MOLECULES D'ADN CODANT LA PROTEINE DE RECEPTEUR DE MELANOCORTINE 3 PROVENANT DE SINGE RHESUS

DNA MOLECULES ENCODING THE MELANOCORTIN 3 RECEPTOR PROTEIN FROM RHESUS MONKEY

Abrégé français

La présente invention concerne des molécules d'ADN de singe rhésus codant la protéine de récepteur de mélanocortine 3, des vecteurs de recombinaison contenant les molécules d'ADN codant MC-3R, des cellules hôtes de recombinaison qui contiennent un vecteur de recombinaison codant MC-3R rhésus, la protéine MC-3R rhésus codée par la molécule d'ADN, et des procédés d'identification d'agonistes et d'antagonistes sélectifs de MC-3R rhésus.

Abrégé anglais

The present invention relates to rhesus monkey DNA molecules encoding the
melanocortin-3 receptor protein, recombinant vectors comprising DNA molecules
encoding rhesus MC-3R, recombinant host cells which contain a recombinant
vectors encoding rhesus MC-3R, the rhesus MC-3R protein encoded by the DNA
molecule, and methods of identifying selective agonists and antagonists of
rhesus MC-3R.

Revendications

WHAT IS CLAIMED:
1. A purified nucleic acid molecule encoding a rhesus
melanocortin 3 receptor protein which comprises the nucleotide sequence
TCTATCTATC TATCTCTCCC TCTCTGGAGA AACTAAAGTC TAGACTGGAC AGCATCCACA
AGAGAAGCAC CTAGAAGGAG AATTTTCCCC AGCAGCTTGC TCAGGACCCT GCAGGAGCCG
CAGCTGGGAC TGGACCTGCT GTTAACCATG AACTCTTCCT GCTGCCTGTC TTCTGTTTCT
CCGATGCTGC CTAACCTCTC TGAGCACCCT GCAGCCCCTC CTGCCAGCAA CCGGAGCGGC
AGTGGGTTCT GTGAGCAGGT CTTCATCAAG CCGGAGGTCT TCCTGGCTCT GGGCATCGTC
AGTCTGATGG AAAACATCCT GGTGATCCTG GCTGTGGTCA GGAATGGCAA CCTGCACTCT
CCCATGTACT TCTTCCTGTG CAGCCTGGCT GCAGCCGACA TGCTGGTGAG CCTGTCCAAC
TCCCTGGAGA CCATCATGAT CGCCGTGATC AACAGCGACT CCCTGACCTT GGAGGACCAG
TTTATCCAGC ACATGGATAA TATCTTCGAC TCTATGATTT GCATCTCCCT GGTGGCCTCC
ATCTGCAACC TCCTGGCCAT TGCCATCGAC AGGTACGTCA CCATCTTCTA TGCCCTTCGG
TACCACAGCA TCATGACAGT GAGGAAAGCC CTCACCTTGA TCGGGGTCAT CTGGGTCTGC
TGCGGCATCT GCGGCGTGAT GTTCATCATC TACTCCGAGA GCAAGATGGT CATCGTGTGT
CTCATCACCA TGTTCTTCGC CATGGTGCTC CTCATGGGCA CCCTATATAT CCACATGTTC
CTCTTCGCCA GGCTCCACGT CCAGCGCATC GCAGTGCTGC CCCCTGCTGG CGTGGTGGCC
CCACAGCAGC ACTCCTGCAT GAAGGGGGCT GTCACCATCA CTATCCTGCT GGGTGTTTTC
ATCTTCTGCT GGGCGCCTTT CTTCCTCCAC CTGGTCCTCA TCATCACCTG CCCCACCAAT
CCCTACTGCA TCTGCTACAC GGCCCATTTC AACACCTACC TGGTTCTCAT CATGTGCAAC
TCCGTCATCG ACCCCCTCAT CTACGCCTTC CGCAGCCTGG AGCTGCGCAA CACGTTCAAG
GAGATTCTCT GCGGCTGCAA CAGCATGAAC TTGGGCTAGG ATGCCCGTGG AGGTGTTCCA
CATCCAGCCA AGAGACAAAA ACAACGCTCA GACGGGACGT AAAAGGGTGT TAGGAGCTGG
AACTGTGCTT GGCTTCGTCT GTAAGCTCGT GGCCCTTTGC AGACGGGACA CGGCGTAGGA
TGGGCTGTCT GTGAGGATCT GTGTGTGGGT AAGTCAGTTT GATCTAGCAC ATAGCCTGGA
AGAATCAGGC AAAGCAGCCC TGAGTGTCAT CTGTGTTCAT TGCTAGGCAC CCAGGGTTTG
TGGCCCCTGC CTGCTTATTG GCTTTGTACC AGTAACTGTG CTTCAAGCCA ACCAGACCGG
AGGGCTCTCG TGAGCAGAAA GAGTGCTTAG ACTTCCGGCA AGCATCCTGG CTCACAGCGG
CCACCTCCTG ACCACTACCG GGAGAGCTTT GCACATATTC TGTGGGAGAT TGAGTGAAGC
CCTGAAAACA ATGTGATATT TGCTGCTCCC TTCCAGAACT TACATCTGTG CCAGCCTCCC
CGAACCCCTG CACAGAGACA TGACCCCCTT CTCCCTGTGC CGTTGTCATG GTTGTTATTA
TTGTTGGAGT TTTGTTCGTT AAAATCTAAG CTTGGGCCCG AACAAAAACT CATCTCAGAA
GAGGATCTGA ATAGCGCCGT CGACCATCAT CATCATCATC ATTGAGTTTA AACGGTCTCC
AGCTTAAGTT TAAACCGCTG ATCAGCCTCG ACTGTGCCTT CTAGTTGCCA GCCATCTGTT
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GTTTGCCCCT CCCCCGTGCC TTCCTTGACC TGGAAGGTGC CACTCCCAC (SEQ ID NO:1).
2. A purified DNA molecule encoding rhesus melanocortin 3
receptor protein wherein said DNA molecule encodes a protein consisting
essentially
of the amino acid sequence
M N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G S G F C
E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V R N G N L H
S P M Y F F L C S L A A A D M L V S L S N S L E T I M I A V I N S D S
L T L E D Q F I Q H M D N I F D S M I C I S L V A S I C N L L A I A I
D R Y V T I F Y A L R Y H S I M T V R K A L T L I G V I W V C C G I C~
G V M F I I Y S E S K M V I V C L I T M F F A M V L L M G T L Y I H M~
F L F A R L H V Q R I A V L P P A G V V A P Q Q H S C M K G A V T I T~
I L L G V F I F C W A P F F L H L V L I I T C P T N P Y C I C Y T A E
F N T Y L V L I M C N S V I D P L I Y A F R S L E L R N T F K E I L C
G C N S M N L G , as set forth in a three-letter abbreviation in SEQ ID NO:2.
3. An expression vector for the expression of a rhesus MC-3R
protein in a recombinant host cell wherein said expression vector comprises a
DNA
molecule which encodes the amino acid sequence of claim 2.
4. An expression vector of claim 3 which is a eukaryotic
expression vector.
5. An expression vector of claim 3 which is a prokaryotic
expression vector.
6. A host cell which expresses a recombinant rhesus MC-3R
protein wherein said host cell contains the expression vector of claim 3.
7. A host cell which expresses a recombinant rhesus MC-3R
protein wherein said host cell contains the expression vector of claim 4.
8. A host cell which expresses a recombinant rhesus MC-3R
protein wherein said host cell contains the expression vector of claim 5.
