor minus or genetically engineered to receptor plus. Culture wells can be prepared. Various test agents may then be added such that each column or part of the grid contains a different test agent. Each well may then be scored for the presence or absence of neurite sprouting. Numerous test agents can be screened for signaling activity in this way.

  The present invention also provides an assay system that can be used according to the above method. Such an assay system can include, for example, an in vitro preparation of the receptor immobilized on a solid support, or, preferably, can include a cell that expresses a receptor protein described herein.

  In addition, the present invention provides host cells and microorganisms and vectors carrying the above-described recombinant nucleic acid molecules, including but not limited to Dmk. As described above, the cells that express the receptor protein are genetically engineered by transfection, transduction, electroporation, microinjection of a nucleic acid encoding Dmk in an appropriate expression vector through a transgenic animal or the like. A receptor can be obtained. In certain embodiments, the host cell carrying the recombinant nucleic acid is an animal cell, such as COS. In another embodiment, the host cell is a bacterium, preferably Escherichia coli.

  The expression vector encoding the receptor can be constructed using any method known to those of skill in the art for insertion of a DNA fragment into a vector. These methods may include in vitro recombinant DNA methods and synthetic techniques and in vivo recombination (genetic recombination). Expression of the nucleic acid sequence encoding the receptor protein or peptide fragment can be regulated by a second nucleic acid sequence such that the receptor protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, receptor expression can be controlled by any promoter / enhancer element known in the art. Promoters that can be used to control receptor expression include long terminal repeats described in Squinto et al. (1991, Cell 65: 1-20); the SV40 early promoter region (Bernoist and Champon, 1981, Nature). 290: 304-310), CMV promoter, M-MuLV 5 'terminal repeat, promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797), herpes thymidine kinase promoter (Wagner). Natl. Acad. Sci. USA 78: 144-1445), the regulatory sequence of the metallothionein gene (Brinster et al., 1982, Na 296: 39-42); a prokaryotic expression vector such as the β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA, 75: 3727-3731), or tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25) (see "Useful Proteins from Recombinant Bacteria" Scientific American, 1980, 242: 74-94). Promoter elements from yeast or other fungi, such as the Gal4 promoter, the ADH (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter, and the like; The following animal transcription regulatory regions that exhibit tissue specificity and have been utilized in transgenic animals: Elastase I gene regulatory region active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639-646; Ornitz et al., 1986) 50: 399-409; MacDonald, 1987, Hepatology 7: 425-515); Insulin gene control region active in pancreatic β cells (Hanahan, 1985, Nature 315: 115-122), Cold Spring Harbor Symp. , An immunoglobulin gene regulatory region active in lymphoid cells (Grosschedi et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318: 533-538; Al xander, et al., 1987, Mol. Cell. Biol. 7: 1436-1444), a mouse mammary tumor virus control region active in testis, breast, lymphoid cells and mast cells (Leder et al., 1986, Cell 45: 485-495), an albumin gene control region active in liver (Pinkert et al.). 1987, Genes and Level., 1: 268-276), α-fetoprotein gene regulatory region active in liver (Krumlauf et al., 1985, Mol. Cell Biol. 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58); α1-antitrypsin gene regulatory region active in liver (Kelsey et al., 1987, Genes and Level. 1: 161-171); β-globin gene regulatory region active in myeloid cells (Mogram et al., 198) Kollias et al., 1986, Cell 46: 89-94); a myelin basic protein gene regulatory region active in oligodendrocytes in the brain (Readhead et al., 1987, Cell 48: 703-712). ); Myosin light chain-2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314: 283-286), and gonadotropin releasing hormone gene regulatory region active in the hypothalamus (Mason et al., 1986, Science 234). : 1372-1378).

