JP2006516387A - Gene whose expression is increased in response to stimulation by corticotropin-releasing hormone - Google Patents

Abstract

The present invention relates generally to the treatment and diagnosis of depression. In particular, the present invention relates to polypeptides as well as polynucleotides encoding these polypeptides, where the polypeptides are shown to play a central role in mediating an endocrine response to adrenocorticotropic hormone releasing hormone. Has been. These polypeptides and polynucleotides are useful in the diagnosis, treatment and / or prevention of depression.

Description

  The present invention relates generally to the treatment and diagnosis of depression. In particular, the present invention relates to polypeptides as well as polynucleotides encoding these polypeptides, where the polypeptides play a central role in mediating cellular responses to corticotropin releasing hormone. It is shown. These polypeptides and polynucleotides are useful in the diagnosis, treatment and / or prevention of depression.

  Recent socio-economic analysis has found that depression is a major cause of disability and a major risk factor for the development of other diseases. In addition, depression is underdiagnosed and undertreated on a global scale. Current antidepressants have proven effective, but have slow onset and side effects. On top of this, it is not yet clear which pharmacological mechanism of action they exert their clinical effect. Hypothesis-driven research based on the corticosteroid receptor hypothesis of depression has led to a new concept that focuses on neuropeptide receptors, particularly corticotropin-releasing hormone (CRH) receptors, as drug targets.

  The 41 amino acid polypeptide adrenocorticotropic hormone releasing hormone (CRH) plays a central role in the regulation of the hypothalamic-pituitary-adrenal system and mediates endocrine responses to various stress factors. Hypothalamic neurons release CRH to the pituitary portal system in response to stress, stimulate pituitary adrenocorticotropin (ACTH) secretion and biosynthesis, resulting in increased adrenal glucocorticoid production (Non-Patent Document 1). ). Some clinical and preclinical studies are directed towards the causal role of CRH system modification in the development of depression (2). Initial studies with CRH in humans have shown that the ACTH response to CRH is blunted in depressed patients and always reflects CRH receptor desensitization associated with increased hypothalamic CRH secretion (non-patent literature). 3; Non-patent document 4). Supporting the blunted ACTH response as a result of increased CRH release is the result of elevated CRH levels in the cerebrospinal fluid of patients with depression. Another result that reinforces this concept of hypersecretion of CRH in depressed states is an increased number of CRH secreting neurons and a decreased number of CRH receptors in suicidal victims with depression (Non-Patent Document 5; Non-Patent Document 6). ).

Two high affinity receptors have been described for CRH (CRH-R1 and CRH-R2), both of which exist in several splice variant forms. Activation of these receptors by CRH results in Gs-mediated stimulation of adenyl cyclase, resulting in increased intracellular cAMP levels. This itself activates cAMP-dependent protein kinase A (PKA), and finally leads to increased cytosolic levels of cAMP and ^{Ca 2+.} Increased cAMP and Ca ²⁺ levels result in the activation of several other additional kinases such as Ca ²⁺ / calmodulin-dependent kinase II (CAMKII) and p42 / p44 mitogen-activated kinase (MAPK). As a result, the Ca ²⁺ / cAMP response element binding protein (CREB) is phosphorylated, which in turn regulates the transcription of genes that contain the cAMP response element (CRE) in the promoter region. Examples of such genes that have been shown to be involved in the regulation of CRH signaling include c-fos, macrophage migration inhibitory factor gene Mif, orphan nuclear receptors Nurr77 and Nurr1.

Despite the fact that the downstream pathway of the CRH activating receptor has been studied in detail in the corticotrophic cell model AtT-20 cells and has led to the identification of numerous genes involved in the signaling cascade, major regions have been explored. Not. Accordingly, it was an object of the present invention to explore the transcriptional response to CRH stimulation at the genome-wide level to identify additional genes involved in the corticotropin releasing hormone receptor activating gene network. The polypeptides thus identified and polynucleotides encoding the polypeptides provide new opportunities for drug development as drug targets by screening techniques, or are useful in the diagnosis, prevention and / or treatment of depression.
De Souza EB 1995 Corticotropin-releasing factor receptor: physology, pharmacology, biochemistry and role in central nervous system. Psychoenuroendocrinology 20: 789-819 Holsboer F 2001 Stress, hypercortisolism and corticosteroid receptors in depression: implications for therapies. J Affective Diode 62: 77-91 Holsboer, F, Gerken A, Stalla GK, Muller OA 1987 Blunted aldosterone and ACTH release after human CRH administration in depressed patents. Am J Phychiatry 144: 229-231 Holsboer F, Gerken A, von Bardeleben U, Grimm W, Beyer H, Muller OA, Stalla GK 1986 Human cortitropine reintroduction of correlation. Biol Psychiatry 21: 601-611 Nemerov CB, Owens MJ, Bissette G, Andorn AC, Stanley M 1988 Reduced corticotropin releasing factor binding sites in the frontier cortex. Arch Gen Psychiatry 45: 577-579 Radsheer FC, Hoogendijk WJ, Stam FC, Tilders FJ, Swab DF 1994 Increased numbers of correlatively intensified neutronics intensified neuron. Neuroendocrinology 60: 436-444

[Summary of Invention]
The present invention relates to the identification of a number of genes involved in the transcriptional response to CRH stimulation at the whole genome level. In particular, it relates to the identification of a number of previously unknown genes encoding proteins that modulate CRH signaling having a sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 and functional analogs thereof.

  As a further aspect, the present invention includes vectors comprising these sequences, host cells containing vectors encoding one of the above sequences, and transgenic non-human animals comprising the polynucleotides or vectors of the invention. To the recombinant use of the above nucleotide sequences.

Provide a number of genes that have not previously been associated with CRH signaling and are therefore useful in methods for identifying compounds that modulate CRH signaling responses in cells or in diagnostic methods for identifying CRH-induced depression in individuals It is also an object of the present invention. In one embodiment, a method of identifying a compound capable of altering a CRH signaling response in a cell comprises contacting the cell with CRH in the presence or absence of the compound and SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, sequence SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 A nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49. Determining the expression level of the nucleotides. In this screening method, expression levels are typically assessed using oligonucleotide probes that bind to the polynucleotides described above, preferably using array technology methods. Thus, as a particular aspect, the present invention provides a method of identifying a compound that modulates a CRH signaling response in a cell, the method comprising contacting the cell with CRH in the presence or absence of the compound; SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49. Determining the expression level of the polynucleotide, wherein the change in the expression profile of these sequences is the CRH signal in the cell. It is indicative of a compound capable of modifying the grayed response.
[Detailed Description of the Invention]
As used herein, the term “compound” or “factor” means a biological or chemical compound such as a simple or complex organic molecule, peptide, protein or oligonucleotide. “Test compound”, as used herein, refers to a “compound” or “factor” that is used in the methods of the invention to assess whether the compound modulates CRH signaling activity.

  “CRH signaling” as used herein refers to a cellular change in gene transcription following activation of adrenocorticotropin releasing hormone receptor by CRH in the cell. It induces a CRH specific gene expression profile. Changes at the transcription level can be assessed at the protein level or at the gene or RNA level.

“CRH responsive activity” as used herein generally refers to a change in a detectable cellular parameter as a result of exposing the cell to CRH. Detectable cellular parameters include, among others, changes in membrane potential, changes in enzyme activity of enzymes that regulate CRH signaling in the cells, changes in the expression level of the protein of the invention or cGMP, cAMP, Ca ²⁺ or IP ₃ Such changes in the amount of second messenger are included.

  The term “analog” or “functional analog” includes at least one amino acid substitution such that the analog retains substantially the same biological activity as the unmodified protein in vivo and / or in vitro. Refers to a modified form of the protein of the present invention.

  The term “functional analog” is intended to encompass “fragments”, “variants”, “degenerate variants”, “analogs” and “homologs” or “chemical derivatives” of the polypeptides of the invention. . Useful chemical derivatives of polypeptides are well known in the art and include, for example, covalent modification of reactive organic sites contained within a polypeptide by secondary chemical moieties. Well known cross-linking reagents are, for example, amino, carboxyl or aldehyde to introduce affinity tags such as biotin, fluorescent dyes, or to bind polypeptides to a solid surface (eg to make an affinity resin). Useful for reacting with residues.

  A polynucleotide or polypeptide variant (s), as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively. A variant of the polynucleotide can be a naturally occurring variant such as a naturally occurring allelic variant, or it can be a variant that is not known to occur naturally. (1) A polynucleotide that differs in nucleotide sequence from another, reference polynucleotide. In general, the differences are limited such that the reference and variant nucleotide sequences are generally very similar and identical in many regions. As described below, changes in the nucleotide sequence of the variant can be silent. That is, they can not alter the amino acid encoded by the polynucleotide. If the modification is limited to this type of silent change, the variant encodes a polypeptide having the same amino acid sequence as the reference. Also, as described below, changes in the nucleotide sequence of the variant can alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Such nucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as described above. (2) A polypeptide that differs in amino acid sequence from another reference polypeptide. In general, the differences are limited such that the reference and variant sequences are generally very similar and identical in many regions. A variant and reference polypeptide can exist in any combination and can differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations.

  The terms “complementary” or “complementarity” as used herein refers to the ability of purine and pyrimidine nucleotides to bind by hydrogen bonding to form a double-stranded nucleic acid molecule. The following base pairs are in a complementary relationship: guanine and cytosine; adenine and thymine; and adenine and uracil. As used herein, “complementary” means that the above relationship applies to substantially all base pairs comprising two single-stranded nucleic acid molecules over the entire length of the molecule. “Partially complementary” refers to the above relationship where one of the two single-stranded nucleic acid molecules is shorter in length than the other so that one part of the molecule remains single-stranded. .

The term “conservative substitution” or “conservative amino acid substitution” is used in parent proteins that do not affect the biological activity of the parent molecule based on the art-recognized substitution possibilities of certain amino acids. Substitution of one or more amino acid residues (see, eg, M. Dayhoff, Atlas of Protein Sequence and Structure , Vol. 5, Supp. 3, pg 345-352, 1978).

  A “fragment thereof” is disclosed herein as a sequence thereof, such that the fragment comprises 5 or more consecutive amino acids, or 10 or more nucleotides in the parent protein or nucleic acid molecule. A fragment, fragment or subregion of a nucleic acid or protein molecule.

  A “functional fragment” as used herein is an isolated subsequence of an amino acid sequence comprising functionally distinct regions such as the proteins disclosed herein or the active site of a receptor, for example. A region or fragment. Functional fragments can be produced by cloning techniques or as natural products of alternative splicing mechanisms.

  The term “homolog” or “homologous” refers to a relationship between different nucleic acid molecules or amino acid sequences, wherein the sequence or molecule is in one or more blocks or regions within the molecule or sequence. Related by partial identity or similarity. An “isolated nucleic acid compound” refers to any RNA or DNA sequence, no matter how constructed or synthesized, that is positionally different from its natural location.

  A “nucleic acid probe” or “probe”, as used herein, is a labeled nucleic acid compound that hybridizes with another nucleic acid compound. “Nucleic acid probe” means a single-stranded nucleic acid sequence that hybridizes to a single-stranded target nucleic acid sequence. The nucleic acid probe can be an oligonucleotide or a nucleotide polymer. A “probe” usually contains a detectable moiety that can be attached to the end (s) of the probe or be within the sequence of the probe.

  The term “primer” is a nucleic acid fragment that functions as an initiation substrate for, for example, enzymatic or synthetic extension of a nucleic acid molecule.

  The term “hybridization” as used herein refers to the process by which a single-stranded nucleic acid molecule binds to a complementary strand by nucleotide base pairing.

  The term “stringency” refers to hybridization conditions. High stringency conditions reject non-homologous base pairing. Low stringency conditions have the opposite effect. Stringency can be modified, for example, by temperature and salt concentration. “Stringent conditions” consisted of 50% formamide, 5 × SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate and 20 μg / ml modified sheared salmon. Refers to overnight incubation at 42 ° C. in a solution comprising sperm DNA, followed by washing the filters in 0.1 × SSC at about 65 ° C. Further suitable hybridization conditions are described in the examples.

