Rhesus monkey nur77

This application claims the benefit of U.S. Provisional Application No. 60/662,197, filed Mar. 16, 2005, the contents of which are incorporated herein by reference in their entirety.

(1) Field of the Invention

The present invention relates to the Macaca mulatta (rhesus monkey) Nur77 (rhNur77) nuclear receptor and the nucleic acid encoding the rhNur77 nuclear receptor. The present invention further relates to methods for identifying analytes that modulate expression or activity of Nur77. Analytes that affect Nur77 expression or activity may be useful for treating or inhibiting inflammatory diseases such as osteoarthritis and various bone, neurological, and prostrate disorders. The rhNur77 may also be useful in treatments for particular cancers such as lung cancer, prostrate cancer, colon cancer, ovarian cancer, or gastric cancer.

(2) Description of Related Art

Nur77 is a transcription factor that belongs to the superfamily of nuclear receptors, which includes receptors for various steroid hormones, retinoids, thyroid hormone, and estradiol. The superfamily further includes a large group of receptors classified as orphan receptors because their natural ligands are not yet known. Nur77 is currently classified as an orphan receptor and has been variously known as HMR, N10, TR3, NP10, GFRP1, NAK-1, NGFIB, or MGC9485. Nur77 is expressed in various peripheral tissues and in some regions of the brain and has been implicated to have a role in cell proliferation, differentiation, and apoptosis.

Nur77 exhibits a close structural relationship to the orphan receptors Nurr1 (Hazel et al., Proc. Natl. Acad. Sci. USA. 85: 8444-8448 (1988); Milbrant, Neuron 1: 183-188 (1998); Ryseck et al., EMBO J. 8: 3327-3335 (1989); Nakai et al., Mol. Endocrinol. 4: 1438-1443 (1990)) and NOR-1 (Ohkura et al., Biochem. Biophys. Res. Comm. 205: 1959-1965 (1994); Maruyama et al., Cancer Lett. 96:117-122 (1995); Hedvat and Irving, Mol. Endocrinol. 9: 1692-1700 (1995)). All three of these orphan nuclear receptors comprise the Nurr subfamily of nuclear receptors which has been characterized as being able to bind to the same cis-acting consensus nucleotide sequence—the NGFI-B response element (NBRE)—to regulate target gene expression (Ohkura et al., Biochem. Biophys. Res. Comm. 205: 1959-1965 (1994); Wilson et al., Science 252: 1296-1300 (1991); Wilson et al., Mol. Cell. Biol. 13: 5794-5804 (1993); Murphy et al., Gene Express. 5: 169-179 (1995)). For example, Nurr1 and Nur77 regulate expression of the Corticotropin Releasing Hormone (CRH) and proopiomelanocortin (POMC) genes by interacting with specific cis-acting sequences in their proximal promoter region.

Like most nuclear receptors of the nuclear receptor superfamily, Nur77 consists of an amino-terminal transactivation function 1 (AF1) near the amino terminus, which enables ligand-independent transcription activation; a core DNA binding domain (DBD) located near the center of the protein, which contains two highly conserved zinc finger motifs and which binds to specific nucleotide sequences; a hinge region, which permits protein flexibility to allow for simultaneous receptor dimerization and DNA binding; a conserved ligand binding domain (LBD) near the carboxy terminus, which includes a dimerization interface; and, a carboxyl-terminal activation function 2 (AF2) near the carboxy terminus, which enables ligand-independent transcription activation. Many nuclear receptors act as dimers, either as homodimers or as heterodimers. The dimerization interface in the LBD, the I-box, has been mapped to a region in the carboxyl terminal part of the LBD that corresponds to helix 10 in the canonical nuclear receptor LBD structure (Perlmann et al., Mol. Endocrinol. 10: 958-966 (1996); Lee et al. Mol. Endocrinol. 12: 325-332 (1998)).

