DNA MOLECULES ENCODING HUMAN NUCLEAR RECEPTOR PROTEINS

MOLECULES D'ADN CODANT POUR DES PROTEINES RECEPTRICES NUCLEAIRES

English 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.

French Abstract

L'invention concerne l'isolement et la caractérisation de molécules d'ADNc codant pour deux protéines réceptrices nucléaires humaines, désignées par nNR1, nNR2 et/ou nNR2-1. L'invention concerne également des vecteurs recombinants, des cellules hôtes recombinantes, des techniques de criblage permettant l'identification de modulateurs de l'activité nNR1, nNR2 et/ou nNR2-1, et la production d'antibiotiques contre nNR1, nNR2 et/ou de nNR2-1, ou des épitopes de ces derniers.

Claims

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.
-39-

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.
-40-

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
-41-

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.
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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
-43-

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
-44-

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.
-45-

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:
-46-

(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:
-47-

(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
-48-

<IMG>
-49-

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.
-50-

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
-51-

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.
-52-

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.
-R53-

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.
-R54-

Description

CA 02301554 2000-02-24
WO 99/10367 PCT/US98/17826
TITLE OF THE I1WENTION
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.
-1-

CA 02301554 2000-02-24
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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.
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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
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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~

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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
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CA 02301554 2000-02-24
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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.
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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.
7

CA 02301554 2000-02-24
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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_

CA 02301554 2000-02-24
WO 99/10367 PCT/US98/17826
(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
9

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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:
2O GAATATGATG ACCCTAATGC AACAATATCT AACATACTAT CCGAGCTTCG
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
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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
15 ATACAAGCGA CGGCTGGACT CAGAGAGCAGCCCATACCTG AGCTTACAAA
TTTCTCCACC TGCTAAAAAG CCATTGACCAAGATTGTCTC ATACCTACTG
GTGGCTGAGC CGGACAAGCT CTATGCCATGCCTCCCCCTG GTATGCCTGA
GGGGGACATC AAGGCCCTGA CCACTCTCTGTGACCTGGCA GACCGAGAGC
TTGTGGTCAT CATTGGCTGG GCCAAGCACATCCCAGGCTT CTCAAGCCTC
20 TCCCTGGGGG ACCAGATGAG CCTGCTGCAGAGTGCCTGGA TGGAAATCCT
CATCCTGGGC ATCGTGTACC GCTCGCTGCCCTACGACGAC AAGCTGGTGT
ACGCTGAGGA CTACATCATG GATGAGGAGCACTCCCGCCT CGCGGGGCTG
CTGGAGCTCT ACCGGGCCAT CCTGCAGCTGGTACGCAGGT ACAAGAAGCT
CAAGGTGGAG AAGGAGGAGT TTGTGACGCTCAAGGCCCTG GCCCTCGCCA
25 ACTCCGATTC CATGTACATC GAGGATCTAGAGGCTGTCCA GAAGCTGCAG
GACCTGCTGC ACGAGGCACT GCAGGACTACGAGCTGAGCC AGCGCCATGA
GGAGCCCTGG AGGACGGGCA AGCTGCTGCTGACACTGCCG CTGCTGCGGC
AGACGGCCGC CAAGGCCGTG CAGCACTTCTATAGCGTCAA ACTGCAGGGC
AAAGTGCCCA TGCACAAACT CTTCCTGGAGATGCTGGAGG CCAAGGCCTG
3O GGCCAGGGCT GACTCCCTTC AGGAGTGGAGGCCACTGGAG CAAGTGCCCT
CTCCCCTCCA CCGAGCCACC AAGAGGCAGCATGTGCATTT CCTAACTCCC
TTGCCCCCTC CCCCATCTGT GGCCTGGGTGGGCACTGCTC AGGCTGGATA
CCACCTGGAG GTTTZ'CCTTCCGCAGAGGGCAGGTTGGCCA AGAGCAGCTT
AGAGGATCTC CCAAGGATGA AAGAATGTCAAGCCATGATG GAAAATGCCC
35 CTTCCAATCA GCTGCCTTCA CAAGCAGGGATCAGAGCAAC TCCCCGGGGA
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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