“Low stringency conditions” include 6 × SSPE (20 × SSPE = 3 M NaCl; 0.2 M NaH ₂ PO ₄ ; 0.02 M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / ml. Incubation overnight at 37 ° C. in a solution comprising salmon sperm blocking DNA; followed by washing with 1 × SSPE, 0.1% SDS at 50 ° C. Furthermore, in order to obtain even lower stringency, washing after stringent hybridization can be performed at higher salt concentrations (eg, 5X SSC). It should be noted here that variations on the above conditions can be implemented by including and / or substituting alternative blocking reagents used to reduce background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA and commercially available proprietary formulations. Inclusion of specific blocking reagents may require modification of the hybridization conditions described above due to compatibility issues.

  The term “fusion protein” as used herein refers to a protein construct that is the result of combining multiple protein domains or linker regions for the purpose of obtaining the combined function of the domains or linker regions. This can be done by molecular cloning of a nucleotide sequence encoding such a domain to yield a new polynucleotide sequence encoding the desired fusion protein. Alternatively, the production of a fusion protein can be performed by chemically linking two proteins.

The term “linker region” or “linker domain” or similar such descriptive terms, as used herein, refers to a polynucleotide or polypeptide sequence used in the construction of a cloning vector or fusion protein. The function of the linker region can include the introduction of a cloning site into the nucleotide sequence, the introduction of a flexible component or space creation region between two protein domains, or the creation of an affinity tag for specific molecular interactions. . The linker region can be introduced into the fusion protein resulting from the selection performed during polypeptide or nucleotide sequence construction.
Screening Methods The present invention relates to screening methods for identifying compounds that modulate corticotropin releasing hormone (CRH) -induced depression and stress. It is based on the identification of a number of genes as downstream modulators of CRH activated CRH receptors. In particular, the present invention provides a method for identifying a compound capable of modifying a CRH signaling response in a cell, said method comprising:
a) contacting the cell with CRH with or without the compound;
b) determining a change in the transcriptional level of at least one protein that modulates corticotropin releasing hormone (CRH) signaling in the cell; and c) comparing the transcriptional level of the protein with and without the compound. ;
(Here, proteins that regulate corticotropin releasing hormone (CRH) signaling are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, (Selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48)
Comprising.

  To determine transcriptional changes at the protein level, the amount of the protein can be determined using techniques known in the art. For example, isoelectric focusing or separation techniques such as SDS-page are used in combination with protein staining techniques such as Coomassie or silver staining. Alternatively, for proteins that are enzymes, the amount in a given solution or tissue extract can be measured or assayed for the catalytic effect that the enzyme provides, which is the conversion of its substrate to a reaction product. For example, for kinases, kinase activity can be assessed using a substrate comprising a kinase-specific phosphorylation site and measuring the phosphorylation of the substrate by introduction of radioactive phosphate into the substrate. This assay can be performed both with and without the compound being tested. For proteins that are not enzymes, other quantification methods are required. For example, transport proteins can be assessed by their binding to the molecules they transport and hormones and toxins by the biological effects they provide.

  To assess transcriptional changes at the gene level, RNA or cDNA can be used directly for detection or can be amplified enzymatically by using PCR or other amplification techniques prior to analysis. Preferably, the analytical method comprises the use of labeled oligonucleotide probes that target the appropriate region of the gene.

  Therefore, in a preferred embodiment, the level of gene transcription is as follows: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, It is evaluated using a probe that binds to a polynucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 48.

  As another aspect, an array of oligonucleotide probes comprising a nucleotide sequence encoding a protein or fragment thereof that modulates CRH signaling can be constructed for efficient screening of gene expression. Array technology methods are well known, have general applicability, and can be used to address a variety of problems in molecular genetics, including gene expression, gene linkage and gene variability (eg: M. Chee et al., Science, Vol 274, pp 610-613 (1996)). Accordingly, it is an object of the present invention to provide a method for identifying a compound capable of modifying a CRH signaling response in a cell, the method comprising: contacting the cell with CRH in the presence or absence of the compound to be tested; and SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: No. 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48. Determining the level of gene transcription using an array of oligonucleotide probes that bind to a polynucleotide encoding a group of polypeptides Comprising that.

In an alternative embodiment, a method for identifying a compound capable of modifying a CRH signaling response in a cell;
a) contacting the cell with CRH with or without the compound; and b) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: Determining the expression level of a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 or SEQ ID NO: 49.

  To assess changes in expression levels, RNA or cDNA can be used directly for detection or can be amplified enzymatically by using PCR or other amplification techniques prior to analysis. Preferably, the analytical method comprises the use of a labeled oligonucleotide probe that targets the appropriate region of the polynucleotide. Accordingly, in a preferred embodiment, the level of gene transcription is as follows: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, It is evaluated using a probe that binds to a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47 or SEQ ID NO: 49.

  In another embodiment, an array of oligonucleotide probes comprising a nucleotide sequence encoding a protein that modulates CRH signaling or a fragment thereof can be constructed for efficient screening of gene expression. In this aspect, the invention provides a method of identifying a compound capable of altering a CRH signaling response in a cell, the method contacting the cell with CRH in the presence or absence of the compound; and SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 25 SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49. Determining the expression level of the polynucleotide comprising. In particular, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, Sequence number 25, Sequence number 27, Sequence number 29, Sequence number 31, Sequence number 33, Sequence number 35, Sequence number 37, Sequence number 39, Sequence number 41, Sequence number 43, Sequence number 45, Sequence number 47 and Sequence number An array of oligonucleotide probes that bind to a polynucleotide having 49 nucleic acid sequences is used.

  As another aspect, an array of oligonucleotide probes comprising a nucleotide sequence encoding a protein or fragment thereof that modulates CRH signaling can be constructed for efficient screening of gene expression. Array technology methods are well known, have general applicability, and can be used to address various problems in molecular genetics including gene expression, gene linkage and gene variability (eg: M. Chee) et al., Science, Vol 274, pp 610-613 (1996)).

If genomic DNA is not used directly, mRNA can be isolated and first strand cDNA synthesis can be performed. A second round of DNA synthesis can be performed for the generation of the second strand. The isolated cDNA can then be obtained by specific PCR amplification. If desired, the double-stranded cDNA can be cloned into any suitable vector, eg, a plasmid, thereby generating a cDNA library. As above, it is possible to screen a cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labeled oligonucleotide probe targeting any suitable region of a gene encoding a protein that modulates CRH signaling. is there. See, for example, PCR Protocols: A Guide to Method and Application , Ed. M.M. Innis et al. , Academic Press (1990).

  Methods for constructing cDNA libraries in appropriate vectors such as plasmids or phage for propagation in prokaryotic or eukaryotic cells are well known to those skilled in the art [see, eg, Maniatis et al. See above]. Suitable cloning vectors are well known and widely available.

  In a further embodiment, changes in gene transcription are determined at the mRNA level. Decreased or increased expression is known in the art for polynucleotide quantification, such as; for example; nucleic acid amplification (eg, by PCR, RT-PCR); RNase protection; Northern blotting and other hybridization methods. It can be measured at the RNA level using any of the well-known methods. Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays. Assay techniques that can be used to determine the presence of a protein derivative or variant comprise, among other things, mass spectrometry.

Accordingly, it is an object of the present invention to provide a method for identifying a compound capable of modifying a CRH signaling response in a cell, said method comprising:
a) contacting the cell with CRH with or without the compound;
b) determining the amount of at least one protein that modulates corticotropin releasing hormone (CRH) signaling in the cell; and c) comparing the amount of the protein with and without the compound;
(Here, proteins that regulate corticotropin releasing hormone (CRH) signaling are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, (Selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48)
Comprising.

  Preferably, the method for assaying the amount of protein that modulates CRH signaling comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, sequence SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42 An antibody that binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48 is used.

  Thus, in another aspect, the invention provides a monospecific antibody that immunologically reacts with a protein that modulates CRH signaling, wherein the protein comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 , SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48. Antibodies raised against the polypeptides of the present invention are obtained by administering the polypeptide or epitope-bearing fragment, analog, or cells expressing them to animals, preferably non-human animals, using routine protocols. be able to. For production of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. Examples include hybridoma technology (Kohler, G. and Milstein, C., Nature (1975) 256: 495-497), trioma technology, human B cell hybridoma technology (Kozbor et al., Immunology Today (1983)). 4:72) and EBV-hybridoma technology (Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

  Techniques for the production of single chain antibodies, such as those described in US Pat. No. 4,946,778, can also be adapted to produce single chain antibodies to the polypeptides of the invention. it can. Also, transgenic mice or other organisms including other mammals can be used to express humanized antibodies.

  The above antibodies can be used to isolate or identify clones expressing the polypeptide or to purify the polypeptide by affinity chromatography.

  Antibodies against the polypeptides of the invention can also be used to treat CRH metabolism related diseases such as CRH-induced stress or depression, among others.

  To determine the amount of protein that modulates CRH signaling, the antibodies of the invention are used in conventional immunological techniques. Suitable immunological techniques are well known to those skilled in the art and include, for example, ELISA, Western blot analysis, competitive or sandwich immunoassays, etc., all of which form antigen-antibody immune complexes as is well known. Where, for the purpose of the assay, the antibody can be detectably labeled, eg, with a radioactive, enzymatic or fluorescent label, or immobilized on an insoluble carrier.

For example, in an ELISA screening format, the antibody is added to a solid phase (eg, the bottom of a microplate) that is coated with either a protein bound to a carrier (such as BSA) or a peptide fragment thereof, and then An anti-immunoglobulin antibody conjugated with a detectable label such as an enzyme, preferably horseradish peroxidase, or a radioisotope such as ¹²⁵ I (eg, when immunization is performed in mice, an anti-mouse immunoglobulin antibody is used) For example, sheep anti-mouse immunoglobulin (Ig)) is added.

  Accordingly, it is an object of the present invention to provide an immunoassay for the determination or detection of proteins that modulate CRH signaling in a sample, the method comprising contacting a sample with an antibody against the protein of the present invention and the antibody and said Determining whether an immune complex is formed with the protein. These methods can be performed on a tissue sample or body fluid sample and generally obtain a sample from the subject's body; contacting the sample with an imaging effective amount of a detectably labeled antibody of the invention And detecting a label to reveal the presence of a protein that modulates CRH signaling in the sample.

  The measuring method using the antibody of the present invention is not particularly limited. A standard curve generated by the use of a standard solution in which the amount of antibody, antigen or antigen-antibody complex corresponding to the amount of antigen to be measured is detected by chemical or physical means and contains a known amount of antigen Any measurement method can be used as long as it is calculated from For example, a turbidimetric method, a competitive method, an immunoassay method and a sandwich method are appropriately used. With respect to sensitivity and specificity, it is particularly preferred to use the following sandwich method.

In a measurement method using a labeling substance, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used as a labeling factor. Examples of radioactive isotopes include ¹²⁵ I, ¹³¹ I, ³ H and ¹⁴ C. The enzyme is usually made detectable by the binding of an appropriate substrate that then catalyzes a detectable reaction. Examples include, for example, beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidase and malate dehydrogenase, preferably horseradish peroxidase. Luminescent substances include, for example, luminol, luminol derivatives, luciferin, aequorin and luciferase. In addition, avidin-biotin systems can also be used to label the antibodies and immunogens of the present invention.

Thus, as a further aspect, the present invention provides:
a) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48 Contacting a cell expressing at least one protein comprising an amino acid sequence selected from with a test compound; and b) comparing the CRH response activity of the cell with or without the compound. Methods are provided for identifying and obtaining compounds that alter CRH signaling response activity.