Unlike most nuclear receptors, the Nurr subfamily of nuclear receptors are encoded by immediate early genes whose expression can be differentially induced in response to a variety of extracellular stimuli such as growth factors (Hazel et al., ibid.; Milbrandt, ibid.), neurotransmitters (Watson and Milbrandt, Mol. Cell. Biol. 9: 4213-4219 (1989)), and polypeptide hormones (Wilson et al., Mol. Cell. Biol. 13: 861-868 (1993); Murphy and Conneely, ibid.; Davis and Lau, Mol. Cell. Biol. 14: 3469-3483 (1994)). Nurr1 and Nur77 are rapidly induced by CR1H in primary pituitary cells, resulting in increased synthesis of POMC (Murphy and Conneely, ibid.). Glucocorticoid repression of the POMC gene is mediated by glucocorticoid receptor dependent inhibition of activation of the POMC gene by Nurr1 and Nur77 (Evans, ibid.; Philips et al., Mol. Cell. Biol. 17: 5952-5959 (1997)) and appears to antagonize negative feedback of ACTH synthesis and secretion by glucocorticoid in pituitary corticotrope cells (Okabe et al., J. Endocrinol. 156: 169-175 (1998). Because NOR-1 possesses an identical DNA binding domain and is capable of binding the same cis-acting consensus sequence as Nurr1 and Nur77, it has been included in the Nurr subfamily. Therefore, the close structural relationship, the identical cis-acting consensus sequence, and the ability of the different members of the Nurr subfamily of transcription factors to functionally complement one another are strong indications that the Nurr subfamily members might have redundancy of function.

Nur77 forms heterodimers with retinoid X receptor (RXR) (Perlmann and Jansson, Genes Dev. 9: 769-782 (1995); Forman et al. (1995); Zetterstrom et al., Science 276: 248-250 (1996)). The unique ability of the Nur77/RXR heterodimer complex to transduce RXR signals establishes a novel response pathway. Heterodimer formation may impart allosteric changes upon the LBD of the Nur77. These allosteric changes may confer transcriptional activities onto the Nur77-RXR heterodimer that are distinct from those of the Nur77 or RXR monomers. This would permit a limited number of regulatory proteins to generate a diverse set of transcriptional responses to multiple hormonal signals.

Nur77 appears to have a role in both apoptosis and cell proliferation. Kolluri et al. (Molec. Cell. Biol. 23: 8651-8667 (2003)), and Wilson et al. (Cancer Res. 63: 5401-5407 (2003)) have shown that Nur77 can induce apoptosis or cellular differentiation in the same cells depending on the stimuli and its cellular location. Cao et al. (Molec. Cell. Biol. 24: 9705-9725 (2004) has shown that RXR regulates Nur77/thyroid hormone receptor 3-dependent apoptosis by modulating its nuclear export and translocation to the mitochondria where it induces apoptosis. Other researchers have shown that parathyroid hormone can regulate Nur77 expression in bone (Tetradis et al., in Biochem. Biophys. Res. Comm. 281: 913-916 (2001)) and LH can regulate Nur77 expression in testicular Leydig cells (Song et al., Endocrinol. 142: 5116-5123 (2001). Promyelocytic leukemia protein PML is a tumor and growth suppresser that inhibits Nur77-mediated transcription by interacting with the DNA-binding domain of Nur77, which prevents it from binding its target promoter (Wu et al., oncogene 21: 3925-3933 (2002). The role of Nur77 in signal transduction has been discussed by Winoto and Littman, Cell 109: S57-S66 (2002).

The present invention provides the Macaca mulatta (rhesus monkey) Nur77 nuclear receptor (rhNur77) and the nucleic acid encoding the rhNur77. The present invention further provides methods for identifying analytes that modulate expression or activity of Nur77. Analytes that affect Nur77 expression or activity may be useful for treating or inhibiting inflammatory diseases such as osteoarthritis and various bone, neurological, and prostrate disorders. The rhNur77 may also be useful in treatments for particular cancers such as lung cancer, prostrate cancer, colon cancer, ovarian cancer, and gastric cancer.

Therefore, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an rhNur77 polypeptide or fragment thereof having the amino acid sequence shown in SEQ ID NO:2 or fragment or subsequence thereof. In a further aspect of the invention, the nucleic acid molecule encoding the rhNur77 comprises the nucleotide sequence of SEQ ID NO:1 or fragment or subsequence thereof. The present invention further provides an isolated nucleic acid encoding the LBD or the DBD of the rhNur77 polypeptide. For example, the present invention provides a nucleic acid encoding the LBD, which comprises the nucleotides from about 997 to about 1698 of SEQ ID NO:1, and a nucleic acid encoding the DBD, which comprises the nucleotides from about 800 to about 996 of SEQ ID NO:1. The present invention further includes nucleic acids comprising SEQ ID NO:1 wherein nucleotide 1435 is a cytosine.

