CA2301554A1 - Dna molecules encoding human nuclear receptor proteins - Google Patents

Abstract

The present invention discloses the isolation and characterization of cDNA
molecules encoding two human nuclear receptor proteins, designated nNR1, nNR2 and/or nNR2-1. Also within the scope of the disclosure are recombinant vectors, recombinant host cells, methods of screening for modulators of nNR1, nNR2 and/or nNR2-1 activity, and production of antibodies against nNR1, nNR2 and/or nNR2-1, or epitopes thereof.

Description

DNA MOLECULES ENCODING HUMAN NUCLEAR
RECEPTOR PROTEINS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. Provisional Application Serial No. 601078,633, filed March 19, 1998 which is a continuation-in-part of U.S. Provisional Application Serial No.
60/062,902, filed October 21, 1997, which is a continuation-in-part of U.S.
Provisional Application Serial No. 60/057,090, filed August 27, 199?. .
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
The present invention relates in part to isolated nucleic acid molecules (polynucleotide) which encode human nuclear receptor proteins, referred to throughout as nNR,I, nNR,2 and/or nNR,2-1. The present invention also relates to recombinant vectors and recombinant hosts which contain a DNA fragment encocling nNR,l, nNR2 and/or nNR2-1, substantially purified forms of associated human nNRl, nNR,2 and/or nNR2-1 protein, human mutant proteins, and methods associated with identifying compounds which modulate nNRI, nNR2 and/or nNR2-1 activity.

BACKGROUND OF THE INVENTION
The nuclear receptor superfamily, which includes steroid hormone receptors, are small chemical ligand-inducible transcription factors which have been shown to play roles in controlling development, differentiation and physiological function. Isolation of cDNA clones encoding nuclear receptors reveal several characteristics.
First, the NH2-terminal regions, which vary in length between receptors, is hypervariable with low homology between family members.
There are three internal regions of conservation, referred to as domain I, II and III. Region I is a cysteine-rich region which is referred to as the DNA binding domain (DBD). Regions II and III are within the COON-terminal region of the protein and is also referred to as the ligand binding domain (LBD). For a review, see Power et al. (1992, Trends in Pluarmc~ceutical Scixnces 13: 318-323).
The lipophilic hormones that activate steroid receptors are known to be associated with human diseases. Therefore, the respective nuclear receptors have been identified as possible targets for therapeutic intervention. For a review of the mechanism of action of various steroid hormone receptors, see Tsai and O'Malley (1994, Annu. Reu. Biochem.
63: 451-486).
Recent work with non-steroid nuclear receptors has also shown the potential as drug targets for therapeutic intervention. This work reports that peroxisome proliferator activated receptor g (PPARg), identified by a conserved DBD region, promotes adipocyte differentiation upon activation and that thiazolidinediones, a class of antidiabetic drugs, function through PPARg (Tontonoz et al., 1994, Cell ?9: 1147-1156;
Lehmann et al.,1995, J. Biol. Chew. 270(22): 12953-12956; Teboul et al., 1995, J. Biol. Chem. 270(47): 28183-2818?). This indicates that PPARg plays a role in glucose homeostasis and lipid metabolism.
Gigu~re, et al. (1988, Nature 331: 91-94) isolated two cDNAs which encode a human nuclear receptor, referred to as hERRl and hEER2. The authors did not assign a ligand and subsequent ligand-inducible function to either of these human nuclear receptors.

Trapp and Holaboer (1996, J. Biol. Chem. 271(17): 9879-9882) show that hERR2 acts as a cell-specific inhibitor of glucocorticoid receptor-mediated gene expression.
It would be advantageous to identify a gene encoding an additional human nuclear receptor protein. A nucleic acid molecule expressing a human nuclear receptor protein will be useful in screening for compounds acting as a modulator of cell differentiation, cell development and physiological function. The present invention addresses and meets these needs by disclosing isolated nucleic acid molecules which express a human nuclear receptor protein which will have a role in cell differentiation and development.
SUMMARY OF THE INVENTION
The present invention relates to isolated nucleic acid molecules (polynucleotides) which encode novel nuclear receptor proteins, preferably human nuclear receptor proteins, such as human nuclear receptor proteins exemplified and referred to throughout this specification as nNRl, nNR2 and/or nlVR2-1.
The present invention also relates to isolated nucleic acid fragments of nNR1 (SE(d ID NO:1) and nNR2 (SEA ID N0:3) which encode mRNA expressing a biologically active novel human nuclear receptor. Any such nucleic acid fragment will encode either a protein or protein fragment comprising at least an intracellular DNA-binding domain and/or ligand binding domain, domains conserved throughout the human nuclear receptor family domain which exist in nNRl (SEQ
ID N0:2) and nNR,2 (SEQ ID N0:4). Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonistss and/or antagonists for nNRl, nNR2 and/or nNR,2-1 function.
The isolated nucleic acid molecule of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
A preferred aspect of the present invention is disclosed in Figure lA-C and SEla ID NO:1, a human cDNA encoding a novel nuclear traps-acting receptor protein, nNRl.
Another preferred aspect of the present invention is disclosed in Figure 4A-C and SE(a ID N0:3, a human cDNA encoding a novel nuclear traps-acting receptor protein, nNR2.
Another preferred aspect of the present invention is disclosed in Figure 7A-C and SEQ ID N0:5, a human cDNA encoding a truncated version of nNR2, referred to as nNR2-1.
The present invention also relates to a substantially purified form of the novel nuclear traps-acting receptor protein, nNRl, which is disclosed in Figures 2A-F and Figure 3 and as set forth in SEQ ID N0:2.
The present invention also relates to biologically active fragments and/or mutants of nNRl as set forth as SEQ ID N0:2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists of nNRl function.
The present invention also relates to a substantially purified form of the novel nuclear traps-acting receptor protein, nNR2, which is disclosed in Figure 5A-H and Figure 6 and as set forth in SEQ ID N0:4.
The present invention also relates to biologically active fragments and/or mutants of nNR2 as set forth as SEQ ID N0:4, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein 4~

fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists of nNR2 function.
A preferred aspect of the present invention is disclosed in Figure 3 and is set forth as SEQ ID N0:2, the amino acid sequence of the novel nuclear trans-acting receptor protein, nNRl.
A preferred aspect of the present invention is disclosed in Figure 6 and is set forth as SEQ ID N0:4, the amino acid sequence of the novel nuclear traps-acting receptor protein, nNR2.
A preferred aspect of the present invention is disclosed in Figure 8 and is set forth as SEQ ID N0:6, the amino acid sequence of a truncated version of nNR2, refereed to as nNR2-1.
The present invention also relates to polyclonal and monoclonal antibodies raised in response to either the human form of nNR,l, nNR,2 and/or nNR2-1 disclosed herein, or a biologically active fragment thereof. It will be especially preferable to raise antibodies against epitopes within the NIiZ terminal domain of nNRl, nNR2 and/or nNR2-1, which show the least homology to other known proteins belonging to the human nuclear receptor superfamily. To this end, the DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of human nNRI, nNR2 and/or nNR2-1. The recombinant proteins, DNA
molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of human nNRl, nNR,2 and/or nNR2-1.
The present invention also relates to isolated nucleic acid molecules which are fusion constructions expressing fusion proteins useful in assays to identify compounds which modulate wild-type human nNRI, nNR,2 and/or nNR,2-1 activity. A preferred aspect of this portion of the invention includes, but is not limited to, glutathione S-tranaferase GST-nNRl and/or GST-nNR2 fusion constructs. These fusion constructs include, but are not limited to, all or a portion of the ligand-binding domain of nNRl, nNR,2 and/or nNR2-1, respectively, as an in-frame fusion at the carboxy terminus of the GST gene. The disclosure of SECd ID NOS:1-4 allow the artisan of ordinary skill to construct any such nucleic acid molecule encoding a GST-nuclear receptor fusion protein. Soluble recombinant GST-nuclear receptor fusion proteins may be expressed in various expression systems, including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using a baculovirus expression vector (e.g., Bac-N-Blue DNA from Invitrogen or pAcG2T from Pharmingen).
It is an object of the present invention to provide an isolated nucleic acid molecule which encodes a novel form of a nuclear receptor protein such as human nNRl and/or human nNR,2, human nuclear receptor protein fragments of full length proteins such as nNR,l, nNR2 and/or nNR2-1, and mutants which are derivatives of SEQ ID N0:2 and SEQ ID N0:4. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonista and/or antagonists for nNRl, nNR2 and/or nIVR2-1 function.
It is a further object of the present invention to provide the human nuclear receptor proteins or protein fragments encoded by the nucleic acid molecules referred to in the preceding paragraph.
It is a further object of the present invention to provide recombinant vectors and recombinant host cells which comprise a nucleic acid sequence encoding human nNRI, nNR2 and/or nNR2-1 or a biological equivalent thereof.
It is an object of the present invention to provide a substantially purified form of nNRl, as set forth in SEQ ID N0:2.
It is an object of the present invention to provide for biologically active fragments and/or mutants of nNRl, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use.
It is an object of the present invention to provide a substantially purified form of nNR2, as set forth in SE4~ ID N0:4.

It is an object of the present invention to provide for biologically active fragments and/or mutants of nNR2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use.
It is also an object of the present invention to provide for nNRl- and/or nNR2-based in-frame fusion constructions, methods of expressing these fusion constructions and biological equivalents disclosed herein, related assays, recombinant cells expressing these constructs and agonistic and/or antagonistic compounds identified through the use DNA molecules encoding human nuclear receptor proteins such as nNRl, nNR2 and/or nNR2-1.
As used herein, "DBD" refers to DNA binding domain.
As used herein, "LBD" refers to ligand binding domain.
As used herein, the term "mammalian host" refers to any mammal, including a human being.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure lA-C shows the nucleotide sequence (SEQ ID
N0:1) which comprises the open reading frame encoding the human nuclear receptor protein, nNRl.
Figure 2A-F shows the nucleotide sequence of the double stranded cDNA molecule (SEQ ID NO:1 and SEQ ID N0:29) which encodes nNRl, and the amino acid sequence of nN'R,1 (SEQ ID N0:2).
The region in bold and underline is the DNA binding domain.
Figure 3 shows the amino acid sequence of nNRl (SEQ ID
N0:2). The region in bold and underline is the DNA binding domain.
Figure 4A-C shows the nucleotide sequence (SEQ ID N0:3) which comprises the open reading frame encoding the human nuclear receptor protein, nNR,2.
Figure 5A-H shows the nucleotide sequence of the double stranded cDNA molecule (SEQ ID NO:1 and SEQ ID N0:29) which encodes nNR2, and the amino acid sequence of nNR,2 (SEQ ID N0:4).
The region in bold and underline is the DNA binding domain.

Figure 6 shows the amino acid sequence of nNR2 (SEQ ID
N0:4). The region in bold and underline is the DNA binding domain.
Figure 7A-C shows the nucleotide sequence (SEQ ID N0:5) which comprises the open reading frame encoding the human nuclear receptor protein, nNR2.
Figure 8 shows the amino acid sequence of nNR2-1, a carboxy-terminal truncated version of nNR2 (SEQ ID N0:6). The region in bold and underline is the DNA binding domain.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to isolated nucleic acid and protein forms which represent nuclear receptors, preferably but not necessarily limited to human receptors. These expressed proteins are novel nuclear receptors and which are useful in the identification of downstream target genes and ligands regulating their activity. The nuclear receptor superfamily is composed of a group of structurally related receptors which are regulated by chemically distinct ligands.
The common structure for a nuclear receptor is a highly conserved DNA
binding domain (DBD) located in the center of the peptide and the ligand-binding domain (LBD) at the COOH-terminus. Eight out of the nine non-variant cysteines form two type II zinc fingers which distinguish nuclear receptors from other DNA-binding proteins. The DBDs share at least 50% to 60°.fv amino acid sequence identity even among the most distant members in vertebrates. The superfamily has been expanded within the past decade to contain approximately 25 subfamilies. An EST
database search using whole peptide sequences of several representative subfamily members was used to identify two human ESTs (GenBank accession numbers h91890 and w26275 for an EST corresponding to nNRI, nNR2 and/or nNR2-1, respectively). The sequence information from each EST was utilized to isolate and characterize the full length cDNA for the gene corresponding to nNR,l (see Figure lA-C and SEQ ID
NO:1) and nNR2 (see Figure 4A-C and SEQ ID N0:3). The cDNA of SEQ
ID NO:1 encodes nNRl, a protein 500 amino acids in length (Figure 3;
SEQ ID N0:2), which has a distinctive DBD structure (Figure 2A-F).
The cDNA of SEQ ID N0:3 encodes nNR,2, a protein 458 amino acids _g_ (Figure 6; SEQ ID N0:4) in length, and also has a distsnctive DBD
structure (Figure 5A-H). The cDNA of SEQ ID N0:5 encodes nNR2-1, a protein 418 amino acids (Figure 8; SEQ ID N0:6) in length which is a carboxy terminal truncated version of nNR,2. The protein nNR2-1 also S has a distinctive DBD structure (Figure 8).
The nNR1 protein shows 95% homology to hERR2 (GiguLre, et al., 1988, Nature 331: 91-94) in the overlapping peptide region.
However, nNRl contains an additional 6? amino acids at the carboxy-terminus in comparison to hERR2. The gene encoding nNRl is located on locus 14q24.3 ~ 14q31, which is the Alzheimer disease gene 3 (AD3) locus. Therefore, nNRl maybe an endogenous modulator of glucocorticoid receptor (GR) in view of data showing that hERR2 represses GR activity. nNR2 and nNR2-1 share 77% and 75% homology, respectively, at the amino acid level to hERR2 (Gigu~re, et al., 1988, Nature 331: 91-94) in the overlapping region. The nNR2 and nNR1 proteins show ??°k homology at the amino acid level. The gene encoding nNR.2 is located on chromosome 1. Both genes are expressed at very low levels in the majority of the tissues exapnined via RT-PCR.
Therefore, the present invention also relates to isolated nucleic acid fragments of nNRl (SEQ ID NO:1) and nNR2 (SEQ ID N0:3) which encode mRNA expressing a biologically active novel human nuclear receptor. Any such nucleic acid fragment will encode either a protein or protein fragment comprising at least an intracellular DNA-binding domain and/or ligand binding domain, domains conserved throughout the human nuclear receptor family domain which exist in nNRl (SEQ ID N0:2) and nNR,2 (SEQ ID N0:4). Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for nNR,l, nNR,2 and/or nNR,2-1 function. Such a nucleic acid fragment is exemplified as an altered version of the DNA fragment encoding nNR2. This DNA molecule (as set forth in SEQ ID N0:5) is identical to SEQ ID N0:3 save for a two nucleotide insertion at nucleotide 1352 of SEQ