Changes in membrane potential can be measured using conventional electrophysiological techniques and, when available, using new high-throughput mass processing methods currently under development. Since changes in membrane potential are usually the result of ionic flow, as an alternative, changes in membrane potential can be obtained from fluo-3, fluo-4, fluo-5N, fura red from suppliers including Molecular Probes. It can be measured indirectly by changes in intracellular ion concentration using ion-sensitive fluorescent dyes, including Sodium Green, SBFI, and other similar probes. DIBAC _{4 (3)} or Di-4-ANEPPS other like, other fluorescent dyes from including suppliers of Molecular Probes can detect the changes in membrane potential. For example, calcium and sodium ion fluxes are thus characterized in real time using fluorescence and fluorescence imaging techniques, including fluorescence microscopy, with or without laser confocal techniques combined with image analysis algorithms be able to.

  In a preferred embodiment, this assay is based on a device called a fluorescence imaging plate reader (FLIPR), Molecular Devices Corporation, which, in its most common form, emits fluorescence emitted by a fluorescein-based dye. Excitation and measurement, which captures the emitted fluorescence by an argon ion laser to provide high power excitation at 488 nm of the fluorophore, an optical system for rapidly scanning the bottom of a 96-384 well plate A highly sensitive cooled CCD camera is used, which also allows the device to deliver a solution of the test agent to the wells of a 96/384 well plate. Also contains a 4-well pipetting head The FLIPR assay is designed to measure fluorescence signals from a population of cells from all 96/384 wells simultaneously, in real time, before, during and after addition of compounds. The

  Accordingly, it is an object of the present invention to provide a FLIPR assay that is used to screen and characterize compounds that are functionally active in modulating a CRH response in a cell, said cell comprising SEQ ID NO: 26, A protein selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 34 is expressed.

  As an alternative, the activity of the cells can be assessed using electrophysiological methods. Thus, proteins that modulate CRH signaling in cells can be characterized using whole cell and single channel electrophysiology.

Accordingly, it is a further object of the present invention to provide a screening method for identifying compounds that modulate CRH signaling response activity in cells, the method comprising:
(A) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48. Contacting a host cell expressing a protein selected from the group with the compound to be tested;
(B) measuring the effect of the test compound on the membrane potential of the cells using electrophysiological techniques; and (c) comparing the CRH response activity of the cells with and without the compound. Alternatively, the host cell in the above screening method expresses a protein selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 34.

  In a preferred embodiment, the host cell is a xenopus oocyte and the electrophysiological measurement consists of measuring membrane current using voltage clamp techniques at a separate membrane potential.

  Changes in the enzyme activity of an enzyme that modulates CRH signaling in the cell can generally be measured or assayed for the catalytic effect the enzyme provides, which is the conversion of its substrate into a reaction product. For example, for kinases, kinase activity can be assessed using a substrate comprising a kinase-specific phosphorylation site and measuring phosphorylation of the substrate. Similarly, for phosphatases, phosphatase activity can be assessed using a phosphorylated substrate and measuring the dephosphorylation of the substrate. These assays can be performed both with and without the compound being tested.

Accordingly, it is an object of the present invention to provide a method for identifying a compound capable of modifying CRH signaling response activity in a cell, said method comprising:
a) contacting a mixture comprising a kinase selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18 with a phosphate source and a suitable kinase substrate;
b) incubating the mixture in the presence or absence of the compound; and measuring the level of phosphorylation of the substrate in the presence of the compound compared to the level of phosphorylation of the substrate in the absence of the test compound Comprising that.

  In the assays of the present invention, the kinase can be provided as a protein or can be provided in the assay mixture as mRNA encoding the kinase. If the assay comprises a cell free component, the kinase is provided as a protein. If the assay is performed in a cellular environment, the kinase can be provided as a protein or mRNA encoding the kinase, where the mRNA is translated and so that the kinase is available in the assay. Produces a kinase protein. It is clear from the examples provided herein that it is simple to obtain mRNA specifying a kinase and inject the mRNA into cells for the production of kinase protein. The kinase can also be provided by expression of a plasmid encoding the kinase protein. Standard molecular biology techniques can be used to construct operable plasmids encoding the kinase protein and to express the plasmid in cells (Sambrook, et al., 1989, Molecular Cloning: A Laboratory). Manual, Cold Spring Harbor Laboratory, New York).

  As described herein, methods for identifying kinase modulators can be performed in vitro when the assay mixture is cell-free, in vitro when live cells are included in the assay, or in vivo in animals. Thus, as one aspect of the present invention, the mixture is contained within a eukaryotic cell and the method of the present invention can be performed, wherein some of the components of the assay mixture are microinjected with the components therein Can be provided exogenously to the cell, and some of the components can be endogenous in the cell.

  The term “endogenous in a cell” as used herein means that the component is naturally produced in the subject cell.

  The term “exogenous to a cell” as used herein means that the component is not naturally present in the subject cell, or is present therein and added to it at a low level.

  When the method of the invention is performed using eukaryotic cells, one or more kinase proteins, kinase substrates and test compounds can be injected into the eukaryotic cells prior to incubation. The cells so injected are then incubated under conditions that promote protein kinase activity, and then the level of protein kinase activity is measured after the incubation period using the assay described herein.

  Eukaryotic cells useful in the methods of the present invention include Xenopus laevis oocytes, Xenopus laevis embryo cells, mammalian cells (such as IOTI / 2 cells), Drosophila melanogaster S2 cells , Any one of Dictyosterium discoideum cells and yeast cells. Even more preferably, the eukaryotic cell is the mouse pituitary corticotroph-derived adenoma cell line cell AtT-20.

  The phosphate source used in the methods of the present invention may be any common phosphate source including, but not limited to, nucleotide triphosphates such as, but not limited to, ATP or GTP. Can be. In a preferred embodiment, the phosphate source has a detectable label attached thereon, which is introduced with the phosphate group to the kinase substrate during the reaction. Thus, a phosphorylated substrate contains a detectable label, whereas a non-phosphorylated substrate can distinguish a phosphorylated kinase substrate from a non-phosphorylated kinase substrate. In another embodiment, the phosphate source does not have a detectable label attached thereon; instead, the phosphorylated kinase substrate recognizes one form of the substrate, eg, by an antibody, while the other Can be distinguished from non-phosphorylated kinase substrates.

  Detectable labels useful in the methods of the invention include any known or previously unknown detectable detectable that is introduced upon introduction of a phosphate group into a kinase substrate as a result of protein kinase activity. Various labels can be included.

Useful labels include, but are not limited to, radioactive labels such as γ ³² P, ³¹ S, and non-radioactive labels such as biotin.

In another aspect, the present invention provides a method for identifying a compound capable of altering CRH signaling response activity in a cell, the method comprising:
a) contacting a mixture comprising a phosphatase selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO: 38 and a suitable phosphorylated substrate;
b) incubating the mixture in the presence or absence of the compound; and measuring the level of phosphorylation of the substrate in the presence of the compound compared to the level of phosphorylation of the substrate in the absence of the test compound Comprising that.

As in the kinase assay, the phosphatase in the assays of the invention can be provided as a protein, or can be provided in the assay mixture as mRNA encoding the phosphatase. The phosphorylated substrate is typically labeled with a detectable phosphate residue. Useful labels include, but are not limited to, radioactive labels such as γ ³² P, ³¹ S, and non-radioactive labels such as biotin. For use in phosphatase activity assay, the substrate, preferably are typically labeled with gamma ^{32 P,} it is phosphorylated by tyrosine or serine residue, consisting of the peptide substrate. In general, phosphorylation can be performed in various ways. Typically, protein tyrosine kinases are used. For example, soluble EGF receptor kinase in combination with ³² P-labeled ATP can be used to phosphorylate tyrosine residues on the peptides of the invention. Such phosphorylation reactions typically proceed for about 2 hours at 30 ° C. or overnight at room temperature. Next, a phosphorylated peptide, hereinafter referred to as “phosphopeptide”, is purified from the phosphorylation reaction mixture. For example, the peptide can be separated from the reaction mixture by the addition of trichloroacetic acid and centrifugation, where the peptide remains in the supernatant. The peptide is generally further purified by column chromatography, for example on C18. The purified phosphopeptide can be lyophilized and stored at -20 ° C prior to use.

  After incubation, phosphopeptides that are not dephosphorylated ("non-dephosphorylated phosphopeptides") are separated from the radioactivity released by dephosphorylation of the phosphopeptide (ie, from free radioactive phosphorus released by dephosphorylation). To do. As used herein, the term “radiophosphorus” includes all forms in which a radioactive phosphorus atom is present on a tyrosine residue and can be removed by dephosphorylation (eg, as a phosphate group). The Typically, separation of non-dephosphorylated phosphopeptides from free radioactive phosphorus liberated by dephosphorylation of the phosphopeptide is achieved by centrifuging after the dephosphorylation reaction has been stopped by the addition of non-radiophosphate and charcoal containing substances. Brought about by separation. Radioactivity in the supernatant is determined by means well known to those skilled in the art. Calculate the phosphatase enzyme activity of the sample being assayed based on the amount of radioactivity initially added to the assay mixture by the phosphopeptide and the amount of radioactivity detected at the end of the assay as the radioactivity released by dephosphorylation can do.

It is also an aspect of the present invention to provide a method for identifying a compound capable of altering CRH signaling response activity in a cell, the method comprising:
a) contacting a cell expressing at least one protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 34 with the test compound. And b) comparing the level of second messengers such as cAMP, cGMP, Ca ²⁺ or IP _{3 in} the cells with and without the compound.

  Second messenger levels can be determined using techniques known in the art in either whole cells or cell extracts comprising one of the above proteins.

  A further method of identifying compounds capable of modifying CRH signaling in a cell is operably linked to a gene promoter or regulatory sequence element characterized in that the gene promoter or regulatory sequence element comprises a transcription factor binding site Based on the use of genes, such as reporter genes, where the transcription factor can regulate CRH signaling in the cell. In a preferred embodiment, the transcription factor capable of regulating CRH signaling in the cell is selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. Accordingly, the present invention provides a recombinant DNA molecule comprising a gene promoter region as defined above. In the recombinant DNA molecule, the promoter region can be operably linked to a nucleic acid molecule encoding a detectable product, such as a reporter gene. The term “operably linked” as used herein means functionally fusing a gene and a promoter in an appropriate frame to express the gene under the control of the promoter. As used herein, the term “reporter gene” refers to a gene product that can be identified using simple inexpensive methods or reagents and operably linked to a promoter region or an active fragment thereof. Means a gene. For example, reporter genes such as firefly luciferase, β-galactosidase, alkaline phosphatase, bacterial chloramphenicol acetyltransferase or green fluorescent protein reporter gene can be used to determine transcriptional activity in the screening assays of the present invention (eg, , Goeddel (ed.), Methods Enzymol., Vol. 185, San Diego: Academic Press, Inc. (1990); see also Sambrook, supra). In a preferred embodiment, the reporter gene is a firefly luciferase gene. The present invention also provides vectors comprising recombinant DNA molecules as defined above, as well as host cells stably transformed with such vectors or generally with the recombinant DNA molecules of the present invention. The term “vector” refers to any carrier of exogenous DNA that is useful for introducing DNA into a host cell for replication and / or proper expression of the exogenous DNA by the host cell. Thus, in a particular embodiment, the vector is an expression vector such as pGL3luc, pBLCAT5 (LMBP2451), pGMCSFlacZ (LMBP2979), pEGFP or pSEAPbasic (DMB3115), where the LMBP and DMB numbers are Belgian Co-ordinated. Refers to the accession number of these expression vectors at Collections of Microorganisms.

In another aspect, the invention provides a method of identifying a compound that modulates CRH signaling activity, the method comprising: (i) contacting a gene promoter region as defined above with a candidate factor; and (Ii) determining whether the candidate factor modulates the expression of a detectable product, such modulation being an indicator of a factor capable of modulating CRH signaling activity . A detectable product refers to either the protein encoded by the gene or the product of a reporter gene such as luciferase, β-galactosidase or green fluorescent protein. Methods for quantifying detectable products are generally known in the art and, in particular, the use of colorimetric substrates when the expressed product is an enzyme, the use of specific antibodies in an RIA or ELISA assay. Or measurement of the level of mRNA transcribed from a gene operably linked to a promoter, where the mRNA can be measured directly or indirectly using standard methods. . Preferably, the gene promoter comprises a transcription factor binding site of a transcription factor selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.
Identification of a nucleic acid sequence encoding a protein capable of modulating CRH signaling In another aspect, the invention relates to an isolated and purified nucleic acid molecule encoding a protein capable of modulating CRH signaling, wherein The nucleic acid molecule is either RNA, DNA, cDNA or genomic DNA.

