JP2001513984A - DNA molecule encoding human nuclear receptor protein - Google Patents

Abstract

  (57) [Summary] The present invention discloses the isolation and characterization of cDNA molecules encoding two human nuclear receptor proteins, termed nNR1, nNR2 and / or nNR2-1. The disclosure also describes recombinant vectors, recombinant host cells, screening methods for modulators of nNR1, nNR2 and / or nNR2-1 activity, and methods for producing antibodies against nNR1, nNR2 and / or nNR2-1 or epitopes thereof. Included in the range.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates, in part, to isolated nucleic acid molecules (polynucleotides encoding human nuclear receptor proteins, generally referred to as nNR1, nNR2 and / or nNR2-1). ). The present invention also provides a DN encoding nNR1, nNR2 and / or nNR2-1.
A recombinant vector and recombinant host containing the A fragment, a substantially purified form of a related human nNR1, nNR2 and / or nNR2-1 protein, a human mutein, and nNR1, nNR2 and / or nNR2-1. Methods related to the identification of compounds that modulate activity.

BACKGROUND OF THE INVENTION [0002] The nuclear receptor superfamily, including steroid hormone receptors, has been shown to play a role in the control of development, differentiation and physiological function,
It is a transcription factor that can be induced by small chemical ligands. Isolation of a cDNA clone encoding a nuclear receptor reveals several features. First, the NH ₂ -terminal region are varied in length by receptors, is highly variable, with a low homology between family members. There are three conserved intermediate regions called domains I, II and III. Region I is a cysteine-rich region called the DNA binding domain (DBD). Regions II and III are located in the COOH-terminal region of the protein and are also called ligand binding domains (LBD). For a review, see Power et al. (19
92, Trends in Pharmaceutical Sciences 13: 318-323).

[0003] Lipophilic hormones that activate steroid receptors are known to be associated with human disease. Thus, each nuclear receptor has been identified as a potential target for therapeutic intervention. For a review of the mechanism of action of various steroid hormone receptors, see Tsai and O'Malley (1994, Annu. Rev. Biochem. 63:
451-486).

[0004] Recent research on non-steroidal nuclear receptors has also shown potential as a drug target for therapeutic intervention. This study shows that peroxisome proliferator-activated receptor g (PPARg), identified by a conserved DBD region, promotes adipocyte differentiation upon activation, and thiazolidinedione, a class of antidiabetic drugs, is mediated through PPARg. Work (Tontonoz et al., 1994, Cell 79: 1147-1156; Lehmann et al.,
1995, J. Biol. Chem. 270 (22): 12953-12956; Teboul et al., 1995, J. Biol. Che.
m. 270 (47): 28183-28187). This indicates that PPARg plays some role in glucose homeostasis and lipid metabolism.

[0005] Giguere et al. (1988, Nature 331: 91-94) have isolated two cDNAs encoding human nuclear receptors designated hERR1 and hEER2. The authors believe that the function that can be induced by the ligand and subsequent ligands is not due to any of these human nuclear receptors.

[0006] Trapp and Holsboer (1996, J. Biol. Chem. 271 (17): 9879-9882) provide hERR
2 has been shown to act as a cell-specific inhibitor of glucocorticoid receptor-mediated gene expression.

[0007] It would be advantageous to be able to identify genes encoding additional human nuclear receptor proteins. Nucleic acid molecules that express human nuclear receptor proteins would be useful in screening for compounds that act as modulators of cell differentiation, cell development and physiological function. The present invention addresses and satisfies these needs by disclosing isolated nucleic acid molecules that express human nuclear receptor proteins that play a role in cell differentiation and development.

SUMMARY OF THE INVENTION [0008] The present invention relates to novel nuclear receptor proteins, preferably human nuclear receptor proteins, such as, for example, referred to as nNR1, nNR2 and / or nNR2-1 throughout the specification. Isolated nucleic acid molecule (polynucleotide) encoding a human nuclear receptor protein).

[0009] The invention also encodes an mRNA that expresses a biologically active human nuclear receptor.
The present invention relates to isolated nucleic acid fragments of nNR1 (SEQ ID NO: 1) and nNR2 (SEQ ID NO: 3). Any such nucleic acid fragment comprises at least an intracellular DNA binding domain and / or a ligand binding domain, a human nuclear receptor family domain present in nNR1 (SEQ ID NO: 2) and nNR2 (SEQ ID NO: 4). It will encode either a protein or protein fragment that contains a domain that is conserved throughout. Any such polynucleotides include, but are not limited to, nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations (these mutations may be diagnostic, Encodes mRNA expressing a therapeutic or prophylactic protein or protein fragment, nNR1, nN
It will be useful for screening for agonists and / or antagonists of R2 and / or nNR2-1 function).

[0010] Isolated nucleic acid molecules of the invention include deoxyribonucleic acid molecules (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single-stranded (coding or non-coding) or double-stranded. And synthetic DNA, such as a synthesized single-stranded polynucleotide. Also, the isolated nucleic acid molecule of the present invention includes:
A ribonucleic acid molecule (RNA) can be included.

[0011] The present invention also relates to recombinant vectors and recombinant hosts (both prokaryotic and eukaryotic) that contain the substantially purified nucleic acid molecules disclosed throughout this specification.

A preferred embodiment of the present invention is disclosed in FIGS. 1A-C and SEQ ID NO: 1 (a human cDNA molecule encoding a novel nuclear transacting receptor protein, nNR1).

Another preferred embodiment of the present invention is disclosed in FIGS. 4A-C and SEQ ID NO: 3 (a human cDNA molecule encoding a novel nuclear transacting receptor protein, nNR2).

Another preferred embodiment of the present invention is disclosed in FIGS. 7A-C and SEQ ID NO: 5 (a human cDNA molecule encoding a truncated version of nNR2, termed nNR2-1).

[0015] The present invention also relates to the novel nuclear trans-acting receptor protein nNR1 in substantially purified form as disclosed in FIGS. 2A-F and FIG. 3 and set forth in SEQ ID NO: 2.

The present invention also includes, but is not limited to, amino acid substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations, the biology of nNR1 as set forth in SEQ ID NO: 2. Active fragments and / or mutants (these mutations may provide a diagnostic or therapeutic or prophylactic protein or protein fragment and may be useful in screening for agonists and / or antagonists of nNR1 function. ).

The present invention also relates to a substantially purified form of the novel nuclear trans-acting receptor protein nNR2, disclosed in FIGS. 5A-H and FIG. 6 and set forth in SEQ ID NO: 4.

[0018] The invention also includes, but is not limited to, amino acid substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations, the biology of nNR2 as set forth in SEQ ID NO: 4. Active fragments and / or mutants (these mutations would provide a diagnostic, therapeutic or prophylactic protein or protein fragment and would be useful in screening for agonists and / or antagonists of nNR2 function) ).

A preferred embodiment of the present invention is disclosed in FIG. 3 and is set forth in SEQ ID NO: 2 (the amino acid sequence of the novel nuclear transacting receptor protein nNR1).

A preferred embodiment of the present invention is disclosed in FIG. 6 and is set forth in SEQ ID NO: 4 (amino acid sequence of the novel nuclear transacting receptor protein nNR2).

A preferred embodiment of the present invention is disclosed in FIG. 8 and comprises SEQ ID NO: 6 (designated nNR2-1, nNR2
Amino acid sequence of the truncated form).

The present invention also relates to the human forms of nNR1, nNR2 and / or nNR2 disclosed herein.
Polyclonal and monoclonal antibodies raised against -1 or biologically active fragments thereof. NNR1, nNR2 and / or nNR2-1 exhibiting the lowest homology to other known proteins belonging to the human nuclear receptor superfamily
NH ₂ cause antibodies to epitope-terminal domain may be particularly preferred. To this end, the DNA molecules, RNA molecules, recombinant proteins and antibodies of the invention are used to screen human nNR1, nNR2 and / or nNR2-1,
Their levels can be measured. The recombinant proteins, DNA molecules, RNA molecules and antibodies are useful in formulating kits suitable for the detection and typing of human nNR1, nNR2 and / or nNR2-1.

The present invention also relates to an isolated nucleic acid molecule that is a fusion construct that expresses a fusion protein useful in assays for identifying compounds that modulate wild-type human nNR1, nNR2, and / or nNR2-1 activity. . In a preferred embodiment of this aspect of the invention there is provided a glutathione S-transferase GST-nNR1 and / or GST-
Includes, but is not limited to, nNR2 fusion constructs. These fusion constructs include n as an in-frame fusion at the carboxy terminus of the GST gene.
Includes, but is not limited to, all or a portion of each of the ligand binding domains of NR1, nNR2 and / or nNR2-1. The disclosure of SEQ ID NOs: 1-4 allows one of skill in the art to construct any such nucleic acid molecule encoding a GST-nuclear receptor fusion protein. Soluble recombinant GST-nuclear receptor fusion protein
Using a baculovirus expression vector (eg, Bac-N-Blue DNA from Invitrogen or pAcG2T from Pharmingen), use Spodoptera frugiperda (Spodopte).
rafrugiperda) (Sf21) can be expressed in various expression systems such as insect cells (Invitrogen).