WO 99/10367 PCT/US98/1~826 ID N0:3. This insertion results in a shifted reading frame and introduction of a TGA termination codon 33 nucleotides from the insertion site, resulting in an open reading frame which encodes the carboxy-truncated nNR2 protein, nNR2-1, as shown in Figure 8 and SEQ
ID NO: 6.
The isolated nucleic acid molecule of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
A preferred aspect of the present invention is disclosed in Figure lA-C and SEQ ID NO:1, a human cDNA encoding a novel nuclear traps-acting receptor protein, nNRI, disclosed as follows:

GTCATTTGGA AGAACTGCAG ACTCC TTCAAAATTA AAGTCAGGTT
ATGGAGAACA TGTATGCTAT GTTCTTGATT GCTTCGCTGA AGAAGCATTG
AAATATATTG GTTTCACCTG GAAAAGGCCA ATATACCCAG TAGAAGAATT
AGAAGAAGAA AGCGTTGCAG AAGATGATGC AGAATTAACA TTAAATAAAG
TGGATGAAGA ATTTGTGGAA GAAGAGACAG ATAATGAAGA AAACTTTATT
GATCTCAACG TTTTAAAGGC CCAGACATAT CACTTGGATA TGAACGAGAC
TGCCAAACAA GAAGATATTT TGGAATCCAC AACAGATGCT GCAGAATGGA
GCCTAGAAGT GGAACGTGTA CTACCGCAAC TGAAAGTCAC GATTAGGACT
GACAATAAGG ATTGGAGAAT CCATGTTGAC CAAATGCACC AGCACAGAAG
TGGAATZGAA TCTGCTCTAA AGGAGACCAA GGGATTTTTG GACAAACTCC
ATAATGAAAT TACTAGGACT TTGGAAAAGA TCAGCAGCCG AGAAAAGTAC
ATCAACAATC AGCCGGGAGC CCATGGAGCA CTGTCCTCAG AGATGCGCAG
GTTAGGCTCA CTGTCTAGGC CAGGCCCACC TTAGTCACTG TGGACTGGCA
ATGGAAGCTC TTCCTGGACA CACCTGCCCT AGCCCTCACC CTGGGGTGGA
AGAGAAATGA GCTTGGCTTG CAACTCAGAC CATTCCACGG AGGCATCCTC

CCCTTCCCTG GGCTGGTGAA TAAAAGTTTCCTGAGGTCAA GGACTTCCTT

TTCCCTGCCA AAATGGTGTC CAGAACTTTGAGGCCAGAGG TGATCCAGTG

ATTTGGGAGC TGCAGGTCAC ACAGGCTGCTCAGAGGGCTG CTGAACAGGA

TGTCCTCGGA CGACAGGCAC CTGGGCTCCAGCTGCGGCTC CTTCATCAAG

S ACTGAGCCGT CCAGCCCGTC CTCGGGCATAGATGCCCTCA GCCACCACAG

CCCCAGTGGC TCGTCCGACG CCAGCGGCGGCTTTGGCCTG GCCCTGGGCA

CCCACGCCAA CGGTCTGGAC TCGCCACCCATGTTTGCAGG CGCCGGGCTG

GGAGGCACCC CATGCCGCAA GAGCTACGAGGACTGTGCCA GCGGCATCAT

GGAGGACTCG GCCATCAAGT GCGAGTACATGCTCAACGCC ATCCCCAAGC

IO GCCTGTGCCT CGTGTGCGGG GACATTGCCTCTGGCTACCA CTACGGCGTG

GCCTCCTGCG AGGCTTGCAA GGCCTTCTTCAAGAGGACTA TCCAAGGGAA

CATTGAGTAC AGCTGCCCGG CCACCAACGAGTGCGAGATC ACCAAACGGA

GGCGCAAGTC CTGCCAGGCC TGCCGCTTCATGAAATGCCT CAAAGTGGGG

ATGCTGAAGG AAGGTGTGCG CCTTGATCGAGTGCGTGGAG GCCGTCAGAA

TTTCTCCACC TGCTAAAAAG CCATTGACCAAGATTGTCTC ATACCTACTG

GTGGCTGAGC CGGACAAGCT CTATGCCATGCCTCCCCCTG GTATGCCTGA

GGGGGACATC AAGGCCCTGA CCACTCTCTGTGACCTGGCA GACCGAGAGC

TTGTGGTCAT CATTGGCTGG GCCAAGCACATCCCAGGCTT CTCAAGCCTC

CATCCTGGGC ATCGTGTACC GCTCGCTGCCCTACGACGAC AAGCTGGTGT

ACGCTGAGGA CTACATCATG GATGAGGAGCACTCCCGCCT CGCGGGGCTG

CTGGAGCTCT ACCGGGCCAT CCTGCAGCTGGTACGCAGGT ACAAGAAGCT

CAAGGTGGAG AAGGAGGAGT TTGTGACGCTCAAGGCCCTG GCCCTCGCCA

GACCTGCTGC ACGAGGCACT GCAGGACTACGAGCTGAGCC AGCGCCATGA

GGAGCCCTGG AGGACGGGCA AGCTGCTGCTGACACTGCCG CTGCTGCGGC

AGACGGCCGC CAAGGCCGTG CAGCACTTCTATAGCGTCAA ACTGCAGGGC

AAAGTGCCCA TGCACAAACT CTTCCTGGAGATGCTGGAGG CCAAGGCCTG

CTCCCCTCCA CCGAGCCACC AAGAGGCAGCATGTGCATTT CCTAACTCCC

TTGCCCCCTC CCCCATCTGT GGCCTGGGTGGGCACTGCTC AGGCTGGATA

CCACCTGGAG GTTTZ'CCTTCCGCAGAGGGCAGGTTGGCCA AGAGCAGCTT

AGAGGATCTC CCAAGGATGA AAGAATGTCAAGCCATGATG GAAAATGCCC

TCCCCAATCC ACGCCCTTCT AGTCCAACCC CCCTCAATGA GAGAGGCAGG

CAGATCTCAC CCAGCACTAG GACACCAGGA GGCCAGGGAA AGCATCTCTG

GCTCACCATG TAACATCTGG CTTGGAGCAA GTGGGTGTTC TGCACACCAG

GCAGCTGCAC CTCACTGGAT CTAGTGTTGC TGCGAGTGAC CTCACTTCAG

AGCCCCTCTA GCAGAGTGGG GCGGAAGTCC TGATGGTTGG TGTCCATGAG

GTGGAAG (SEQ .
ID N0:1) Another preferred aspect of the present invention is disclosed in Figure 4A-C and SEQ ID N0:3, a human cDNA encoding a novel nuclear traps-acting receptor protein, nNR,2, disclosed as follows:

GCGGGCCGCC AGTGTGGTGG AATTCGGCTT GTCACTAGGA GAACATTTGT

GTTAATTGCA CTGTGCTCTG TCAAGGAAAC TTTGATTTAT AGCTGGGGTG

CACAAATAAT GGTTGCCGGT CGCACATGGA TTCGGTAGAA CTTTGCCTTC

CTGAATCTTT TTCCCTGCAC TACGAGGAAG AGCTTCTCTG CAGAATGTCA

AACAAAGATC GACACATTGA TTCCAGCTGT TCGTCCTTCA TCAAGACGGA

ACCTTCCAGC CCAGCCTCCC TGACGGACAG CGTCAACCAC CACAGCCCTG

GTGGCTCTTC AGACGCCAGT GGGAGCTACA GTTCAACCAT GAATGGCCAT

CAGAACGGAC TTGACTCGCC ACCTCTCTAC CCTTCTGCTC CTATCCTGGG

AGGTAGTGGG CCTGTCAGGA AACTGTATGA TGACTGCTCC AGCACCATTG

TTGAAGATCC CCAGACCAAG TGTGAATACA TGCTCAACTC GATGCCCAAG

AGACTGTGTT TAGTGTGTGG TGACATCGCT TCTGGGTACC ACTATGGGGT

AGCATCATGT GAAGCCTGCA AGGCATTCTT CAAGAGGACA ATTCAAGGCA

ATATAGAATA CAGCTGCCCT GCCACGAATG AATGTGAAAT CACAAAGCGC

AGACGTAAAT CCTGCCAGGC TTGCCGCTTC ATGAAGTGTT TAAAAGTGGG

CATGCTGAAA GAAGGGGTGC GTCTTGACAG AGTACGTGGA GGTCGGCAGA

AGTACAAGCG CAGGATAGAT GCGGAGAACA GCCCATACCT GAACCCTCAG

CTGGTTCAGC CAGCCAAAAA GCCATATAAC AAGATTGTCT CACATTTGTT

GGTGGCTGAA CCGGAGAAGA TCTATGCCAT GCCTGACCCT ACTGTCCCCG

ACAGTGACAT CAAAGCCCTC ACTACACTGT GTGACTTGGC CGACCGAGAG

TTGGTGGTTA TCATTGGATG GGCGAAGCAT ATTCCAGGCT TCTCCACGCT

GTCCCTGGCG GACCAGATGA GCCTTCTGCA GAGTGCTTGG ATGGAAATTT

TGATCCTTGG TGTCGTATAC CGGTCTCTTT CATTTGAGGA TGAACTTGTC

TATGCAGACG ATTATATAAT GGACGAAGAC CAGTCCAAAT TAGCAGGCCT

TCTTGATCTA AATAATGCTA TCCTGCAGCT GGTAAAGAAA TACAAGAGCA

TGAAGCTGGA AAAAGAAGAA TTTGTCACCC TCAAAGCTAT AGCTCTTGCT

AATTCAGACT CCATGCACAT AGAAGATGTT GAAGCCGTTC AGAAGCTTCA

GGATGTCTTA CATGAAGCGC TGCAGGATTA TGAAGCTGGC CAGCACATGG

AAGACCCTCG TCGAGCTGGC AAGATGCTGA TGACACTGCC ACTCCTGAGG

CAGACCTCTA CCAAGGCCGT GCAGCATTTC TACAACATCA AACTAGAAGG

CAAAGTCCCA ATGCACAAAC TTTTTTTGGA AATGTTGGAG GCCAAGGTCT

S GACTAAAAGC TCCCTGGGCC TTCCCATCCT TCATGTTGAA AAAGGGAAAA

TAAACCCAAG AGTGATGTCG AAGAAACTTA GAGTTTAGTT AACAACATCA

AAAATCAACA GACTGCACTG ATAATTTAGC AGCAAGACTA TGAAGCAGCT

TTCAGATTCC TCCATAGGTT CCTGATGAGT TCTTTCTACT TTCTCCATCA

TCTTCTTTCC TCTTTCTTCC CACATTTCTC TTTCTCTTTA TTTTTTCTCC

lO TTTTCTTCTT TCACCTCCCT TATTTCTTTG CTTCTTTCAT TCCTAGTTCC

CATTCTCCTT TATTTTCTTC CCGTCTGCCT GCCTTCTTTC TTTTCTTTAC

CTACTCTCAT TCCTCTCTTT TCTCATCCTT CCCCTTTTTT CTAAATTTGA

AATAGCTTTA GTTTAAAAAA AAAAATCCTC CCTTCCCCCT TTCCTTTCCC

TTTCTTTCCT TTTTCCCTTT CCTTTTCCCT TTCCTTTCCT TTCCTCTTGA

AGGTCTCTAA CTGAAGAGAG ATGGAAGCCA GCCCTGCCAA AGGATGGAGA

TCCATAATAT GGATGCCAGT GAACTTATTG TGAACCATAC CGTCCCCAAT

GACTAAGGAA TCAAAGAGAG AGAACCAACG TTCCTAAAAG TACAGTGCAA

CATATACAAA TTGACTGAGT GCAGTATTAG ATTTCATGGG AGCAGCCTCT

TCCATCTAGA TCAGTTACAG CCATTTGATT CCTTAATTGT TTTTTCAAGT

CTTCCAGGTA TTTGTTAGTT TAGCTACTAT GTAACTTTTT CAGGGAATAG

TTTAAGCTTT ATTCATTCAT GCAATACTAA AGAGAAATAA GAATACTGCA

ATTTTGTGCT GGCTTTGAAC AATTACGAAC AATAATGAAG GACAAATGAA

2S TCCTGAAGGA AGATTTTZ'AAAAATG TTTCTTCTTA CAAATGGAGA

TTTTTTTGTA CCAGCTTTAC CACTTTTCAG CCATTTATTA ATATGGGAAT

TTAACTTACT CAAGCAATAG TTGAAGGGAA GGTGCATATT ATCACGGATG

CAATTTATGT TGTGTGCCAG TCTGGTCCCA AACATCAATT TCTTAACATG

AGCTCCAGTT TACCTAAATG TTCACTGACA CAAAGGATGA GATTACACCT

TCCGTAGAAT TGTCAGGAGT GCACCTCTCT ACTTGGGAGG TACAATTGCC

ATATGATTTC TAGCTGCCAT GGTGGTTAGG AATGTGATAC TGCCTGTTTG

CAAAGTCACA GACCTTGCCT CAGAAGGAGC TGTGAGCCAG TATTCATTTA

AGAGAATTCC ACCACACTGG CGGCCCGCGC TTGAT (SEQ
ID N0:3).