In particular, the present invention includes:
(A) a nucleic acid molecule encoding a protein that modulates CRH signaling with at least 70% identity to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 48;
(B) a nucleic acid molecule complementary to the polynucleotide of (a);
(C) a nucleic acid molecule comprising at least 15 contiguous bases of the polynucleotide of (a) or (b);
(D) a nucleic acid molecule that hybridizes under stringent conditions to the polynucleotide molecule of (a) or (b); and (e) the nucleotide sequence of any of the polynucleotides of (a)-(d) Included is an isolated and purified nucleic acid molecule comprising a member selected from the group consisting of nucleic acid molecules encoding proteins that modulate CRH signaling comprising nucleotide sequences that are degenerate as a result of the genetic code. Is done.

  Sequence identified as SEQ ID NO: 45, SEQ ID NO: 47 or SEQ ID NO: 49 without altering the identity of the encoded amino acid (s) or protein product due to the degeneracy of the genetic code One skilled in the art will recognize that multiple “silent” substitutions of nucleotide base pairs can be introduced into the. All such substitutions are intended to be within the scope of the present invention.

  In a further aspect, the present invention relates to a human purine permease polynucleotide. Such polynucleotides have at least 70% identity, preferably at least 80%, over the entire length of the amino acid sequence to an amino acid sequence selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 48 Included are isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide having identity, more preferably at least 90% identity, and even more preferably at least 95% identity. In this connection, polypeptides with at least 97% identity are highly preferred, while those with at least 98-99% identity are more highly preferred and those with at least 99% identity are most preferred Highly preferred. Such polynucleotides include a polynucleotide consisting essentially of a polynucleotide sequence selected from SEQ ID NO: 45, SEQ ID NO: 47 or SEQ ID NO: 49.

Thus, as a further aspect, the present invention provides:
a) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferred to an amino acid sequence selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 48 A nucleotide sequence encoding a polypeptide having at least 95% identity, even more preferably at least 97-99% identity;
b) to a polynucleotide selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49, at least 70% identity, preferably at least 80% identity over the entire length of the polynucleotide; Nucleotide sequences having preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 97-99% identity;
c) at least 70% identity, preferably at least 80% identity over the entire coding region of the polynucleotide to a polynucleotide selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49 More preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 97-99% identity; and d) SEQ ID NO: 45, SEQ ID NO: 47 and An isolated polynucleotide comprising a nucleotide sequence consisting of a polynucleotide selected from the group consisting of SEQ ID NO: 49 is provided.

  Polynucleotides as outlined above are provided in particular for use in the screening or diagnostic methods of the invention (in particular for identifying (compounds) capable of modulating CRH signaling in cells or To diagnose altered CRH metabolism in an individual).

  Identity or similarity is between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing sequences, as is known in the art. It is a relationship. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. Both identity and similarity can be easily calculated (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genomic Prot. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Human Press Analysis in Molecular Bi logy, von Heinje, G., Academic Press, 1987;. and Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds, M Stockton Press, New York, 1991). There are numerous ways to measure identity and similarity between two polynucleotides or two polypeptide sequences, but both terms are well known to those skilled in the art (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Math., 48, 1073). Commonly used methods for determining identity or similarity between sequences include Carillo, H. et al. , And Lipman, D.M. (1988) SIAM J. et al. Applied Math. , 48, 1073 are included, but not limited thereto. Preferred methods for determining identity are designed to give the greatest match between the sequences tested. The method for determining identity and similarity is organized in a computer program. Preferred computer program methods for determining identity and similarity between two sequences include the GCG program package (Devereux, J., et al., (1984) Nucleic Acids Research 12 (1), 387), BLASTP. , BLASTN and FASTA (Atschul, SF et al., (1990) J. Molec. Biol. 215, 403), but are not limited thereto.

  Nucleic acid sequences encoding a protein or fragment thereof capable of modulating CRH activity include tissues such as, but not limited to, brain, heart, kidney, pancreas, liver and skin Can be isolated from The sequence can also be isolated from mammals other than humans and mice. Other cells and cell lines may also be suitable for use to isolate mammalian purine permease cDNA. Selection of appropriate cells can be done by screening for CRH modulating activity in cell extracts or in whole cell assays, as described herein. Cells that possess CRH modulating activity in any one of these assays can be suitable for the isolation of purine permease DNA or mRNA.

Any of a variety of methods known in the art can be used for molecular cloning of the DNA encoding the protein of the present invention. As one method, mRNA is isolated and first strand cDNA synthesis is performed. A second round of DNA synthesis can be performed for the generation of the second strand. The isolated cDNA can then be obtained by specific PCR amplification of the DNA fragment by designing degenerate oligonucleotide primers from the purified protein amino acid sequence that modulates CRH signaling. If desired, the double-stranded cDNA can be cloned into any suitable vector, eg, a plasmid, thereby generating a cDNA library. Another method is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, sequence SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 Alternatively, screening a cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labeled oligonucleotide probe targeting any suitable region of SEQ ID NO: 49. See, for example, PCR Protocols: A Guide to Method and Application , Ed. M.M. Innis et al. , Academic Press (1990).

  Methods for constructing cDNA libraries in appropriate vectors such as plasmids or phage for propagation in prokaryotic or eukaryotic cells are well known to those skilled in the art [see, eg, Maniatis et al. See above]. Suitable cloning vectors are well known and widely available.

It will be readily apparent to those skilled in the art that other types of libraries, as well as libraries constructed from other cells or cell types, can be useful for isolating the nucleic acid sequences of the present invention. Other types of libraries include cDNA libraries from other cells, from human and non-mouse organisms, and genomic DNA libraries, including YAC (yeast artificial chromosome) and cosmid libraries, It is not limited to these. Construction of a genomic DNA library can be performed by standard techniques well known in the art. A well-known genomic DNA library construction technique is described in T.W. Maniatis et al. Molecular Cloning: A Laboratory Manual , 2d Ed. Chap. 14 (1989).

  In many cases, the skilled artisan will recognize that the isolated cDNA sequence is incomplete in that the region encoding the polypeptide is missing at the 5 'end of the cDNA. This is inherently low “processivity” (a measure of the ability of an enzyme to remain bound to the template during the polymerization reaction), which fails to complete a DNA copy of the mRNA template during first strand cDNA synthesis. The result of reverse transcriptase, which is an enzyme having

Several methods available and well known to those skilled in the art for obtaining full-length cDNAs or extending short cDNAs, such as the rapid amplification of cDNA ends (RACE) (Frohman et al., 1988, PNAS USA 85, 8998-9002), or based on recent modifications of this technology, exemplified by Marathon ^™ technology (Clontech Laboratories Inc.).

  In order to clone a polynucleotide encoding the protein of the present invention by the above method, the amino acid sequence of the polypeptide encoded by the nucleic acid sequence may be necessary. In order to do this, the protein of the invention can be purified and the partial amino acid sequence can be determined by an automatic sequenator. Although it is not necessary to determine the entire amino acid sequence, a linear sequence of two regions of 6-8 amino acids from the protein is determined for the production of primers for PCR amplification of partial DNA fragments.

Once suitable amino acid sequences are identified, DNA sequences that can encode them are synthesized. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid, and therefore the amino acid sequence must be encoded by one of a set of similar DNA oligonucleotides. Can do. Only one member of the set is identical to the polynucleotide sequence of the present invention and can hybridize to DNA encoding the desired protein even in the presence of mismatched DNA oligonucleotides. DNA isolated by these methods can be used to screen DNA libraries from invertebrate and vertebrate sources from various cell types and to isolate homologous genes.
Polypeptides In a further aspect, the present invention relates to a substantially pure form of a polypeptide that modulates CRH signaling, wherein the polypeptide is encoded by an isolated and purified nucleic acid molecule of the present invention. . In a preferred embodiment, the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, and functional analogs thereof.

  The proteins of the present invention include all possible amino acid variants, including the polypeptides encoded by the molecules encoded by the nucleic acids of the present invention and having conservative amino acid changes.

Those skilled in the art will recognize that proteins that modulate CRH signaling can be obtained by multiple recombinant DNA techniques including, for example, hybridization, polymerase chain reaction (PCR) amplification, or de novo DNA synthesis (eg, T. Maniatis et al. al. Molecular Cloning: A Laboratory Manual , 2d Ed. Chap. 14 (1989)).

  Purified biologically active proteins that modulate CRH signaling can have several different physical forms. The polypeptides of the present invention can exist as full-length nascent or unprocessed polypeptides, or as partially processed polypeptides or combinations of processed polypeptides. Full-length nascent polypeptides can be post-translationally modified by, inter alia, specific proteolytic cleavage events that result in the formation of fragments of the full-length nascent polypeptide. The physical association of a fragment, or multiple fragments, can have full biological activity associated with the protein of the invention; however, the degree of CRH modulating activity can vary between individual fragments.

Also preferred in this aspect of the invention are fragments characterized by the structural or functional properties of the polypeptide. In this respect, preferred embodiments of the present invention include α-helix and α-helix forming region, β-sheet and β-sheet-forming region, turn and turn-forming region, coil and coil-forming region, hydrophilic region, hydrophobicity of the polypeptide of the present invention. Includes fragments comprising a neutral region, an α amphipathic region, a β amphipathic region, a flexible region, a surface forming region, a substrate binding region, a high antigenic index region, and combinations of such fragments Is done. Preferred regions are those that mediate the activity of the polypeptides of the invention. Most highly preferred in this regard are chemical, biological, or chemical of the response modulator polypeptides of the present invention, including those with similar or improved activity, or with reduced undesirable activity. Fragments with other activities.
Recombinant expression of a polynucleotide encoding a protein that modulates CRH activity Alternatively, the polynucleotide of the invention can be recombinantly produced by molecular cloning into an expression vector containing a suitable promoter and other suitable transcriptional regulatory elements. It can be introduced into prokaryotic or eukaryotic host cells to produce proteins that are expressed and modulate CRH signaling. Such manipulation techniques are described in Maniatis, T .; et al. , Fully described above and well known in the art.

  Thus, as a further aspect, the invention provides an expression vector for expression of a protein that regulates CRH signaling in a recombinant host, wherein the vector comprises a nucleic acid sequence encoding a protein that regulates CRH signaling and its Contains functional analogs. In a further preferred embodiment of the present invention, the expression vector comprises a protein that regulates CRH signaling, having a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, and functional analogs thereof. Or a genomic DNA encoding a protein that modulates CRH signaling.

Expression vectors are defined herein as DNA sequences required for transcription of cloned copies of genes and translation of their mRNAs in a suitable host. Such vectors are true in a variety of hosts such as bacteria, including E. coli , cyanobacteria, plant cells, insect cells, amphibian cells, fungal cells, including yeast cells, and animal cells. It can be used to express nuclear genes.

  Specially designed vectors allow reciprocation of DNA between hosts such as bacteria-yeast or bacteria-animal cells or bacteria-fungal cells or bacteria-invertebrate cells. A properly constructed expression vector: contains an origin of replication for autonomous replication in the host cell, a selectable marker, a limited number of useful restriction enzyme sites, a high copy number potential, and an active promoter Can do. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and to initiate RNA synthesis. A strong promoter is one that initiates mRNA at a high frequency. Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.

  An isolated and purified nucleic acid molecule of the invention that encodes a protein that modulates CRH signaling can be cloned into an expression vector for expression in a recombinant host cell. Recombinant host cells include cell lines derived from bacteria such as Escherichia coli, fungal cells such as yeast, amphibian cells such as xenopus oocytes, humans, cows, pigs, monkeys and rodents. Prokaryotic organisms, including but not limited to mammalian cells, and insect cells including but not limited to cell lines derived from Drosophila and silkworm Or it can be eukaryotic. Cell lines derived from mammalian species that may be suitable and commercially available include CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1. (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH / 3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CCL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), L cells, neuroblastoma, glial cells and HEK-293 (ATCC CRL 1573) are included, but are not limited to these.