A novel form of nuclear receptor protein, such as human nNR1 and / or nNR2, nNR1,
Provided is an isolated nucleic acid molecule encoding a human nuclear receptor protein fragment of a full-length protein, such as nNR2 and / or nNR2-1, and a mutant that is a derivative of SEQ ID NO: 2 and SEQ ID NO: 4. This is one object of the present invention. Any such polynucleotides include, but are not necessarily limited to, nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations (these mutations may be diagnostic, It encodes mRNA that expresses a therapeutic or prophylactic protein or protein fragment and may be useful for screening for agonists and / or antagonists of nNR1, nNR2 and / or nNR2-1 function).

It is another object of the present invention to provide a human nuclear receptor protein or protein fragment encoded by the nucleic acid molecule described in the preceding paragraph.

It is another object of the present invention to provide recombinant vectors and recombinant host cells comprising a nucleic acid sequence encoding human nNR1, 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 nNR1 (as set forth in SEQ ID NO: 2).

Biologically active fragments and / or mutants of nNR1, including but not limited to amino acid substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations It is an object of the present invention to provide (these mutations provide a diagnostic, therapeutic or prophylactic protein or protein fragment).

It is an object of the present invention to provide a substantially purified form of nNR2 (as set forth in SEQ ID NO: 4).

[0030] Biologically active fragments and / or mutants of nNR2, including but not limited to amino acid substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations It is an object of the present invention to provide (these mutations provide a diagnostic, therapeutic or prophylactic protein or protein fragment).

[0031] In-frame fusion constructs based on nNR-1 and / or nNR-2, methods for expressing these fusion constructs and the bioisosteres disclosed herein, related assays,
Recombinant cells expressing these constructs, and nNR1, nNR2 and / or nNR2
It is also an object of the present invention to provide agonist and / or antagonist compounds identified by the use of a DNA molecule encoding a human nuclear receptor protein such as -1.

“DBD” as used in the present invention means a DNA binding domain.

“LBD” as used in the present invention means a ligand binding domain.

The term “mammalian host” as used herein refers to any mammal, including humans.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to isolated nucleic acid and protein forms corresponding to nuclear receptors, preferably, but not necessarily, human receptors. . These expressed proteins are novel nuclear receptors and are useful for identifying downstream target genes and ligands that regulate their activity. The nuclear receptor superfamily consists of a group of structurally related receptors that are regulated by chemically different ligands. Common structures for nuclear receptors are a highly conserved DNA binding domain (DBD) located in the middle of the peptide, and a COOH-terminal ligand binding domain (LBD). Eight of the nine non-mutated cysteines form two type II zinc fingers that distinguish nuclear receptors from other DNA binding proteins. The DBD has at least 50% to 60% amino acid sequence homology even between the farthest members in vertebrates. During the last decade, the superfamily has been expanded to include about 25 subfamilies. A search of the EST database using the entire peptide sequence of some representative subfamily members revealed that for the ESTs corresponding to the two human ESTs (nNR1, nNR2 and / or nNR2-1 respectively, GenBank accession numbers h91890 and w26275). ) Was identified. Using sequence information from each EST, n
The full-length cDNAs of the genes corresponding to NR1 (see FIGS. 1A-C and SEQ ID NO: 1) and nNR2 (see FIGS. 4A-C and SEQ ID NO: 3) were isolated and characterized. The cDNA of SEQ ID NO: 1 encodes a 500 amino acid long protein nNR1 (FIG. 3, SEQ ID NO: 2) with a unique DBD structure (FIGS. 2A-F). The cDNA of SEQ ID NO: 3 encodes a 458 amino acid long protein nNR2 (FIG. 6, SEQ ID NO: 4), and also has a unique DBD structure (FIGS. 5A-H). The cDNA of SEQ ID NO: 5 encodes a 418 amino acid long protein, nNR2-1 (FIG. 8, SEQ ID NO: 6), which is a carboxy-terminal truncated form of nNR2. The protein nNR2-1 has a unique DBD structure (FIG. 8).

[0036] The nNR1 protein binds to hERR2 (Giguere et al., 1988,
(Nature 331: 91-94). However, nNR1 has hER
Compared to R2, it contains an additional 67 amino acids at the carboxy terminus. The gene encoding nNR1 is the Alzheimer's disease gene 3 (AD3) locus, 14q24.3-1
Located at 4q31. Thus, considering data showing that hEER2 suppresses glucocorticoid receptor (GR) activity, nNR1 is
) May be an endogenous modulator. nNR2 and nNR2-1 have 77% and 75% homology to hERR2 (Giguere et al., 1988, Nature 331: 91-94) at the amino acid level, respectively, in the overlap region. The nNR2 and nNR1 proteins show 77% homology at the amino acid level. The gene encoding nNR2 is located on the first chromosome. Both genes are expressed at very low levels in most tissues examined by RT-PCR.

Thus, the present invention also expresses a novel biologically active human nuclear receptor
The present invention relates to isolated nucleic acid fragments of nNR1 and nNR2 that encode mRNA (SEQ ID NO: 1 and SEQ ID NO: 3, respectively). Any such nucleic acid fragment spans at least the intracellular DNA binding domain and / or ligand binding domain, the human nuclear receptor family domain present in nNR1 (SEQ ID NO: 2) and nNR2 (SEQ ID NO: 4). It will encode either a protein or protein fragment that contains a conserved domain. Any such polynucleotides include, but are not limited to, nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations (these mutations may be diagnostic, It encodes mRNA that expresses a therapeutic or prophylactic protein or protein fragment and may be useful for screening for agonists and / or antagonists of nNR1, nNR2 and / or nNR2-1 function). Such a nucleic acid fragment is exemplified as a modified form of the DNA fragment encoding nNR2. This DNA molecule (described in SEQ ID NO: 5) is identical to SEQ ID NO: 3 except for a two nucleotide insertion at nucleotide 1352 of SEQ ID NO: 3. This insertion results in a shift in the reading frame and the introduction of a TGA termination codon 33 nucleotides from the insertion site, resulting in a carboxy truncation as shown in FIG. 8 and SEQ ID NO: 6.
Provides an open reading frame encoding the nNR2 protein (nNR2-1).

The isolated nucleic acid molecules of the present invention include deoxyribonucleic acid molecules (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single-stranded (coding or non-coding) or double-stranded. And synthetic DNA, such as a synthesized single-stranded polynucleotide. Also, the isolated nucleic acid molecule of the present invention includes:
A ribonucleic acid molecule (RNA) can be included.

[0039] The present invention also relates to recombinant vectors and recombinant hosts (both prokaryotic and eukaryotic) containing the substantially purified nucleic acid molecules disclosed throughout this specification.

A preferred embodiment of the present invention is disclosed in FIGS. 1A-C and SEQ ID NO: 1 (a human cDNA encoding a novel nuclear trans-acting receptor protein nNR1) (as follows):

[0041]

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[0042]

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Another preferred embodiment of the invention is disclosed in FIGS. 4A-C and SEQ ID NO: 3 (a human cDNA encoding a novel nuclear transacting receptor protein nNR2) (as follows):

[0044]

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[0045]

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The present invention also relates to an isolated and purified form of the DNA molecule that encodes a truncated form of nNR2, referred to as nNR2-1. This cDNA molecule is set forth in SEQ ID NO: 5 and is disclosed below:

[0047]

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[0048]

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[0049] The present invention also provides a novel nuclear trans-acting receptor protein nN in substantially purified form as set forth in FIGS. 2A-F and FIG. 3 and set forth in SEQ ID NO: 2, as follows.
About R1:

[0050]

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The present invention also includes, but is not limited to, amino acid substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations, the biology of nNR1 as set forth in SEQ ID NO: 2. Active fragments and / or mutants (these mutations may provide a diagnostic or therapeutic or prophylactic protein or protein fragment and may be useful in screening for agonists and / or antagonists of nNR1 function. ).