  Expression vectors containing a gene insert encoding the receptor can be prepared by three general approaches: (a) DNA-DNA hybridization, (b) the presence or absence of "marker" gene function, and (c) insertion. Can be identified by expression of the sequence. In a first approach, the presence of a foreign gene inserted into the expression vector can be detected by DNA-DNA hybridization using a probe containing a sequence homologous to the inserted gene. In a second approach, the function of certain “marker” gene functions (eg, thymidine kinase activity, resistance to antibiotics, transformed phenotype, inclusion body formation in baculovirus, etc.) caused by insertion of a foreign gene in a vector, Based on the presence or absence, a recombinant vector / host system can be identified and selected. For example, if the gene encoding the receptor is inserted within the marker gene sequence of the vector, recombinants containing the gene insert can be identified by the absence of marker gene function. In a third approach, a recombinant expression vector can be identified by assaying the foreign gene product expressed by the recombinant vector. Such assays may be based, for example, on the physical or functional properties of the gene product encoding the receptor (eg, by binding of the receptor to a neurotrophic factor or to an antibody that recognizes the receptor directly). The cells of the invention may transiently or, preferably, constitutively and permanently express the receptor or a portion thereof.

  In a preferred embodiment, the present invention relates to the expression of a receptor as described herein or a part thereof, and further to the use of an immediate gene promoter [eg the fos or jun promoter (Gllman et al., 1986, Mol. Cell. Biol. 6: 4305-4316)]. When such cells are exposed to a ligand that binds to the receptor, binding secondarily induces transcription of the immediate promoter. Using such cells, the transcriptional activity of the immediate gene promoter is measured, for example, by nuclear run-off analysis, Northern blot analysis, or the level of the gene controlled by the promoter is measured. By doing so, receptor / ligand binding can be detected. Using the immediate gene promoter, fos or jun, or a gene that confers hygromycin resistance (Murphy and Estratidis, 1987, Proc. Natl. Acad. Sci. USA) for detection or measurement of neurotrophic activity. 84: 8277-8281), any known reporter gene such as chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (neo), β-galactosidase, β-glucuronidase, β-galactosidase and the like ( However, (but not limited to) the expression of any detectable gene product can be controlled.

Further, the cells used in the assay system of the present invention may or may not be cells of the nervous system. For example, in a detailed, non-limiting embodiment of the invention, growth factor dependent fibroblasts can be used as the basis for a signaling assay system. Fibroblast cell lines that are growth factor-dependent in serum-free medium (eg, as described in Zham and Goldfalb, 1986, Mol. Cell, Biol. 6: 3544-1544) can be transfected with the receptor-encoding gene. . This uses, for example, the CaPO ₄ transfection protocol using the DNA of a CMV-promoter-based expression vector containing the dmk gene (5 μg) and the expression vector containing the hygromycin resistance gene (1 μg). After about 48 hours, the cells can then be selected for hygromycin resistance and positive transfectants identified. The cells can then be cultured in the presence of hygromycin for about 3 weeks, and the resistant colonies pooled. The cells can then be placed on a tissue culture plate coated with poly-D-lysine and human fibronectin and grown in DMEM with 10% calf serum for about 4 hours to bind the cells to the plate. The serum-containing medium can then be aspirated and the cells can be washed about three times with PBS to remove any remaining serum. The cells were then cultured in serum-free defined medium (8 mM sodium bicarbonate, 15 mM HEPES, 4 × 10 ⁻⁶ MnCl ₂ , 3 mM histidine, 10 ⁻⁵ M ethanolamine, 10 ⁻⁷ M sodium selenite, 5 mg transferrin / liter). (A 3: 1 mixture of DMEM and HamsF12 supplemented with 200 mg bovine serum albumin-linolenic acid conjugate per liter, gentamicin, penicillin, and streptomycin, 20 mM L-glutamine). Cells generated in this way and then incubated with factors capable of binding Dmk are expected to grow and proliferate after approximately 5 days of culture (replace media and growth factors every 48 hours). Obtain; however, cells treated with an irrelevant ligand at 100 ng / ml or in serum-free medium should not grow.

  Further insight into the physiological role of Dmk comes from its ligand definition. Since the kinase domain of the Dmk protein appears to be related to other receptor tyrosine kinases, this protein appears to be involved in signal transduction in the cell in which it is expressed. Thus, Dmk binding ligands screened using the receptors described herein can be used to induce signal transduction in naturally occurring Dmk expressing cells. The cells include cells in muscle tissue, heart, spleen, ovaries and retina, as well as cells that have been engineered to express the Dmk protein. Such a ligand may promote the growth or survival of such a cell.