  Accordingly, in a further aspect, the present invention relates to a recombinant host cell containing a recombinantly cloned nucleic acid molecule encoding a protein that modulates CRH signaling or a functional analog thereof. In a further embodiment, the recombinant host cell of the invention is genomic DNA or is selected from the group consisting of (SEQ ID NO: 45); (SEQ ID NO: 47); (SEQ ID NO: 49); and functional analogs thereof. A nucleic acid molecule having a nucleotide sequence.

  Expression vectors can be introduced into host cells by any one of numerous techniques including, but not limited to, transformation, transfection, protoplast fusion, lipofection and electroporation. Cells containing the expression vector are expanded clonally and analyzed to determine if they produce a protein that modulates CRH signaling. Identification of host cell clones that express permease includes, but is not limited to, immunological reactivity with antibodies to polypeptides of the invention and the presence of mammalian purine permease activity associated with the host cell. Not, it can be done by several means.

  Accordingly, the present invention also includes culturing a host cell of the invention under conditions that allow expression of a protein that modulates CRH signaling from an expression vector as outlined herein. It also relates to methods of expression of proteins that modulate CRH signaling in cells. The protein of the invention can be synthesized as a fusion protein comprising a protein of interest as a translational fusion with another protein or peptide that can be removed by direct expression or by self, enzymatic or chemical cleavage. it can. Thus, as a particular aspect, the present invention provides a protein of the invention, wherein the polypeptide is part of a fusion protein.

Expression as a fusion protein is often found in the production of certain peptides in recombinant systems to extend life span, increase the yield of the desired peptide, or provide a convenient means of purifying the protein. . This is especially true when expressing mammalian proteins in prokaryotic hosts. Various peptidases are known that cleave polypeptides at specific sites (eg, enterokinase and thrombin) or digest peptides from the amino or carboxy terminus of a peptide chain (eg, diaminopeptidase). In addition, certain chemicals (eg, cyanogen bromide) cleave polypeptide chains at specific sites. Those skilled in the art will recognize the necessary modifications to the amino acid sequence (and the synthetic or semi-synthetic coding sequence if recombinant means are used) to introduce site-specific internal cleavage sites. For example, P.I. Carter, "Site Specific Proteolysis of Fusion Proteins", Chapter 13, Protein Purification: From Molecular Mechanisms to Large Scale Processes, American Chemical Society, Washington, D. C. (1990).

  Further, for example, using a mammalian cell that already contains in its genome a nucleic acid molecule that encodes a protein that regulates CRH signaling as described above, but does not express it or does not express properly, eg due to a weak promoter, In order to induce its expression, a regulatory sequence such as a strong promoter can be introduced into the mammalian cell in the immediate vicinity of the endogenous nucleic acid molecule encoding the purine permease polypeptide.

  As such, recombinant host cells containing a polynucleotide encoding a protein that modulates CRH signaling under the control of heterologous transcriptional and / or regulatory sequences or proteins are another aspect of the invention.

  In this context, the term “regulatory sequence” is used to increase the expression of purine permease polypeptide due to its integration into the cell's genome in the immediate vicinity of the gene encoding the CRH regulatory protein. Means a nucleic acid molecule capable of Such regulatory sequences induce expression of genes encoding promoters, enhancers, inactivating silencer intron sequences, 3′UTR and / or 5′UTR coding regions, proteins and / or RNA stabilizing elements, CRH regulatory proteins. Regulatory proteins that can make or cause, such as nucleic acid molecules encoding transcription factors or other gene expression control elements known to activate gene expression and / or increase the amount of gene product. The introduction of the regulatory sequence results in increased expression and / or induction of a polypeptide that modulates CRH signaling, resulting in an increased amount of the polypeptide in the cell. Accordingly, the present invention aims to provide de novo and / or increased expression of polypeptides that modulate CRH signaling.

  Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in numerous standard laboratory manuals such as Davis, Basic Methods In Molecular Biology (1986). In particular, it is believed that polypeptides that modulate CRH signaling can actually be expressed by host cells that lack recombinant vectors.

Furthermore, the expression of the polynucleotide of the present invention can also be performed using a synthetic mRNA produced in vitro. Synthetic mRNA or mRNA isolated from cells capable of modulating CRH signaling is efficient in a variety of cell-free systems including, but not limited to, wheat germ extract and reticulocyte extract. Microinjection into frog oocytes that can be translated and efficiently translated in cell-based systems, including but not limited to, microinjection into frog oocytes Is generally preferred.
Transgenic non-human animals The present invention also includes a transgenic non-human animal, preferably transgenic, comprising introduction of a polynucleotide or vector of the present invention into germ cells, embryonic cells, stem cells or eggs or cells derived therefrom. It also relates to a method of manufacturing a mouse. The non-human animal can be used according to the screening methods of the invention described herein and can be a non-transgenic healthy animal or has a phosphate uptake or reabsorption disorder, preferably CRH signaling. It may have a disorder due to at least one mutation in the modulating protein. Such transgenic animals are well suited for drug pharmacological studies, for example in connection with mutant forms of the above polypeptides. Production of transgenic embryos and their screening are described in, for example, A. L. Joyner Ed. , Gene Targeting, A Practical Approach (1993), Oxford University Press. Embryonic embryo membrane DNA can be analyzed, for example, using Southern blots with appropriate probes; see above.

  Preferably, the transgenic non-human animals of the invention further comprise at least one inactivated wild type allele of the gene encoding the corresponding mammalian CRH regulatory protein; see above. This embodiment allows for the study of the interaction of various mutant forms of the polypeptides of the invention, for example with respect to the development of clinical symptoms of diseases associated with impaired CRH metabolism. All applications described above with respect to transgenic animals also apply to animals carrying 2, 3 or more transgenes (eg, encoding neutral endopeptidase (NEP)). It may also be desirable to inactivate proteins that modulate CRH signaling expression or function at some stage in the development and / or life of a transgenic animal. This can be accomplished, for example, by using a tissue-specific, developmental and / or cell-regulatory and / or inducible promoter that directs the expression of antisense or ribozymes to RNA transcripts that encode proteins capable of regulating CRH signaling. As above; see also above. Suitable induction systems are described, for example, by Gossen and Bujard (Proc. Natl. Acad. Sci. 89 USA (1992), 5547-5551) and Gossen et al. For example, tetracycline-regulated gene expression as described by (Trends Biotech. 12 (1994), 58-62). Similarly, the expression of mutant proteins that modulate CRH signaling can be controlled by such regulatory elements.

  Furthermore, the present invention also relates to a transgenic mammalian cell containing the nucleic acid molecule of the invention or part thereof (preferably stably integrated into its genome), wherein the transcription of the nucleic acid molecule or part thereof. And / or expression results in a decrease in the synthesis of proteins that modulate CRH signaling.

  In a preferred embodiment, the reduction is effected by antisense, sense, ribozyme, co-suppression and / or dominant mutant effects. “Antisense” and “antisense nucleotide” refer to a DNA or RNA construct that prevents expression of a naturally occurring gene product.

  The provision of the polynucleotides of the present invention opens up the possibility of producing transgenic non-human animals having reduced levels of proteins as described above and thus having a defect in phosphate metabolism. Techniques for how to accomplish this are well known to those skilled in the art. These include, for example, expression of antisense RNA, ribozymes, molecules that combine antisense and ribozyme functions, and / or molecules that provide a co-suppressive effect; see also above. When using antisense methods to reduce the amount of protein that modulates CRH signaling in a cell, the nucleic acid molecule encoding the antisense RNA is preferably of homologous origin with respect to the animal species used for transformation. However, it is also possible to use nucleic acid molecules that exhibit a high degree of homology to endogenous nucleic acid molecules that encode proteins that modulate CRH signaling. In this case, the homology is preferably higher than 80%, in particular higher than 90% and even more preferably higher than 95%. Reduced synthesis of the protein of the invention in transgenic mammalian cells can result in, for example, alteration of adenine reabsorption. In transgenic animals comprising such cells, this can result in various physiological, developmental and / or morphological changes.

Accordingly, the present invention also relates to a transgenic non-human animal comprising the above-described transgenic cell. These include, for example, the following features:
(A) disruption of the endogenous gene (s) encoding a protein capable of modulating CRH signaling;
(B) expression of at least (one) antisense RNA and / or ribozyme against a transcript comprising a polynucleotide of the invention;
(C) sense of the polynucleotide of the invention and / or expression of untranslated mRNA;
(D) expression of the antibody of the present invention;
(E) introduction of a functional or non-functional copy of a regulatory sequence of the invention; or (f) the presence of a stable or transient presence of foreign DNA resulting in at least one of introduction of a recombinant DNA molecule or vector of the invention. Therefore, it can show a defect in CRH metabolism compared to wild type animals.

  Using the polypeptides of the invention, their encoding polynucleotides and vectors, it is now possible to study the efficacy of drugs in vivo and in vitro for specific mutations in proteins that modulate CRH signaling of patients and affected phenotypes. Is possible. Furthermore, mutant forms of the polypeptides of the invention may be used to determine the pharmacological profile of a drug that may be effective in treating disorders associated with CRH metabolism, particularly in ameliorating CRH-induced stress or depression. It can then be used for further drug identification and manufacture.

Accordingly, the present invention also includes administering to a mammalian subject a therapeutically effective amount of a polypeptide, polynucleotide or vector encoding a protein capable of modulating CRH signaling of the present invention. It is also understood to relate to methods of preventing, treating or ameliorating conditions associated with disorders of CRH metabolism, including body related disorders.
Diagnostic Assays The present invention further relates to the use of the polynucleotides of the present invention as diagnostic reagents. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 21 SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 Alternatively, detection of a mutant form of a gene characterized by the polynucleotide of SEQ ID NO: 49 is susceptible to a disease or disorder resulting from insufficient expression, overexpression or altered spatial or temporal expression of the gene A diagnostic means is provided which can add or specify a diagnosis of the length. Individuals carrying mutations in the gene can be detected at the DNA level by various techniques.

Accordingly, the present invention provides:
(A) determining the presence or absence of a mutation in the polynucleotide of the present invention; and (b) diagnosing a pathological condition or susceptibility to the pathological condition based on the presence or absence of the mutation. It is understood that it provides a method of diagnosing a pathological condition or susceptibility to a pathological condition in a subject associated with a disorder of CRH activity. Nucleic acids for diagnosis can be obtained from a subject's cells, such as blood, urine, saliva, tissue biopsy or autopsy material. Genomic DNA can be used directly for detection or can be amplified enzymatically by using PCR or other amplification techniques prior to analysis. RNA or cDNA can also be used as well. Deletions and insertions can be detected by a change in the size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled mammalian purine permease nucleotide sequences. Preferably, matching sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting points. DNA sequence differences can also be detected by the presence or absence of denaturing agents, by modification of electrophoretic mobility of DNA fragments in capillary electrophoresis columns or gels, or by direct DNA sequencing (see, for example, Myers et al., Science). (1985) 230: 1242). Sequence changes at specific positions can also be demonstrated by nuclease protection assays such as specific restriction endonucleases, RNases and S1 protection or chemical cleavage methods (Cotton et al., Proc Natl Acad Sci USA (1985). 85: 4397-4401). In another embodiment, an array of oligonucleotide probes comprising a nucleotide sequence encoding a protein capable of modulating CRH activity or a fragment thereof can be constructed, for example, for efficient screening of gene mutations. . Array technology methods are well known, have general applicability, and can be used to address various problems in molecular genetics, including gene expression, gene linkage, and gene variability (eg: M. Chee et al., Science, Vol 274, pp 610-613 (1996))).