The present invention also relates to a substantially purified form of the novel nuclear trans-acting receptor protein nNR2 disclosed in FIGS. 5A-H and FIG. 6 and set forth in SEQ ID NO: 4 (as follows):

[0053]

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The present invention also includes, but is not limited to, amino acid substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations, the biology of nNR2 as set forth in SEQ ID NO: 4. Active fragments and / or mutants (these mutations may provide diagnostic, therapeutic or prophylactic proteins or protein fragments and may be useful in screening for agonists and / or antagonists of nNR2 function. ). For this purpose, an example of such a protein is the nN described in FIG. 8 and SEQ ID NO: 6 (as follows).
A carboxy-terminal truncated version of R2 (referred to as nNR2-1) is included:

[0055]

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The present invention also relates to an isolated nucleic acid molecule that is a fusion construct that expresses a fusion protein useful in an assay to identify compounds that modulate wild-type human nNR1, nNR2 and / or nNR2-1 activity. . In a preferred embodiment of this aspect of the invention there is provided a glutathione S-transferase GST-nNR1 and / or GST-
Includes, but is not limited to, nNR2 fusion constructs. These fusion constructs include n as an in-frame fusion at the carboxy terminus of the GST gene.
Includes, but is not limited to, all or a portion of each of the ligand binding domains of NR1, nNR2 and / or nNR2-1. The disclosure of SEQ ID NOs: 1-4 allows one of skill in the art to construct any such nucleic acid molecule encoding a GST-nuclear receptor fusion protein. Soluble recombinant GST-nuclear receptor fusion protein
Using a baculovirus expression vector (eg, Bac-N-Blue DNA from Invitrogen or pAcG2T from Pharmingen), use Spodoptera frugiperda (Spodopte).
rafrugiperda) (Sf21) can be expressed in various expression systems such as insect cells (Invitrogen).

The isolated nucleic acid molecules of the present invention include deoxyribonucleic acid molecules (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single-stranded (coding or non-coding) or double-stranded. And synthetic DNA, such as a synthesized single-stranded polynucleotide. Also, the isolated nucleic acid molecule of the present invention includes:
A ribonucleic acid molecule (RNA) can be included.

It is known that there is a significant amount of overlap between the various codons encoding a particular amino acid. Accordingly, the present invention also relates to DNA sequences encoding RNA that include alternative codons encoding the final translation of the same amino acid as shown below.

A = Ala = alanine: codons GCA, GCC, GCG, GCU C = Cys = cysteine: codon UGC, UGU D = Asp = aspartic acid: codon GAC, GAU E = Glu = glutamic acid: codon GAA, GAG F = Phe = phenylalanine: codon UCC, UUU G = Gly = glycine: codon GGA, GGC, GGG, GGU H = His = histidine: codon CAC, CAU I = Ile = isoleucine: codon AUA, AUC, AUU K = Lys = lysine: Codon AAA, AAG L = Leu = Leucine: Codon UUA, UUG, CUA, CUC, CUG, CUU M = Met = Methionine: Codon AUG N = Asp = Asparagine: Codon AAC, AAU P = Pro = Proline: Codon CCA, CCC , CCG, CCU Q = Gln = glutamine: codon CAA, CAGR = Arg = arginine: codon AGA, AGG, CGA, CGC, CGG, CGU S = Ser = serine: codon AGC, AGU, UCA, UCC, UCG, UCU T = Thr = Threonine: Codons ACA, ACC, ACG, ACU V = Val = Valine: GUA, GUC, GUG, GUU W = Trp = Tryptophan: Codon UGG Y = Tyr = Tyrosine: Codon UAC, UAU

Thus, the present invention discloses codon duplication that can result in differences in DNA molecules expressing the same protein. For purposes herein, sequences that carry one or more substituted codons are defined as degenerate variants. Also, mutations in the DNA sequence or translated protein that do not substantially alter the ultimate physical properties of the expressed protein are included within the scope of the invention. For example, leucine to valine,
Substitution of lysine for arginine or glutamine for asparagine will not cause a change in functionality in the polypeptide.

It is known that a DNA sequence encoding a peptide can be modified to encode a peptide having properties that differ from the naturally occurring peptide. DN
Methods for altering the A sequence include, but are not limited to, site-directed mutagenesis. Properties that are altered include, but are not limited to, for example, changes in the affinity of an enzyme for a substrate or a receptor for a ligand.

As used herein, “purified” and “isolated” means that the nucleic acid, protein or respective fragment thereof has been substantially removed from its in vivo environment, It is a pure means that allows the skilled person the opportunity of various means (eg, sequencing nucleotides for nucleic acid fragments, restriction digestion, site-directed mutagenesis and subcloning into expression vectors, and the production of polyclonal, monoclonal antibodies). Obtaining a substantial amount of protein or protein fragments,
(Including, but not limited to, amino acid sequencing and peptide digestion). Thus, the nucleic acids claimed in the present invention can be present in whole cells or in cell lysates or in partially purified or substantially purified form.
A nucleic acid is considered to be substantially purified if it has been purified from surrounding contaminants. Thus, a nucleic acid sequence isolated from a cell is considered substantially purified if it has been purified from cellular components by standard methods, while a chemically synthesized nucleic acid sequence has If purified from a chemical precursor, it is considered substantially purified.

[0063] The present invention also relates to recombinant vectors and recombinant hosts (both prokaryotic and eukaryotic) containing the substantially purified nucleic acid molecules disclosed throughout this specification.

Thus, the present invention also relates to a method of expressing nNR1, nNR2 and / or nNR2-1 and their biological equivalents (as disclosed in the present invention), these recombinantly expressed genes Assays using the products, cells expressing these gene products, and agonist and / or antagonist compounds identified by use of assays utilizing these recombinant forms [via direct contact with LBD, or DBD or Direct or with a ligand that interacts with the wild-type transcription complex, where nNR1, nNR2 and / or nNR2-1 act in trans, thereby modulating cell differentiation or cell development Including, but not limited to, one or more modulators of human nNR1, nNR2 and / or nNR2-1 via indirect contact No] concerning.

As used herein, a “biologically active equivalent” or “functional derivative” of wild-type human nNR1, nNR2 and / or nNR2-1 is a wild-type human nNR1, nNR2 and / or nNR2- It has a biological activity substantially similar to one biological activity. The term “functional derivative” refers to “fragments” of wild-type human nNR1, nNR2 and / or nNR2-1 proteins,
It is intended to include "mutant", "variant", "degenerate variant", "analog" and "homolog" or "chemical derivative". The term “fragment” refers to any polypeptide subset of wild-type human nNR1 or nNR2. The term "mutant" refers to a molecule that may be substantially similar to the wild-type form but has different biological properties. Such altered properties include altered substrate binding, altered substrate affinity, and biological properties of human nNR1, nNR2 and / or nNR2-1 or human nNR1, nNR2 and / or nNR2-1 functional derivatives. Includes, but is not limited to, altered sensitivity to compounds that affect activity. The term "variant" refers to a molecule that is substantially similar in structure and function to the entire wild-type protein or a fragment thereof. Certain molecules are wild-type human nNR1, nN
"Substantially similar" to an R2 and / or nNR2-1 like protein is when both molecules have a substantially similar structure, or when both molecules have similar biological activities. It is. Thus, if the two molecules have substantially similar activities, even if one structure of the molecules is not found in the other, or the two amino acid sequences are not identical Even if they are, they are considered variants. The term "analog" refers to a molecule that is substantially similar in function to the full length human nNR1, nNR2 and / or nNR2-1 proteins or biologically active fragments thereof.

For cloning human nNR1, nNR2 and / or nNR2-1, any of a variety of methods can be used. These methods include, but are not limited to, the following techniques. (1) RACE PCR cloning technology (Fro
hman et al., 1988, Proc. Natl. Acad. Sci. 85: 8998-9002). 5 'and / or 3' RACE can be performed to obtain the full length cDNA sequence. This method involves the use of gene-specific oligonucleotide primers for PCR amplification of human nNR1, nNR2 and / or nNR2-1 cDNA. These gene-specific primers are designed through the identification of EST (expressing tag sequences) nucleotide sequences identified by searching a large number of publicly available nucleic acid and protein databases. (2) Human nNR1, nNR2 and / or nNR2-1 containing c in a suitable expression vector system
Direct functional expression of human nNR1, nNR2 and / or nNR2-1 cDNA after DNA library construction. (3) Contains human nNR1, nNR2 and / or nNR2-1 constructed in a bacteriophage or plasmid shuttle vector with a labeled degenerate oligonucleotide probe designed from the amino acid sequence of human nNR1, nNR2 and / or nNR2-1 protein Screening of cDNA library. (4) human nNR1, nNR2 and / or constructed in bacteriophage or plasmid shuttle vector with partial cDNA encoding human nNR1, nNR2 and / or nNR2-1 protein
Screening of a cDNA library containing nNR2-1. This partial cDNA is the human nNR1,
human by designing degenerate oligonucleotide primers from amino acid sequences known for other kinases related to the nNR2 and / or nNR2-1 proteins
Obtained by specific PCR amplification of nNR1, nNR2 and / or nNR2-1 DNA fragments. (Five
3.) Human nN constructed in bacteriophage or plasmid shuttle vector with partial cDNA encoding human nNR1, nNR2 and / or nNR2-1 proteins.
Screening of a cDNA library containing R1, nNR2 and / or nNR2-1. Also,
This method may involve the use of gene-specific oligonucleotide primers for PCR amplification of human nNR1, nNR2 and / or nNR2-1 cDNA identified as EST as described above. (6) Design of 5 ′ and 3 ′ gene specific oligonucleotides by using SEQ ID NO: 1 as a template. Thereby, the full-length cDNA can be made by known PCR techniques, or a portion of the coding region can be made by these same known PCR techniques to produce multiple types of cDNA and / or genomic libraries. It is possible to create and isolate a portion of the coding region used as a probe to screen one to isolate the full length form of the nucleotide sequence encoding human nNR1, nNR2 and / or nNR2-1. is there.