The present invention further provides an isolated polypeptides comprising an amino acid sequence of SEQ ID NO:2. In particular, the present invention provides a polypeptide comprising amino acids 1 to 598 of SEQ ID NO:2, and fragments thereof, for example; a polypeptide comprising the LBD, preferably, amino acids from about 332 to about 566 of SEQ ID NO:2; or, a polypeptide comprising the DBD, preferably amino acids from about 265 to about 332 of SEQ ID NO:2. The present invention further includes polypeptides comprising SEQ ID NO:2 wherein amino acid 479 is a leucine.

The present invention further provides an antibody which binds a polypeptide comprising an amino acid sequence of SEQ ID NO:2. In further aspects, the antibody is a polyclonal antibody or a monoclonal antibody.

The present invention further still provides a vector comprising a nucleic acid encoding an rhNur77 polypeptide or fragment thereof which comprises an amino acid sequence of SEQ ID NO:2. Preferably, the nucleic acid encoding the rhNur77 comprises the sequence of SEQ ID NO:1 or fragment or subsequence thereof. In particular aspects of the vector, the vector comprises a nucleic acid encoding the LBD or the DBD of the rhNur77. In further aspects of the above vector, the nucleic acid encodes a fusion or chimeric protein, which comprises the rhNur77 polypeptide, the LBD of the rhNur77, or the DBD of the Nur77 fused to a heterologous protein. In particular aspects of the vector, the fusion protein comprises the rhNur77 LBD fused to the GAL4 yeast transcription activation factor DBD or glucocorticoid receptor (GR) DBD; or, the H1 alpha helix of the Nur77 LBD fused to the GAL4 or GR LBD and the remainder of the Nur77 LBD fused to the herpesvirus VP16 activation domain. In a further still aspect of the above vector, the present invention further provides a vector wherein the nucleic acid encoding the rhNur77 polypeptide or fragment thereof, or fusion protein, is operably linked to a heterologous promoter. The heterologous promoter can be a constitutive promoter or an inducible promoter.

The present invention further still provides a cell comprising a nucleic acid encoding an rhNur77 polypeptide or fragment thereof having an amino acid sequence of SEQ ID NO:2 wherein the nucleic acid is operably linked to a heterologous constitutive or inducible promoter which enables expression of the rhNur77 or fragment thereof in the cell. Preferably, the nucleic acid comprises the sequence of SEQ ID NO:1 or a fragment or subsequence thereof. For example, the nucleic acid can comprise nucleotides from about 997 to about 1698 of SEQ ID NO:1 (wherein nucleotide 1435 is a thymidine or cytosine), which encode the rhNur77 LBD, or nucleotides from about 800 to about 996 of SEQ ID NO:1, which encode the rhNur77 DBD.

In a further aspect of the above cell, the nucleic acid encodes a fusion or chimeric protein comprising the rhNur77 polypeptide, the Nur77 LBD or the Nur77 DBD fused to a heterologous protein. In particular aspects of the above cell, the fusion protein comprises the rhNur77 LBD fused to the GAL4 yeast transcription activation factor DBD or GR DBD; or, the H1 alpha helix of the LBD fused to the GAL4 or GR LBD and the remainder of the LBD fused to the herpesvirus VP16 activation domain. In a further still aspect of the above cell, the present invention provides a vector wherein the nucleic acid encoding the rhNur77 polypeptide or fragment thereof, or fusion protein, is operably linked to a heterologous constitutive or inducible promoter.

In a further aspect of the above cell, the cell further includes a second nucleic acid encoding a reporter gene, which encodes an assayable product, wherein the nucleic acid encoding the reporter gene is operably linked to a promoter responsive to the rhNur77. In further aspects of the above cell, the rhNur77 responsive promoter comprises one or more copies of a Nur77 responsive sequence, for example, an NGFI-B (Nur77)-responsive element (NBRE), a Nur77 response element (NurRE) or an RA response element (βRARE) in the RARβ promoter. Examples of Nur77 responsive promoters include the promoter for the gene encoding NGFI-B protein, the gene encoding the pro-opiomelanocortin (POMC), or the gene encoding the retinoid acid receptor (RAR), or a minimal promoter such as the CMVie or HSV TK promoter operably linked to one or more copies of a Nur77 responsive sequences.