  Diagnostic assays provide a method of diagnosing or determining disease susceptibility by detecting mutations in genes encoding CRH regulatory proteins according to the methods described. Further, such diseases may be detected by a method comprising determining an abnormally decreased or increased level of polypeptide or mRNA from a sample obtained from a subject, as well as from the sample as compared to normal structure. Diagnosis can be made by determining the presence of the derivative. Decreased or increased expression is known in the art for polynucleotide quantification, such as; for example; nucleic acid amplification (eg, by PCR, RT-PCR); RNase protection; Northern blotting and other hybridization methods. It can be measured at the RNA level using any of the well-known methods. Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays. Assay techniques that can be used to determine the presence of a protein derivative or variant comprise, among other things, mass spectrometry.

Thus, as another aspect, the present invention provides:
(A) determining the presence or expression level of a polypeptide of the invention or a derivative thereof in a biological sample; and (b) pathological symptoms based on the presence or level of expression of the polypeptide or derivative thereof. Alternatively, a method of diagnosing susceptibility to a pathological condition or symptom in a subject associated with a disorder of prolonged CRH exposure comprising diagnosing susceptibility to a pathological condition is provided.

In particular, the present invention provides a method of diagnosing a CRH-induced gene expression profile in an individual, the method comprising:
a) obtaining a biological sample of the individual; and b) determining the amount of at least one protein that modulates corticotropin releasing hormone (CRH) signaling in the biological sample;
(Here, proteins that regulate corticotropin releasing hormone (CRH) signaling are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, (Selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48)
Comprising. In an alternative embodiment, the method of diagnosing a CRH-induced expression profile is not limited to at least one protein of the present invention, but is a protein identified as being involved in CRH signaling, ie, SEQ ID NO: 2, SEQ ID NO: 4 SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: Expression level of a group of proteins having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48. Requires simultaneous evaluation.

  Preferably, the amount of the protein is preferably at the protein level, preferably using antibodies that bind to it, or preferably SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, Sequence number 14, Sequence number 16, Sequence number 18, Sequence number 20, Sequence number 22, Sequence number 24, Sequence number 26, Sequence number 28, Sequence number 30, Sequence number 32, Sequence number 34, Sequence number 36, Sequence number 38, determined at the level of gene transcription using a probe that binds to a polynucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48 . In the simultaneous assessment of a group of proteins involved in CRH signaling, the level of gene expression is analyzed using microarray technology.

  Alternatively, the CRH-inducible gene expression profile is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 43 It is determined by evaluating the level of gene transcription of a gene comprising a nucleic acid sequence selected from the group consisting of No. 45, SEQ ID No. 47 or SEQ ID No. 49. Methods for determining the level of gene transcription have been described above, and preferred embodiments are SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39 Use of a probe that binds, preferably selectively binds to a polynucleotide selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 or SEQ ID NO: 49 or complements thereof. Become. In another embodiment, an array of oligonucleotide probes comprising a nucleotide sequence of the present invention or a fragment thereof can be constructed for efficient screening of the level of gene transcription in a sample of an individual.

In a further aspect, the present invention provides:
(A) the nucleotide sequence of the polynucleotide of the present invention, preferably SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 or a fragment thereof;
(B) a nucleotide sequence complementary to that of (a);
(C) a polypeptide of the invention, preferably SEQ ID NO: 46, sequence code: 48 or a fragment thereof; or (d) against a polypeptide of the invention, preferably SEQ ID NO: 46 or SEQ ID NO: It relates to a diagnostic kit comprising antibodies against 48 polypeptides and optionally suitable means for detection.

  In any such kit, it is understood that (a), (b), (c) or (d) can comprise a substantial component. Such kits are useful in diagnosing diseases or susceptibility to diseases, particularly CRH metabolism related disorders such as CRH-induced stress or depression.

  The nucleotide sequences of the present invention are also useful for chromosomal location confirmation. These sequences can specifically target and hybridize to specific locations on individual human chromosomes. The mapping of related sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene related diseases. Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data is, for example, V.I. McKusick, Mendelian Inheritance in Man (available online through the Johns Hopkins University Welch Medical Library). Next, the relationship between the gene mapped to the same chromosomal region and the disease is identified by linkage analysis (co-inheritance of physically adjacent genes). The gene of the present invention maps to human chromosome 15.

  Differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If the mutation is found in some or all of the affected individuals but not in normal individuals, the mutation is likely a causative factor of the disease.

  The nucleotide sequences of the present invention are also useful for tissue localization. Such techniques allow the determination of the expression pattern of the polypeptides of the invention in tissues by detection of the mRNA that encodes them. These techniques include in situ hybridization techniques and nucleotide amplification techniques such as PCR. Such techniques are well known in the art. The results from these studies provide an indication of the normal function of the polypeptide in the organism.

  The polypeptides of the invention or fragments thereof or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the polypeptides of the invention. The term “immunospecific” means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.

  Thus, as another aspect, the present invention provides monospecific antibodies that are immunologically reactive with mammalian purine permeases. In a preferred embodiment, the antibody is immunologically reactive with a polypeptide having an amino acid sequence selected from the group consisting of (SEQ ID NO: 46); (SEQ ID NO: 48); and functional analogs thereof, or The antibody inhibits the activity of a protein that modulates CRH signaling.

  Antibodies raised against the polypeptides of the present invention are obtained by administering the polypeptide or epitope-bearing fragment, analog, or cells expressing them to animals, preferably non-human animals, using routine protocols. be able to. For production of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. Examples include hybridoma technology (Kohler, G. and Milstein, C., Nature (1975) 256: 495-497), trioma technology, human B cell hybridoma technology (Kozbor et al., Immunology Today (1983) 4:72). ) And EBV-hybridoma technology (Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

  Techniques for the production of single chain antibodies, such as those described in US Pat. No. 4,946,778, can also be adapted to produce single chain antibodies to the polypeptides of the invention. it can. Also, transgenic mice or other organisms including other mammals can be used to express humanized antibodies.

  The above antibodies can be used to isolate or identify clones expressing the polypeptide or to purify the polypeptide by affinity chromatography.

  Antibodies against the polypeptides of the invention can also be used to treat CRH metabolism-related disorders.

In a further aspect, the present invention comprises the polypeptide of the present invention or a fragment thereof, and various portions of the constant region of the immunoglobulin heavy or light chain of various subclasses (IgG, IgM, IgD, IgE). It relates to a soluble fusion protein produced by genetic engineering. Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgG I, in which case the fusion takes place in the hinge region. In a particular embodiment, the Fc portion can be easily removed by introduction of a cleavage sequence that can be cleaved by, for example, blood coagulation factor Xa. Furthermore, the invention relates to a process for the production of these fusion proteins by genetic engineering and to their use for drug screening, diagnosis and therapy. A further aspect of the invention also relates to a polynucleotide encoding such a fusion protein. Examples of fusion protein technology can be found in International Patent Applications Nos. WO94 / 29458 and WO94 / 22914.
Therapeutic Utility A further aspect of the present invention relates to an immunological / vaccine formulation (composition) that, when introduced into a mammalian host, induces an immunological response in said mammal against said polypeptide of the present invention, wherein The composition comprises a polypeptide or polynucleotide of the present invention. The vaccine formulation can further comprise a suitable carrier. Since the polypeptide can be broken down in the stomach, it is preferably administered parenterally (eg subcutaneous, intramuscular, intravenous or intradermal injection). Formulations suitable for parenteral administration include aqueous or non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the recipient; and precipitation Aqueous and non-aqueous sterile suspensions can be included that can contain inhibitors or thickeners. The formulations can be given in unit-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a lyophilized state requiring only the addition of a sterile liquid carrier just prior to use. Vaccine formulations can also include adjuvant systems to increase the immunogenicity of the formulation, such as oil-in-water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

  In yet another aspect, expression of a gene encoding a protein that modulates CRH signaling can be inhibited using expression blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or administered externally (eg, O'Connor, J. Neurochem (1991) 56: 560; Oligodeoxynucleotides as (See Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Alternatively, oligonucleotides can be provided that form a triple helix ("triplex") with the gene (eg, Lee et al., Nucleic Acids Res (1979) 6: 3703; Cooney et al., Science (1988)). ) 241: 456; see Dervan et al., Science (1991) 251: 1360). These oligomers can be administered per se or the relevant oligomers can be expressed in vivo. A synthetic antisense or triplex oligonucleotide can comprise a modified base or a modified backbone. Examples of the latter include methyl phosphonate, phosphorothioate or peptide nucleic acid backbone. Such backbones are introduced into antisense or triplex oligonucleotides to provide protection from nuclease degradation and are well known in the art. Antisense and triplex molecules synthesized with these and / or other modified backbones also form part of the present invention.

  Another method for inhibiting the expression of a target gene in a cell is to introduce RNA having partial or fully double-stranded characteristics into the cell or into the extracellular environment. Inhibition is specific in that it selects a nucleotide sequence from a portion of the target gene to produce an inhibitory RNA. The RNA can comprise one or more strands of polymerized ribonucleotides; it can comprise modifications to either the phosphate-sugar backbone or the nucleoside. A double-stranded structure can be formed by a single self-complementary RNA strand or two complementary strands. Inhibition is sequence specific in that the nucleotide sequence corresponding to the double stranded region of RNA is a target for gene inhibition. RNA containing a nucleotide sequence that is identical to a portion of the target sequence is preferred. An example of RNA inhibition technology can be found in international patent application WO 99/32619.

  Furthermore, the expression of proteins that regulate CRH signaling can be inhibited by using ribozymes specific for the mRNA sequence encoding the protein. Ribozymes are catalytically active RNAs that are natural or synthetic (see, eg, Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6 (4), 527-33). Synthetic ribozymes are designed to specifically cleave the above mRNA at selected positions, thereby preventing translation of the mRNA into a functional polypeptide. Ribozymes can be synthesized with a natural ribose phosphate backbone and natural bases, as normally present in RNA molecules. Alternatively, ribozymes can be synthesized with a non-natural backbone to provide protection from ribonuclease degradation (eg 2'-O-methyl RNA) and can contain modified bases.

  Several methods are also available for treating abnormal symptoms associated with insufficient expression of proteins that modulate CRH signaling. One method involves administering to a subject a therapeutically effective amount of a compound that activates a polypeptide of the invention, ie, an agonist as described above, in combination with a pharmaceutically acceptable carrier. To alleviate abnormal symptoms. Alternatively, gene therapy can be used to effect endogenous production of mammalian purine permease by associated cells in a subject. For example, the polynucleotides of the invention can be designed for expression in replication defective retroviral vectors, as described above. A retroviral expression vector containing RNA encoding the polypeptide of the invention is then isolated and the packaging cell now produces infectious viral particles containing the gene of interest. Can be introduced into the packaging cells transduced in. These production cells can be administered to a subject for in vivo cell design and expression of the polypeptide in vivo. For an overview of gene therapy, see Chapter 20, Gene Therapies and other Molecular Genetics-based Therapeutic Approaches (and references cited therein), Human Molecular Genetics, and T Strach. reference. Another method is to administer a therapeutic amount of a polypeptide of the invention in combination with a suitable pharmaceutical carrier.

  In a further aspect, the present invention relates to therapeutically treating a polypeptide, agonist / antagonist peptide or small molecule compound, such as a soluble form of the polypeptide of the present invention, in combination with a pharmaceutically acceptable carrier or excipient. Pharmaceutical compositions comprising an effective amount are provided. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The present invention further relates to pharmaceutical packs and kits comprising one or more containers packed with one or more of the components of the above-described composition of the present invention. The polypeptides and other compounds of the invention can be used alone or in conjunction with other compounds such as therapeutic compounds.

  The composition is adapted to the route of administration, eg by systemic or oral route. Preferred forms of systemic administration include injection, typically by intravenous injection. Other injection routes such as subcutaneous, intramuscular or intraperitoneal can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acid or other solvents. Furthermore, oral administration may also be possible if the polypeptide of the invention or other compound can be formulated into an enteric or encapsulated formulation. Administration of these compounds can also be topical and / or local in the form of patches, ointments, plasters, gels and the like.

  The required dosage range will depend on the choice of the peptide or other compound of the invention, the route of administration, the nature of the formulation, the nature of the subject's symptoms, and the judgment of the attending physician. However, suitable dosages are in the range of 0.1-100 μg / kg of subject. However, a wide variation in the required dosage is expected given the diversity of available compounds and the different efficiencies of the various routes of administration. For example, oral administration is expected to require a higher dosage than administration by intravenous injection. These dosage level variations can be adjusted using standard laboratory routines for optimization, as is well understood in the art.