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9. A host cell of claim 6 wherein said rhesus MC-3R protein is
overexpressed from said expression vector.
10. A host cell of claim 7 wherein said rhesus MC-3R protein is
overexpressed from said expression vector.
11. A host cell of claim 8 wherein said rhesus MC-3R protein is
overexpressed from said expression vector.
12. A subcellular membrane fraction obtained from the host cell of
claim 9 which contains recombinant rhesus MC-3R protein.
13. A subcellular membrane fraction obtained from the host cell of
claim 10 which contains recombinant rhesus MC-3R protein.
14. A subcellular membrane fraction obtained from the host cell of
claim 11 which contains recombinant rhesus MC-3R protein.
15. A purified DNA molecule which consists of the nucleotide
sequence
TCTATCTATC TATCTCTCCC TCTCTGGAGA AACTAAAGTC TAGACTGGAC AGCATCCACA
AGAGAAGCAC CTAGAAGGAG AATTTTCCCC AGCAGCTTGC TCAGGACCCT GCAGGAGCCG
CAGCTGGGAC TGGACCTGCT GTTAACCATG AACTCTTCCT GCTGCCTGTC TTCTGTTTCT
CCGATGCTGC CTAACCTCTC TGAGCACCCT GCAGCCCCTC CTGCCAGCAA CCGGAGCGGC
AGTGGGTTCT GTGAGCAGGT CTTCATCAAG CCGGAGGTCT TCCTGGCTCT GGGCATCGTC
AGTCTGATGG AAAACATCCT GGTGATCCTG GCTGTGGTCA GGAATGGCAA CCTGCACTCT
CCCATGTACT TCTTCCTGTG CAGCCTGGCT GCAGCCGACA TGCTGGTGAG CCTGTCCAAC
TCCCTGGAGA CCATCATGAT CGCCGTGATC AACAGCGACT CCCTGACCTT GGAGGACCAG
TTTATCCAGC ACATGGATAA TATCTTCGAC TCTATGATTT GCATCTCCCT GGTGGCCTCC
ATCTGCAACC TCCTGGCCAT TGCCATCGAC AGGTACGTCA CCATCTTCTA TGCCCTTCGG
TACCACAGCA TCATGACAGT GAGGAAAGCC CTCACCTTGA TCGGGGTCAT CTGGGTCTGC
TGCGGCATCT GCGGCGTGAT GTTCATCATC TACTCCGAGA GCAAGATGGT CATCGTGTGT
CTCATCACCA TGTTCTTCGC CATGGTGCTC CTCATGGGCA CCCTATATAT CCACATGTTC
CTCTTCGCCA GGCTCCACGT CCAGCGCATC GCAGTGCTGC CCCCTGCTGG CGTGGTGGCC
CCACAGCAGC ACTCCTGCAT GAAGGGGGCT GTCACCATCA CTATCCTGCT GGGTGTTTTC
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ATCTTCTGCT GGGCGCCTTT CTTCCTCCAC CTGGTCCTCA TCATCACCTG CCCCACCAAT
CCCTACTGCA TCTGCTACAC GGCCCATTTC AACACCTACC TGGTTCTCAT CATGTGCAAC
TCCGTCATCG ACCCCCTCAT CTACGCCTTC CGCAGCCTGG AGCTGCGCAA CACGTTCAAG
GAGATTCTCT GCGGCTGCAA CAGCATGAAC TTGGGCTAGG ATGCCCGTGG AGGTGTTCCA
CATCCAGCCA AGAGACAAAA ACAACGCTCA GACGGGACGT AAAAGGGTGT TAGGAGCTGG
AACTGTGCTT GGCTTCGTCT GTAAGCTCGT GGCCCTTTGC AGACGGGACA CGGCGTAGGA
TGGGCTGTCT GTGAGGATCT GTGTGTGGGT AAGTCAGTTT GATCTAGCAC ATAGCCTGGA
AGAATCAGGC AAAGCAGCCC TGAGTGTCAT CTGTGTTCAT TGCTAGGCAC CCAGGGTTTG
TGGCCCCTGC CTGCTTATTG GCTTTGTACC AGTAACTGTG CTTCAAGCCA ACCAGACCGG
AGGGCTCTCG TGAGCAGAAA GAGTGCTTAG ACTTCCGGCA AGCATCCTGG CTCACAGCGG
CCACCTCCTG ACCACTACCG GGAGAGCTTT GCACATATTC TGTGGGAGAT TGAGTGAAGC
CCTGAAAACA ATGTGATATT TGCTGCTCCC TTCCAGAACT TACATCTGTG CCAGCCTCCC
CGAACCCCTG CACAGAGACA TGACCCCCTT CTCCCTGTGC CGTTGTCATG GTTGTTATTA
TTGTTGGAGT TTTGTTCGTT AAAATCTAAG CTTGGGCCCG AACAAAAACT CATCTCAGAA
GAGGATCTGA ATAGCGCCGT CGACCATCAT CATCATCATC ATTGAGTTTA AACGGTCTCC
AGCTTAAGTT TAAACCGCTG ATCAGCCTCG ACTGTGCCTT CTAGTTGCCA GCCATCTGTT
GTTTGCCCCT CCCCCGTGCC TTCCTTGACC TGGAAGGTGC CACTCCCAC (SEQ ID NO:1).
16. A purified rhesus MC-3R protein which consists of the amino

acid sequence
M N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G S G F C
E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V R N G N L H
S P M Y F F L C S L A A A D M L V S L S N S L E T I M I A V I N S D S
L T L E D Q F I Q H M D N I F D S M I C I S L V A S I C N L L A I A I
D R Y V T I F Y A L R Y H S I M T V R K A L T L I G V I W V C C G I C
G V M F I I Y S E S K M V I V C L I T M F F A M V L L M G T L Y I H M
F L F A R L H V Q R I A V L P P A G V V A P Q Q H S C M K G A V T I T
I L L G V F I F C W A P F F L H L V L I I T C P T N P Y C I C Y T A H
F N T Y L V L I M C N S V I D P L I Y A F R S L E L R N T F K E I L C
G C N S M N L G , as set forth in three letter abbreviation in SEQ ID NO:2.
17. The purified DNA molecule of claim 15 which consists of a
nucleotide sequence from nucleotide 148 to nucleotide 1119 of SEQ ID NO:1.
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18. An expression vector for the expression of a rhesus MC-3R
protein in a recombinant host cell wherein said expression vector comprises a
DNA
molecule of claim 17.
19. An expression vector of claim 18 which is a eukaryotic
expression vector.
20. An expression vector of claim 18 which is a prokaryotic
expression vector.
21. A host cell which expresses a recombinant rhesus MC-3R
protein wherein said host cell contains the expression vector of claim 19.
22. A host cell which expresses a recombinant rhesus MC-3R
protein wherein said host cell contains the expression vector of claim 20.
23. A host cell which expresses a recombinant rhesus MC-3R
protein wherein said host cell contains the expression vector of claim 21.
24. A subcellular membrane fraction obtained from the host cell of
claim 22 which contains recombinant rhesus MC-3R protein.
25. A subcellular membrane fraction obtained from the host cell of
claim 23 which contains recombinant rhesus MC-3R protein.
26. A process for the expression of a rhesus MC-3R protein in a
recombinant host cell, comprising:
(a) transfecting the expression vector of claim 3 into a suitable host
cell; and,
(b) culturing the host cells of step (a) under conditions which allow
expression of the rhesus MC-3R protein from the expression vector.