  As noted above, the present invention relates to tyrosine kinase receptors that are likely to be expressed in denervated muscle. According to the present invention, a disease or disorder can be identified by measuring altered levels of the receptor in cells and tissues using probes capable of recognizing these receptors. Such diseases or disorders can then be treated with ligands that bind to these receptors. Such diseases include, but are not limited to, diseases in which atrophic or dystrophic changes in muscle are the underlying pathological findings. For example, muscular atrophy is denervation due to nerve damage (loss of contact with the nerve by the muscle); degenerative, metabolic or inflammatory neuropathy (eg, Guillain-Barre syndrome), peripheral neuropathy, or environmental toxicants or It can result from nerve damage caused by drugs. In another embodiment, the muscle atrophy results from denervation due to motor neuron damage. Such motor neuron diseases include adult motor neuron diseases including amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease); juvenile and juvenile spinal atrophy, and multifocal disease Examples include, but are not limited to, autoimmune motor neuropathy due to multifocal conduction block. In another embodiment, the muscle wasting results from chronic disuse. Such nonuse atrophy can result from conditions including, but not limited to: paralysis due to stroke, spinal cord injury; skeleton due to injury (eg, fracture, sprain, or dislocation) or prolonged bed rest. Immobilization. In yet another embodiment, the muscle wasting results from metabolic stress or malnutrition. The following are non-limiting examples: cachexia of cancer and other chronic diseases, fasting or rhabdomyolysis, endocrine disorders (eg, but not limited to thyroid disorders and diabetes) . Muscle atrophy can also result from muscular dystrophy syndrome. Examples include, but are not limited to: Duchenne, Becker, tonicity, fascioscapulohumeral, Emery-Dreifuss, ocular pharynx, scapulohumeral, limb girdle and congenital types and hereditary peripheral muscle. Dystrophy known as injury. In a further embodiment, the muscle atrophy is due to a congenital muscle injury. Examples include, but are not limited to: benign congenital hypotension, central core disease, nemarin myopathy, and myotubular (central nucleus) myopathy. In addition, Dmk and its related ligands can be used in the treatment of acquired (toxic or inflammatory) myopathy. Muscle disorders resulting from inflammatory diseases of the muscle include, but are not limited to, polymyositis and dermatomyositis. Toxic myopathy can be due to drugs. The following are non-limiting examples: adiodarone, chloroquine, clofibrate, colchicine, doxorubicin, ethanol, hydroxyquinone, organophosphates, perihexiline, and vincristine.

  Without wishing to be bound by theory, preliminary mapping of Dmk in mice revealed that this gene maps to mouse chromosome 4 in a region homologous to human chromosome 9q. Variants in mice associated with this region of chromosome 4 include signs of shaker syndrome (hearing loss, head-tossing, circling, and hyperactivity). "Wi" mutations (whirlers) that result in the inclusion of (Wane) (Lane, PW, 963, J, Hered. 54; 263-266). Another mutation in mice associated with this region of chromosome 4 is the "vc" mutation (vacillans), which is associated with signs of violent tremors when walking and shaking of the hind legs (Sirlin, JL. , 1956, J. Genet. 54: 42-48).

  In humans, a disease known as idiopathic torsion disorder (ITD) is associated with a gene mapped by linkage analysis to human chromosome 9q band 34. The disease is characterized by persistent involuntary muscle contractions that frequently cause torsion and repetitive movements or abnormal postures.

  If a defect in dmk is found to be associated with these diseases, the present invention may prove useful in gene therapy for the replacement of such genes in situ. Alternatively, probes utilizing unique segments of the Dmk gene may prove useful as diagnostics for such disorders.

  The present invention provides a method of diagnosing a neurological disorder or other disorder in a patient, comprising comparing the level of Dmk expression in a patient sample with the level of Dmk expression in a comparative sample from a healthy subject. . In this way, a difference in the level of expression of Dmk in the patient, as compared to a healthy individual, indicates that the disorder in the patient may be primarily or secondarily associated with Dmk metabolism. The patient sample can be any cell, tissue, or body fluid, but is preferably muscle tissue or cerebrospinal fluid.