In order to isolate DNA encoding nNR1, nNR2 and / or nNR2-1 or homologs of nNR1, nNR2 and / or nNR2-1, other types of libraries and other cell or species types may be used. One skilled in the art will readily recognize that the constructed library may be useful. Other types of libraries include cells or cell lines other than human cells or tissues (eg, mouse cells, rodent cells, or other cells that may contain DNA encoding nNR1, nNR2 and / or nNR2-1). CDNA libraries derived from any vertebrate host), including but not limited to. In addition, nNR1, nNR2 and / or nNR2-1 genes and homologs can be used in vertebrate genomic libraries, such as mouse genomic libraries, rodent genomic libraries and associated human genomic DNA libraries, including but not limited to ) Can be isolated by hybridization screening based on oligonucleotides or polynucleotides.

A cell or cell line that has nNR1, nNR2 and / or nNR2-1 activity has a suitable cD
One skilled in the art will readily recognize that NA libraries can be prepared. nNR1, nNR2
Selection of cells or cell lines for use in preparing a cDNA library for the isolation of cDNA encoding and / or nNR2-1 first determines cell-related nNR1, nNR2 and / or nNR2-1 activity. (This can be done using any known assay available for such purpose).

Preparation of a cDNA library can be performed by standard techniques well known in the art. Well-known cDNA library construction techniques include, for example, Sambro
ok et al., 1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor L
aboratory, Cold Spring Harbor, New York. Also, complementary DNA
Libraries can be obtained from a number of 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 nNR1, nNR2 and / or nNR2-1 can be isolated from a suitable genomic DNA library. Construction of a genomic DNA library can be performed by standard techniques well known in the art. Well-known genomic DNA library construction techniques are described in Sambrook et al., Supra.

To clone a human nNR1, nNR2 and / or nNR2-1 gene by one of these preferred methods, the amino acid or DNA sequence of human nNR1, nNR2 and / or nNR2-1 or a homologous protein is May be necessary. To achieve this, the human nNR1, nNR2 and / or nNR2-1 protein or homologous protein can be purified and the partial amino acid sequence determined by an automatic sequencer. Although it is not necessary to determine the entire amino acid sequence, partial human nNR1,
For PCR amplification of nNR2 and / or nNR2-1 DNA fragments, it is possible to determine the linear sequence of two regions of 6-8 amino acids. Once you have identified the appropriate amino acid sequence,
A DNA sequence capable of encoding them is synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid. Thus, the amino acid sequence may be encoded by any set of similar DNA oligonucleotides. Only one member of the set comprises human nNR1, nNR2 and / or nNR2-
It is identical to one sequence. However, other members of the set will be able to hybridize to human nNR1, nNR2 and / or nNR2-1 DNA, even in the presence of mismatched DNA oligonucleotides. The mismatched DNA oligonucleotide still encodes human nNR1, nNR2 and / or nNR2-1
It can hybridize to human nNR1, nNR2 and / or nNR2-1 DNA to a degree sufficient to allow identification and isolation of the DNA. Alternatively, by searching one or more available genomic databases, the nucleotide sequence of the region of the expressed sequence can be identified. From the cDNA library or cDNA population, find the cDNA
To perform PCR amplification of NA, gene-specific primers can be used. As described above, a suitable nucleotide sequence for use in a PCR-based method can be obtained from SEQ ID NO: 1 and used to isolate overlapping 5 'and 3' RACE products, A full-length sequence encoding nNR1, nNR2, and / or nNR2-1 can be generated, or a portion of a nucleotide sequence encoding human nNR1, nNR2, and / or nNR2-1 can be isolated to obtain a cDNA or genomic live sequence. Human nNR1, nNR, used as a probe to screen one or more of the rally
It is possible to isolate full-length sequences encoding 2 and / or nNR2-1 or human nNR1, nNR2 and / or nNR2-1 like proteins.

In an exemplary method, the human nNR1, nNR2 and / or nNR2-1 of the present invention are
Full-length cDNA was generated by PCR scanning a human cDNA library with oligonucleotide primers from ESTs showing homology to hERR2. Briefly, a random and oligo dT primed cDNA library described herein consisting of approximately 4 million primary clones was constructed in the plasmid vector pBluescript (Stratagene, LaJolla, CA). The primary clone was subdivided into 188 pools, 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. The respective cDNA libraries distributed in the 96-well plate are scanned using primer pairs of the 5 'and 3' portions of the EST. The initial positive pool is identified with the EST primer. The corresponding full-length cDNA clone is recovered by inverse PCR using back-to-back primer pairs designed from the EST. Thus, the primers walk away from each other during PCR, resulting in the amplification of a population of linearized plasmid DNA molecules corresponding to the EST. The linear DNA was ligated and the circularized DNA was transformed into bacterial competent cells to obtain a cDNA clone. Usually, four positive clones for each gene were used for sequence analysis due to possible mutations during long PCR reactions. The consensus DNA
The sequence was considered a wild type DNA sequence. Has the same DNA sequence as the consensus sequence
To obtain a cDNA clone, the gene was recloned by PCR using gene-specific primers spanning the entire open reading frame. This method does not rely on the use of a cDNA library with directionally cloned inserts, but does not require that the cDNA be constructed in a plasmid vector such as pBluescript.
A Requires library. Using this method, full-length cDNA molecules corresponding to human nNR1, nNR2 and / or nNR2-1 were identified.

To express recombinant human nNR1, nNR2, and / or nNR2-1 in mammalian cells, various mammalian expression vectors can be used. An expression vector is defined in the present invention as a DNA sequence necessary for transcription of cloned DNA in an appropriate host and translation of their mRNA. Such vectors can be used to express eukaryotic DNA in various hosts such as bacteria, cyanobacteria, plant cells, insect cells, animal cells, and the like. Specially designed vectors allow the shuttle of DNA between hosts, such as bacteria-yeast or bacteria-animal cells. A properly constructed expression vector should contain an origin of replication for autonomous replication in a host cell, a selectable marker, a certain number of useful restriction enzyme sites, a potentially high copy number and an active promoter. is there. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. Strong promoters are those that result in frequent initiation of mRNA. Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, specially designed plasmids or viruses.

Commercially available mammalian expression vectors that may be suitable for expressing recombinant human nNR1, nNR2 and / or nNR2-1 include pcDNA3.1 (Invitrogen), pLITMU
S28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI
, PcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMC1neo (Stratagene),
pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC37593), pBPV-
1 (8-2) (ATCC37110), pdBPV-MMTneo (342-12) (ATCC37224), pRSVgpt (ATCC37
199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC37146), pUCTag (ATCC37460)
And 1ZD35 (ATCC37565), but are not limited thereto.

For expressing recombinant human nNR1, nNR2 and / or nNR2-1 in bacterial cells, various bacterial expression vectors can be used. Commercially available bacterial expression vectors that may be suitable for expression of recombinant human nNR1, nNR2 and / or nNR2-1 include pQE (Qiagen), pET11a (Novagen), lambda gt11 (Invitrogen)
) And pKK223-3 (Pharmacia), but are not limited thereto.

For expressing recombinant human nNR1, nNR2 and / or nNR2-1 in fungal cells, various fungal cell expression vectors can be used. Recombinant human nNR1, nN
Commercially available fungal cell expression vectors that may be suitable for expression of R2 and / or nNR2-1 include pYES2 (Invitrogen) and Pichia expression vectors (Invitrogen), It is not limited to these.

To express a recombinant receptor in insect cells, various insect cell expression vectors can be used. Commercially available insect cell expression vectors that may be suitable for recombinant expression of human nNR1, nNR2 and / or nNR2-1 include pBlu
Including but not limited to eBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).

In order to express human nNR1, nNR2 and / or nNR2-1 in a recombinant host cell, an expression vector containing a DNA encoding a human nNR1, nNR2 and / or nNR2-1 like protein is used. be able to. Recombinant host cells can be prokaryotic or eukaryotic, including bacteria such as E. coli, fungal cells such as yeast, human, bovine, porcine, monkey and germ cells. Mammalian cells, including, but not limited to, cell lines derived from teeth, and Drosophila (Dr.
osophila) and insect cells, including, but not limited to, cell lines derived from silkworms. The cell lines derived from mammalian species which may be suitable are commercially available, L cells ^{LM (TK -) (ATCC CCL1.3} ), L cells LM (ATCC
CCL1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 8
6), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651)
, CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH / 3T3 (ATCC CRL 1658), H
including, but not limited to, eLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), and CPAE (ATCC CCL 209) Not something.