The present invention also relates to an isolated and purified DNA molecule which encodes a truncated version of nNR,2 referred to as nNR2-1. This cDNA molecule is set forth in SEQ ID N0:5 and is disclosed as follows:
S GCGGGCCGCC AGTGTGGTGG AATTCGGCTT GTCACTAGGAGAACATTTGT

GTTAATTGCA CTGTGCTCTG TCAAGGAAAC TTTGATTTATAGCTGGGGTG

CACAAATAAT GGTTGCCGGT CGCACATGGA TTCGGTAGAACTTTGCCTTC

CTGAATCTTT TTCCCTGCAC TACGAGGAAG AGCTTCTCTGCAGAATGTCA

AACAAAGATC GACACATTGA TTCCAGCTGT TCGTCCTTCATCAAGACGGA

lO ACCTTCCAGC CCAGCCTCCC TGACGGACAG CGTCAACCACCACAGCCCTG

GTGGCTCTTC AGACGCCAGT GGGAGCTACA GTTCAACCATGAATGGCCAT

CAGAACGGAC TTGACTCGCC ACCTCTCTAC CCTTCTGCTCCTATCCTGGG

AGGTAGTGGG CCTGTCAGGA AACTGTATGA TGACTGCTCCAGCACCATTG

TTGAAGATCC CCAGACCAAG TGTGAATACA TGCTCAACTCGATGCCCAAG

AGCATCATGT GAAGCCTGCA AGGCATTCTT CAAGAGGACAATTCAAGGCA

ATATAGAATA CAGCTGCCCT GCCACGAATG AATGTGAAATCACAAAGCGC

AGACGTAAAT CCTGCCAGGC TTGCCGCTTC ATGAAGTGTTTAAAAGTGGG

CATGCTGAAA GAAGGGGTGC GTCTTGACAG AGTACGTGGAGGTCGGCAGA

CTGGTTCAGC CAGCCAAAAA GCCATATAAC AAGATTGTCTCACATTTGTT

GGTGGCTGAA CCGGAGAAGA TCTATGCCAT GCCTGACCCTACTGTCCCCG

ACAGTGACAT CAAAGCCCTC ACTACACTGT GTGACTTGGCCGACCGAGAG

TTGGTGGTTA TCATTGGATG GGCGAAGCAT ATTCCAGGCTTCTCCACGCT

TGATCCTTGG TGTCGTATAC CGGTCTCTTT CATTTGAGGATGAACTTGTC

TATGCAGACG ATTATATAAT GGACGAAGAC CAGTCCAAATTAGCAGGCCT

TCTTGATCTA AATAATGCTA TCCTGCAGCT GGTAAAGAAATACAAGAGCA

TGAAGCTGGA AAAAGAAGAA TTTGTCACCC TCAAAGCTATAGCTCTTGCT

GGATGTCTTA CATGAAGCGC TGCAGGATTA TGAAGCTGGCCAGCACATGG

AGAAGACCCT CGTCGAGCTG GCAAGATGCT GATGACACTGCCACTCCTGA

GGCAGACCTC TACCAAGGCC GTGCAGCATT TCTACAACATCAAACTAGAA

GGCAAAGTCC CAATGCACAA ACTTZ"I"I'TTGGAAATGTTGGAGGCCAAGGT

AATAAACCCA AGAGTGATGT CGAAGAAACT TAGAGTTTAGTTAACAACAT

CAAAAATCAA CAGACTGCAC TGATAATTTA GCAGCAAGACTATGAAGCAG

CTTTCAGATT CCTCCATAGG TTCCTGATGA GTTCTTTCTACTTTCTCCAT

CATCTTCTTT CCTCTTTCTT CCCACATTTC TCTTTCTCTTTATTTTTTCT

CCTTTTCTTC TTTCACCTCC CTTATTTCTT TGCTTCTTTCATTCCTAGTT

CCCATTCTCC TTTATTTTCT TCCCGTCTGC CTGCCTTCTTTCTTTTCTTT

ACCTACTCTC ATTCCTCTCT TTTCTCATCC TTCCCCTTTTTTCTAAATTT

GAAATAGCTT TAGTTTAAAA AAAAAAATCC TCCCTTCCCCCTTTCCTTTC

CCTTTCTTTC CTTTTTCCCT TTCCTTTTCC CTTTCCTTTCCTTTCCTCTT

lO GACCTTCTTT CCATCTTTCT TTTTCTTCCT TCTGCTGCTGAACTTTTAAA

AGAGGTCTCT AACTGAAGAG AGATGGAAGC CAGCCCTGCCAAAGGATGGA

GATCCATAAT ATGGATGCCA GTGAACTTAT TGTGAACCATACCGTCCCCA

ATGACTAAGG AATCAAAGAG AGAGAACCAA CGTTCCTAAAAGTACAGTGC

AACATATACA AATTGACTGA GTGCAGTATT AGATTTCATGGGAGCAGCCT

TTTCCATCTA GATCAGTTAC AGCCATTZGA TTCCTTAATTGTTTTTTCAA

GTCTTCCAGG TATTTGTTAG TTTAGCTACT ATGTAACTTTTTCAGGGAAT

AGTTTAAGCT TTATTCATTC ATGCAATACT AAAGAGAAATAAGAATACTG

CAATTTTGTG CTGGCTTTGA ACAATTACGA ACAATAATGAAGGACAAATG

2O AATCCTGAAG GAAGAT'I~I'AAAAATGTTT TGTTTCTTCTTACAAATGGA

GATTTTTTTG TACCAGCTTT ACCAC AGCCATTTATTAATATGGGA

ATTTAACTTA CTCAAGCAAT AGTTGAAGGG AAGGTGCATATTATCACGGA

TGCAATTTAT GTTGTGTGCC AGTCTGGTCC CAAACATCAATTTCTTAACA

TGAGCTCCAG TTTACCTAAA TGTTCACTGA CACAAAGGATGAGATTACAC

GATCCGTAGA ATTGTCAGGA GTGCACCTCT CTACTTGGGAGGTACAATTG

CCATATGATT TCTAGCTGCC ATGGTGGTTA GGAATGTGATACTGCCTGTT

TGCAAAGTCA CAGACCTTGC CTCAGAAGGA GCTGTGAGCCAGTATTCATT

TAAGAGAATT CCACCACACT GGCGGCCCGC GCTTGAT
(SEQ ID
N0:5) 30 The present invention also relates to a substantially purified form of the novel nuclear trans-acting receptor protein, nNRl, which is shown in as set Figures forth 2A-F and in SEQ
Figure 3 ID N0:2, and disclosed as follows:

MSSDDRHLGS BCGSFIKTEP SSPSSGIDAL SHHSPSGSSDASGGFGLALG

RLCLVCGDIA SGYHYGVASC EACKAFFKRT IQGNIEYSCP ATNECEITKR
RRKSCQACRF MKCLKVGMLK EGVRLDRVRG GRQKYKRRLD SESSPYLSLQ
ISPPAKKPLT KIVSYLLVAE PDKLYAMPPP GMPEGDIKAL TTLCDLADRE
LWIIGWAKH IPGFSSLSLG DQMSLLQSAW MEILILGIVY RSLPYDDKLV
S YAEDYINmEE HSRLAGLLEL YRAILQLVRR YKKLKVEKEE FVTLKALALA
NSDSMYIEDL EAVQKLQDLL HEALQDYELS QRHEEPWRTG KLLLTLPLLR
QTAAKAVQHF YSVKLQGKVP MHKLFLEMLE AKAWARADSL QEWRPLEQVP.
SPLHRATKRQ HVHFLTPLPP PPSVAWVGTA QAGYHLEVFL PQRAGWPRAA
( SEQ ID NO : 2 ) .
The present invention also relates to biologically active fragments and/or mutants of nNR1 as set forth as SEQ ID N0:2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists of nNRl function.
The present invention also relates to a substantially purified form of the novel nuclear trans-acting receptor protein, nNR2, which is shown in Figure 5A-H and Figure 6 and as set forth in SEQ ID N0:4, disclosed as follows:
MDSVELCLPE SFSLHYEEEL LCRMSNKDRH IDSSCSSFIK TEPSSPASLT
DSVNHHSPGG SSDASGSYSS TMNGHQNGLD SPPLYPSAPI LGGSGPVRKL
YDDCSSTIVE DPQTKCEYML NSMPKRLCLV CGDIASGYHY GVASCEACKA
FFKRTIQGNI EYSCPATNEC EITKRRRKSC QACRFMKCLK VGMLKEGVRL
DRVRGGRQKY KRRIDAENSP YLNPQLVQPA KKPYNKIVSH LLVAEPEKIY
AMPDPTVPDS DIKALTTLCD LADRELWII GWAKHIPGFS TLSLADQMSL
LQSAWMEILI LGWYRSLSF EDELWADDY IMDEDQSKLA GLLDLNNAIL
QLVKKYKSMK LEKEEFVTLK AIALANSDSM HIEDVEAVQK LQDVLHEALQ
DYEAGQHMED PRRAGKMLMT LPLLRQTSTK AVQHFYNIKL EGKVPMHKLF
LEMLEAKV (SEQ ID N0:4).
The present invention also relates to biologically active fragments and/or mutants of nNR,2 as set forth as SEA ID N0:4, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carbozy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists of nNR2 function.
To this end, an example of such a protein is the carboxy-terminal truncated version of nNR,2, referred to as nNR2-1 and described in Figure 8 and set forth as SEQ ID N0:6, as follows:
MDSVELCLPE SFSLHYEEEL LCRMSNKDRH IDSSCSSFIK TEPSSPASLT
DSVNHHSPGG SSDASGSYSS TMNGHQNGLD SPPLYPSAPI LGGSGPVRKL.
YDDCSSTIVE DPQTKCEYML NSMPKRLCLV CGDIASGYHY GVASCEACKA
FFKRTIQGNI EYSCPATNEC EITKRRRKSC QACRFMKCLK VGMLKEGVRL
DRVRGGRQKY KRRIDAENSP YLNPQLVQPA KKPYNKIVSH LLVAEPEKIY
AMPDPTVPDS DIKALTTLCD LADRELVVII GWAKHIPGFS TLSLADQMSL
LQSAWMEILI LGVVYRSLSF EDELVYADDY IMDEDQSKLA GLLDLNNAIL
QLVKKYKSMK LEKEEFVTLK AIALANSDSM HIEDVEAVQK LQDVLHEALQ
DYEAGQHMEK TLVELARC (SEQ ID N0:6).
The present invention also relates to isolated nucleic acid molecules which are fusion constructions expressing fusion proteins useful in assays to identify compounds which modulate wild-type human nNRl, nNR2 and/or nNR,2-1 activity. A preferred aspect of this portion of the invention includes, but is not limited to, glutathione S-transferase GST-nNRl and/or GST-nNR2 fusion constructs. These fusion constructs include, but are not limited to, all or a portion of the ligand-binding domain of nNRl, nNR2 and/or nNR2-1, respectively, as an in-frame fusion at the carboxy terminus of the GST gene. The disclosure of SEQ ID NOS:1-4 allow the artisan of ordinary skill to construct any such nucleic acid molecule encoding a GST-nuclear receptor fusion protein. Soluble recombinant GST-nuclear receptor fusion proteins may be expressed in various expression systems, including Spodoptera frugiperdac (St21) insect cells (Invitrogen) using a baculovirus expression vector (e.g., Bac-N-Blue DNA from Invitrogen or pAcG2T from Pharmingen).
The isolated nucleic acid molecule of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
It is known that there is a substantial amount of redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those DNA
sequences encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid, as shown below:
A=Ala=Alanine: codons GCA, GCC, GCG, GCU
C=Cys=Cysteine: codons UGC, UGU
D=Asp=Aspartic acid: codons GAC, GAU
E=Glu=Glutamic acid: codons GAA, GAG
F=Phe=Phenylalanine: codons UUC, UUU
G=Gly=Glycine: codons GGA, GGC, GGG, GGU
H=His =Histidine: codons CAC, CAU
I=Ile =Isoleucine: codons AUA, AUC, AUU
K=Lys=Lysine: codons AAA, AAG
L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU
M=Met=Methionine: codon AUG
N=Asp=Asparagine: codons AAC, AAU
P=Pro=Proline: codons CCA, CCC, CCG, CCU
Q=Gln=Glutamine: codons CAA, CAG
R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU
S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU
T=Thr=Threonine: codons ACA, ACC, ACG, ACU
V=Val=Valine: codons GUA, GUC, GUG, GUU
W=Trp=Tryptophan: codon UGG
Y=Tyr=Tyrosine: codons UAC, UAU
Therefore, the present invention discloses codon redundancy which may result in differing DNA molecules expressing an identical protein. For purposes of this specification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide. Methods of altering the DNA sequences include but are not limited to site directed mutagenesis.
Examples of altered properties include but are not limited to changes in the amity of an enzyme for a substrate or a receptor for a ligand.
As used herein, "purified" and "isolated" are utilized interchangeably to stand for the proposition that the nucleic acid, protein, or respective fragment thereof in question has been substantially removed from its in viuo environment so that it may be manipulated by the skilled artisan, such as but not limited to nucleotide sequencing, restriction digestion, site-directed mutagenesis, and subcloning into expression vectors for a nucleic acid fragment as well as obtaining the protein or protein fragment in pure quantities so as to afford the opportunity to generate polyclonal antibodies, monoclonal antibodies, amino acid sequencing, and peptide digestion. Therefore, the nucleic acids claimed herein may be present in whole cells or in cell lysates or in a partially purified or substantially purified form. A
nucleic acid is considered substantially purified when it is purified away from environmental contaminants. Thus, a nucleic acid sequence isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
Therefore, the present invention also relates to methods of expressing nNRl, nNR,2 and/or nNR2-1 and biological equivalents disclosed herein, assays employing these recombinantly expressed gene products, cells expressing these gene products, and agonistic and/or antagonistic compounds identified through the use of assays utilizing these recombinant forms, including, but not limited to, one or more modulators of the human nNRl, nNR2 and/or nNR2-1 either through direct contact LBD or through direct or indirect contact with a ligand which either interacts with the DBD or with the wild-type transcription complex which either nNR,l, nNR2 and/or. nNR2-I interacts in traps, thereby modulating cell differentiation or cell development.
Aa used herein, a "biologically active equivalent" or "functional derivative" of a wild-type human nNRl, nNR2 and/or nNR2-1 possesses a biological activity that is substantially similar to the biological activity of the wild type human nNRl, nNR2 and/or nNR2-1.
The term "functional derivative" is intended to include the "fragments,"
"mutants," "variants," "degenerate variants," "analogs" and "homologues" or to "chemical derivatives" of the wild type human nNRl, nTTR2 and/or nNR2-1 protein. The term "fragment" is meant to refer to any polypeptide subset of wild-type human nNRl or nNR,2. The term "mutant" is meant to refer to a molecule that may be substantially similar to the wild-type form but possesses distinguishing biological characteristics. Such altered characteristics include but are in no way limited to altered substrate binding, altered substrate affinity and altered sensitivity to chemical compounds affecting biological activity of the human nNRI, nNR2 and/or nNR2-1 or human nNRl, nNR2 and/or nNR,2-1 functional derivatives. The term "variant" is meant to refer to a molecule substantially similar in structure and function to either the entire wild-type protein or to a fragment thereof. A molecule is "substantially similar" to a wild-type human nNRl, nNR,2 and/or nNR,2-1-like protein if both molecules have substantially similar structures or if both molecules possess similar biological activity.
Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical. The term "analog" refers to a molecule substantially similar in function to either the full-length human nNRl, nNR,2 and/or nNR2-1 protein or to a biologically active fragment thereof.
Any of a variety of procedures may be used to clone human nNR,l, nI~TR,2 and/or nNR,2-1. These methods include, but are not limited to, (1) a RACE PCR cloning technique (Frohman, et al., 1988, Proc. Nactl. Acad. Sci. USA 85: 8998-9002). 5' and/or 3' RACE may be performed to generate a full-length cDNA sequence. This strategy involves using gene-specific oligonucleotide primers for PCR
amplification of human nNRl, nNR2 and/or nNR,2-1 cDNA. These gene-specific primers are designed through identification of an expressed sequence tag (EST) nucleotide sequence which has been identified by searching any number of publicly available nucleic acid and protein databases; (2) direct functional expression of the human nNRI, nNR2 and/or nNR,2-1 cDNA following the construction of a human nNRl, nNR2 and/or nNR2-1-containing cDNA library in an appropriate expression vector system; (3) screening a human nNRl, nNR2 and/or nNR2-1-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labeled degenerate oligonucleotide probe designed from the amino acid sequence of the human nNRl, nNR2 and/or nNR,2-1 protein; (4) screening a human nNRl, nNR2 andlor nNR,2-1-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the human nNRl, nNR,2 and/or nNR,2-1 protein. This partial cDNA is obtained by the specific PCR amplification of human nNR,l, nNR2 and/or nNR2-1 DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence known for other kinases which are related to the human nNRl, nNR2 and/or nNR2-1 protein; (5) screening a human nNR,l, nNR2 and/or nNR,2-1-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the human nNR,l, nNR2 and/or nNR2-1 protein. This strategy may also involve using gene-specific oligonucleotide primers for PCR amplification of human nNR,l, nNR2 and/or nNR,2-1 cDNA identified as an EST as described above; or (6) designing 5' and 3' gene specific oligonucleotides using SEQ ID NO: 1 as a template so that either the full-length cDNA may be generated by known PCR techniques, or a portion of the coding region may be generated by these same known PCR techniques to generate and isolate a portion of the coding region to use as a probe to screen one of numerous types of cDNA and/or genomic libraries in order to isolate a full-length version of the nucleotide sequence encoding human nNRl;
nNR2 and/or nNR2-1.
It is readily apparent to those skilled in the art that other types of libraries, as well as libraries constructed from other cell types-or species types, may be useful for isolating a nNR,l, nNR2 and/or nNR,2-1-encoding DNA or a nNRl, nNR2 and/or nNR,2-1 homologue. Other types of libraries include, but are not limited to, cDNA libraries derived from other cells or cell lines other than human cells or tissue such as marine cells, rodent cells or any other such vertebrate host which may contain nNRI, nNR2 and/or nNR2-1-encoding DNA. Additionally a nNRI, nNR2 and/or nNR2-1 gene and homologues may be isolated by oligonucleotide- or polynucleotide-based hybridization screening of a vertebrate genomic library, including but not limited to, a marine genomic library, a rodent genomic library, as well as concomitant human genomic DNA libraries.
It is readily apparent to those skilled in the art that suitable cDNA libraries may be prepared from cells or cell lines which have nNR,I, nNR2 and/or nNR2-1 activity. The selection of cells or cell lines for use in preparing a cDNA library to isolate a cDNA encoding nNRl, nNR,2 and/or nNR2-1 may be done by first measuring cell-associated nNRl, nNR2 and/or nNft2-1 activity using any known assay available for such a purpose.
Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found for example, in Sambrook et al., 1989, Moleculacr Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Complementary DNA
libraries may also be obtained from numerous commercial sources, including but not limited to Clontech Laboratories, Inc. and Stratagene.
It is also readily apparent to those skilled in the art that DNA encoding human nNftl, nNR,2 and/or nNR2-1 may also be isolated from a suitable genomic DNA library. Construction of genomic DNA
libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techniques can be found in Sambrook, et al., supra.

In order to clone the human nNR,l, nNR,2 and/or nNR2-1 gene by one of the preferred methods, the amino acid sequence or DNA
sequence of human nNRl, nNR2 and/or nNR2-I or a homologous protein may be necessary. To accomplish this, the nNRl, nNR2 andJor nNR,2-1 protein or a homologous protein may be purified and partial amino acid sequence determined by automated sequenators. It is not necessary to determine the entire amino acid sequence, but the linear sequence of two regions of 6 to 8 amino acids can be determined for the PCR amplification of a partial human nNRl, nNR,2 and/or nNR,2-1 DNA
fragment. Once suitable amino acid sequences have been identified, the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the human nNR.l, nNR2 and/or nNR2-1 sequence but others in the set will be capable of hybridizing to human nNRl, nNR2 andlor nNR2-1 DNA even in the presence of DNA
oligonucleotides with nusmatches. The mismatched DNA
oligonucleotidea may still sufficiently hybridize to the human nNRI, nNR2 and/or nNR2-1 DNA to permit identification and isolation of human nNRl, nNR2 and/or nNR2-1 encoding DNA. Alternatively, the nucleotide sequence of a region of an expressed sequence may be identified by searching one or more available genomic databases. Gene-specific primers may be used to perform PCR amplification of a cDNA of interest from either a cDNA library or a population of cDNAs. As noted above, the appropriate nucleotide sequence for use in a PCR-based method may be obtained from SEQ ID NO: 1, either for the purpose of isolating overlapping 5' and 3' RACE products for generation of a full-length sequence coding for human nNR,l, nNR2 and/or nNR2-1, or to isolate a portion of the nucleotide sequence coding for human nNRl, nNR2 and/or nNR2-1 for use as a probe to screen one or more cDNA- or genomic-based libraries to isolate a full-length sequence encoding human nNRI, nNR2 and/or nNR2-1 or human nNRl, nNR2 and/or nNR2-1-like proteins.
_ 2,3 -In an exemplified method, the human nNR,l, nNR2 and/or nNR,2-1 full-length cDNA of the present invention were generated by PCR scanning human cDNA libraries with oligonucleotide primers generated from ESTs showing homology to hERR2. Briefly, random and oligo dT primed cDNA libraries as described herein which consist of approximately 4 million primary clones were constructed in the plasmid vector pBluescript (Stratagene, LaJolla, CA). The primary clones were subdivided into 188 pools with each pool containing -20,000 clones. Each pool was amplified separately and the resulting plasmid pools were collected and transferred into two 96-well plates. Primer pairs from the 5' and 3' portion of an EST are used to scan the respective cDNA library distributed in a 96-well plate. Initial positive pools are identified with EST primers. Corresponding full length cDNA clones were retrieved via inverse PCR using primer pairs designed from the EST which are back IS to back against each other. Therefore, the primers walk away from each other during the PCR reaction, resulting in amplification of a population of linearized plasmid DNA molecules corresponding to the EST. cDNA clones were obtained by ligating linear DNA and transforming the circularized DNA into bacteria competent cells.
Usually, four positive clones for each gene were used for sequence analysis because of the possibility of mutation during long PCR
reactions. The consensus DNA sequence is considered as the wild type DNA sequence. Recloning of the gene through PCR using gene specific primers covering the whole open reading frame was done so as to obtain a cDNA clone which has an identical DNA sequence to the consensus sequence. This procedure does not depend upon using a cDNA library with directionally cloned inserts, but does require cDNA libraries constructed in a plasmid vector, such as pBluescript. This procedure was utilized to identify full length cDNA molecules representing human nNRl, nNR2 and/or nlVR2-1.
A variety of mammalian expression vectors may be used to express recombinant human nNRl, nNR2 and/or nNR2-1 in mammalian cells. Expression vectors are defined herein as DNA
sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, blue green algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA
sequence that directs RNA polymerase to bind to DNA and initiate RNA
synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
Commercially available mammalian expression vectors which may be suitable for recombinant human nNRI, nNR2 and/or nNR2-1 expression, include but are not limited to, pcDNA3.1 (Invitrogen), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV 1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pR,SVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC
37460), and 1ZD35 (ATCC 37565).
A variety of bacterial expression vectors may be used to express recombinant human nNR,i, nNR2 and/or nNR2-1 in bacterial cells. Commercially available bacterial expression vectors which may be suitable for recombinant human nNRl, nNR,2 and/or nNR,2-1 expression include, but are not limited to pQE (Qiagen), pETlla (Novagen), lambda gtll (Invitrogen), and pKK223-3 (Phartnacia).
A variety of fungal cell expression vectors may be used to express recombinant human nNR,l, nNR2 and/or nNR2-1 in fungal cells. Commercially available fungal cell expression vectors which may be suitable for recombinant human nNR,I, nNR2 and/or nNR,2-1 expression include but are not limited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).