  One type of probe that can be used is an anti-Dmk antibody or a fragment thereof that contains the binding domain of the antibody.

  According to the present invention, the Dmk protein, or a fragment or derivative thereof, can be used as an immunogen to generate anti-Dmk antibodies. By providing the production of relatively large amounts of Dmk protein using recombinant techniques for protein synthesis (based on the Dmk nucleic acid sequences of the present invention), the problem of limited amounts of Dmk has been eliminated.

  To further improve the likelihood of generating an anti-Dmk immune response, the amino acid sequence of Dmk can be analyzed to identify portions of the molecule that may be associated with increased immunogenicity. For example, this amino acid sequence is subjected to computer analysis to surface epitopes showing computer generated plots of Dmk hydrophilicity, surface probability, flexibility, antigenic index, amphipathic helix, amphiphilic sheet, and secondary structure Can be identified. Alternatively, the deduced amino acid sequences of Dmk from different species can be compared and regions of relatively non-homology identified. These heterologous regions are more likely to be immunogenic across various species.

  For preparation of monoclonal antibodies to Dmk, any technique which provides for the production of antibody molecules by continuous cell lines in culture can be used. For example, hybridoma technology originally developed by Kohler and Milstein (1975, Nature 256: 495-497), and trioma technology, human B cell hybridoma technology (Kozbor et al., 1983, Immunology Today 4:72), and human EBV-hybridoma technology for producing monoclonal antibodies (Cole et al., 1985, "Monoclonal Antibodies and Cancer Therapy" Alan R. Lis, Inc., pages 77-96) and the like are within the scope of the invention.

  Monoclonal antibodies for therapeutic use can be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies can be prepared using any of a number of techniques known in the art (eg, Teng et al., 1983, Proc. Natl. Acad. Sci. USA 80: 7308-7312; Kozbor et al., 1983, Immunology Today). 4: 72-79; Olson et al., 1982, Meth, Enzymol, 92: 3-16). Chimeric antibody molecules containing a mouse antigen binding domain with a human constant region can be prepared (Morrison et al., 1984, Proc, Natl. Acad. Sci, USA 81: 6851, Takeda et al., 1985, Nature. 314: 452).

  Various procedures known in the art can be used for the production of polyclonal antibodies to the epitope of Dmk. For the production of antibodies, various host animals can be immunized by injection of the Dmk protein, or a fragment or derivative thereof. The following are non-limiting examples: rabbits, mice, rats, and the like. Various adjuvants, depending on the host species, can be used to increase the immunological response. The following are non-limiting examples of adjuvants: Freund's adjuvant (complete and incomplete), mineral gels (eg, aluminum hydroxide), surfactants (eg, lysolecithin), pluronic polyols, polyanions, peptides, Oily emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants (eg, BCG (Bacille Calmette-Guerin)) and Corynebacterium parvum.

  Molecular clones of antibodies to the Dmk epitope can be prepared by known techniques. Using a recombinant DNA method (eg, Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) to encode the nucleic acid antibody or antigen binding region encoding the nucleic acid binding region. I can do it.

  Antibody molecules can be purified by known techniques. For example, immunoadsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof.

The present invention provides antibody molecules and fragments of such antibody molecules. Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include, but are not limited to, the following: F (ab ') ₂ fragments produced by pepsin digestion of antibody molecules; reducing disulfide bridges of F (ab') ₂ fragments Fab ′ fragments that can be generated by treating an antibody molecule with papain and a reducing agent.

  The probes described above can be used experimentally to identify cells or tissues that have not previously been shown to express Dmk. In addition, these methods can be used to identify Dmk expression by abnormal tissues (eg, malignant tumors). In another embodiment, these methods involve the expression of Dmk in a cell, fluid, or tissue from a patient suffering from a disorder and in a compared cell, fluid, or tissue from a healthy individual. For comparison, it can be used diagnostically. Fluid is taken to mean in particular any body fluid other than blood or cerebrospinal fluid. Differences in the level of expression of Dmk in patients compared to healthy individuals indicate that the disorder in the patient may be, primarily or secondary, related to Dmk metabolism. For example, an increase in the level of Dmk may indicate that the disorder in the patient is associated with increased sensitivity to normal levels of the Dmk-binding ligand, or the patient has low Dmk-binding ligand levels, thereby compensating for the number of receptors. It can be suggested that it increases.