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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
1S 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
2O 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
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
30 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 N0:3).
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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
1S AGACTGTGTT TAGTGTGTGG TGACATCGCT TCTGGGTACCACTATGGGGT
AGCATCATGT GAAGCCTGCA AGGCATTCTT CAAGAGGACAATTCAAGGCA
ATATAGAATA CAGCTGCCCT GCCACGAATG AATGTGAAATCACAAAGCGC
AGACGTAAAT CCTGCCAGGC TTGCCGCTTC ATGAAGTGTTTAAAAGTGGG
CATGCTGAAA GAAGGGGTGC GTCTTGACAG AGTACGTGGAGGTCGGCAGA
20 AGTACAAGCG CAGGATAGAT GCGGAGAACA GCCCATACCTGAACCCTCAG
CTGGTTCAGC CAGCCAAAAA GCCATATAAC AAGATTGTCTCACATTTGTT
GGTGGCTGAA CCGGAGAAGA TCTATGCCAT GCCTGACCCTACTGTCCCCG
ACAGTGACAT CAAAGCCCTC ACTACACTGT GTGACTTGGCCGACCGAGAG
TTGGTGGTTA TCATTGGATG GGCGAAGCAT ATTCCAGGCTTCTCCACGCT
2S GTCCCTGGCG GACCAGATGA GCCTTCTGCA GAGTGCTTGGATGGAAATTT
TGATCCTTGG TGTCGTATAC CGGTCTCTTT CATTTGAGGATGAACTTGTC
TATGCAGACG ATTATATAAT GGACGAAGAC CAGTCCAAATTAGCAGGCCT
TCTTGATCTA AATAATGCTA TCCTGCAGCT GGTAAAGAAATACAAGAGCA
TGAAGCTGGA AAAAGAAGAA TTTGTCACCC TCAAAGCTATAGCTCTTGCT
3O AATTCAGACT CCATGCACAT AGAAGATGTT GAAGCCGTTCAGAAGCTTCA
GGATGTCTTA CATGAAGCGC TGCAGGATTA TGAAGCTGGCCAGCACATGG
AGAAGACCCT CGTCGAGCTG GCAAGATGCT GATGACACTGCCACTCCTGA
GGCAGACCTC TACCAAGGCC GTGCAGCATT TCTACAACATCAAACTAGAA
GGCAAAGTCC CAATGCACAA ACTTZ"I"I'TTGGAAATGTTGGAGGCCAAGGT
3S CTGACTAAAA GCTCCCTGGG CCTTCCCATC CTTCATGTTGAAAAAGGGAA
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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
1S CTAATTAGAC AACTTAAGCA ACGTTGCATC GGCTGCTTCTTATCATTGCT
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
2S CTACAGTGAC TCTGAGTAGT CACATATATA AGCACTGCACATGAGATATA
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
3S THANGLDSPP MFAGAGLGGT PCRKSYEDCA SGIMEDSAIKCEYMLNAIPK
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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
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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
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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
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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
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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

CA 02301554 2000-02-24
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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
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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.

CA 02301554 2000-02-24
WO 99/10367 PCT/US98/17826
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 -

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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

CA 02301554 2000-02-24
WO 99/10367 PCT/US98/17826
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).
- 25 -

CA 02301554 2000-02-24
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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

CA 02301554 2000-02-24
WO 99/10367 PCTNS98/17826
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_

CA 02301554 2000-02-24
WO 99/10367 PCT/US98/17826
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
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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
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CA 02301554 2000-02-24
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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_

CA 02301554 2000-02-24
WO 99/10367 PCT/US98/178Z6
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.
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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
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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

CA 02301554 2000-02-24
WO 99110367 PCTNS98/17826
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.

CA 02301554 2000-02-24
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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
2O CCCCGCACAC GAGGCACAGG CGCTTGGGGA TGGCGTTGAG CATGTTACTT
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
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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
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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.
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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 _