27. A purified rhesus melanocortin 3 receptor protein which
comprises the amino acid sequence as follows:
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M N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G S G F C
E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V R N G N L H
S P M Y F F L C S L A A A D M L V S L S N S L E T I M I A V I N S D S
L T L E D Q F I Q H M D N I F D S M I C I S L V A S I C N L L A I A I
D R Y V T I F Y A L R Y H S I M T V R K A L T L I G V I W V C C G I C
G V M F I I Y S E S K M V I V C L I T M F F A M V L L M G T L Y I H M
F L F A R L H V Q R I A V L P P A G V V A P Q Q H S C M K G A V T I T
I L L G V F I F C W A P F F L H L V L I I T C P T N P Y C I C Y T A H
F N T Y L V L I M C N S V I D P L I Y A F R S L E L R N T F K E I L C
G C N S M N L G , as set forth in a three-letter abbreviation in SEQ ID NO:2.
28. A purified rhesus melanocortin 3 receptor protein which
consists of the amino acid sequence as set forth in SEQ ID NO:2.
29. A method for determining whether a substance is capable of
binding to rhMC-3R comprising:
(a) providing test cells by transfecting cells with an expression
vector that directs the expression of rhMC-3R in the cells;
(b) exposing the test cells to the substance;
(c) measuring the amount of binding of the substance to rhMC-3R;
(d) comparing the amount of binding of the substance to rhMC-3R
in the test cells with the amount of binding of the substance to control cells
that have
not been transfected with rhMC-3R.
30. A method for determining whether a substance is capable of
activating rhMC-3R comprising:
(a) providing test cells by transfecting cells with an expression
vector that directs the expression of rhMC-3R in the cells;
(b) exposing the test cells to the substance;
(c) measuring the amount of accumulated intracellular cAMP;
(d) comparing the amount of cAMP in the test cells in response to
the substance with the amount of cAMP in test cells that have not been exposed
to the
substance.
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31. A method of identifying a substance which modulates MC-3R
receptor activity, comprising:
(a) combining a test substance in the presence and absence of a MC-3R
receptor protein wherein said MC-3R receptor protein comprises the amino acid
sequence as set forth in SEQ ID NO:2; and,
(b) measuring and comparing the effect of the test substance in the
presence and absence of the MC-3R receptor protein.
32. A method for determining whether a substance is a potential
agonist or antagonist of MC-3R comprising:
(a) transfecting or transforming cells with an expression vector of
claim 3 that directs expression of MC-3R in the cells, resulting in test
cells;
(b) allowing the test cells to grow for a time sufficient to allow
MC-3R to be expressed;
(c) exposing the cells to a labeled ligand of MC-3R in the presence
and in the absence of the substance;
(d) measuring the binding of the labeled ligand to MC-3R; where
if the amount of binding of the labeled ligand is less in the presence of the
substance
than in the absence of the substance, then the substance is a potential
agonist or
antagonist of MC-3R.
33. A method for determining whether a substance is capable of
binding to MC-3R comprising:
(a) transfecting or transforming cells with an expression vector of
claim 3 that directs the expression of MC-3R in the cells, resulting in test
cells;
(b) exposing the test cells to the substance;
(c) measuring the amount of binding of the substance to MC-3R;
(d) comparing the amount of binding of the substance to MC-3R in
the test cells with the amount of binding of the substance to control cells
that have not
been transfected with MC-3R;
wherein if the amount of binding of the substance is greater in the test
cells as compared to the control cells, the substance is capable of binding to
MC-3R.
34. A method for determining whether a substance is capable of
binding to MC-3R comprising:
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(a) transfecting or transforming cells with an expression vector of
claim 3 that directs the expression of MC-3R in the cells, resulting in test
cells;
(b) preparing membranes containing MC-3R from the test cells and
exposing the membranes to a ligand of MC-3R under conditions such that the
ligand
binds to the MC-3R in the membranes;
(c) subsequently or concurrently to step (b), exposing the
membranes from the test cells to a substance;
(d) measuring the amount of binding of the ligand to the MC-3R in
the membranes in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to MC-3R in the
membranes in the presence and the absence of the substance where a decrease in
the
amount of binding of the ligand to MC-3R in the membranes in the presence of
the
substance indicates that the substance is capable of binding to MC-3R.
35. A method for determining whether a substance is capable of
binding to MC-3R comprising:
(a) transfecting or transforming cells with an expression vector of
claim 3 that directs the expression of MC-3R in the cells, resulting in test
cells;
(b) preparing membranes containing MC-3R from the test cells and
exposing the membranes from the test cells to the substance;
(c) measuring the amount of binding of the substance to the MC-
3R in the membranes from the test cells;
(d) comparing the amount of binding of the substance to MC-3R in
the membranes from the test cells with the amount of binding of the substance
to
membranes from control cells that have not been transfected with MC-3R, where
if
the amount of binding of the substance to MC-3R in the membranes from the test
cells is greater than the amount of binding of the substance to the membranes
from
the control cells, then the substance is capable of binding to MC-3R.
36. A method of identifying agonists of MC-3R comprising:
(a) transfecting or transforming cells with a first expression vector
of claim 3 which directs expression of MC-3R and a second expression vector
which
directs the expression of a promiscuous G-protein, resulting in test cells;
(b) exposing the test cells to a substance that is a suspected agonist
of MC-3R;
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(c) measuring the level of inositol phosphates in the cells; where
an increase in the level of inositol phosphates in the cells as compared to
the level of
inositol phosphates in the cells in the absence of the suspected agonist
indicates that
the substance is an agonist of MC-3R.
37. A method of identifying antagonists of MC-3R comprising:
(a) transfecting or transforming cells with a first expression vector
of claim 3 which directs expression of MC-3R and a second expression vector
which
directs the expression of a promiscuous G-protein, resulting in test cells;
(b) exposing the test cells to a substance that is an agonist of MC-
3R;
(c) subsequently or concurrently to step (b), exposing the test cells
to a substance that is a suspected antagonist of MC-3R;
(d) measuring the level of inositol phosphates in the cells; where a
decrease in the level of inositol phosphates in the cells in the presence of
the
suspected antagonist as compared to the level of inositol phosphates in the
cells in the
absence of the suspected antagonist indicates that the substance is an
antagonist of
MC-3R.
38. A method of identifying antagonists of MC-3R as recited in
claim 37 wherein the first and second expression vectors of step (a) are
replaced with
a single expression vector which expresses a chimeric MC-3R protein fused at
its C-
terminus to a promiscuous G-protein.
39. An antibody that binds specifically to MC-3R protein wherein
the MC-3R receptor protein comprises the amino acid sequence as set forth in
SEQ
1D NO:2.
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Description

CA 02349950 2001-05-08
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TITLE OF THE INVENTION
DNA MOLECULES ENCODING THE MELANOCORTIN 3 RECEPTOR
PROTEIN FROM RHESUS MONKEY
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to U.S. Serial No. 60/107,725,
filed November 9, 1998, which is hereby incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to rhesus monkey (Macaca mulatta)
DNA molecules encoding the melanocortin-3 receptor protein belonging to the
rhodopsin sub-family of G-protein coupled receptors, recombinant vectors
comprising
DNA molecules encoding rhesus MC-3R, recombinant host cells which contain a
recombinant vector encoding rhesus MC-3R, the rhesus MC-3R protein encoded by
the DNA molecule, and methods of identifying selective agonists and
antagonists of
rhesus MC-3R.