  The invention further provides for the use of a soluble receptor (extracellular domain) to counter the effect of the ligand on Dmk expressing cells. For example, if a cognate is found to have undesirable mitogenic properties, such blocking is desired.

The present invention provides a novel tyrosine kinase called denervated muscle kinase "Dmk".

(6. Example: Cloning of cDNA encoding Dmk)
(6.1 Materials and Methods)
Tyrosine kinase homology domains were identified based on alignments by Hanks et al. (1988) Science 241, 42-52. The highly conserved regions Asp-Leu-Ala-Ala-Arg-Asn (SEQ ID NO: 7) and Asp-Val-Trp-Ser-Tyr-Gly (SEQ ID NO: 13) design the following degenerate oligonucleotide primers: Used to:
5'-TCTTGACTCGAGAYYTNGCNGCNMGNAA-3 '(SEQ ID NO: 8)
5'-GAATTCGAGCTCCCRTANSWCCANACRTC-3 '(SEQ ID NO: 10)
This was used to prime a PCR reaction using denervated muscle cDNA. The resulting amplified DNA fragment was cloned by insertion into a plasmid, sequenced, and the DNA sequence compared to that of all known tyrosine kinases. cDNA templates were generated by reverse transcription of denervated muscle tissue RNA using oligo d (T) primers. The PCR reaction was performed at a primer annealing temperature of 40 ° C. Aliquots of the PCR reaction were subjected to electrophoresis on agarose gel.

  The size-selected amplified DNA fragments from these PCR reactions were cloned into plasmids as follows: Each PCR reaction was reamplified as described above, digested with XhoI and SacI, and the The site at was cut (see below). XhoI / SacI-cleaved DNA was purified with the Magic PCR kit (from Promega), cloned into a compatible XhoI / SacI site in the Bluescript II SK (+) plasmid, and electroporated by DH10B E.C. E. coli and then transformants were plated on selective agar. Ampicillin resistant bacterial colonies from the PCR transformation were inoculated into 96-well microtiter plates and used for PCR using vector primers (T3 and T7) flanking the tyrosine kinase insert, and these PCR fragments were used. And analyzed by sequencing.

  One of the cloned fragment sequences contained a segment of the novel tyrosine kinase domain, designated Dmk. PCR primers were generated using the sequence of the PCR-derived fragment corresponding to Dmk, and a longer Dmk-specific fragment was obtained by the RACE procedure. Using these longer Dmk probes as hybridization probes, full-length Dmk cDNA clones were obtained from a rat denervated skeletal muscle cDNA library. DNA was sequenced using an ABI 373A DNA sequencer and TaqDyeoxy Terminator Cycle Sequencing Kit (Applied Biosystem, Inc., Foster City, CA). The sequence of Dmk (FIG. 1; SEQ ID NO: 1) has a high degree of homology with members of the trk family of proteins. It was also found to be similar to Jennings et al., Torpedo RTK found intramuscularly.

(6.2 Results and discussion)
Oligonucleotide primers corresponding to conserved regions of known tyrosine kinase molecules were used to amplify and clone DNA sequences encoding novel orphan tyrosine kinase receptor molecules. The amino acid sequences of representatives of the branches of the tyrosine kinase family, and homologous regions within the catalytic domains of these proteins, were used in the design of degenerate oligonucleotide primers. These primers were then used to initiate a PCR reaction using the rat denervated muscle cDNA library as a template. The resulting amplified DNA fragment was then cloned into a Bluescript II SK (+) plasmid, sequenced, and the DNA sequence compared to that of a known tyrosine kinase. The sequence of the PCR fragment encoding the novel tyrosine kinase, designated Dmk, was used to obtain further flanking DNA sequences. A DNA fragment containing the Dmk sequence was used as a probe to obtain a cDNA clone from a denervated skeletal muscle library. This clone encodes a novel tyrosine kinase receptor with a high degree of homology to members of the trk family of proteins. It was also found to be homologous to the Jorning et al. Torpedo RTK.