The expression vector can be introduced into a host cell by any one of a number of techniques including, but not limited to, transformation, transfection, protoplast fusion and electroporation. . The cells containing the expression vector are individually analyzed to determine if they produce human nNR1, nNR2 and / or nNR2-1 protein. The identification of cells expressing human nNR1, nNR2 and / or nNR2-1 can be determined by immunoreactivity with anti-human nNR1, nNR2 and / or nNR2-1 antibodies, labeled ligand binding and host cell-associated human nNR1, nNR2 and / Or nN
This can be done by several means, including, but not limited to, determining the presence of R2-1 activity.

The cloned human nNR1, nNR2 and / or nNR2-1 cDNA obtained by the above-described method can be used to prepare an expression vector containing a suitable promoter and other suitable transcription control elements (
For example, recombinantly expressed by molecular cloning into pcDNA3.1, pQE, pBlueBacHis2 and pLITMUS28), introduced into prokaryotic or eukaryotic host cells, and treated with recombinant human nNR1, nNR2 and / or nNR2- 1 can be produced. Techniques for such manipulations are described in Sambrook et al., Supra, are described in detail in the Examples section, and are well known and readily available to those skilled in the art.

The expression of human nNR1, nNR2 and / or nNR2-1 DNA can be carried out using in vitro obtained synthetic mRNA. Synthetic mRNA can be efficiently translated in a variety of cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, Microinjection into frog oocytes is preferred, as it can be efficiently translated in a cell-based system that includes microinjection into it.

To determine a human nNR1, nNR2, and / or nNR2-1 cDNA sequence that gives optimal levels of human nNR1, nNR2, and / or nNR2-1, include, but are not limited to: 2.) cDNA molecules can be constructed: cDNA fragments containing the full length open reading frame of human nNR1, nNR2 and / or nNR2-1, and encoding only the specific domain of the protein or the rearranged domain of the protein Various constructs containing the cDNA portion. All constructs are human nNR1
, NNR2 and / or nNR2-1 cDNA can be designed to contain all or part of the 5 'and / or 3' untranslated region or not at all. The expression level and activity of human nNR1, nNR2 and / or nNR2-1 can be measured after introducing these constructs alone or in combination into a suitable host cell. Human nNR1, nNR2 and / or give optimal expression in transient assays
Alternatively, after determining the nNR2-1 cDNA cassette, the nNR1, nNR2 and / or nNR2-
1 cDNA constructs can be transformed into various expression vectors, including but not limited to expression vectors for expression in mammalian cells, insect cells, oocytes, bacteria and yeast cells, including but not limited to recombinant viruses. Introduce.

The present invention also relates to the human forms of nNR1, nNR2 and / or nNR2 disclosed herein.
Polyclonal and monoclonal antibodies raised against -1 or biologically active fragments thereof. NNR1, nNR2 and / or nNR2-1 exhibiting the lowest homology to other known proteins belonging to the human nuclear receptor superfamily
NH ₂ cause antibodies to epitope-terminal domain may be particularly preferred.

The use of immunoaffinity columns made with monoclonal or polyclonal antibodies specific for full-length nNR1, nNR2 and / or nNR2-1 proteins or for polypeptide fragments of nNR1, nNR2 and / or nNR2-1 proteins Recombinant nNR1, nNR2 and / or nNR2-1 proteins can be separated from other cellular proteins. In addition, polyclonal or monoclonal antibodies can be raised against synthetic peptides (usually about 9 to about 25 amino acids in length) derived from a portion of the protein set forth in SEQ ID NO: 2. Human nNR1, nNR2 and / or nNR2
Monospecific antibodies to -1 can be purified from mammalian antisera containing antibodies reactive to human nNR1, nNR2 and / or nNR2-1, or can be obtained from Kohler and Mils.
It is prepared as a monoclonal antibody reactive with human nNR1, nNR2 and / or nNR2-1 using the technique of tein (1975, Nature 256: 495-497). A monospecific antibody for use in the present invention is defined as multiple antibody species or a single antibody species having uniform binding properties to human nNR1, nNR2 and / or nNR2-1. Homogeneous binding, as used in the present invention, means that the antibody species can bind to a particular antigen or epitope (eg, associated with nNR1, nNR2 and / or nNR2-1 described above). Human nNR1, nNR2
And / or antibodies specific for the nNR2-1 protein can be used to treat animals such as mice, rats, guinea pigs, rabbits, goats, horses, etc., in the presence or absence of an immune adjuvant, with appropriate concentrations of human nNR1, / Or nNR2-1 protein or human
It is produced by immunization with a synthetic peptide made from part of nNR1, nNR2 and / or nNR2-1.

Prior to the first immunization, pre-immune serum is collected. Each animal receives from about 0.1 mg to about 1000 mg of nNR1, nNR2 and / or nNR2-1 protein together with an acceptable immune adjuvant. Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum sediment, water-in-oil emulsions (containing Corynebacterium parvum and tRNA). It is not limited. The primary immunization comprises administering human nNR1, nNR2 and / or nNR2-1 protein or peptide fragment thereof, preferably in complete Freund's adjuvant, subcutaneously (SC), intraperitoneally (IP) or both at multiple sites. . Blood is collected from each animal at regular intervals (preferably weekly) and antibody titers are determined. After the first immunization, the animals may or may not receive booster injections. Animals receiving booster injections generally receive an equivalent amount of human nNR1, nNR2 and / or nNR2-1 in Freund's incomplete adjuvant by the same route. Booster injections are given approximately every 3 weeks until maximal titer is obtained. Approximately 7 days after each booster immunization, or approximately every week after a single immunization, the animals are bled, the serum collected, and an aliquot stored at approximately -20 ° C.

A monoclonal antibody (mAb) reactive to human nNR1, nNR2 and / or nNR2-1 is used to immunize inbred mice, preferably Balb / c, with human nNR1, nNR2 and / or nNR2-1 proteins. Prepared by Approximately 1 mg to about 100 mg, preferably about 10 mg, of human nNR1, nNR2, and / or nNR2-1 protein in about 0.5 ml of buffer or saline is contained in an equal amount of the acceptable adjuvant. Or immunize by SC route. Complete Freund's adjuvant is preferred. The mice are primed on day 0 and rested for about 3 to about 30 weeks. Immunized mice receive one or more booster immunizations of about 1 to about 100 mg of human nNR1, nNR2 and / or nNR2-1 in a buffer solution such as phosphate buffered saline by the intravenous (IV) route. By removing the spleen from the immunized mouse by standard methods known in the art,
Lymphocytes, preferably spleen lymphocytes, are obtained from antibody-positive mice. Hybridoma cells are produced by mixing the splenic lymphocytes with a suitable fusion partner (preferably, myeloma cells) under conditions that permit stable hybridoma formation. Fusion partners can include, but are not limited to, mouse myeloma P3 / NS1 / Ag4-1, MPC-11, S-194 and Sp2 / 0, with Sp2 / 0 being preferred. The antibody producing cells and myeloma cells are fused at a concentration of about 30% to about 50% in polyethylene glycol having a molecular weight of about 1000. According to methods known in the art, the fused hybridoma cells
Selection is performed by growth in Dulbecco's modified Eagle's basal medium (DMEM) supplemented with hypoxanthine, thymidine and aminopterin. Supernatant fluid was collected from the growth positive wells at about 14, 18, and 21 days and used as antigens human nNR1, nNR2 and / or
Screen for antibody production by immunoassay such as solid phase immunoradioassay (SPIRA) using nNR2-1. The culture fluid is also tested in an Ouchterlony precipitation assay to determine the isotype of the mAb. Hybridoma cells from antibody-positive wells were isolated from MacPherson, 1973, Soft Agar Techniques, Ti
ssue Culture Methods and Applications, Kruse and Paterson, Academic P
Cloning by techniques such as ress soft agar technology.

About 2 × 10 ⁶ to about 6 × 10 ⁶ hybridoma cells are injected into pristine, prime-primed Balb / c mice about 4 days after the priming (about 0.5 ml / mouse). By doing so, a monoclonal antibody is produced in vivo. About 8 to 1 for cell introduction
Two days later, ascites is collected and the monoclonal antibody is purified by techniques known in the art.

In vitro production of anti-human nNR1, nNR2 and / or nNR2-1 mAb is achieved by growing the hybridoma in DMEM containing about 2% fetal calf serum to provide a sufficient amount of the specific Get mAb. The mAb is purified by techniques known in the art.

The antibody titer of ascites or hybridoma cultures can be determined by various methods including, but not limited to, sedimentation, passive agglutination, enzyme-linked immunosorbent (ELISA) and radioimmunoassay (RIA) techniques. Determined by serologic or immunological assays. A similar assay is used to detect the presence of human nNR1, nNR2 and / or nNR2-1 in body fluids or tissue and cell extracts.

Using the above methods for producing monospecific antibodies, human nNR1, nNR2 and / or nNR2-1 peptide fragments or full length human nNR1, nNR2 and / or nNR2
It is easily recognized by those skilled in the art that an antibody specific to -1 can be produced.