A variety of insect cell expression vectors may be used to express recombinant receptor in insect cells. Commercially available insect cell expression vectors which may be suitable for recombinant expression of human nNRl, nNR.2 and/or nNR,2-1 include but are not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T
(Pharmingen).
An expression vector containing DNA encoding a human nNRl, nNR2 and/or nNR2-1-like protein may be used for expression of human nNRl; nNR2 and/or nNR2-1 in a recombinant host cell.
Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila- and silkworm-derived cell lines. Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL
171) and CPAE (ATCC CCL 209).
The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation.
The expression vector-containing cells are individually analyzed to determine whether they produce human nNRl, nNR2 and/or nNR,2-1 protein. Identification of human nNRI, nNR2 and/or nNR2-1 expressing cells may be done by several means, including but not limited to immunological reactivity with anti-human nNRl, nNR2 and/or nNR2-1 antibodies, labeled ligand binding and the presence of host cell-associated human nNRl, nNR2 and/or nNR,2-1 activity.
The cloned human nNRl, nNR2 and/or nNR,2-1 cDNA
obtained through the methods described above may be recombinantly expressed by molecular cloning into an expression vector (such as pcDNA3.1, pQE, pBlueBacHis2 and pLITMUS28) containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant human nNRl, nNR,2 and/or nNR2-1. Techniques for such manipulations can be found described in Sambrook, et aL, supra, are discussed at length in the Example section and are well known and easily available to the artisan of ordinary skill in the art.
Expression of human nNR,I; nNR2 and/or nNR2-1 DNA
may also be performed using in uitro produced synthetic mRNA.
Synthetic mRNA can be eiBciently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as eiBciently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.
To determine the human nNR,I, nNR2 and/or nNR2-1 cDNA sequences) that yields optimal levels of human nNRl, nNR,2 and/or nNR2-1, cDNA molecules including but not limited to the following can be constructed: a cDNA fragment containing the full-length open reading frame for human nNRi, nNR2 and/or nNR,2-1 as well as various constructs containing portions of the cDNA encoding only specific domains of the protein or rearranged domains of the protein. All constructs can be designed to contain none, all or portions of the 5' and/or 3' untranslated region of a human nNR,l, nNR2 and/or nNR,2-1 cDNA. The expression levels and activity of human nNRl, nNR2 and/or nNR,2-1 can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells. Following determination of the human nNRI, nNR,2 and/or nNR,2-1 cDNA cassette yielding optimal expression in transient assays, this nNRl, nhTR2 and/or nNR2-1 cDNA construct is transferred to a variety of expression vectors (including recombinant viruses), including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, bacteria, and yeast cells.
The present invention also relates to polyclonal and monoclonal antibodies raised in response to either the human form of nNRl, nNR2 and/or nNR,2-1 disclosed herein, or a biologically active -27_ fragment thereof. It will be especially preferable to raise antibodies against epitopes within the NHa terminal domain of nNRl, nNR2 and/or nNR2-1, which show the least homology to other known proteins belonging to the human nuclear receptor auperfamily.
Recombinant nNR,l, nNR,2 and/or nNR,2-1 protein can be separated from other cellular proteins by use of an immunoa~nity column made with monoclonal or polyclonal antibodies specific for full-length nNRI, nNR2 and/or nNR2-1 protein, or polypeptide fragments of nNRl, nNR2 and/or nNR,2-1 protein. Additionally, polyclonal or monoclonal antibodies may be raised against a synthetic peptide (usually from about 9 to about 25 amino acids in length) from a portion of the protein as disclosed in SEQ ID N0:2. Monospecific antibodies to human nNR,l, nNR2 and/or nNR2-1 are purified from mammalian antisera containing antibodies reactive against human nNRl, nNR,2 and/or nNR2-1 or are prepared as monoclonal antibodies reactive with human nNRl, nNR.2 and/or nNR,2-1 using the technique of Kohler and Milstein (1975, Nature 256: 495-497). Monoapecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for human nNRl, nNR,2 and/or nNR2-1. Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with human nNR,I, nNR,2 and/or nNR2-1, as described above. Human nNRl, nNR2 and/or nNR2-1-specific antibodies are raised by immunizing animals such as mice, rata, guinea pigs, rabbits, goats, horses and the like, with an appropriate concentration of human nNRl, nNR2 and/or nNR,2-1 protein or a c synthetic peptide generated from a portion of human nNRl, nNR2 and/or nNR,2-1 with or without an immune adjuvant.
Preimmune serum is collected prior to the first e: 30 immunization. Each animal receives between about 0.1 mg and about e: 1000 mg of human nNRl, nNR2 and/or nNR2-1 protein associated with Di an acceptable immune adjuvant. Such acceptable adjuvanta include, i but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium paruum and tRNA. The initial immunization consists of human nNRl, nNR2 and/or nNR2-1 protein or peptide fragment thereof in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally (IP) or both. Each animal is bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunization. Those animals receiving booster injections are generally given an equal amount of human nNRl, nNR2 and/or nNR2-1 in Freund's incomplete adjuvant by the same route. Booster injections are given at about three week intervals until maximal titers are obtained. At about ? days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about -20°C.
Monoclonal antibodies (mAb) reactive with human nNRl, nNR2 and/or nNR2-1 are prepared by immunizing inbred mice, preferably Balb/c, with human nNR,l, nNR2 and/or nNR2-1 protein.
The mice are immunized by the IP or SC route with about 1 mg to about 100 mg, preferably about 10 mg, of human nNRl, nNR2 and/or nNR2-1 protein in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred. The mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 1 to.about 100 mg of human nNRl, nNR,2 and/or nNR,2-1 in a buffer solution such as phosphate buffered saline by the intravenous (IV) route. Lymphocytes, from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas. Fusion partners may include, but are not limited to:
mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's _ 29 -Modified Eagles Medium (DMEM) by procedures known in the art.
Supernatant fluids are collected form growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using S human nNRI, nNR2 and/or nNR2-1 as the antigen. The culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, 1973, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press.
Monoclonal antibodies are produced in viao by injection of pristine primed Balb/c mice, approximately 0.5 ml per mouse, with about 2 x 108 to about 6 x 108 hybridoma cells about 4 days after priming.
Ascites fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
In vitro production of anti-human nNRI, nNR2 and/or nNR,2-1 mAb is carried out by growing the hybridoma in DMEM
containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb. The mAb are purified by techniques known in the art.
Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of human nNR,l, nNR2 and/or nNR2-1 in body fluids or tissue and cell extracts.
It is readily apparent to those skilled in the art that the above described methods for producing monospecific antibodies may be utilized to produce antibodies specific for human nNRl, nNR2 and/or nNR2-1 peptide fragments, or full-length human nNRl, nNR,2 and/or nNR2-1.
Human nNRl, nNR2 and/or nNR2-1 antibody amity columns are made, for example, by adding the antibodies to Afligel-10 (Biorad), a gel support which is pre-activated with N--3p_ hydroxysuccinimide esters such that the antibodies form covalent linkages with,the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HCl (pH 8).
The column is washed with water followed by 0.23 M glycine HCl (pH
2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extracts containing full-length human nNRl, nNR,2 and/or nNR2-1 or human nNR,l, nNR2 and/or nNR2-1 protein fragments are slowly passed through the column. The column is then washed with phosphate buffered saline until the optical density (A2g0) falls to background, then the protein is eluted with 0.23 M
glycine-HCl (pH 2.6). The purified human nNRl, nNR2 and/or nNR2-1 protein is then dialyzed against phosphate buffered saline.
Levels of human nNRl, nNR,2 and/or nNR2-1 in host cells is quantified by a variety of techniques including, but not limited to, immunoaffinity and/or ligand affinity techniques. nNRl, nNR2 and/or nNR2-1-specific affinity beads or nNR,l, nNR2 and/or nNR2-1-specific antibodies are used to isolate ~S-methionine labeled or unlabelled nNRl, nNR2 and/or nNR2-1. Labeled nNRl, nNR2 and/or nNR2-1 protein is analyzed by SDS-PAGE. Unlabelled nNR,l, nNR2 and/or nNR2-1 protein is detected by Western blotting, ELISA or RIA assays employing either nNRI, nNR2 and/or nNR2-1 protein specific antibodies andlor antiphosphotyrosine antibodies.
Following expression of nNRl, nNR2 and/or nNR,2-I in a host cell, nNRI, nNR2 and/or nNR,2-1 protein may be recovered to provide nlVRl, nNR,2 and/or nNR2-1 protein in active form. Several nNRl, nNR2 and/or nNR2-1 protein purification procedures are available and suitable for use. Recombinant nNRl, nNR,2 and/or nNR2-1 protein may be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography.

The present invention is also directed to methods for screening for compounds which modulate the expression of DNA or RNA encoding a human nNRl, nNR2 and/or nNR2-1 protein.
Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules.
Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding human nNR,l, nNR2 and/or nNR2-1, or the function of human nNRi, nNR,2 and/or nNR2-1. Compounds that modulate the expression of DNA or RNA encoding human nNR,l, nNR2 and/or nNR2-1 or the biological function thereof may be detected by a variety of assays. The assay may be a simple "yes/no" assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a teat sample with the levels of expression or function in a standard sample. Kits containing human nNRl, nNR2 and/or nNR2-1, antibodies to human nNRl, nNR,2 and/or nNR2-1, or modified human nNRl, nNR2 and/or nNR2-1 may be prepared by known methods for such uses.
The DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of human nNRl, nNR2 and/or nNR2-1. The recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of human nNR,l, nNR2 and/or nNR2-1. Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as recombinant nNRI, nNR2 and/or nNR,2-1 or anti-nNRl, nNR2 and/or nNR2-1 antibodies suitable for detecting human nNRl, nNR2 and/or nNR2-1. The carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the Like.
Pharmaceutically useful compositions comprising modulators of human nNRl, nNR2 and/or nNR2-1 may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein, DNA, RNA, modified human nNRl, nNR2 and/or nNR2-1, or either nNRl, nNR2 and/or nNR2-1 agonists or antagonists.
Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat ar diagnose disorders. The effective amount may vary according to a variety of factors such $s the individual's condition, weight, sex and age. Other factors include the mode of administration.
The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
The term "chemical derivative" describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages. Alternatively, co-administration or sequential administration of other agents may be desirable.
The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds identified according to this invention as the active ingredient can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
Advantageously, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
Furthermore, compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forma of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.
The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.
The following examples are provided to illustrate the present invention without, however, limiting the same hereto.

EXAMPLE 1:
Isolation and Characterization of DNA Fragments Encoding nNRl, nNR2 and/or nIVR2-1 The DNA sequences from several representative subfamilies (Gigu~re, et al., 1988, Nature 331: 91-94) were used to query the EST

database by using the Two ESTs Blastn program. (Genbank accession number h91890 (nNRl) and w26275 (nNR2)) were identified with homology to human ER,R2at DNA sequence level.

EST h91890 is disclosed herein as SEQ ID N0:7 and is as set forth:

CTTTTTAGGA GGTGGAGAAA TTTGTAAGCT CAGGTATGGG CTGCTCTCTG

AGTCCAGCCG TCGCTTGTAT TTCTGACGGC CTCCACGCAC TCGATCAAGG

CGCACACCTT CCTTCAGCAT CCCCACTTTG AGGCATTTCA TGAAGCGGCA

GGCCTGGCAG GACTTGCGCC TCCGTTTGGT GATCTCGCAC TCGTTGGTGG

CCGGGCAGCT GTACTCAATG TTCCCTTGGA TAGTCCTCTT GAAGAAGGCC

TTGCAAGCCT CGCAGGAGGC CCACGCGTNA GTGGTAGCCA GAGNAAATGT

CGCACTTGGA TGGGCCGAGT CCTCCATGGA TGGCCGCTGG CAACAGTTCC

TCG (SEQ ID N0:7).

EST w26275 is disclosed herein as SEQ
ID N0:8 and is as set forth:

CNNNNNNNNN NNNTTTTNNT GCCTAAAGTG GTACCCNGAA GNGATGTCAC

CACACACTAA ACACAGTCTC TTGGGCATCG AGTTGAGCAT GTATTCACAC

TTGGTCTGGG GATCTTCAAC AATGGTGCTG GAGCAGTCAT CATACAGTTT

CCTGACAGGC CCACTACCTC CCAGGATAGG AGCAGAAGGG TAGAGAGGTG

GCGAGTCAAG TCCGTTCTGA TGGCCATTCA TGGTTGAACT GTAGCTCCCA

CTGGCGTCTG AAGAGCCACC AGGGCTGTGG TGGTTGACGC TGTCCGTCAG

GGAGGCTGGG CTGGAAGGTT CCGTCTTGAT GAAGGACGAA CAGCTGGAAT

CAATGTGTCG ATCTTTGTTT GGACATTCTG CAGAGAAGCT CTTCCTCCGT

NGTGCAGGGA AAAAGATTCA GGAAGGCAAA GTTCTTCCCG AATCCATGTG

CGACCGGAAA CCATTATTTG NGCACCCCAG CTATTAATCA AAGTTCCTTG

ACAGAGACAG GGCAATTACA NAATGTCTCC TNTNGGGGAT CAACTGTTCN

GTATZTJNNNN N

rf~~NN TT ( SEQ ID NO : 8 ) .
Primer pairs 5'-TGAGTCCAGCCGTCGCTTGTAT-3' (ERR4F1; SEQ ID N0:9), 5'-TGCAAGCCTCGCAGGAGGCC-3' (ERR4iFl; SEQ ID NO:10), and 5'-GGCCTTCTTCAAGAGGACTATC-3'(ERR4R1; SEQ ID N0:11) were designed from h91890;
5'-AAAGATCGACACATTGATTCC-3' (ERRSF; SEQ ID N0:12), 5'-GACTTGACTCGCCACCTCTC-3' (ERR5iF; SEQ ID N0:13) and 5'-GTTCTGATGGCCATTCATGGT-3' (ERRSR; SEQ ID N0:14) were designed from W26275. Primer pairs ERR4F/ERR4R and ERRSF/ERRSR
were used to scan cDNA made from testis, fetal brain, prostate and placenta first before scanning cDNA libraries made from those cDNA
and distributed in 96-well plates. Primers for nNRl produced a PCR
product from testis cDNA, while primers for nNR2 generated a PCR
product a cDNA library generated from fetal brain, prostate and placenta mRNA. Therefore, a cDNA library made from testis with >2.5 kb insert was used for nNRl positive pool identification, and A4 and G8 gave the PCR product of expected size. Inverse PCR using ERR4iF1 and ERR4R1 were performed on positive pools and DNA fragments of about 6.0 kb were amplified. The DNA fragment was purified using fdiagen gel extraction kit. Phosphorylation, self ligation and transformation of the purified DNA was carried out. DNA mini-preps from four individual clones were used in automated sequencing with gene specific and vector primers. Since a PCR-induced mutation is possible in long PCR reactions, nNR.l was re-subcloned in to the PCR2.1 vector (Invitrogen) using a PCR fragment amplified by a 5'-primer 5'-GAATATGATGACCCTAATGCA-3' (SEQ ID N0:15) and a 3'-primer 5'-CTTCCACCTCATGGACACCAA-3' (SEQ ID N0:16) on the positive A4 pool. One out of the four TA-clones showed no mutation through sequencing confirmation. DNA sequence analysis was performed using the ABI PRISM''M. dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerise, FS (Perkin Elmer, Norwalk, CT). DNA sequence analysis was performed with M13 forward/reverse primers and gene specific sequencing primers manufactured by GIBCO BRL (Gaithersburg, MD). Sequence assembly and analysis were performed with SEQUENCHERTM 3.0 (Gene Codes Corporation, Ann Arbor, MI). Ambiguities and/or discrepancies between automated base calling in sequencing reads were visually examined and edited to the correct base call. Several .regions were resequenced after initial automated or visual calling. Four oligonucleotidea close to the regions with potential sequence ambiguities were utilized ([R,1F1] 5'-CAT TCC ACG GAG GCA TCC TC-3' (SEQ ID
N0:23); CR1F2] 5'-CCA AGG CCG TGC AGC ACT TC-3' (SEQ ID
N0:24); [R1R1] 5'-GAC AGC CTC TAG ATC CTC GAT-3' (SEQ ID
N0:25); and, [R,1R2] 5' ATC ATG GCT TGA CAT TCT TTC-3' (SEQ ID
N0:26) and automated sequencing was performed. The final nucleotide sequence encoding NRl is shown as set forth in Figure lA-C and as set forth as SEQ ID NO:1 For nNR2, a cDNA library made from fetal brain with >2.5 kb insert was used. Positive pools C1, F7 and G6 were identified and used in inverse PCR with primer pairs ERR5iF/ERRSR. A PCR fragment of 6.0 kb was amplified from C1. The same methodology as described herein for nNR1 was applied to isolation, characterization and sequencing of. a nNR,2 cDNA. The cDNA fragment cloned into pCR2.1 vector was amplified by 5'-primer 5'-GTTAATTGCACTGTGCTCTG-3' (SEQ ID N0:17) and 3'-primer 5'-AGTGTGGTGGAATTCTCTTA-3' (SEQ ID N0:18).
Primer pairs Xft2F3 (5'-AGCTCTTGCTAATTCAGAC-3' [SEQ
ID N0:27]) and XR2R4 (5'-TCAACATGAAGGATGGGAAGG-3' [SEQ ID
N0:28]) were used in DNA sequence analysis (performed using the ABI
PRISMTM dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase, FS (Perkin Elmer, Norwalk, CT)) of the carboxy region of nNR2. DNA sequence analysis was performed with M13 forward/reverse primers and gene specific sequencing primers customarily manufactured by GTBCO BRL (Gaithersburg, MD).
Sequence assembly and analysis were performed with SEQUENCHERTM 3.0 (Gene Codes Corporation, Ann Arbor, MI).
Ambiguities and/or discrepancies between automated base calling in sequencing reads were visually examined and edited to the correct base call. Resequencing of the ligand binding domain showed a new open reading frame that was confirmed with the XR2F3/ XR2ft4 primers.

WO 99/103b7 PCT/US98/17826 The nNR2 peptide coded by the complete open reading frame has 40 extra amino acids at C-terminus compared to nNR2-1 and is similar in length to its closest related member hERR2.
In order to identify the genome map position of the genes, primers in the 3' non-coding region were designed. Forwarding primer 5'-TCTAGTGTTGCTGCGAGTGAC-3' (SEfa ID N0:19) and reversing primer 5'-CTTCCACCTCATGGACACCAA-3' (SEQ ID N0:20) were used for nNRl, while forwarding primer 5'-GTCTGACTAAAAGCTCCCTG-3' (SEQ ID N0:21) and reversing primer 5'-GAAGATGATGGAGAAAGTAGA-3' (SEfd ID N0:22) were used for nNR2. PCR scanning was performed on the 83 clones of the Stanford radiation hybrid panel (Cox et al., 1990, Science, 250:245:250).
The PCR results were scored and submitted to the Stanford Genome Center for linkage analysis. The results indicate that nNRl is located on locus 14q24.3 ~ 14q31 and nNR2 is located on chromosome 1.
_ 3g _

Claims (53)

WHAT IS CLAIMED:

1. A purified DNA molecule encoding a human nNR1 protein wherein said protein comprises the amino acid sequence as follows:
MSSDDRHLGS SCGSFIKTEP SSPSSGIDAL SHHSPSGSSD ASGGFGLALG
THANGLDSPP MFAGAGLGGT PCRKSYEDCA SGIMEDSAIK CEYMLNAIPK
RLCLVCGDIA SGYHYGVASC EACKAFFKRT IQGNIEYSCP ATNECEITKR
RRKSCQACRF MKCLKVGMLK EGVRLDRVRG GRQKYKRRLD SESSPYLSLQ
ISPPAKKPLT KIVSYLLVAE PDKLYAMPPP GMPEGDIKAL TTLCDLADRE
LVVIIGWAKH IPGFSSLSLG DQMSLLQSAW MEILILGIVY RSLPYDDKLV
YAEDYIMDEE HSRLAGLLEL YRAILQLVRR YKKLKVEKEE FVTLKALALA
NSDSMYIEDL EAVQKLQDLL HEALQDYELS QRHEEPWRTG KLLLTLPLLR
QTAAKAVQHF YSVKLQGKVP MHKLFLEMLE AKAWARADSL QEWRPLEQVP
SPLHRATKRQ HVHFLTPLPP PPSVAWVGTA QAGYHLEVFL PQRAGWPRAA, as set forth in three-letter abbreviation in SEQ ID NO:2.

2. An expression vector for expressing a human nNR1 protein in a recombinant host cell wherein said expression vector comprises a DNA molecule of claim 1.

3. A host cell which expresses a recombinant human nNR1 protein wherein said host cell contains the expression vector of claim 2.

4. A process for expressing a human nNR1 protein in a recombinant host cell, comprising:
(a) transfecting the expression vector of claim 2 into a suitable host cell; and, (b) culturing the host cells of step (a) under conditions which allow expression of said the human nNR1 protein from said expression vector.

5. A purified DNA molecule encoding a human nNR1 protein wherein said protein consists of the amino acid sequence as follows:

MSSDDRHLGS SCGSFIKTEP SSPSSGIDAL SHHSPSGSSD ASGGFGLALG
THANGLDSPP MFAGAGLGGT PCRKSYEDCA SGIMEDSAIK CEYMLNAIPK
RLCLVCGDIA SGYHYGVASC EACKAFFKRT IQGNIEYSCP ATNECEITKR
RRKSCQACRF MKCLKVGMLK EGVRLDRVRG GRQKYKRRLD SESSPYLSLQ
ISPPAKKPLT KIVSYLLVAE PDKLYAMPPP GMPEGDIKAL TTLCDLADRE
LVVIIGWAKH IPGFSSLSLG DQMSLLQSAW MEILILGIVY RSLPYDDKLV
YAEDYIMDEE HSRLAGLLEL YRAILQLVRR YKKLKVEKEE FVTLKALALA
NSDSMYIEDL EAVQKLQDLL HEALQDYELS QRHEEPWRTG KLLLTLPLLR
QTAAKAVQHF YSVKLQGKVP MHKLFLEMLE AKAWARADSL QEWRPLEQVP
SPLHRATKRQ HVHFLTPLPP PPSVAWVGTA QAGYHLEVFL PQRAGWPRAA, as set forth in three-letter abbreviation in SEQ ID NO:2.

6. An expression vector for expressing a human nNR1 protein in a recombinant host cell wherein said expression vector comprises a DNA molecule of claim 5.

7. A host cell which expresses a recombinant human nNR1 protein wherein said host cell contains the expression vector of claim 6.

8. A process for expressing a human nNR1 protein in a recombinant host cell, comprising:
(a) transfecting the expression vector of claim 6 into a suitable host cell; and, (b) culturing the host cells of step (a) under conditions which allow expression of said the human nNR1 protein from said expression vector.

9. A purified DNA molecule encoding a human nNR1 protein wherein said DNA molecule comprises the nucleotide sequence as set forth in SEQ ID NO:1, as follows:

GAATATGATG ACCCTAATGC AACAATATCT AACATACTAT CCGAGCTTCG
GTCATTTGGA AGAACTGCAG ATTTTCCTCC TTCAAAATTA AAGTCAGGTT
ATGGAGAACA TGTATGCTAT GTTCTTGATT GCTTCGCTGA AGAAGCATTG
AAATATATTG GTTTCACCTG GAAAAGGCCA ATATACCCAG TAGAAGAATT
AGAAGAAGAA AGCGTTGCAG AAGATGATGC AGAATTAACA TTAAATAAAG
TGGATGAAGA ATTTGTGGAA GAAGAGACAG ATAATGAAGA AAACTTTATT
GATCTCAACG TTTTAAAGGC CCAGACATAT CACTTGGATA TGAACGAGAC
TGCCAAACAA GAAGATATTT TGGAATCCAC AACAGATGCT GCAGAATGGA
GCCTAGAAGT GGAACGTGTA CTACCGCAAC TGAAAGTCAC GATTAGGACT
GACAATAAGG ATTGGAGAAT CCATGTTGAC CAAATGCACC AGCACAGAAG
TGGAATTGAA TCTGCTCTAA AGGAGACCAA GGGATTTTTG GACAAACTCC
ATAATGAAAT TACTAGGACT TTGGAAAAGA TCAGCAGCCG AGAAAAGTAC
ATCAACAATC AGCCGGGAGC CCATGGAGCA CTGTCCTCAG AGATGCGCAG
GTTAGGCTCA CTGTCTAGGC CAGGCCCACC TTAGTCACTG TGGACTGGCA
ATGGAAGCTC TTCCTGGACA CACCTGCCCT AGCCCTCACC CTGGGGTGGA
AGAGAAATGA GCTTGGCTTG CAACTCAGAC CATTCCACGG AGGCATCCTC
CCCTTCCCTG GGCTGGTGAA TAAAAGTTTC CTGAGGTCAA GGACTTCCTT
TTCCCTGCCA AAATGGTGTC CAGAACTTTG AGGCCAGAGG TGATCCAGTG
ATTTGGGAGC TGCAGGTCAC ACAGGCTGCT CAGAGGGCTG CTGAACAGGA
TGTCCTCGGA CGACAGGCAC CTGGGCTCCA GCTGCGGCTC CTTCATCAAG
ACTGAGCCGT CCAGCCCGTC CTCGGGCATA GATGCCCTCA GCCACCACAG
CCCCAGTGGC TCGTCCGACG CCAGCGGCGG CTTTGGCCTG GCCCTGGGCA
CCCACGCCAA CGGTCTGGAC TCGCCACCCA TGTTTGCAGG CGCCGGGCTG
GGAGGCACCC CATGCCGCAA GAGCTACGAG GACTGTGCCA GCGGCATCAT
GGAGGACTCG GCCATCAAGT GCGAGTACAT GCTCAACGCC ATCCCCAAGC
GCCTGTGCCT CGTGTGCGGG GACATTGCCT CTGGCTACCA CTACGGCGTG
GCCTCCTGCG AGGCTTGCAA GGCCTTCTTC AAGAGGACTA TCCAAGGGAA
CATTGAGTAC AGCTGCCCGG CCACCAACGA GTGCGAGATC ACCAAACGGA
GGCGCAAGTC CTGCCAGGCC TGCCGCTTCA TGAAATGCCT CAAAGTGGGG
ATGCTGAAGG AAGGTGTGCG CCTTGATCGA GTGCGTGGAG GCCGTCAGAA
ATACAAGCGA CGGCTGGACT CAGAGAGCAG CCCATACCTG AGCTTACAAA
TTTCTCCACC TGCTAAAAAG CCATTGACCA AGATTGTCTC ATACCTACTG

GTGGCTGAGC CGGACAAGCT CTATGCCATG CCTCCCCCTG GTATGCCTGA
GGGGGACATC AAGGCCCTGA CCACTCTCTG TGACCTGGCA GACCGAGAGC
TTGTGGTCAT CATTGGCTGG GCCAAGCACA TCCCAGGCTT CTCAAGCCTC
TCCCTGGGGG ACCAGATGAG CCTGCTGCAG AGTGCCTGGA TGGAAATCCT
CATCCTGGGC ATCGTGTACC GCTCGCTGCC CTACGACGAC AAGCTGGTGT
ACGCTGAGGA CTACATCATG GATGAGGAGC ACTCCCGCCT CGCGGGGCTG
CTGGAGCTCT ACCGGGCCAT CCTGCAGCTG GTACGCAGGT ACAAGAAGCT
CAAGGTGGAG AAGGAGGAGT TTGTGACGCT CAAGGCCCTG GCCCTCGCCA
ACTCCGATTC CATGTACATC GAGGATCTAG AGGCTGTCCA GAAGCTGCAG
GACCTGCTGC ACGAGGCACT GCAGGACTAC GAGCTGAGCC AGCGCCATGA
GGAGCCCTGG AGGACGGGCA AGCTGCTGCT GACACTGCCG CTGCTGCGGC
AGACGGCCGC CAAGGCCGTG CAGCACTTCT ATAGCGTCAA ACTGCAGGGC
AAAGTGCCCA TGCACAAACT CTTCCTGGAG ATGCTGGAGG CCAAGGCCTG
GGCCAGGGCT GACTCCCTTC AGGAGTGGAG GCCACTGGAG CAAGTGCCCT
CTCCCCTCCA CCGAGCCACC AAGAGGCAGC ATGTGCATTT CCTAACTCCC
TTGCCCCCTC CCCCATCTGT GGCCTGGGTG GGCACTGCTC AGGCTGGATA
CCACCTGGAG GTTTTCCTTC CGCAGAGGGC AGGTTGGCCA AGAGCAGCTT
AGAGGATCTC CCAAGGATGA AAGAATGTCA AGCCATGATG GAAAATGCCC
CTTCCAATCA GCTGCCTTCA CAAGCAGGGA TCAGAGCAAC TCCCCGGGGA
TCCCCAATCC ACGCCCTTCT AGTCCAACCC CCCTCAATGA GAGAGGCAGG
CAGATCTCAC CCAGCACTAG GACACCAGGA GGCCAGGGAA AGCATCTCTG
GCTCACCATG TAACATCTGG CTTGGAGCAA GTGGGTGTTC TGCACACCAG
GCAGCTGCAC CTCACTGGAT CTAGTGTTGC TGCGAGTGAC CTCACTTCAG
AGCCCCTCTA GCAGAGTGGG GCGGAAGTCC TGATGGTTGG TGTCCATGAG
GTGGAAG (SEQ ID NO:1).