  Polypeptides used for treatment can also be made endogenously in a subject in a therapy often referred to as “gene therapy” as described above. Thus, for example, cells from a subject can be engineered with a polynucleotide, such as DNA or RNA, to encode the polypeptide ex vivo and, for example, by use of a retroviral plasmid vector. These cells are then introduced into the subject.

The present invention will be better understood by reference to the following experimental details, which will be understood by those of ordinary skill in the art, which are merely illustrative of the invention as more fully described in the claims that follow. Is easy to understand. Furthermore, various publications are cited throughout this application. The disclosures of these publications are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Experimental method Cell culture and sample preparation-AtT-20 cells were purchased from ATCC and Dulbecco's modification containing 10% fetal calf serum, 5% horse serum and 4.5 g / L D-glucose Maintained at 37 ° C. in 5% CO ₂ in humidified air in Eagle medium (Invitrogen Life Technologies). For experiments, cells were seeded in 25 cm ² flasks. The medium was changed after 48 hours and the cells were replaced with 0, 0 in either 0.1% DMSO, 1 μM CRH (Sigma) in DMSO, 1 μM R121919 in DMSO or 1 μM CRH + 1 μM R121919 in DMSO. Processed for 5, 1, 2, 4, 8 and 24 hours. Incubation was stopped by aspirating the incubation medium and adding 3 ml Trizol (Invitrogen Life Technologies) for cell lysis. Total RNA was extracted with Trizol according to manufacturer's instructions. 100 μg of total RNA was further purified on the column with DNase I treatment using the Rneasy kit (Qiagen).
Microarray hybridization-cRNA was prepared as follows. Reverse transcription was performed for 1 hour at 42 ° C. with 10 μg of total RNA using T7-oligo (dT) ₂₄ primer and Superscript II RT (Invitrogen Life Technologies). Second strand cDNA synthesis was performed at 16 ° C. for 2 hours using Escherichia coli DNA polymerase I, DNA ligase and RNase H (Invitrogen Life Technologies). Following phenol-chloroform extraction using a phase-locked gel (Eppendorf), in vitro transcription was performed at 37 ° C. for 6 hours using a Bioarray high yield RNA transcript labeling kit (Enzo Diagnostics) with biotin-labeled ribonucleotides. cRNA samples were purified on Qiagen Rneasy columns followed by fragmentation at 95 ° C. for 35 minutes. cRNA yield was between 50-100 μg. Samples were processed on GeneChips (Affymetrix, Santa Clara, CA). To examine the quality of each sample, 5 μg of labeled cRNA was performed on the Test2 array. The actual experiment was performed on a mouse genome U74Av2 array containing probe sets that probe approximately 12,000 full-length mouse genes and EST clusters from the UniGene database (Build 74). Hybridization was performed with 15 μg cRNA for 16 hours at 45 ° C. under continuous rotation. Arrays were stained with streptavidin / phycoerythrin (SAPE) at the Affymetrix fluidics station, followed by anti-streptavidin antibody and secondary SAPE staining. The array was then scanned with a HP-laser scanner and the data analyzed with Microarray Suite software (Affymetrix). Neither scaling nor normalization was done at this stage. The quality of the experiment was evaluated based on the present call rate across all samples, which averaged 47.06 ± 2.45%. The cytoplasmic β-actin and GAPDH 5 ′ / 3 ′ ratios were 1.10 ± 0.08 and 0.93 ± 0.05, respectively.
Data analysis and gene selection Raw intensities on each array were aligned using the following global average algorithm:

Basically, this alignment defines the average intensity of one array relative to the average measured across all arrays, corrects for each array variation in hybridization, washing and staining, and finally a reasonable comparison between the arrays. Enable. The data polished after alignment was analyzed using weighted spectral mapping. Weighted spectral mapping is an unsupervised multivariate analysis method that includes double-centering of data combined with specialized visualizations representing the two highest principal components. Double centering removes the “size” component of the array data, but this information is reintroduced in the visualization by the area of the symbol representing the size of each sample and gene. This method allows for the reduction of large microarray datasets and provides a means to visually examine and thereby identify genes and / or clusters of interest in the data (7). Further detailed analysis was performed using the OmniViz program. All records that were not present in all experiments were excluded and all signals less than 20 were set to 20. The gene expression fold difference between CRH, R121919 and CRH + R121919 treatment was calculated at each time point. These calculations used the signal at the corresponding time point in the DMSO-treated sample to calculate the ratio.
Quantitative RT-PCR-microarray data was verified using real-time PCR analysis. First strand cDNA synthesis was performed with 0.5 μg of total RNA using random hexamer primers and Superscript II RT (Invitrogen Life Technologies). Quantitative PCR was performed on an ABIPrism 7700 cycler (Applied Biosystems) using a Taqman PCR kit. Generate standard curve of threshold cycle for logarithm of concentrations of β-actin, c-fos, Crh-R1, Crh-R2, Rgs2 and gene of interest (see Table 2 for sequences) using serial dilutions of cDNA did. A linear regression line calculated from a standard curve allowed determination of transcript levels in RNA samples from different time points.
Results The transcriptional response to CRH was studied in a mouse AtT-20 pituitary corticotroph derived adenoma cell line expressing CRH-R1. CRH-R1 was readily detectable by both real-time quantitative RT-PCR (RTq) and Western blot, but CRH-R2 expression could not be observed in AtT-20 cells. To identify CRH-R1 specific responses, cells were exposed to 1 μM CRH in the presence of 1 μM CRH, 1 μM CRH-R1 specific antagonist R121919, and to R121919 only. The transcriptional response was followed over time up to 24 hours after the first dose. To assess treatment effects, c-fos mRNA levels were determined by RTq with RNA from different treatments and time points before performing array experiments. Consistent with previous reports, exposure to CRH caused a temporary surge in c-fos transcription (see Figure 1) (8; 9), with levels already falling after 0.5-1 hour. This response was almost completely suppressed in the presence of R121919. Interestingly, c-fos expression levels induced by 0.1% DMSO were, however, 5-10 fold lower compared to expression induced by CRH.

  All time points were analyzed on a microarray containing about 12000 mouse genes and ESTs. Global analysis of expression profiles using spectral mapping (so-called unmonitored methods) suggested a time of progression that accounted for most of the observed changes in gene expression (see FIG. 2). Metabolite accumulation and progressive cell culture are additional factors, but cell cycle synchronization in these cultures induced by the addition of fresh media and serum may explain this phenomenon . Spectral map analysis also shows that the CRH-treated sample differs from the other samples mainly at the initial time point (from 0.5 hours to 2 hours), and the overall difference in expression is very small after 8 hours. Indicated. Because of this obvious effect of time, expression measurements were analyzed against those found at the corresponding time points of DMSO treated control samples. A regulated gene was defined as showing more than twice the change in transcript level at any one time point. The OmniViz Treescape view was used to select 111 genes that met this criterion that showed differential expression after treatment with CRH compared to treatment with antagonists. Twenty-six of the 111 genes were “early responders” that already showed a 2-fold change after 30 minutes of treatment with CRH. Thirty-two genes were “intermediate responders” that responded 1-2 hours after treatment, and 53 genes were “late responders” that responded 2 hours or more after treatment (FIG. 3). reference). These responses were suppressed by the CRH-R1 antagonist R121919. Among the initial responders were known players in the downstream pathway of CRH-R1, such as the transcription factors Nurr1, Nurr77, Jun-B, which demonstrated the assay.

Interesting novel players to be identified include transcription factors (eg, hair / enhancer-of-split associated 1 (Hey1), nuclear factor regulated by interleukin 3 (NFIL3), cAMP response element modulator (CREM) and prostate Specific ets transcription factor (Pse)), receptor and channel regulators (eg Ras-related GTP binding protein (GEM) and receptor (calcitonin) activity-modifying protein 3 (RAMP3)), secretory peptides (eg adrenomedullin) , Calcitonin, cholecystokinin) and proteins involved in intracellular signaling (eg, G protein signaling regulator 2 (Rgs2), cAMP-specific phosphodiesterase 4B (Pde4b), inositol 1,4 5-3 phosphate receptor _(IP 3 R1) and a regulatory subunit phosphatidylinositol 3-kinase, p85) and the like. Other interesting regulated genes comprise the period homolog Per1, fibroblast growth factor receptor 2 (Fgfr2), serum / glucocorticoid-regulated kinase and serum-inducible kinase (FIG. 4). Interestingly, all responders identified according to the above criteria were up-regulated after exposure to CRH. This induction is transient and almost all of the induced genes return to baseline after 4-8 hours. Many of the induced transcripts encode proteins that exert negative feedback on CRH-R1 signaling (eg, Pde4, Rgs2, CREM, etc.) and appear to contribute to the temporal nature of induction. In addition to this negative feedback, other mechanisms such as desensitization of CRF ₁ by phosphorylation and internalization contribute to the transient nature of transcription induction. In this regard, it is interesting to note that CRF challenge rapidly down-regulates CRF ₁ mRNA in rat pituitary cells. However, no alteration of CRF ₁ mRNA could be detected in pituitary-derived AtT-20 cells exposed to 1 μM CRF. Confirmation of the microarray data was performed using quantitative real-time PCR analysis on the same samples used in the hybridization experiments and in replicate experiments. The level of regulation and time course identified by the microarray was consistent with that observed by quantitative PCR as shown for Rgs2 in FIG. These genes tested show the level of induction compared to the untreated sample in FIG.
Description A CRH-R1 specific antagonist was used to identify a transcriptional pathway downstream of CRH-R1 in AtT-20 cells. Our results are consistent with activation of several second messengers such as cAMP and Ca ²⁺ upon stimulation with CRH. Some of the transcriptional responses can be explained by phosphorylation of CREB and subsequent transcription of genes downstream of the cAMP response element. These sequences have been found, for example, in Per1, Nurr1, CREM-ICER, c-Fos promoters. Furthermore, the kinetic profile of induction of these genes is consistent with the maximum transcription rate observed by CREB 0.5 hours after cAMP formation. Induction of CREM-ICER constitutes a negative feedback mechanism in weakening the transcriptional response to cAMP. Of interest is the reported induction of CREM-ICER in response to acute stress in the medial lobe of the pituitary gland. Mice deficient in CREM-ICER show a chronic increase in β-endorphin levels, suggesting that CREM-ICER induction may be involved in the regulation of gene expression in response to stress (10). Our results suggest that CREM-ICER is directly involved in the regulation of CRH signaling and, as a consequence, removal of CREM-ICER may result in an altered response to stress signals. Another novel putative negative feedback regulator of CRH signaling is Rgs2. We have identified two single CRE motifs in the promoter of the human RGS2 gene that give a possible explanation for the early response behavior of this gene upon stimulation with CRH. In support of our results, there are recent reports showing that both phosphoinositide signaling and cAMP induce rapid and transient increases in Rgs2 mRNA in human astrocytoma and neuroblastoma cells . The Rgs2 protein is a selective inhibitor of _Gqα function. Recently, RGS2, rather than by acting on G _alpha, by directly inhibiting adenylyl cyclase type III activity has been shown to reduce cAMP production induced by odorants. Although Rgs2 was initially identified as an immediate early response gene in activated T lymphocytes, studies in Rgs2-deficient mice have shown that it is in the regulation of stress-related behaviors as these mice show increased anxiety and aggressiveness. Suggest that also plays a role (11). Induction of cAMP-specific phosphodiesterase 4B (Pde4b) can also be classified under negative feedback, which directly attenuates the cAMP signal. Another important second messenger generated upon stimulation with CRH is Ca ²⁺ . CRH causes extracellular Ca ²⁺ influx stimulated by steady state depolarization through voltage-gated Ca ²⁺ channels and by release from an inositol 1,4,5-phosphate (InsP3) sensitive Ca ²⁺ pool. It has been shown to increase intracellular Ca ²⁺ concentration (12). Both the InsP3 receptor and the p85 regulatory subunit of phosphatidylinositol 3-kinase appear to be up-regulated, explaining the offset mechanism of long-term Ca ²⁺ signaling. Up-regulation of the low molecular weight G protein kir / Gem is also directed towards long-term decay of Ca ²⁺ signaling. Recent studies have shown that Gem regulates Ca ²⁺ channel expression at the cell membrane by the β auxiliary subunit. Increased levels of Gem have been shown to inhibit Ca ²⁺ -induced exocytosis, and it has been suggested that Gem may have a protective effect against Ca ²⁺ overload. Intracellular Ca ²⁺ also, in addition to its role as a second messenger, has also been shown to play an important role in regulating gene expression. Interesting is the regulation of NFIL3 / E4BP4 by calcineurin / NFAT and CaM kinase signaling, explaining the increase in NFIL3 mRNA levels upon CRH treatment. In B lymphocytes, NFIL3 expression is induced by interleukin 3 through both the Raf mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. In this cell type, NFIL3 suppresses apoptosis in synergy with the Bcl-xL-dependent pathway. Our data suggest a role for NFIL3 in preventing apoptosis of AtT-20 cells.