For example, Affigel- a gel support that has been pre-activated with N-hydroxysuccinimide ester such that the antibody forms a covalent bond with the agarose gel bead support.
By adding the antibody to 10 (Biorad), human nNR1, nNR2 and / or nNR2
Prepare a -1 antibody affinity column. The antibody is then bound to the gel via an amide bond at the spacer arm. Then, the remaining activated ester was
Quench with ethanolamine HCl (pH 8). The column is washed with water and then with 0.23 M glycine HCl (pH 2.6) to remove unconjugated antibodies or foreign proteins. The column is then equilibrated in phosphate buffered saline (pH 7.3) and full length human nNR1, nNR2 and / or nNR2-1 or human nNR1, nNR2 and / or nNR
The cell culture supernatant or cell extract containing the R2-1 protein fragment is passed slowly through the column. The column is then washed with phosphate buffered saline until the optical density (A ₂₈₀ ) drops to background, and the protein is eluted with 0.23 M glycine-HCl (pH 2.6). The purified human nNR1, nNR2 and / or nNR2-
1 Dialyze protein against phosphate buffered saline.

[0092] The levels of human nNR1, nNR2 and / or nNR2-1 in the host cells are quantified by a variety of techniques, including but not limited to immunoaffinity and / or ligand affinity techniques. nNR1, nNR2 and / or nNR2-1
Is used to isolate ³⁵ S-methionine labeled or unlabeled nNR1, nNR2 and / or nNR2-1 using affinity beads specific for or NNR1, nNR2 and / or nNR2-1. Labeled human nNR1, nNR2 and / or nNR2-1 proteins are analyzed by SDS-PAGE. Unlabeled nNR1, nNR2 and / or nNR2-1 protein is detected by Western blot, ELISA or RIA assay using an antibody specific for nNR1, nNR2 and / or nNR2-1 protein and / or anti-phosphotyrosine antibody.

After expression of nNR1, nNR2 and / or nNR2-1 in a host cell, the nNR1, nNR2 and / or nNR2-1 proteins are recovered and the active forms of nNR1, nNR2 and / or nNR2-1 proteins are recovered. Can be obtained. Some nNR1, nNR2 and / or
nNR2-1 protein purification methods are available and suitable for use. Recombinant nNR1 from cell lysates and extracts or from conditioned media by various combinations of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography or alone , NNR2 and / or nNR2-1 protein can be purified.

The present invention also relates to a screening method for a compound that modulates the expression of DNA or RNA encoding human nNR1, nNR2 and / or nNR2-1 protein. Compounds that modulate these activities may be DNA, RNA, peptides, proteins or non-proteinaceous organic molecules. The compound is human
Expression of DNA or RNA encoding nNR1, nNR2 and / or nNR2-1 or human
It can be modulated by enhancing or attenuating the function of nNR1, nNR2 and / or nNR2-1. Compounds that modulate the expression of DNA or RNA encoding human nNR1, nNR2 and / or nNR2-1 or their biological function can be detected by various assays. The assay may be a simple "yes / no" assay to determine if there is a change in expression or function. The assay can be made quantitative by comparing the expression or function of a test sample to the level of expression or function in a standard sample. Human nNR1, nNR2 and / or nNR2-1, human nNR1, nNR2 and / or
Or antibodies to nNR2-1, or modified human nNR1, nNR2 and / or
Production of a kit containing nNR2-1 can be performed by known methods for such uses.

Using the DNA molecules, RNA molecules, recombinant proteins and antibodies of the present invention, human nN
R1, nNR2 and / or nNR2-1 can be screened and their levels measured. The recombinant protein, DNA molecule, RNA molecule and antibody are human nNR1
, NNR2 and / or nNR2-1 are useful in formulating a kit suitable for detection and typing. Such a kit comprises at least one container sealed (c
It will include compartmentalized carriers suitable for containing in a lose confinement. The carrier may further comprise a reagent (eg, a recombinant nNR1, nNR2 and / or nNR2-1, or an anti-human nNR1, nNR2 and / or nNR2-1 antibody suitable for detecting human nNR1, nNR2 and / or nNR2-1). Will be included. In addition, the carrier may contain detection means such as a labeled antigen or an enzyme substrate.

A pharmaceutically useful composition comprising a modulator of human nNR1, nNR2 and / or nNR2-1 can be formulated according to known methods, such as mixing with a pharmaceutically acceptable carrier. Specific examples of such carriers and formulation methods are described in Remington's Pharmaceuticals.
tical Sciences. In order to form a pharmaceutically acceptable composition suitable for effective administration, such a composition should comprise the protein, DNA, RNA, modified human
It will contain an effective amount of nNR1, nNR2 and / or nNR2-1, or nNR1, nNR2 and / or nNR2-1 agonist or antagonist.

The therapeutic or diagnostic composition of the present invention is administered to an individual in an amount sufficient to treat or diagnose a disorder. The effective amount can vary depending on various factors such as the condition, weight, sex, age and the like of the individual. Other factors include the mode of administration.

The pharmaceutical composition can be administered to an individual by various routes such as subcutaneous, topical, oral, and intramuscular.

The term “chemical derivative” refers to a molecule that contains additional chemical moieties that are not normally part of the base molecule. Such moieties can improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively, the moiety may reduce unwanted side effects of the base molecule or reduce the toxicity of the base molecule. Specific examples of such moieties are described in various publications such as Remington's Pharmaceutical Sciences.

The compounds identified according to the methods disclosed herein can be used alone at appropriate doses. Alternatively, simultaneous or sequential administration of other substances may be desirable.

The present invention also provides a suitable topical for use in the novel therapeutic methods of the invention.
It has the purpose of providing oral, systemic and parenteral pharmaceutical formulations. Compositions containing a compound identified according to the present invention as an active ingredient can be administered in a wide variety of therapeutic dosage forms in conventional administration vehicles. For example, the compound may be a tablet,
Oral dosage forms such as capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups, emulsions or It can be administered by injection. Similarly, they can be administered intravenously (both bolus and infusion), intraperitoneal, subcutaneous, topical (occlus
It can also be administered in the presence or absence of (ion) or in an intramuscular form, all of which can employ forms well known to those skilled in the pharmaceutical arts.

The compounds of the present invention may be administered alone in a daily dose, or may be administered in a total daily dose of 2, 3 or
It may be advantageous to administer in four divided doses daily. Further, the compounds for the present invention may be administered in intranasal form by topical use of a suitable intranasal vehicle, or by the transdermal route using transdermal skin patch forms well known to those skilled in the art. Can be. Of course, when administered in the form of a transdermal delivery system, the dosage administration will be continuous rather than intermittent throughout the dosage regimen.

In the case of combination therapy with two or more active agents in separate dosage forms, the active agents can be administered simultaneously, or each of them can be administered separately, at staggered times .

Dosage regimens using the compounds of the invention may include the 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; The choice will depend on various factors, including and the particular compound used. 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 and accurate drug concentrations within the range that yield efficacy without toxicity require a kinetic-based plan of drug availability to the target site. this is,
Includes considerations regarding drug distribution, equilibrium, and elimination.

The following examples are for illustrating the present invention, and the present invention is not limited to these examples.

Example 1 Isolation and Characterization of DNA Fragments Encoding nNR1, nNR2 and / or nNR2-1 To query the EST database by using the Blastn program, from several representative subfamilies DNA sequence of Giguere et al., 1988, Nature 3
31: 91-94). Two ESTs (Genbank accession numbers h91890 (nNR1) and w26275 (nNR2)) were identified by homology to human ERR2 at the DNA sequence level.