10. A DNA molecule of claim 9 which comprises from about nucleotide 950 to about nucleotide 2452 of SEQ ID NO:1.

11. An expression vector for expressing a human nNR1 protein wherein said expression vector comprises a DNA molecule of claim 9.

12. An expression vector for expressing a human nNR1 protein wherein said expression vector comprises a DNA molecule of claim 11.

13. A host cell which expresses a recombinant human nNR1 protein wherein said host cell contains the expression vector of claim 11.

14. A host cell which expresses a recombinant human nNR1 protein wherein said host cell contains the expression vector of claim 12.

15. A process for expressing a human nNR1 protein in a recombinant host cell, comprising:
(a) transfecting the expression vector of claim 11 into a suitable host cell; and, (b) culturing the host cells of step (a) under conditions which allow expression of said the human nNR1 protein from said expression vector.

16. A purified DNA molecule encoding a human nNR1 protein wherein said DNA molecule consists of the nucleotide sequence as set forth in SEQ ID NO:1, as follows:

GAATATGATG ACCCTAATGC AACAATATCT AACATACTAT CCGAGCTTCG
GTCATTTGGA AGAACTGCAG ATTTTCCTCC TTCAAAATTA AAGTCAGGTT
ATGGAGAACA TGTATGCTAT GTTCTTGATT GCTTCGCTGA AGAAGCATTG
AAATATATTG GTTTCACCTG GAAAAGGCCA ATATACCCAG TAGAAGAATT
AGAAGAAGAA AGCGTTGCAG AAGATGATGC AGAATTAACA TTAAATAAAG
TGGATGAAGA ATTTGTGGAA GAAGAGACAG ATAATGAAGA AAACTTTATT
GATCTCAACG TTTTAAAGGC CCAGACATAT CACTTGGATA TGAACGAGAC
TGCCAAACAA GAAGATATTT TGGAATCCAC AACAGATGCT GCAGAATGGA
GCCTAGAAGT GGAACGTGTA CTACCGCAAC TGAAAGTCAC GATTAGGACT

GACAATAAGG ATTGGAGAAT CCATGTTGAC CAAATGCACC AGCACAGAAG

TGGAATTGAA TCTGCTCTAA AGGAGACCAA GGGATTTTG GACAAACTCC

ATAATGAAAT TACTAGGACT TTGGAAAAGA TCAGCAGCCG AGAAAAGTAC

ATCAACAATC AGCCGGGAGC CCATGGAGCA CTGTCCTCAG AGATGCGCAG

GTTAGGCTCA CTGTCTAGGC CAGGCCCACC TTAGTCACTG TGGACTGGCA

ATGGAAGCTC TTCCTGGACA CACCTGCCCT AGCCCTCACC CTGGGGTGGA

AGAGAAATGA GCTTGGCTIG CAACTCAGAC CATTCCACGG AGGCATCCTC

CCCTTCCCTG GGCTGGTGAA TAAAAGTTTC CTGAGGTCAA GGACTTCCTT

TTCCCTGCCA AAATGGTGTC CAGAACTTTC AGGCCAGAGG TGATCCAGTG

ATTTGGGAGC TGCAGGTCAC ACAGGCTGCT CAGAGGGCTG CTGAACAGGA

TGTCCTCGGA CGACAGGCAC CTGGGCTCCA GCTGCGGCTC CTTCATCAAG

ACTGAGCCGT CCAGCCCGTC CTCGGGCATA GATGCCCTCA GCCACCACAG

CCCCAGTGGC TCGTCCGACG CCAGCGGCGG CTTTGGCCTG GCCCTGGGCA

CCCACGCCAA CGGTCTGGAC TCGCCACCCA TGTTTGCAGG CGCCGGGCTG

GGAGGCACCC CATGCCGCAA GAGCTACGAG GACTGTGCCA GCGGCATCAT

GGAGGACTCG GCCATCAAGT GCGAGTACAT GCTCAACGCC ATCCCCAAGC

GCCTGTGCCT CGTGTGCGGG GACATZGCCT CTGGCTACCA CTACGGCGTG

GCCTCCTGCG AGGCTZGCAA GGCCTTCTTC AAGAGGACTA TCCAAGGGAA

CATTGAGTAC AGCTGCCCGG CCACCAACGA GTGCGAGATC ACCAAACGGA

GGCGCAAGTC CTGCCAGGCC TGCCGCTTCA TGAAATGCCT CAAAGTGGGG

ATGCTGAAGG AAGGTGTGCG CCTTGATCGA GTGCGTGGAG GCCGTCAGAA

ATACAAGCGA CGGCTGGACT CAGAGAGCAG CCCATACCTG AGCTTACAAA

TTTCTCCACC TGCTAAAAAG CCATTGACCA AGATTGTCTC ATACCTACTG

GTGGCTGAGC CGGACAAGCT CTATGCCATG CCTCCCCCTG GTATGCCTGA

GGGGGACATC AAGGCCCTGA CCACTCTCTG TGACCTGGCA GACCGAGAGC

TTGTGGTCAT CATTGCTGG GCCAAGCACA TCCCAGGCTT CTCAAGCCTC

TCCCTGGGGG ACCAGATGAG CCTGCTGCAG AGTGCCTGGA TGGAAATCCT

CATCCTGGGC ATCGTGTACC GCTCGCTGCC CTACGACGAC AAGCZGGTGT

ACGCTGAGGA CTACATCATG GATGAGGAGC ACTCCCGCCT CGCGGGGCTG

CTGGAGCTCT ACCGGGCCAT CCTGCAGCTG GTACGCAGGT ACAAGAAGCT

CAAGGTGGAG AAGGAGGAGT TTGTGACGCT CAAGGCCCTG GCCCTCGCCA

ACTCCGATTC CATGTACATC GAGGATCTAG AGGCTGTCCA GAAGCTGCAG

GACCTGCTGC ACGAGGCACT GCAGGACTAC GAGCTGAGCC AGCGCCATGA

GGAGCCCTGG AGGACGGGCA AGCTGCTGCT GACACZGCCG CTGCTGCGGC

AGACGGCCGC CAAGGCCGTG CAGCACTTCT ATATAGCGTCAA ACTGCAGGGC

AAAGTGCCCA TGCACAAACTCT CTTCCTGGAG ATGCTGGAGG CCAAGGCCTG

GGCCAGGGCT GACTCCCTTC AGGAGTGGAG GCCACTGGAG CAAGTGCCCT

CTCCCCTCCA CCGAGCCACC AAGAGGCAGC ATGTGCATTT CCTAACTCCC

TTGCCCCCTC CCCCATCTGT GGCCTGGGTG GGCACTGCTC AGGCTGGATA

CCACCTGGAG GTTTTCCTTC CGCAGAGGGC AGGTZGGCCA AGAGCAGCTT

AGAGGATCTC CCAAGGATGA AAGAATGTCA AGCCATGATG GAAAATGCCC

CTTCCAATCA GCTGCCTTCA CAAGCAGGGA TCAGAGCAAC TCCCCGGGGA

TCCCCAATCC ACGCCCTTCT AGTCCAACCC CCCTCAATGA GAGAGGCAGG

CAGATCTCAC CCAGCACTAG GACACCAGGA GGCCAGGGAA AGCATCTCTG

GCTCACCATG TAACATCTGG CTTGGAGCAA GTGGGTGTTC TGCACACCAG

GCAGCTGCAC CTCACTGGAT CTAGTGTTGC TGCGAGTGAC CTCACTTCAG

AGCCCCTCTA GCAGAGTGGG GCGGAAGTCC TGATGGTTGG TGTCCATGAG

GTGGAAG (SEQ ID NO:1).

17. A DNA molecule of claim 16 which consists of nucleotide 950 to about nucleotide 2452 of SEQ ID NO:1.

18. An expression vector for expressing a human nNR1 protein wherein said expression vector comprises a DNA molecule of claim 16.

19. An expression vector for expressing a human nNR1 protein wherein said expression vector comprises a DNA molecule of claim 17.

20. A host cell which expresses a recombinant human nNR1 protein wherein said host cell contains the expression vector of claim 18.

21. A host cell which expresses a recombinant human nNR1 protein wherein said host cell contains the expression vector of claim 19.

22. A process for expressing a human nNR1 protein in a recombinant host cell, comprising:

(a) transfecting the expression vector of claim 18 into a suitable host cell; and, (b) culturing the host cells of step (a) under conditions which allow expression of said the human nNR1 protein from said expression vector.

23. A purified DNA molecule encoding a human nNR2 protein wherein said protein comprises the amino acid sequence as follows:
MDSVELCLPE SFSLHYEEEL LCRMSNKDRH IDSSCSSFIK TEPSSPASLT
DSVNHHSPGG SSDASGSYSS TMNGHQNGLD SPPLYPSAPI LGGSGPVRKL
YDDCSSTIVE DPQTKCEYML NSMPKRLCLV CGDIASGYHY GVASCEACKA
FFKRTIQGNI EYSCPATNEC EITKRRRKSC QACRFMKCLK VGMLKEGVRL
DRVRGGRQKY KRRIDAENSP YLNPQLVQPA KKPYNKIVSH LLVAEPEKIY
AMPDPTVPDS DIKALTTLCD LADRELWII GWAKHIPGFS TLSLADQMSL
LQSAWMEILI LGVVYRSLSF EDELVYADDY IMDEDQSKLA GLLDLNNAIL
QLVKKYKSMK LEKEEFVTLK AIALANSDSM HIEDVEAVQK LQDVLHEALQ
DYEAGQHIKED PRRAGKMLMT LPLLRQTSTK AVQHFYNIKL EGKVPMHKLF
LEMLEAKV, as set forth in three-letter abbreviation in SEQ ID NO:4.

24. An expression vector for expressing a human nNR2 protein in a recombinant host cell wherein said expression vector comprises a DNA molecule of claim 23.

25. A host cell which expresses a recombinant human nNR2 protein wherein said host cell contains the expression vector of claim 24.

26. A process for expressing a human nNR2 protein in a recombinant host cell, comprising:

(a) transfecting the expression vector of claim 24 into a suitable host cell; and, (b) culturing the host cells of step (a) under conditions which allow expression of said the human nNR1 protein from said expression vector.

27. A purified DNA molecule encoding a human nNR2 protein wherein said protein consists of the amino acid sequence as follows:
MDSVELCLPE SFSLHYEEEL LCRMSNKDRH IDSSCSSFIK TEPSSPASLT
DSVNHHSPGG SSDASGSYSS TMNGHQNGLD SPPLYPSAPI LGGSGPVRKL
YDDCSSTIVE DPQTKCEYML NSMPKRLCLV CGDIASGYHY GVASCEACKA
FFKRTIQGNI EYSCPATNEC EITKRRRKSC QACRFMKCLK VGMLKEGVRL
DRVRGGRQKY KRRIDAENSP YLNPQLVQPA KKPYNKIVSH LLVAEPEKIY
AMPDPTVPDS DIKALTTLCD LADRELWII GWAKHIPGFS TLSLADQMSL
LQSAWMEILI LGVWRSLSF EDELVYADDY IMDEDQSKLA GLLDLNNAIL
QLVKKYKSMK LEKEEFVTLK AIALANSDSM HIEDVEAVQK LQDVLHEALQ
DYEAGQHMED PRRAGKMLMT LPLLRQTSTK AVQHFYNIKL EGKVPMHKLF
LEMLEAKV, as set forth in three letter code as SEQ ID NO 4.

28. An expression vector for expressing a human nNR2 protein in a recombinant host cell wherein said expression vector comprises a DNA molecule of claim 27.