  FIG. 1 shows the nucleotide sequence of the Dmk clone (SEQ ID NO: 2).

7. Examples: Identification of Additional Tyrosine Kinases
Novel Dmk sequences were used to obtain homologous segments in the currently available tyrosine kinase receptors in combination with other homologous segments. For example, an alignment of Torpedo trk-related kinase and Dmk shows the following conserved protein segments:
Asp-Val-Trp-Ala-Tyr-Gly (SEQ ID NO: 3)
This homology "box" is not present on any other mammalian tyrosine kinase receptor. Degenerate oligonucleotides based essentially on this "box" can be used to identify novel tyrosine kinase receptors, in combination with either known or novel tyrosine kinase homology segments.

(7.1 Materials and Methods)
Based on regions highly conserved between Dmk and Torpedo TRK Asp-Val-Trp-Ala-Tyr-Gly (SEQ ID NO: 3) and homologous known regions such as SEQ ID NO: 5, 7, 9 or 11 Additional primers are used to design degenerate oligonucleotide primers used to initiate a PCR reaction with the cDNA. A cDNA template is generated by reverse transcription of tissue RNA using oligo d (T) or other suitable primers. An aliquot of the PCR reaction is subjected to electrophoresis on an agarose gel. The resulting amplified DNA fragment is inserted into a plasmid, cloned, sequenced, and the DNA sequence compared to the sequence of all known tyrosine kinases.

  The size-selected amplified DNA fragments from these PCR reactions were cloned into plasmids as follows: Each PCR reaction was re-amplified as in Example 1 above and used to cut sites at the ends of the primers. Digest with XhoI and SacI (see below).

  The XhoI / SacI digested DNA was cloned into a compatible XhoI / SacI site in the plasmid and E. coli was electroporated. E. coli, and then transformants are plated on a selective agar medium. Ampicillin-resistant bacterial colonies from the PCR transformation are inoculated into 96-well microtiter plates, and each colony from these PCR clones is analyzed by sequencing plasmid DNA purified by standard plasmid miniprep procedures.

  The cloned fragment containing a segment of the novel tyrosine kinase domain is used as a probe for hybridization to obtain a full-length cDNA clone from a cDNA library.

(8. Example: Tmk tissue-specific expression)
(8.1 Materials and Methods)
A 680 nt fragment containing the tyrosine kinase domain of Dmk was radiolabeled and used for Northern analysis of various rat tissue-specific RNAs. Rat tissue-specific RNA was fractionated by electrophoresis on a 1% agarose-formaldehyde gel and subsequently transferred using 10 × SSC using the capillary action of a nylon membrane. RNA was cross-linked to the membrane by UV irradiation, and the radiolabeled Dmk _{probe, 0.5MNaPO 4 (pH7), 1} % bovine serum albumin (Fraction V, Sigma), 7 % SDS, lmM EDTA, and 100 ng / m1 greater Hybridized at 65 ° C. in the presence of sonicated denatured salmon sperm DNA. The filters were washed at 65 ° C. with 2 × SSC, 0.1% SDS and subjected to autoradiography at −70 ° C. for 5 days with one intensifying screen, X-ray film. Ethidium bromide staining of the gel indicated that equal levels of total RNA were being assayed against different samples.

(8.2 Results)
The Dmk probe hybridized strongly in adult tissues (FIG. 2) to 7 kb transcripts from denervated skeletal muscle and weakly to transcripts from normal muscle, retina, ovary, heart and spleen. Weaker levels of expression could also be found in liver, kidney and lung. It also hybridizes weakly to approximately 6 kb shorter Dmk transcripts in the cerebrum, spinal cord and cerebellum.

  In embryonic tissue (FIG. 3), Dmk transcripts can be found in the body, spinal cord, placenta, and head (at E12 and E13).