In the present invention, EST h91890 is disclosed as SEQ ID NO: 7 (as follows):

[0108]

Embedded image

[0109] In the present invention, EST w26275 is disclosed as SEQ ID NO: 8 (as follows):

[0110]

Embedded image

Primer pair 5′-TGAGTCCAGCCGTCGCTTGTAT-3 ′ (ERR4F1; SEQ ID NO: 9), 5′-T
GCAAGCCTCGCAGGAGGCC-3 '(ERR4iF1; SEQ ID NO: 10) and 5'-GGCCTTCTTCAAGAGG
ACTATC-3 '(ERR4R1; SEQ ID NO: 11) was designed from h91890, and 5'-AAAGATCGACACATTG
ATTCC-3 '(ERR5F; SEQ ID NO: 12), 5'-GACTTGACTCGCCACCTCTC-3'(ERR5iF; SEQ ID NO: 13) and 5'-GTTCTGATGGCCATTCATGGT-3 '(ERR5R; SEQ ID NO: 14) are W262
Designed from 75. CDNA libraries prepared from testis, fetal brain, prostate and placenta are first scanned using primer pairs ERR4F / ERR4R and ERR5F / ERR5R, and then cDNA libraries prepared from those cDNAs and distributed in 96-well plates Was scanned. Primers for nNR1 provided PCR products from testis cDNA, while primers for nNR2 provided PCR products, a cDNA library made from fetal brain, prostate and placental mRNA. Therefore, a testis-generated cDNA library with an insert of> 2.5 kb was used to identify the nNR1-positive pool. A4 and G8 gave PCR products of the expected size. Inverse PCR using ERR4iF1 and ERR4R1 was performed on the positive pool to amplify a DNA fragment of about 6.0 kb. The DNA fragment was purified using a Qiagen gel extraction kit. The purified DNA was phosphorylated, self-ligated and transformed. DNA minipreps from four clones were used in automated sequencing with gene-specific vector primers. 5'-primer 5'-GAATATGATGACCCTA due to possible PCR-induced mutations during long PCR reactions
ATGCA-3 '(SEQ ID NO: 15) and 3'-primer 5'-CTTCCACCTCATGGACACCAA-3'
Using the PCR fragment amplified on the positive A4 pool by (SEQ ID NO: 16),
Resubcloned into CR2.1 vector (Invitrogen). One of those four TA clones showed no mutations by sequencing confirmation. AmpliTaq
DNA sequence analysis was performed using the ABI PRISM ^™ Dye Terminator Cycle Sequencing Simple Reaction Kit with DNA polymerase FS (Perkin Elmer, Norwalk, CT). DNA sequence analysis was performed with the M13 forward / reverse primer and a gene-specific sequencing primer (GIBCO BRL, Gaithersburg, MD). Sequence collection and analysis were performed using SEQUENCHER ^™ 3.0 (Gene Codes Corporation, Ann Ar
bor, MI). Ambiguities and / or differences between automatic base calls in reading the sequence were visually inspected and corrected for correct base calls. After the first automatic or visual call, some regions were resequenced. Four oligonucleotides near the region with potential sequence ambiguity ([R1
F1] 5′-CAT TCC ACG GAG GCA TCC TC-3 ′ (SEQ ID NO: 23); [R1F2] 5′-CCA AGG C
CG TGC AGC ACT TC-3 '(SEQ ID NO: 24); [R1R1] 5'-GAC AGC CTC TAG ATC CTC G
Automatic sequencing was performed using AT-3 '(SEQ ID NO: 25) and [R1R2] 5'-ATC ATG GCT TGA CAT TCT TTC-3' (SEQ ID NO: 26). The final nucleotide sequence encoding NR1 is shown in FIGS. 1A-C and is set forth in SEQ ID NO: 1.

In the case of nNR2, a cDNA library made from fetal brain with> 2.5 kb insert was used. Positive pools C1, F7 and G6 were identified and primer pair ERR5iF
Used in inverse PCR with / ERR5R. A ~ 6.0 kb PCR fragment was amplified from C1. The same methods described herein for nNR1 were applied to the isolation, characterization and sequencing of nNR2 cDNA. The cDNA fragment cloned into the pCR2.1 vector was amplified with 5'-primer 5'-GTTAATTGCACTGTGCTCTG-3 '(SEQ ID NO: 17) and 3'-primer 5'-AGTGTGGTGGAATTCTCTTA-3' (SEQ ID NO: 18).

Primer pair XR2F3 (5′-AGCTCTTGCTAATTCAGAC-3 ′ [SEQ ID NO: 27]) and XR2R
4 (5'-TCAACATGAAGGATGGGAAGG-3 '[SEQ ID NO: 28]) was added to the carboxy region of nNR2.
DNA sequence analysis (performed using the ABI PRISM ^™ Dye Terminator Cycle Sequencing Simple Reaction Kit with AmpliTaq DNA Polymerase FS (Perkin Elmer, Norwalk, Conn.)). DNA sequence analysis was performed with the M13 forward / reverse primer and gene-specific sequencing primers (usually produced by GIBCO BRL, Gaithersburg, MD). Sequence collection and analysis can be performed using SEQ
UENCHER ^™ 3.0 (Gene Codes Corporation, Ann Arbor, MI). Ambiguities and / or differences between automatic base calls in reading the sequence were visually inspected and corrected for correct base calls. Resequencing of the ligand binding domain revealed a new open reading frame confirmed with the XR2F3 / XR2R4 primers. The nNR2 peptide encoded by the complete open reading frame, compared to nNR2-1, has 40 additional amino acids at the C-terminus and is the member most closely related to it, hERR2, Are similar in nature.

To confirm the location of the genomic map of the gene, a primer in the 3 'non-coding region was designed. nNR1 has a forward primer 5'-TCTAGTGTTGCTGCGAGTGA
C-3 '(SEQ ID NO: 19) and reverse primer 5'-CTTCCACCTCATGGACACCAA-3'
(SEQ ID NO: 20), and forward primer 5′-GTCTGACTAAAAGCTC was used for nNR2.
CCTG-3 '(SEQ ID NO: 21) and reverse primer 5'-GAAGATGATGGAGAAAGTAGA-
3 '(SEQ ID NO: 22) was used. Stanford Radiation Hybrid Panel (Cox et al., 19
90, Science, 250: 245-250). PCR scanning was performed on 83 clones. The PCR results were scored and submitted to the Stanford Genome Center for linkage analysis. The results indicate that nNR1 is located on loci 14q24.3-14Q31 and nNR2 is located on chromosome 1.

[Brief description of the drawings]

FIG. 1A shows the nucleotide sequence (SEQ ID NO: 1) containing the open reading frame encoding the human nuclear receptor protein nNR1.

FIG. 1B shows the nucleotide sequence (SEQ ID NO: 1) containing the open reading frame encoding the human nuclear receptor protein nNR1.

FIG. 1C shows the nucleotide sequence (SEQ ID NO: 1) containing the open reading frame encoding the human nuclear receptor protein nNR1.

FIG. 2A shows the nucleotide sequence of a double-stranded cDNA molecule encoding nNR1 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR1 (SEQ ID NO: 2). The underlined bold region is the DNA binding domain.

FIG. 2B shows the nucleotide sequence of the double-stranded cDNA molecule encoding nNR1 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR1 (SEQ ID NO: 2). The underlined bold region is the DNA binding domain.

FIG. 2C shows the nucleotide sequence of the double-stranded cDNA molecule encoding nNR1 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR1 (SEQ ID NO: 2). The underlined bold region is the DNA binding domain.

FIG. 2D shows the nucleotide sequence of the double-stranded cDNA molecule encoding nNR1 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR1 (SEQ ID NO: 2). The underlined bold region is the DNA binding domain.

FIG. 2E shows the nucleotide sequence of the double-stranded cDNA molecule encoding nNR1 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR1 (SEQ ID NO: 2). The bold and underlined region is the DNA binding domain.

FIG. 2F shows the nucleotide sequence of the double-stranded cDNA molecule encoding nNR1 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR1 (SEQ ID NO: 2). The underlined bold region is the DNA binding domain.

FIG. 3 shows the amino acid sequence of nNR1 (SEQ ID NO: 2). The bold and underlined region is the DNA binding domain.

FIG. 4A shows the nucleotide sequence (SEQ ID NO: 3) containing the open reading frame encoding the human nuclear receptor protein nNR2.

FIG. 4B shows the nucleotide sequence (SEQ ID NO: 3) containing the open reading frame encoding the human nuclear receptor protein nNR2.

FIG. 4C shows the nucleotide sequence (SEQ ID NO: 3) containing the open reading frame encoding the human nuclear receptor protein nNR2.

FIG. 5A shows the nucleotide sequence of a double-stranded cDNA molecule encoding nNR2 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR2 (SEQ ID NO: 4). The underlined bold region is the DNA binding domain.

FIG. 5B shows the nucleotide sequence of the double-stranded cDNA molecule encoding nNR2 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR2 (SEQ ID NO: 4). The underlined bold region is the DNA binding domain.

FIG. 5C shows the nucleotide sequence of the double-stranded cDNA molecule encoding nNR2 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR2 (SEQ ID NO: 4). The underlined bold region is the DNA binding domain.

FIG. 5D shows the nucleotide sequence of the double-stranded cDNA molecule encoding nNR2 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR2 (SEQ ID NO: 4). The underlined bold region is the DNA binding domain.

FIG. 5E shows the nucleotide sequence of the double-stranded cDNA molecule encoding nNR2 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR2 (SEQ ID NO: 4). The underlined bold region is the DNA binding domain.

FIG. 5F shows the nucleotide sequence of the double-stranded cDNA molecule encoding nNR2 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR2 (SEQ ID NO: 4). The underlined bold region is the DNA binding domain.

FIG. 5G shows the nucleotide sequence of the double-stranded cDNA molecule encoding nNR2 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR2 (SEQ ID NO: 4). The bold and underlined region is the DNA binding domain.

FIG. 5H shows the nucleotide sequence of the double-stranded cDNA molecule encoding nNR2 (SEQ ID NO: 1 and SEQ ID NO: 29) and the amino acid sequence of nNR2 (SEQ ID NO: 4). The underlined bold region is the DNA binding domain.