BACKGROUND OF THE INVENTION
Melanocortin receptors belong to the rhodopsin sub-family of G-
protein coupled receptors (GPCR's). Five different subtypes are known. These
melanocortin receptors bind and are activated by peptides such as a-, (3, or y-
melanocyte stimulating hormones (a-, ~i-, y-MSH) derived from the pro-
opiomelanocortin (POMC) gene. A wide range of physiological functions are
believed to be mediated by meianocortin peptides and their receptors.
U.S. Patent No. 5,532,347, issued on July 2, 1996, to Cone and
Mountjoy discloses human and mouse DNA molecules which encode MC-1R (also
known in the art as a-MSH-R). The expressed human protein contains 317 amino
acids.
U.S. Patent No. 5,280,112 (issued January 18, 1994) and U.S. Patent
No. 5,554,729 (issued September 10, 1996), both to Cone and Mountjoy, disclose
human and mouse DNA molecules which encode MC-2R (also known in the art as
ACTH-R). The human MC-2R protein contains 297 amino acids.
Mountjoy, et al. (1992, Science 257: 1248-1251) describe DNA
molecules and the concomitant protein for human MC-1R and human MC-2R.

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Chhaj lani, et al. ( 1992, FEBS Letters 309: 417-420) also disclose a
human DNA molecule comprising an open reading frame which encodes human
MC1-R .
Roselli-Rehfuss, et al, (1993, Proc. Natl. Acad. Sci 90: 8856-8860)
disclose a cDNA clone encoding rat MC-3R cDNA.
U. S. Patent No. 5,622,860 (issued April 22, 1997) and U.S. Patent No.
5,703,220 (issued December 30, 1997) to Yamada and Gantz, disclose DNA
molecules which encode human MC-3R and human MC-4R, respectively (see also
Gantz, et al., 1993, J. Biol. Chem. 268( 11 ): 8246-8250).
A DNA molecule encoding human MC-SR was also disclosed by
Mountjoy, et al. (1994, Mol. Endocrin. 8: 1298-1308).
Chhajlani, et al. (1993, Biochem. Biophys. Res. Comm., 195(2): 866-
873) disclose a DNA molecule which the authors state encodes MC-SR. This clone
was initially designated MC2.
Fathi, et al. (1995, Neurochemical Research 20(1):107-113) also
disclose a DNA molecule thought to encode human MC-SR. There are several
sequence discrepancies when compared to the DNA molecule disclosed by
Chhajlani,
et al., id.
Griffon, et al. (1994, Biochem. Biophys. Res. Comm., 200(2): 1007-
1014) disclose DNA clones from human and rat which encode MC-SR. The human
DNA sequence agrees with the human DNA sequence disclosed in Fathi et al. id.
Gantz, et al. (1994, Biochem. Biophys. Res. Comm., 200(3): 11214-
11220; see also U.S. Patent No. 5,710,265, issued January 20, 1998 to Yamada
and
Gantz) and Labbe, et al. (1994, Biochemistry 33: 4543-4549) disclose DNA
clones
from mouse which encode MC-SR.
Barrett, et al. (1994, J. Mol. Endocrin. 12: 203-213) disclose DNA
clones from sheep which encode MC-SR.
In rodents, MC-4R has been implicated as a key regulator of feeding
behavior which regulates body weight through studies with peptide agonists and
antagonists (Fan et al., 1997, Nature 385: 165-168) and with a MC-4R knock-out
mouse (Huszar et al., 1997, Cell 88: 131-141).
Compounds that bind to such receptors were previously identified by
binding to human and/or rodent receptors and evaluated for their efficacy in
rodents.
However, the neuroendocrine process can differ between rodents and man. It is
also
expected that some compounds exhibit different binding affinities for
different
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species homologues of the same receptor (Fong et al., 1992, J.Biol. Chem.
267:25666-
25671; Hartig et al., 1992, TIPS 13:152-159).
Before compounds can be selected as a drug candidate it is first
evaluated for a physiological effect in rodents and then in the rhesus
primate. It is
often that one compound may be effective in one animal species but not in
another.
Previously, it has been impossible to determine if the failure was due to an
altered
melanocortin pathway in different species, or due to a compound's having a
lower
affinity for one particular species. Past protocols required the use of a
rhesus brain
membrane to determine the in vitro biochemical activity of compounds, if such
protocol could be successfully employed.
It is desirable to correlate in vivo data with in vitro biochemical
activity of compounds.
It is also desirable to first select compounds that are active for the
rhesus receptor in vitro.
It is also desirable to identify compounds which can determine the
relevance of receptor targets in rhesus and allow selection of novel drugs to
treat
obesity.
It is further desirable to discover new drugs which effect
pathophysiological processes by modulating the effects in rhesus to identify
melanocortin active process in primates, followed by human clinical trials.
The present invention addresses and meets these needs by disclosing
an isolated nucleic acid fragment which expresses a form of rhesus MC-3R,
recombinant vectors which house this nucleic acid fragment, recombinant host
cells
which expresses rhesus MC-3R and/or a biologically active equivalent, and
pharmacological properties of this rhesus MC-3R protein.
SUMMARY OF THE INVENTION
The present invention relates to an isolated nucleic acid molecule
(polynucleotide) which encodes a novel rhesus monkey (Macaca mulatta)
melanocortin-3 receptor (rhMC-3R). The nucleic acid molecules of the present
invention are substantially free from other nucleic acids.
The present invention relates to an isolated nucleic acid molecule
(polynucleotide) which encodes mRNA which expresses a novel rhesus MC-3R, this
DNA molecule comprising the nucleotide sequence disclosed herein as SEQ 113
NO:1.
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The present invention also relates to biologically active fragments or
mutants of SEQ 1D NO:1 which encodes mRNA expressing a novel rhesus MC-3R.
Any such biologically active fragment and/or mutant will encode either a
protein or
protein fragment which at least substantially mimics the pharmacological
properties
S of a wild-type MC-3R protein, including but not limited to the rhesus MC-3R
receptor
protein as set forth in SEQ ID N0:2. 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 MC-
3R
function.
A preferred aspect of this portion of the present invention is disclosed
in Figure 1 A - 1 B, a rhesus cDNA molecule encoding a novel MC-3R (SEQ 1D
NO:1 ).
The isolated nucleic acid molecules 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.
The present invention also relates to subcellular membrane fractions of
the recombinant host cells (both prokaryotic and eukaryotic as well as both
stably and
transiently transformed cells) which contain the proteins encoded by the
nucleic acids
of the present invention. These subcellular membrane fractions will comprise
either
wild-type or mutant forms of rhesus melanocortin-3 receptor proteins at levels
substantially above endogenous levels and hence will be useful in various
assays
described throughout this specification.
The present invention also relates to a substantially purified form of
the rhesus melanocortin-3 receptor protein, which comprises the amino acid
sequence
disclosed in Figure 2 and set forth as SEQ m N0:2. A preferred aspect of the
present
invention is disclosed in Figure 2 and is set forth as SEQ m N0:2, the amino
acid
sequence of the novel rhesus melanocortin-3 receptor protein.
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The present invention also relates to biologically active fragments
and/or mutants of the rhesus melanocortin-3 receptor protein comprising the
amino
acid sequence 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
pmtein
fragments of diagnostic, therapeutic or prophylactic use and would be useful
for
screening for agonists and/or antagonists for MC-3R function.
The present invention also relates to assays to screen or select for
various modulators of MC-3R activity, methods of expressing the MC-3R protein
and
biological equivalents disclosed herein, recombinant host cells which comprise
DNA
constructs which express these receptor proteins, and compounds identified
through
these assays which act as agonists or antagonists of MC-3R activity.