  CRH is the most effective ACTH secretagogue. Unfortunately, the microarray used was not examined for POMC levels. However, cholecystokinin (CCK) and several other prepropeptide mRNAs, such as two calcitonin peptide family members, adrenomedullin (ADM) and calcitonin (CT), are found to be upregulated after CRH administration. It was issued. Also interesting in this respect is the upregulation of RAMP3. RAMP controls calcitonin receptor-like receptor (CRLR) transport and glycosylation. In the case of RAMP3, along with CRLR, it has been shown to produce ADM receptors. Upregulation of this gene indicates that AtP-20 cells to ADM or other extracellular stimuli after exposure to CRH so that it is unclear whether RAMP3 may regulate other G-coupled receptors May play a role in regulating the responsiveness of. CCK is secreted by AtT-20 cells, but induction of its expression by CRH has not been reported so far (13). However, the interaction between CCK and CRH has been studied in detail and has been shown in panic attacks, depression, anxiety and gastric emptying (14-19). Most of these experiments point towards the role of CRH in mediating the central effects of CCK. Our data suggest that CRH may further function as a CCK secretagogue. The situation very similar to that of CCK seems to be the same with adrenomedullin. ADM is expressed in the pituitary and has been shown to affect basal and CRH-stimulated ACTH release in animals and thus its potential role in regulating the hypothalamic-pituitary-adrenal system (20-23). Current expression data indicate that CRH induces ADM and CT. Furthermore, recent results indicate that circulating adrenomedullin is increased in Cushing's disease and that the pituitary gland may represent a site of increased production of ADM (24), with CRH inducing ADM. Suggest that there is a possibility.

  In summary, we elucidated part of the corticotropin-releasing hormone receptor 1-activating gene network and identified several novel targets for this signaling cascade. Our results elicit the need for further experiments to elucidate the function of these transcriptional responses to CRH stimulation at both the cellular and whole organism level.

Table 1. List of proteins that regulate CRH signaling.

C as assessed by quantitative RT-PCR normalized to β-actin mRNA levels (considered as 100%) in AtT-20 cells treated with DMSO, CRH, CRH + R121919 or R121919 at different time points (hours) -Fos mRNA levels. Correspondence analysis applied to normalized microarray data for all time points and treatments. Squares indicate different samples, while circles indicate genes. The distance between the squares is a measure of the similarity between the samples. A positive association between a given sample and a gene (ie, up-regulation of that gene in that particular sample) results in the positioning of that gene and sample on the common line through the centroid (shown in cross). Correspondence analysis clearly identifies time as the primary identifier between samples. Furthermore, the effect of treatment with CRH can be identified as being most noticeable at the initial time point. A heat map showing genes that have changed during CRH treatment. Values were calculated by dividing the intensity of each sample by the intensity of the DMSO sample at the corresponding time point. These calculated ratio with log ₂ scale units converted to (using on a _log 2 scale) the color ramp. In this way, different timings of expression induction become apparent. Genes that show a 2-fold change after 30 minutes of treatment with CRH are called “early responders”, “intermediate responders” show changes 1-2 hours after treatment, and “late responders” are 2 hours or more A response will be shown later. Overview of selection of genes induced by CRH classified by pathway or function as described herein. Values were calculated by dividing the intensity of each sample by the intensity of the DMSO sample at the corresponding time point. These calculated ratios are converted to color ramps using log ₂ scale units and shown in the heat map. Induction of Rgs2 by CRH in AtT-20 cells. Induction is calculated relative to the level observed in AtT-20 cells prior to any treatment. The array data obtained with Rgs2 is shown above. Below, the level of Rgs2 mRNA as measured by quantitative RT-PCR on the same sample used for the array experiment and as measured in a repeat experiment is shown.

[Sequence Listing]

Claims (34)

  An isolated polynucleotide that encodes a protein that modulates corticotropin releasing hormone (CRH) signaling and having an amino acid sequence selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 48.   2. The isolated polynucleotide of claim 1, wherein the polynucleotide is mRNA, DNA or cDNA.   An isolated polynucleotide encoding a protein that modulates corticotropin releasing hormone (CRH) signaling, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49. A polynucleotide comprising   An isolated polynucleotide encoding a protein that modulates corticotropin releasing hormone (CRH) signaling, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49. A polynucleotide comprising:   An isolated polynucleotide encoding a protein that modulates corticotropin releasing hormone (CRH) signaling, the protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 48 .   A vector comprising the isolated polynucleotide of any one of claims 1-4.   The vector of claim 6, wherein the polynucleotide is operably linked to an expression control sequence.   A host cell capable of expressing a protein that regulates corticotropin releasing hormone (CRH) signaling and having an amino acid sequence selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 48.   9. A host cell according to claim 8 transfected with a vector comprising a regulatory sequence.   The host cell according to claim 8 transfected with the vector according to claim 6 or 7. a) contacting the cells with CRH with or without compounds;
b) determining the amount of at least one protein that modulates corticotropin releasing hormone (CRH) signaling in the cell; and c) comparing the amount of the protein with and without the compound;
Comprising
Here, the protein that regulates the corticotropin releasing hormone (CRH) signaling is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: Selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48,
A method of identifying a compound capable of altering a CRH signaling response in a cell.   12. The method of claim 11, wherein the cell is a eukaryotic cell such as the mouse pituitary corticotroph-derived adenoma cell line AtT-20.   The amount of protein that regulates CRH signaling is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, Sequence number 22, Sequence number 24, Sequence number 26, Sequence number 28, Sequence number 30, Sequence number 32, Sequence number 34, Sequence number 36, Sequence number 38, Sequence number 40, Sequence number 42, Sequence number 44, Sequence number The method according to claim 11 or 12, which is determined using an antibody that binds to a polypeptide comprising an amino acid sequence selected from the group consisting of 46 and SEQ ID NO: 48.   The amount of protein that regulates CRH signaling is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, Sequence number 22, Sequence number 24, Sequence number 26, Sequence number 28, Sequence number 30, Sequence number 32, Sequence number 34, Sequence number 36, Sequence number 38, Sequence number 40, Sequence number 42, Sequence number 44, Sequence number The method according to claim 11 or 12, which is determined by evaluating the level of gene transcription of a gene encoding an amino acid sequence selected from the group consisting of 46 and SEQ ID NO: 48.   The level of gene transcription is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 15. The method of claim 14, wherein the method is evaluated using a probe that binds to a polynucleotide encoding an amino acid sequence selected from the group consisting of 48.   The method according to claim 14 or 15, wherein the level of gene expression is analyzed using microarray technology.   The level of gene expression is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 17. The method of claim 16, wherein the method is evaluated using an array of oligonucleotide probes that bind to a polynucleotide encoding a group of polypeptides having 48 amino acid sequences. a) contacting the cells with or without the test compound; and b) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, A method of identifying a compound capable of altering a CRH signaling response in a cell comprising determining the expression level of a gene encoding a polypeptide having the amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48.   The gene expression level is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, Sequence number 23, Sequence number 25, Sequence number 27, Sequence number 29, Sequence number 31, Sequence number 33, Sequence number 35, Sequence number 37, Sequence number 39, Sequence number 41, Sequence number 43, Sequence number 45, Sequence number 19. The method of claim 18, wherein the method is determined using an array of oligonucleotide probes that bind to a polynucleotide having the nucleic acid sequence of 47 and SEQ ID NO: 49. a) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48 Contacting a cell expressing at least one protein comprising an amino acid sequence selected from with a test compound; and b) comparing the cell's CRH response activity with or without the compound. A method of identifying a compound capable of altering CRH signaling response activity.   The cells are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24. , SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48 21. The method of claim 20, wherein a group of proteins having a sequence is expressed.   The method of claim 21, wherein CRH response activity is assessed as a transcriptional change at the gene level.   23. The method of claim 21 or 22, wherein CRH response activity is assessed using microarray technology.   The cells are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24. SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48. 24. A method according to any one of claims 20 to 23, which is a host cell capable of expressing at least one protein having an amino acid sequence selected from the group.   25. The method of claim 24, wherein the host cell is transfected with at least one vector comprising regulatory sequences.   Host cells are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48 25. The method of claim 24, wherein the method is transfected with at least one vector comprising a polynucleotide sequence encoding an amino acid sequence selected from the group. a) obtaining a biological sample of the individual; and b) determining the amount of at least one protein that modulates corticotropin releasing hormone (CRH) signaling in the biological sample;
(Here, proteins that regulate corticotropin releasing hormone (CRH) signaling are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, (Selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 48)
For diagnosing CRH-induced depression in an individual comprising.   28. The method of claim 27, wherein the biological sample is a body fluid or tissue sample.   The amount of protein that regulates CRH signaling is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, Sequence number 22, Sequence number 24, Sequence number 26, Sequence number 28, Sequence number 30, Sequence number 32, Sequence number 34, Sequence number 36, Sequence number 38, Sequence number 40, Sequence number 42, Sequence number 44, Sequence number 29. The method of claim 27 or 28, wherein the method is determined using an antibody that binds to a polypeptide comprising an amino acid sequence selected from the group consisting of 46 and SEQ ID NO: 48.   The amount of protein that regulates CRH signaling is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, Sequence number 22, Sequence number 24, Sequence number 26, Sequence number 28, Sequence number 30, Sequence number 32, Sequence number 34, Sequence number 36, Sequence number 38, Sequence number 40, Sequence number 42, Sequence number 44, Sequence number 29. The method according to claim 27 or 28, which is determined by evaluating the level of gene transcription of a gene encoding an amino acid sequence selected from the group consisting of 46 and SEQ ID NO: 48.   The level of gene transcription is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, Sequence number 24, sequence number 26, sequence number 28, sequence number 30, sequence number 32, sequence number 34, sequence number 36, sequence number 38, sequence number 40, sequence number 42, sequence number 44, sequence number 46 and sequence number 31. The method of claim 30, wherein the method is evaluated using a probe that binds to a polynucleotide encoding an amino acid sequence selected from the group consisting of 48.   32. A method according to claim 30 or 31, wherein the level of gene transcription is analyzed using microarray technology.   The level of gene transcription is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, Sequence number 24, sequence number 26, sequence number 28, sequence number 30, sequence number 32, sequence number 34, sequence number 36, sequence number 38, sequence number 40, sequence number 42, sequence number 44, sequence number 46 and sequence number 35. The method of claim 32, wherein the method is analyzed using an array of oligonucleotide probes that bind to a polynucleotide encoding a group of polypeptides having 48 amino acid sequences.   The level of gene transcription is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, Sequence number 23, Sequence number 25, Sequence number 27, Sequence number 29, Sequence number 31, Sequence number 33, Sequence number 35, Sequence number 37, Sequence number 39, Sequence number 41, Sequence number 43, Sequence number 45, Sequence number 35. The method of claim 32, wherein the method is analyzed using an array of oligonucleotide probes that bind to a polynucleotide having the nucleic acid sequence of 47 and SEQ ID NO: 49.

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