  High expression of human Dmk in muscle and nervous tissue indicates that the present invention or Dmk-related ligands may affect the nervous system, particularly motor neurons (discussion, see above) and affect neuromuscular junctions. It suggests that it can be used to treat neuropathy. In addition, high expression of Dmk in heart tissue indicates that the present invention, or a ligand associated with Dmk, may be used in the treatment of heart disease, and that, for example, muscle loss during or after heart disease. Suggest that it may have prophylactic use (discussion, see above) in the prevention of The expression of Dmk in retinal tissue suggests that the present invention may be used to treat disorders related to the retina, including but not limited to retinitis pigmentosa. Expression of Dmk in the ovaries indicates that Dmk, or a ligand related to Dmk, may be useful in treating ovarian-related diseases or disorders. Finally, expression of Dmk in the spleen suggests that Dmk, or a ligand related to Dmk, may be useful in the treatment of diseases or disorders associated with the spleen.

(95. Deposit of microorganism)
The following clones were deposited on July 13, 1993 with the Amrican Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852).

(Accession number)
pBluescript SK-containing dmk
The invention is not limited within the scope of the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Various references are cited herein (the disclosures of which are incorporated by reference in their entirety).
(Sequence listing)

Figure 1-1 shows the dmk nucleic acid and deduced amino acid (single letter code) sequence. The nucleotide sequence encoding mature Dmk begins around nucleotide 192. Figure 1-2 shows the dmk nucleic acid and deduced amino acid (single letter code) sequence. The nucleotide sequence encoding mature Dmk begins around nucleotide 192. Figures 1-3 show dmk nucleic acid and deduced amino acid (single letter code) sequences. The nucleotide sequence encoding mature Dmk begins around nucleotide 192. Figures 1-4 show dmk nucleic acid and deduced amino acid (single letter code) sequences. The nucleotide sequence encoding mature Dmk begins around nucleotide 192. Figures 1-5 show dmk nucleic acid and deduced amino acid (single letter code) sequences. The nucleotide sequence encoding mature Dmk begins around nucleotide 192. Figures 1-6 show the dmk nucleic acid and deduced amino acid (single letter code) sequences. The nucleotide sequence encoding mature Dmk begins around nucleotide 192. Figures 1-7 show the dmk nucleic acid and deduced amino acid (single letter code) sequences. The nucleotide sequence encoding mature Dmk begins around nucleotide 192. Figures 1-8 show dmk nucleic acid and deduced amino acid (single letter code) sequences. The nucleotide sequence encoding mature Dmk begins around nucleotide 192. Figures 1-9 show dmk nucleic acid and deduced amino acid (single letter code) sequences. The nucleotide sequence encoding mature Dmk begins around nucleotide 192. FIG. 2 shows a Northern blot showing the distribution of Dmk in early development. Lane 1: whole fetus E9; lane 2: whole fetus E11; lane 3: placenta E11; lane 4: fetal head E12; lane 5: fetal body E12; lane 6: fetal spinal cord E12; lane 7: placenta E12; Lane 9: fetal body E13; lane 10: fetal brain E17; lane 11: fetal brain Pl; lane 12: fetal brain P10; lane 13: fetal brain P19; lane 14: adult brain; lane 15: adult muscle Lane 16: adult denervated muscle, where the day of fertilization is day El and the day of birth is day Pl. FIG. 3 shows a Northern blot showing the distribution of Dmk in adult tissues. Lane 1: brain; lane 2: olfactory bulb; lane 3: cortex; lane 4: hippocampus; lane 5: thalamus / hypothalamus; lane 6: midbrain; lane 7: hindbrain; lane 8: cerebellum; Lane 10: thymus; lane 11: spleen; lane 12: liver; lane 13: kidney; lane 14: lung; lane 15: sciatic nerve; lane 16: retina; lane 17: heart; Lane 20: denervated muscle.

Claims (1)

(A) the nucleic acid sequence shown in FIG. 1 or (b) a part of the sequence, which encodes the extracellular domain, transmembrane portion, or intracellular domain of the amino acid sequence shown in FIG. 1 or (c) 2.) A substantially purified recombinant nucleic acid molecule which is part of the sequence and comprises a sequence encoding the extracellular domain, transmembrane portion and intracellular domain of the amino acid sequence shown in FIG.

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