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 vertebrate MC-3R activity. A preferred
aspect
of this portion of the invention includes, but is not limited to, glutathione
S-
transferase (GST)-MC-3R fusion constructs which include, but are not limited
to,
either the intracellular domain of rhesus MC-3R as an in-frame fusion at the
carboxy
terminus of the GST gene, or the extracellular and transmembrane ligand
binding
domain of MC-3R fused to the amino terminus of GST, or the extracellular and
transmembrane domain of MC-3R fused to an immunoglobulin gene by methods
known to one of ordinary skill in the art. Soluble recombinant GST-MC-3R
fusion
proteins may be expressed in various expression systems, including Spodoptera
frugiperda (Sfl1) insect cells (Invitrogen) using a baculovirus expression
vector
(pAcG2T, Pharmingen).
Therefore, the present invention also relates to assays to screen or
select for various modulators of MC-3R activity, methods of expressing the
rhesus
MC-3R protein and biological equivalents disclosed herein, recombinant host
cells
which comprise DNA constructs which express these receptor proteins, and
compounds identified through these assays which act as agonists or antagonists
of
MC-3R activity.
It is an object of the present invention to provide an isolated nucleic
acid molecule which encodes a novel form of rhesus MC-3R, or rhesus fragments
MC-3R fragments, mutants or derivatives of SEQ 1D N0:2. Any such
polynucleotide
includes but is not necessarily limited to nucleotide substitutions,
deletions, additions,
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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 vertebrate MC-3R function.
It is a further object of the present invention to provide the rhesus MC-
3R 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
rhesus MC-3R or a biological equivalent thereof.
It is an object of the present invention to provide a substantially
purified form of the rhesus MC-3R protein, 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 the rhesus MC-3R protein, such as set forth in SEQ
m
N0:2, including but not necessarily limited to amino acid substitutions,
deletions,
additions, amino terminal truncations and caxboxy-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 MC-
3R-based assays to select for modulators of this receptor protein. These
assays are
preferably cell based assays whereby a DNA molecule encoding MC-3R is
transfected or transformed into a host cell, this recombinant host cell is
allowed to
grow for a time sufficient to express MC-3R prior to use in various assays
described
herein.
It is a further object to provide for membrane preparations from host
cells transfected or transformed with a DNA molecule encoding MC-3R for use in
assays to select for modulators of MC-3R activity.
It is also an object of the present invention to provide for MC-3R-
based in-frame fusion constructions, methods of expressing these fusion
constructs,
biological equivalents disclosed herein, related assays, recombinant cells
expressing
these constructs, and agonistic and/or antagonistic compounds identified
through the
use of the nucleic acid encoding vertebrate MC-3R protein as well as the
expressed
protein.
It is also an object of the present invention to provide for MC-3R-
based assays to select for modulators of this receptor protein. These assays
are
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preferably cell based assays whereby a DNA molecule encoding rhMC-3R is
transfected or transformed into a host cell, this recombinant host cell is
allowed to
grow for a time sufficient to express MC-3R prior to use in various assays
described
herein.
It is a further object to provide for membrane preparations from host
cells transfected or transformed with a DNA molecule encoding rhMC-3R for use
in
assays to select for modulators of MC-3R activity.
As used herein, "rh" or refers to --rhesus--.
As used herein, "MC-3R" refers to --melanocortin 3 receptor--.
As used herein, "GPCR" refers to --G-protein coupled receptor--.
Whenever used herein, the term "mammalian host" will refer to any
mammal, including a human being.
BRIEF DECRIPTION OF THE FIGURES
Figure 1 A and Figure 1 B show the nucleotide sequence which encodes
rhesus MC-3R, as set forth in SEQ ID NO:1.
Figure 2 shows the one-letter designation of the amino acid sequence
of rhesus MC-3R; as also set forth in SEQ ID N0:2 as a three letter
designation.
Figure 3A and Figure 3B show characterization of rhesus MC-3R.
Figure 3A shows inhibition of (125]~p_a_MSH binding by NDP-a-MSH, y2-MSH
or Shu-9119. Figure 3B shows stimulation of cAMP synthesis in response to NDP-
a-
MSH or y2-MSH. Each curve represents a typical experiment with duplicate
measurements. Average data from multiple experiments are listed in Table 2.
Figure 4 shows the comparison of binding affinity (ICSO) and activation
potency (ECSO) for the MC-3R from 2 species. Abbreviations are as follows: A,
human
ACTH(1-24); a, a-MSH; y, y2-MSH; M, MT-II; N, NDP-a-MSH.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an isolated nucleic acid molecule
(polynucleotide) which encodes a novel rhesus monkey (Macaca mulatta)
melanocortin-3 receptor (rhMC-3R). The nucleic acid molecules of the present
invention are substantially free from other nucleic acids. For most cloning
purposes,
DNA is a preferred nucleic acid.
The present invention relates to an isolated nucleic acid molecule
(polynucleotide) which encodes mRNA which expresses a novel rhesus MC-3R, this

CA 02349950 2001-05-08
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DNA molecule comprising the nucleotide sequence disclosed herein as SEQ )17
NO:1, shown herein as follows:
TCTATCTATC TATCTCTCCC TCTCTGGAGA AACTAAAGTC TAGACTGGAC AGCATCCACA
AGAGAAGCAC CTAGAAGGAG AATTTTCCCC AGCAGCTTGC TCAGGACCCT GCAGGAGCCG
S CAGCTGGGAC TGGACCTGCT GTTAACCATG AACTCTTCCT GCTGCCTGTC TTCTGTTTCT
CCGATGCTGC CTAACCTCTC TGAGCACCCT GCAGCCCCTC CTGCCAGCAA CCGGAGCGGC
AGTGGGTTCT GTGAGCAGGT CTTCATCAAG CCGGAGGTCT TCCTGGCTCT GGGCATCGTC
AGTCTGATGG AAAACATCCT GGTGATCCTG GCTGTGGTCA GGAATGGCAA CCTGCACTCT
CCCATGTACT TCTTCCTGTG CAGCCTGGCT GCAGCCGACA TGCTGGTGAG CCTGTCCAAC
lO TCCCTGGAGA CCATCATGAT CGCCGTGATC AACAGCGACT CCCTGACCTT GGAGGACCAG
TTTATCCAGC ACATGGATAA TATCTTCGAC TCTATGATTT GCATCTCCCT GGTGGCCTCC
ATCTGCAACC TCCTGGCCAT TGCCATCGAC AGGTACGTCA CCATCTTCTA TGCCCTTCGG
TACCACAGCA TCATGACAGT GAGGAAAGCC CTCACCTTGA TCGGGGTCAT CTGGGTCTGC
TGCGGCATCT GCGGCGTGAT GTTCATCATC TACTCCGAGA GCAAGATGGT CATCGTGTGT
IS CTCATCACCA TGTTCTTCGC CATGGTGCTC CTCATGGGCA CCCTATATAT CCACATGTTC
CTCTTCGCCA GGCTCCACGT CCAGCGCATC GCAGTGCTGC CCCCTGCTGG CGTGGTGGCC
CCACAGCAGC ACTCCTGCAT GAAGGGGGCT GTCACCATCA CTATCCTGCT GGGTGTTTTC
ATCTTCTGCT GGGCGCCTTT CTTCCTCCAC CTGGTCCTCA TCATCACCTG CCCCACCAAT
CCCTACTGCA TCTGCTACAC GGCCCATTTC AACACCTACC TGGTTCTCAT CATGTGCAAC
ZO TCCGTCATCG ACCCCCTCAT CTACGCCTTC CGCAGCCTGG AGCTGCGCAA CACGTTCAAG
GAGATTCTCT GCGGCTGCAA CAGCATGAAC TTGGGCTAGG ATGCCCGTGG AGGTGTTCCA
CATCCAGCCA AGAGACAAAA ACAACGCTCA GACGGGACGT AAAAGGGTGT TAGGAGCTGG
AACTGTGCTT GGCTTCGTCT GTAAGCTCGT GGCCCTTTGC AGACGGGACA CGGCGTAGGA
TGGGCTGTCT GTGAGGATCT GTGTGTGGGT AAGTCAGTTT GATCTAGCAC ATAGCCTGGA
ZS AGAATCAGGC AAAGCAGCCC TGAGTGTCAT CTGTGTTCAT TGCTAGGCAC CCAGGGTTTG
TGGCCCCTGC CTGCTTATTG GCTTTGTACC AGTAACTGTG CTTCAAGCCA ACCAGACCGG
AGGGCTCTCG TGAGCAGAAA GAGTGCTTAG ACTTCCGGCA AGCATCCTGG CTCACAGCGG
CCACCTCCTG ACCACTACCG GGAGAGCTTT GCACATATTC TGTGGGAGAT TGAGTGAAGC
CCTGAAAACA ATGTGATATT TGCTGCTCCC TTCCAGAACT TACATCTGTG CCAGCCTCCC
3O CGAACCCCTG CACAGAGACA TGACCCCCTT CTCCCTGTGC CGTTGTCATG GTTGTTATTA
TTGTTGGAGT TTTGTTCGTT AAAATCTAAG CTTGGGCCCG AACAAAAACT CATCTCAGAA
GAGGATCTGA ATAGCGCCGT CGACCATCAT CATCATCATC ATTGAGTTTA AACGGTCTCC
AGCTTAAGTT TAAACCGCTG ATCAGCCTCG ACTGTGCCTT CTAGTTGCCA GCCATCTGTT
GTTTGCCCCT CCCCCGTGCC TTCCTTGACC TGGAAGGTGC CACTCCCAC~SEQ 1D NO:1~.