29. A host cell which expresses a recombinant human nNR1 protein wherein said host cell contains the expression vector of claim 28.

30. A process for expressing a human nNR2 protein in a recombinant host cell, comprising:

(a) transfecting the expression vector of claim 28 into a suitable host cell; and, (b) culturing the host cells of step (a) under conditions which allow expression of said the human nNR1 protein from said expression vector.

31. A purified DNA molecule encoding a human nNR2 protein wherein said DNA molecule comprises the nucleotide sequence as set forth in SEQ ID NO:3, as follows:

GCGGGCCGCC AGTGTGGTGG AATTCGGCTT GTCACTAGGA GAACATTTGT
GTTAATIGCA CTGTGCTCTG TCAAGGAAAC TTTGATTTAT AGCTGGGGTG
CACAAATAAT GGTTGCCGGT CGCACATGGA TTCGGTAGAA CTTTGCCTTC
CTGAATCTTT TTCCCTGCAC TACGAGGAAG AGCTTCTCTG CAGAATGTCA
AACAAAGATC GACACATTGA TTCCAGCTGT TCGTCCTTCA TCAAGACGGA
ACCTTCCAGC CCAGCCTCCC TGACGGACAG CGTCAACCAC CACAGCCCTG
GTGGCTCTTC AGACGCCAGT GGGAGCTACA GTTCAACCAT GAATGGCCAT
CAGAACGGAC TTGACTCGCC ACCTCTCTAC CCTTCTGCTC CTATCCTGGG
AGGTAGTGGG CCTGTCAGGA AACTGTATGA TGACTGCTCC AGCACCATTG
TTGAAGATCC CCAGACCAAG TGTGAATACA TGCTCAACTC GATGCCCAAG
AGACTGTGTT TAGTGTGTGG TGACATCGCT TCTGGGTACC ACTATGGGGT
AGCATCATGT GAAGCCTGCA AGGCATTCTT CAAGAGGACA ATTCAAGGCA
ATATAGAATA CAGCTGCCCT GCCACGAATG AATGTGAAAT CACAAAGCGC
AGACGTAAAT CCTGCCAGGC TTGCCGCTTC ATGAAGTGTT TAAAAGTGGG
CATGCTGAAA GAAGGGGTGC GTCTTGACAG AGTACGTGGA GGTCGGCAGA
AGTACAAGCG CAGGATAGAT GCGGAGAACA GCCCATACCT GAACCCTCAG
CTGGTTCAGC CAGCCAAAAA GCCATATAAC AAGATTGTCT CACATTTGTT
GGTGGCTGAA CCGGAGAAGA TCTATGCCAT GCCTGACCCT ACTGTCCCCG

ACAGTGACAT CAAAGCCCTC ACTACACTGT GTGACTTGGC CGACCGAGAG
TTGGTGGTTA TCATTGGATG GGCGAAGCAT ATTCCAGGCT TCTCCACGCT
GTCCCTGGCG GACCAGATGA GCCTTCTGCA GAGTGCTTGG ATGGAAATTT
TGATCCTTGG TGTCGTATAC CGGTCTCTTT CATTTGAGGA TGAACTTGTC
TATGCAGACG ATTATATAAT GGACGAAGAC CAGTCCAAAT TAGCAGGCCT
TCTTGATCTA AATAATGCTA TCCTGCAGCT GGTAAAGAAA TACAAGAGCA
TGAAGCTGGA AAAAGAAGAA TTTGTCACCC TCAAAGCTAT AGCTCTTGCT

32. A DNA molecule of claim 31 which comprises from about nucleotide 126 to about nucleotide 1382 of SEQ ID NO:3.

33. An expression vector for expressing a human nNR2 protein wherein said expression vector comprises a DNA molecule of claim 31.

34. An expression vector for expressing a human nNR2 protein wherein said expression vector comprises a DNA molecule of claim 32.

35. A host cell which expresses a recombinant human nNR2 protein wherein said host cell contains the expression vector of claim 33.

36. A host cell which expresses a recombinant human nNR2 protein wherein said host cell contains the expression vector of claim 34.

37. A process for expressing a human nNR2 protein in a recombinant host cell, comprising:

(a) transfecting the expression vector of claim 33 into a suitable host cell; and, (b) culturing the host cells of step (a) under conditions which allow expression of said the human nNR1 protein from said expression vector.

38. A purified DNA molecule encoding a human nNR2 protein wherein said DNA molecule consists of the nucleotide sequence as set forth in SEQ ID NO:3, as follows:

GCGGGCCGCC AGTGTGGTGG AATTCGGCTT GTCACTAGGA GAACATTTGT

GTTAATTGCA CTGTGCTCTG TCAAGGAAAC TTTGATTTAT AGCTGGGGTG

CACAAATAAT GGTTGCCGGT CGCACATGGA TTCGGTAGAA CTTTGCCTTC

CTGAATCTTT TTCCCTGCAC TACGAGGAAG AGCTTCTCTG CAGAATGTCA

AACAAAGATC GACACATTGA TTCCAGCTGT TCGTCCTTCA TCAAGACGGA

ACCTTCCAGC CCAGCCTCCC TGACGGACAG CGTCAACCAC CACAGCCCTG

GTGGCTCTTC AGACGCCAGT GGGAGCTACA GTTCAACCAT GAATGGCCAT

CAGAACGGAC TTGACTCGCC ACCTCTCTAC CCTTCTGCTC CTATCCTGGG

AGGTAGTGGG CCTGTCAGGA AACTGTATGA TGACTGCTCC AGCACCATTG

TTGAAGATCC CCAGACCAAG TGTGAATACA TGCTCAACTC GATGCCCAAG

AGACTGTGTT TAGTGTGTGG TGACATCGCT TCTGGGTACC ACTATGGGGT

AGCATCATGT GAAGCCTGCA AGGCATTCTT CAAGAGGACA ATTCAAGGCA

ATATAGAATA CAGCTGCCCT GCCACGAATG AATGTGAAAT CACAAAGCGC

AGACGTAAAT CCTGCCAGGC TTGCCGCTTC ATGAAGTGTT TAAAAGTGGG

CATGCTGAAA GAAGGGGTGC GTCTTGACAG AGTACGTGGA GGTCGGCAGA

AGTACAAGCG CAGGATAGAT GCGGAGAACA GCCCATACCT GAACCCTCAG

CTGGTTCAGC CAGCCAAAAA GCCATATAAC AAGATTGTCT CACATTTGTT

GGTGGCTGAA CCGGAGAAGA TCTATGCCAT GCCTGACCCT ACTGTCCCCG

ACAGTGACAT CAAAGCCCTC ACTACACTGT GTGACTTGGC CGACCGAGAG

TTGGTGGTTA TCATTGGATG GGCGAAGCAT ATTCCAGGCT TCTCCACGCT

GTCCCTGGCG GACCAGATGA GCCTTCTGCA GAGTGCTTGGA TGGAAATTT

TGATCCTTGG TGTCGTATAC CGGTCTCTTT CATTTGAGGA TGAACTTGTC

TATGCAGACG ATTATATAAT GGACGAAGAC CAGTCCAAAT TAGCAGGCCT

TCTTGATCTA AATAATGCTA TCCTGCAGCT GGTAAAGAAA TACAAGAGCA

TGAAGCTGGA AAAAGAAGAA TTTGTCACCC TCAAAGCTAT AGCTCTTGCT

AATTCAGACT CCATGCACAT AGAAGATGTT GAAGCCGTTC AGAAGCTTCA

GGATGTCTTA CATGAAGCGC TGCAGGATTA TGAAGCTGGC CAGCACATGG

AAGACCCTCG TCGAGCTGGC AAGATGCTGA TGACACTGCC ACTCCTGAGG

CAGACCTCTA CCAAGGCCGT GCAGCATTTC TACAACATCA AACTAGAAGG

CAAAGTCCCA ATGCACAAAC TTTTTTTGGA AATGTTGGAG GCCAAGGTCT

GACTAAAAGC TCCCTGGGCC TTCCCATCCT TCATGTTGAA AAAGGGAAAA

TAAACCCAAG AGTGATGTCG AAGAAACTTA GAGTTTAGTT AACAACATCA

AAAATCAACA GACTGCACTG ATAATTTAGC AGCAAGACTA TGAAGCAGCT

TTCAGATTCC TCCATAGGTT CCTGATGAGT TCTTTCTACT TTCTCCATCA

TCTTCTTTCC TCTTTCTTCC CACATTTCTC TTTCTCTTTA TTTTTTCTCC

TTZTCTTCTT TCACCTCCCT TATTTCTTTG CTTCTTTCAT TCCTAGTTCC

CATTCTCCTT TATTTTCTTC CCGTCTGCCT GCCTTCTTTC TTTTCTTTAC

CTACTCTCAT TCCTCTCTTT TCTCATCCTT CCCCTTTTTT CTAAATTTGA

AATAGCTTTA GTTTAAAAAA AAAAATCCTC CCTTCCCCCT TTCCTTTCCC.

TTTCTTTCCT TTTTCCCTTT CCTTTTCCCT TTCCTTTCCT TTCCTCTTGA

CCTTCTTTCC ATCTTTCTTT TTCTTCCTTC TGCTGCTGAA CTTTTAAAAG

AGGTCTCTAA CTGAAGAGAG ATGGAAGCCA GCCCTGCCAA AGGATGGAGA

TCCATAATAT GGATGCCAGT GAACTTATTG TGAACCATAC CGTCCCCAAT

GACTAAGGAA TCAAAGAGAG AGAACCAACG TTCCTAAAAG TACAGTGCAA

CATATACAAA TTGACTGAGT GCAGTATTAG ATTTCATGGG AGCAGCCTCT

AATTAGACAA CTTAAGCAAC GTTGCATCGG CTGCTTCTTA TCATTGCTTT

TCCATCTAGA TCAGTTACAG CCATTTGATT CCTTAATTGT TTTTTCAAGT

CTTCCAGGTA TTTGTTAGTT TAGCTACTAT GTAACTTTTT CAGGGAATAG

TTTAAGCTTT ATTCATTCAT GCAATACTAA AGAGAAATAA GAATACTGCA

ATTTTGTGCT GGCTTTGAAC AATTACGAAC AATAATGAAG GACAAATGAA

TCCTGAAGGA AGATTTTTAA AAATGTTTTG TTTCTTCTTA CAAATGGAGA

TTTTTTTGTA CCAGCTTTAC CACTTZTCAG CCATTTATTA ATATGGGAAT

TTAACTTACT CAAGCAATAG TTGAAGGGAA GGTGCATATT ATCACGGATG

CAATTTATGT TGTGTGCCAG TCTGGTCCCA AACATCAATT TCTTAACATG

AGCTCCAGTT TACCTAAATG TTCACTGACA CAAAGGATGA GATTACACCT

ACAGTGACTC TGAGTAGTCA CATATATAAG CACTGCACAT GAGATATAGA

TCCGTAGAAT TGTCAGGAGT GCACCTCTCT ACTTGGGAGG TACAATTGCC

ATATGATTTC TAGCTGCCAT GGTGGTTAGG AATGTGATAC TGCCTGTTTG

CAAAGTCACA GACCTTGCCT CAGAAGGAGC TGTGAGCCAG TATTCATTTA

AGAGAATTCC ACCACACTGG CGGCCCGCGC TTGAT (SEQ ID NO:3).

39. A DNA molecule of claim 38 which consists of nucleotide 126 to about nucleotide 1382 of SEQ ID NO:3.

40. An expression vector for expressing a human nNR2 protein wherein said expression vector comprises a DNA molecule of claim 38.

41. An expression vector for expressing a human nNR2 protein wherein said expression vector comprises a DNA molecule of claim 39.

42. A host cell which expresses a recombinant human nNR2 protein wherein said host cell contains the expression vector of claim 40.

43. A host cell which expresses a recombinant human nNR2 protein wherein said host cell contains the expression vector of claim 41.

44. A process for expressing a human nNR2 protein in a recombinant host cell, comprising:
(a) transfecting the expression vector of claim 40 into a suitable host cell; and, (b) culturing the host cells of step (a) under conditions which allow expression of said the human nNR2 protein from said expression vector.

45. A purified human nuclear receptor protein which comprises the amino acid sequence as set forth in SEQ ID NO:2.

46. A purified human nuclear receptor protein which consists of the amino acid sequence as set forth in SEQ ID NO:2.

47. A purified human nuclear receptor protein produced by the method of claim 4.

48. A purified human nuclear receptor protein which comprises the amino acid sequence as set forth in SEQ ID NO:4.

49. A purified human nuclear receptor protein which consists of the amino acid sequence as set forth in SEQ ID NO:4.

50. A purified human nuclear receptor protein produced by the method of claim 22.

51. A method for determining whether a substance is capable of binding to a human nuclear receptor protein comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of the human nuclear receptor protein in the cells;
(b) exposing the test cells to the substance;
(c) measuring the amount of binding of the substance to the human nuclear receptor protein;
(d) comparing the amount of binding of the substance to the human nuclear receptor in the test cells with the amount of binding of the substance to control cells that have not been transfected with the human nuclear receptor.

52. The method of claim 51 wherein the human nuclear receptor comprises an amino acid sequence as set forth in SEQ ID NO:2.

53. The method of claim 51 wherein the human nuclear receptor comprises an amino acid sequence as set forth in SEQ ID NO:4.