FIG. 6 shows the amino acid sequence of nNR2 (SEQ ID NO: 4). The underlined bold region is the DNA binding domain.

FIG. 7A shows the nucleotide sequence (SEQ ID NO: 5) containing the open reading frame encoding the human nuclear receptor protein nNR2.

FIG. 7B shows the nucleotide sequence containing the open reading frame encoding the human nuclear receptor protein nNR2 (SEQ ID NO: 5).

FIG. 7C shows the nucleotide sequence (SEQ ID NO: 5) containing the open reading frame encoding the human nuclear receptor protein nNR2.

FIG. 8 shows the amino acid sequence of nNR2-1, a carboxy-terminal truncated form of nNR2 (SEQ ID NO: 6). The underlined bold region is the DNA binding domain.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] February 28, 2000 (2000.2.28)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

Embedded image A purified DNA molecule that encodes a human nNR1 protein comprising (described in three letter abbreviations in SEQ ID NO: 2).

Embedded image A purified DNA molecule encoding the human nNR1 protein consisting of (listed in three letter abbreviations in SEQ ID NO: 2).

Embedded image

Embedded image A purified DNA molecule encoding human nNR1 protein, comprising (SEQ ID NO: 1).

Embedded image

Embedded image A purified DNA molecule encoding the human nNR1 protein, consisting of (SEQ ID NO: 1).

Embedded image A purified DNA molecule that encodes a human nNR2 protein, including (listed in three letter abbreviations in SEQ ID NO: 4).

Embedded image A purified DNA molecule encoding a human nNR2 protein consisting of (listed in three letter abbreviations in SEQ ID NO: 4).

Embedded image

Embedded image A purified DNA molecule encoding human nNR2 protein, comprising (SEQ ID NO: 3).

Embedded image

Embedded image A purified DNA molecule encoding the human nNR2 protein, consisting of (SEQ ID NO: 3).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12Q 1/68 A C12P 21/02 C12N 15/00 ZNAA C12Q 1/68 5/00 A ( 31) Priority claim number 60 / 078,633 (32) Priority date March 19, 1998 (March 19, 1998) (33) Priority claim country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), CA, JP, USF terms (reference) 4B024 AA01 AA11 BA41 BA63 CA01 HA01 HA11 4B063 QA18 QQ79 QQ96 QR48 QS33 4B064 AG20 CA19 CC24 DA01 DA13 4B065 AA93Y AB01 AC14 BA02 CA24 CA25 CA44 CA46 4H045 AA10 AA11 BA10 CA40 DA50 EA20 EA50

Claims (44)

[Claims] 1. An amino acid sequence A purified DNA molecule that encodes a human nNR1 protein comprising (described in three letter abbreviations in SEQ ID NO: 2). 2. An expression vector comprising the DNA molecule according to claim 1 for expressing human nNR1 protein in a recombinant host cell. 3. A recombinant human nNR1 containing the expression vector according to claim 2.
A host cell that expresses the protein. 4. A method for expressing a human nNR1 protein in a recombinant host cell, comprising: (a) transfecting the expression vector according to claim 2 into a suitable host cell; A method comprising culturing the host cell of step (a) under conditions allowing expression of said human nNR1 protein from an expression vector. 5. An amino acid sequence A purified DNA molecule encoding the human nNR1 protein consisting of (listed in three letter abbreviations in SEQ ID NO: 2). 6. An expression vector for expressing a human nNR1 protein in a recombinant host cell, comprising the DNA molecule according to claim 5. 7. A recombinant human nNR1 containing the expression vector according to claim 6.
A host cell that expresses the protein. 8. A method for expressing human nNR1 protein in a recombinant host cell, comprising: (a) transfecting the expression vector according to claim 6 into a suitable host cell; A method comprising culturing the host cell of step (a) under conditions allowing expression of said human nNR1 protein from an expression vector. 9. Nucleotide sequence (as described in SEQ ID NO: 1) Embedded image A purified DNA molecule encoding human nNR1 protein, comprising (SEQ ID NO: 1). 10. The nucleotide of SEQ ID NO: 1 from about 950 to about 24 nucleotides.
10. The DNA molecule of claim 9, comprising 52. An expression vector for expressing a human nNR1 protein, comprising the DNA molecule according to claim 9. An expression vector for expressing a human nNR1 protein, comprising the DNA molecule according to claim 11. 13. A recombinant human comprising the expression vector according to claim 11.
A host cell that expresses the nNR1 protein. A recombinant human comprising the expression vector according to claim 12.
A host cell that expresses the nNR1 protein. 15. A method for expressing human nNR1 protein in a recombinant host cell, comprising: (a) transfecting the expression vector according to claim 11 into a suitable host cell; A) culturing the host cells of step (a) under conditions allowing expression of said human nNR1 protein from said expression vector. 16. Nucleotide sequence (as set forth in SEQ ID NO: 1) Embedded image A purified DNA molecule encoding the human nNR1 protein, consisting of (SEQ ID NO: 1). 17. Nucleotide 950 to about nucleotide 2452 of SEQ ID NO: 1.
17. The DNA molecule according to claim 16, consisting of: An expression vector for expressing a human nNR1 protein, comprising the DNA molecule according to claim 16. 19. An expression vector for expressing a human nNR1 protein, comprising the DNA molecule according to claim 17. 20. A recombinant human comprising the expression vector according to claim 18.
A host cell that expresses the nNR1 protein. 21. A recombinant human comprising the expression vector according to claim 19.
A host cell that expresses the nNR1 protein. 22. A method for expressing human nNR1 protein in a recombinant host cell, comprising: (a) transfecting the expression vector according to claim 18 into a suitable host cell; A) culturing the host cells of step (a) under conditions allowing expression of said human nNR1 protein from said expression vector. 23. Amino acid sequence embedded image A purified DNA molecule that encodes a human nNR2 protein, including (listed in three letter abbreviations in SEQ ID NO: 4). 24. An expression vector for expressing a human nNR2 protein in a recombinant host cell, comprising the DNA molecule according to claim 23. 25. A recombinant human comprising the expression vector according to claim 24.
A host cell that expresses the nNR2 protein. 26. A method for expressing a human nNR2 protein in a recombinant host cell, comprising: (a) transfecting the expression vector according to claim 24 into a suitable host cell; A) culturing the host cells of step (a) under conditions allowing expression of said human nNR1 protein from said expression vector. 27. Amino acid sequence embedded image A purified DNA molecule encoding a human nNR2 protein consisting of (listed in three letter abbreviations in SEQ ID NO: 4). 28. An expression vector for expressing a human nNR2 protein in a recombinant host cell, comprising the DNA molecule according to claim 27. 29. A recombinant human comprising the expression vector according to claim 28.
A host cell that expresses the nNR1 protein. 30. A method for expressing human nNR2 protein in a recombinant host cell, comprising: (a) transfecting the expression vector according to claim 28 into a suitable host cell; A) culturing the host cells of step (a) under conditions allowing expression of said human nNR1 protein from said expression vector. 31. Nucleotide sequence (as set forth in SEQ ID NO: 3) Embedded image A purified DNA molecule encoding human nNR2 protein, comprising (SEQ ID NO: 3). 32. The DNA molecule of claim 31, comprising about nucleotide 126 to about nucleotide 1382 of SEQ ID NO: 3. 33. An expression vector for expressing a human nNR2 protein, comprising the DNA molecule according to claim 31. 34. An expression vector for expressing a human nNR2 protein, comprising the DNA molecule according to claim 32. 35. A recombinant human comprising the expression vector according to claim 33.
A host cell that expresses the nNR2 protein. 36. A recombinant human comprising the expression vector according to claim 34.
A host cell that expresses the nNR2 protein. 37. A method for expressing human nNR2 protein in a recombinant host cell, comprising: (a) transfecting the expression vector according to claim 33 into a suitable host cell; A) culturing the host cells of step (a) under conditions allowing expression of said human nNR1 protein from said expression vector. 38. Nucleotide sequence (as set forth in SEQ ID NO: 3) Embedded image A purified DNA molecule encoding the human nNR2 protein, consisting of (SEQ ID NO: 3). 39. Nucleotide 126 to about nucleotide 1382 of SEQ ID NO: 3
39. The DNA molecule of claim 38, consisting of: 40. An expression vector for expressing a human nNR2 protein, comprising the DNA molecule according to claim 38. 41. An expression vector for expressing a human nNR2 protein, comprising the DNA molecule according to claim 39. 42. A recombinant human comprising the expression vector according to claim 40.
A host cell that expresses the nNR2 protein. 43. A recombinant human comprising the expression vector according to claim 41.
A host cell that expresses the nNR2 protein. 44. A method for expressing a human nNR2 protein in a recombinant host cell, comprising: (a) transfecting the expression vector according to claim 40 into a suitable host cell; A) culturing the host cells of step (a) under conditions allowing expression of said human nNR2 protein from said expression vector.