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The above-exemplified isolated DNA molecule, shown in Figure lA-
1B and set forth as SEQ ID NO:1, contains 1909 nucleotides. This DNA molecule
contains an open reading frame from nucleotide 148 to nucleotide 1116, with a
"TAG" termination codon at nucleotides 1117-1119. This open reading frame
encodes a rhesus MC-3R protein 323 amino acids in length, as shown in Figure 2
and
as set forth in SEQ ID N0:2.
The present invention also relates to biologically active fragments or
mutants of SEQ ID NO:I which encodes mRNA expressing a novel rhesus MC-3R.
Any such biologically active fragment and/or mutant will encode either a
protein or
protein fragment which at least substantially mimics the pharmacological
properties
of a wild-type MC-3R protein, including but not limited to the rhesus MC-3R
receptor
protein as set forth in SEQ ID N0:2. 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 MC-
3R
fimction.
A preferred aspect of this portion of the present invention is disclosed
in Figure 1 A - 1 B, a rhesus cDNA molecule encoding a novel MC-3R (SEQ ID
NO:1 ).
The isolated nucleic acid molecules of the present invention may
include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and
complementary DNA (cDNA), which may be single (coding or noncoding strand) or
double stranded, as well as synthetic DNA, such as a synthesized, single
stranded
polynucleotide. The isolated nucleic acid molecule of the present invention
may also
include a ribonucleic acid molecule (RNA).
It is known that there is a substantial amount of redundancy in the
various codons which code for specific amino acids. Therefore, this invention
is
also directed to those DNA sequences encode RNA comprising alternative codons
which code for the eventual translation of the identical amino acid, as shown
below:
A=Ala=Aianine: codons GCA, GCC, GCG, GCU
C=Cys=Cysteine: codons UGC, UGU
D=Asp=Aspartic acid: codons GAC, GAU
E=GIu=Glutamic acid: codons GAA, GAG
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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
S 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 defmed as a degenerate variation. Also included within the scope of
this
invention are mutations either in the DNA sequence or the translated protein
which do not substantially alter the ultimate physical properties of the
expressed
protein. For example, substitution of valine for leucine, arginine for lysine,
or
asparagine for glutamine may not cause a change in functionality of the
polypeptide.
It is known that DNA sequences coding for a peptide may be altered so
as to code for a peptide having properties that are different than those of
the naturally
occurnng 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 affinity of an enzyme for a substrate or a
receptor for a
ligand.
Any of a variety of procedures may be used to clone rhesus MC-3R.
These methods include, but are not limited to, ( 1 ) a RACE PCR cloning
technique
(Frohman, et al., 1988, Proc. Natl. Acad Sci. USA 85: 8998-9002). 5' and/or 3'
RACE may be performed to generate a full-length cDNA sequence. This strategy
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involves using gene-specific oligonucleotide primers for PCR amplification of
rhesus
MC-3R 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 rhesus MC-3R cDNA following the
construction
of a rhesus MC-3R-containing cDNA library in an appropriate expression vector
system; (3) screening a rhesus MC-3R-containing cDNA library constructed in a
bacteriophage or plasrnid shuttle vector with a labeled degenerate
oligonucleotide
probe designed from the amino acid sequence of the rhesus MC-3R protein; (4)
screening a rhesus MC-3R-containing cDNA library constructed in a
bacteriophage
or plasmid shuttle vector with a partial cDNA encoding the rhesus MC-3R
protein.
This partial cDNA is obtained by the specific PCR amplification of rhesus MC-
3R
DNA fragments through the design of degenerate oligonucleotide primers from
the
amino acid sequence known for other kinases which are related to the rhesus MC-
3R
protein; (5) screening a rhesus MC-3R-containing cDNA library constructed in a
bacteriophage or plasmid shuttle vector with a partial cDNA or oligonucleotide
with
homology to a mammalian MC-3R protein. This strategy may also involve using
gene-specific oligonucleotide primers for PCR amplification of rhesus MC-3R
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 RACE techniques, or a portion of the coding
region may be generated by these same known RACE techniques to generate and
isolate a portion of the coding region to use as a probe to screen one of
numerous
types of cDNA and/or genomic libraries in order to isolate a full-length
version of the
nucleotide sequence encoding rhesus MC-3R.
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 rhesus MC-3R-encoding DNA or a rhesus MC-3R
homologue. Other types of libraries include, but are not limited to, cDNA
libraries
derived from other rhesus cells.
It is readily apparent to those skilled in the art that suitable cDNA
libraries may be prepared from cells or cell lines which have MC-3R activity.
The
selection of cells or cell lines for use in preparing a cDNA library to
isolate a cDNA
encoding rhesus MC-3R may be done by first measuring cell-associated MC-3R
activity using any known assay available for such a purpose.
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Preparation of cDNA libraries can be performed by standard
techniques well known in the art. WeII known cDNA library construction
techniques
can be found for example, in Sambrook et al., 1989, Molecular 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
rhesus MC-3R may also be isolated from a suitable genomic DNA library.
Construction of genomic DNA libraries can be performed by standard techniques
I O well known in the art. Well known genomic DNA library construction
techniques
can be found in Sambrook, et al., supra.
In order to clone the rhesus MC-3R gene by one of the preferred
methods, the amino acid sequence or DNA sequence of rhesus MC-3R or a
homologous protein may be necessary. To accomplish this, the MC-3R 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 rhesus MC-3R 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 rhesus
MC-3R
sequence but others in the set will be capable of hybridizing to rhesus MC-3R
DNA
even in the presence of DNA oligonucleotides with mismatches. The mismatched
DNA oligonucleotides may still sufficiently hybridize to the rhesus MC-3R DNA
to
permit identification and isolation of rhesus MC-3R 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 rhesus MC-3R, or to isolate a portion of the
nucleotide
sequence coding for rhesus MC-3R for use as a probe to screen one or more cDNA-
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or genomic-based libraries to isolate a full-length sequence encoding rhesus
MC-3R
or rhesus MC-3R-like proteins.
Included in the present invention are DNA sequences that hybridize to
SEQ 117 NO:1 under stringent conditions. By way of example, and not
limitation, a
procedure using conditions of high stringency is as follows: Prehybridization
of filters
containing DNA is carried out for 2 hours to overnight at 65°C in
buffer composed of
6X SSC, SX Denhardt's solution, and 100 ltg/ml denatured salmon sperm DNA.
Filters are hybridized for 12 to 48 hrs at 65°C in prehybridization
mixture containing
100 pg/ml denatured salmon sperm DNA and 5-20 X 106 cpm of 32P-labeled probe.
Washing of filters is done at 37°C for 1 hr in a solution containing 2X
SSC, 0.1%
SDS. This is followed by a wash in O.1X SSC, 0.1% SDS at 50°C for 45
min. before
autoradiography. Other procedures using conditions of high stringency would
include
either a hybridization step carried out in SXSSC, SX Denhardt's solution, 50%
formamide at 42°C for 12 to 48 hours or a washing step carried out in
0.2X SSPE,
1 S 0.2% SDS at 65°C for 30 to 60 minutes.
Reagents mentioned in the foregoing procedures for carrying out high
stringency hybridization are well known in the art. Details of the composition
of
these reagents can be found in, e.g., Sambrook et al., 1989, Molecular
Cloning: A
Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New
York.
In addition to the foregoing, other conditions of high stringency which may be
used
are well known in the art.
Melanocortin receptors belong to the rhodopsin sub-family of GPCR's.
However, several features in the rhMC-3R are shared with all other receptors
and are
absent in most other GPCR's, including the EN motif in TM1, the lack of Cys in
the
loop between TM2 and TM3 or between TM4 and TMS, the MxxxxxxxY motif in
TMS, and the DPxxY motif in TM7. Since all melanocortin receptors lack Cys
residues in the extracellular loops that are present in other members of the
rhodopsin
sub-family, interhelical disulfide bond (e.g., between the Cys residues near
the top of
TM3 and TMS) may play the same function as interloop disulfide bond in most
other
GPCR's.
The present invention also relates to a substantially purified form of
the rhesus melanocortin-3 receptor protein, which comprises the amino acid
sequence
is disclosed in Figure 2 and set forth as SEQ ID N0:2.
The present invention also relates to biologically active fragments
and/or mutants of the rhesus melanocortin-3 receptor protein comprising the
amino
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acid sequence set forth as SEQ )D 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
S screening for agonists and/or antagonists for MC-3R function.
A preferred aspect of the present invention is disclosed in Figure 2 and
is set forth as SEQ 1D N0:2, and as herein set forth as follows:
M N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G S G F C
E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V R N G N L H
lO S P M Y F F L C S L A A A D M L V S L S N S L E T I M I A V I N S D S
L T L E D Q F I Q H M D N I F D S M I C I S L V A S I C N L L A I A I
D R Y V T I F Y A L R Y H S I M T V R K A L T L I G V I W V C C G I C
G V M F I I Y S E S K M V I V C L I T M F F A M V L L M G T L Y I H M
F L F A R L H V Q R I A V L P P A G V V A P Q Q H S C M K G A V T I T
IS I L L G V F I F C W A P F F L H L V L I I T C P T N P Y C I C Y T A H
F N T Y L V L I M C N S V I D P L I Y A F R S L E L R N T F K E I L C
G C N S M N L G (SEQ ID N0:2), comprises the amino acid sequence of wild type
rhesus melanocortin-3 receptor protein.
Following expression of MC-3R in a host cell, MC-3R protein may be
20 recovered to provide MC-3R protein in active form. Several MC-3R protein
purification procedures are available and suitable for use. Recombinant MC-3R
protein may be purified from cell lysates and extracts by various combinations
of, or
individual application of salt fractionation, ion exchange chromatography,
size
exclusion chromatography, hydroxylapatite adsorption chromatography and
2S hydrophobic interaction chromatography. In addition, recombinant MC-3R
protein
can be separated from other cellular proteins by use of an immunoaffinity
column
made with monoclonal or polyclonal antibodies specific for full-length MC-3R
pmtein, or polypeptide fragments of MC-3R protein.
The present invention also relates to isolated nucleic acid molecules
30 which are fusion constructions expressing fusion proteins useful in assays
to identify
compounds which modulate wild-type vertebrate MC-3R activity. A preferred
aspect
of this portion of the invention includes, but is not limited to, glutathione
S-
transferase (GST)-MC-3R fusion constructs which include, but are not limited
to,
either the intracellular domain of rhesus MC-3R as an in-frame fusion at the
carboxy
35 terminus of the GST gene or the extracellular and transmembrane ligand
binding
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domain of MC3R fused to an GST or immunoglobulin gene by methods known to one
of ordinary skill in the art. Recombinant GST-MC-3R fusion proteins may be
expressed in various expression systems, including Spodoptera frugiperda (SfII
}
insect cells (Invitrogen) using a baculovirus expression vector (pAcG2T,
S Pharmingen).
The present invention also relates to subcellular membrane fractions
from the recombinant host cells (both prokaryotic and eukaryotic as well as
both
stably and transiently transformed cells) which contain the nucleic acids of
the present
invention. These subcellular membrane fractions will comprise either wild-type
or
mutant forms of rhesus melanocortin-3 receptor proteins at levels
substantially above
endogenous levels and hence will be useful in various assays described
throughout
this specification.
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. The
nucleic
acid molecules of the present invention encoding rhMC-3R, in whole or in part,
can
be linked with other DNA molecules, i.e, DNA molecules to which the rhMC-3R
are
not naturally linked, to form "recombinant DNA molecules" containing the
receptor.
The novel DNA sequences of the present invention can be inserted into vectors
which
comprise nucleic acids encoding a rhMC-3R or a functional equivalent. These
vectors may be comprised of DNA or RNA; for most cloning purposes DNA vectors
are preferred. Typical vectors include plasmids, modified viruses,
bacteriophage and
cosmids, yeast artificial chromosomes and other forms of episomal or
integrated DNA
that can encode a rhMC-3R. It is well within the skilled artisan to determine
an
appropriate vector for a particular gene transfer or other use.
For example, a variety of mammalian expression vectors may be used
to express recombinant rhesus MC-3R in mammalian cells. Expression vectors are
defined herein as DNA sequences that are required for the transcription of
cloned
DNA and the translation of their mRNAs in an appropriate host. Such vectors
can be
used to express eukaryotic DNA in a variety of hosts such as bacteria, blue
green
algae, plant cells, insect cells and animal cells. Specifically designed
vectors allow
the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal
cells.
An appropriately constructed expression vector should contain: an origin of
replication for autonomous replication in host cells, selectable markers, a
limited
number of useful restriction enzyme sites, a potential for high copy number,
and
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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
S designed plasmids or viruses. Commercially available mammalian expression
vectors
which may be suitable for recombinant rhesus MC-3R expression, include but are
not
limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega),
pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs),
pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMC 1 neo (Stratagene),
pXTI (Stratagene), pSGS (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2)
(ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199),
pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and
~,ZD35 (ATCC 37565).
Also, a variety of bacterial expression vectors may be used to express
recombinant rhesus MC-3R in bacterial cells. Commercially available bacterial
expression vectors which may be suitable for recombinant rhesus MC-3R
expression
include, but are not limited to pCR2.1 (Invitrogen), pETI la (Novagen), lambda
gtl l
(Invitrogen), and pKK223-3 (Pharmacia).
In addition, a variety of fungal cell expression vectors may be used to
express recombinant rhesus MC-3R in fungal cells. Commercially available
fungal
cell expression vectors which may be suitable for recombinant rhesus MC-3R
expression include but are not limited to pYES2 (Invitrogen) and Pichia
expression
vector (Invitrogen).
Also, 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 rhesus MC-3R
include
but are not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T
(Pharmingen).
Expression of rhesus MC-3R DNA may also be performed using in
vitro produced synthetic mRNA. Synthetic mRNA can be efficiently translated in
various cell-free systems, including but not limited to wheat germ extracts
and
reticulocyte extracts, as well as efficiently translated in cell based
systems, including
but not limited to microinjection into frog oocytes, with microinjection into
frog
oocytes being preferred.
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To determine the rhesus MC-3R cDNA sequences) that yields optimal
levels of rhesus MC-3R, cDNA molecules including but not limited to the
following
can be constructed: a cDNA fragment containing the full-length open reading
frame
for rhesus MC-3R 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 rhesus MC-3R cDNA. The expression levels and activity
of
rhesus MC-3R can be determined following the introduction, both singly and in
combination, of these constructs into appropriate host cells. Following
determination
of the rhesus MC-3R cDNA cassette yielding optimal expression in transient
assays,
this MC-3R 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.
As with many receptor proteins, it is possible to modify many of the
amino acids, particularly those which are not found in the ligand binding
domain, and
still retain substantially the same biological activity as the original
receptor. Thus this
invention includes modified rhMC-3R polypeptides which have amino acid
deletions,
additions, or substitutions but that still retain substantially the same
biological activity
as rhMC-3R. It is generally accepted that single amino acid substitutions do
not
usually alter the biological activity of a protein (see, e.g., Molecular
Biology of the
Gene, Watson et al., 1987, Fourth Ed., The Benjamin/Cummings Publishing Co.,
Inc.,
page 226; and Cunningham & Wells, 1989, Science 244:1081-1085). Accordingly,
the present invention includes isolated nucleic acid molecules and expressed
MC-3R
proteins wherein one amino acid substitution is generated and which this
protein
retains substantially the same biological activity as wild-type rhMC-3R. The
present
invention also includes isolated nucleic acid molecules and expressed MC-3R
proteins wherein two or more amino acid substitution is generated wherein this
protein retains substantially the same biological activity as wild-type rhMC-
3R. In
particular, the present invention includes embodiments where the above-
described
substitutions are conservative substitutions. In particular, the present
invention
includes embodiments where the above-described substitutions do not occur in
the
ligand-binding domain of rhMC-3R.
Therefore, this invention also includes vectors containing an rhMC-3R
gene, host cells containing the vectors, and methods of making substantially
pure
rhMC-3R protein comprising the steps of introducing the rhMC-3R gene into a
host
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cell, and cultivating the host cell under appropriate conditions such that
rhMC-3R is
produced. The rhMC-3R so produced may be harvested from the host cells in
conventional ways. Therefore, the present invention also relates to methods of
expressing the rhesus MC-3R protein and biological equivalents disclosed
herein,
assays employing these gene products, recombinant host cells which comprise
DNA
constructs which express these receptor proteins, and compounds identified
through
these assays which act as agonists or antagonists of MC-3R activity.
The cloned rhesus MC-3R cDNA obtained through the methods
described above may be recombinantly expressed by molecular cloning into an
expression vector (such as pcDNA3.neo, pcDNA3.1, pCR2.l, pBlueBacHis2 or
pLITMUS28) containing a suitable promoter and other appropriate transcription
regulatory elements, and transferred into prokaryotic or eukaryotic host cells
to
produce recombinant rhesus MC-3R. 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. Therefore, another aspect of the present invention includes host cells
that have
been engineered to contain and/or express DNA sequences encoding the rhMC-3R.
Such recombinant host cells can be cultured under suitable conditions to
produce
rhMC-3R or a biologically equivalent form. 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. Therefore, an expression vector
containing DNA encoding a rhesus MC-3R-like protein may be used for expression
of
rhesus MC-3R 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. For instance, one insect
expression
system utilizes Spodoptera frugiperda (Sf21 ) insect cells (Invitrogen) in
tandem with
a baculovirus expression vector (pAcG2T, Pharmingen). Also, 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-K1 (ATCC CCL
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61 ), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2),
C 1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171 ) and
CPAE (ATCC CCL 209).
The assays described herein as well as protein purification schemes
can be carried out with cells that have been transiently or stably transfected
or
transformed with an expression vector which directs expression of MC-3R. 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. Transformation is meant to encompass a genetic change to
the
target cell resulting from an incorporation of DNA. Transfection is meant to
include
any method known in the art for introducing MC-3R into the test cells. For
example,
transfection includes calcium phosphate or calcium chloride mediated
transfection,
lipofection, infection with a retroviral construct containing MC-3R, and
electroporation. The expression vector-containing cells are individually
analyzed to
determine whether they produce human MC-3R protein. Identification of human
MC-3R expressing cells may be done by several means, including but not limited
to
immunological reactivity with anti-human MC-3R antibodies, labeled ligand
binding
and the presence of host cell-associated human MC-3R activity.
The specificity of binding of compounds showing affinity for rhMC-
3R is shown by measuring the affinity of the compounds for recombinant cells
expressing the cloned receptor or for membranes from these cells. Expression
of the
cloned receptor and screening for compounds that bind to rhMC-3R or that
inhibit the
binding of a known, radiolabeled ligand of rhMC-3R to these cells, or
membranes
prepared from these cells, provides an effective method for the rapid
selection of
compounds with high affinity for rhMC-3R. Such ligands need not necessarily be
radiolabeled but can also be nonisotopic compounds that can be used to
displace
bound radiolabeled compounds or that can be used as activators in fiinctional
assays.
Compounds identified by the above method are likely to be agonists or
antagonists of
rhMC-3R and may be peptides, proteins, or non-proteinaceous organic molecules.
Accordingly, the present invention is directed to methods for screening
for compounds which modulate the expression of DNA or RNA encoding a MC-3R
protein as well as compounds which effect the function of the MC-3R protein.
Methods for identifying agonists and antagonists of other receptors are well
known in
the art and can be adapted to identify agonists and antagonists of MC-3R. For
example, Cascieri et al. (1992, Molec. Pharmacol. 41:1096-1099) describe a
method
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for identifying substances that inhibit agonist binding to rat neurokinin
receptors and
thus are potential agonists or antagonists of neurokinin receptors. The method
involves transfecting COS cells with expression vectors containing rat
neurokinin
receptors, allowing the transfected cells to grow for a time sufficient to
allow the
S neurokinin receptors to be expressed, harvesting the transfected cells and
resuspending the cells in assay buffer containing a known radioactively
labeled
agonist of the neurokinin receptors either in the presence or the absence of
the
substance, and then measuring the binding of the radioactively labeled known
agonist
of the neurokinin receptor to the neurokinin receptor. If the amount of
binding of the
known agonist is less in the presence of the substance than in the absence of
the
substance, then the substance is a potential agonist or antagonist of the
neurokinin
receptor. Where binding of the substance such as an agonist or antagonist to
MC-3R
is measured, such binding can be measured by employing a labeled substance or
agonist. The substance or agonist can be labeled in any convenient manner
known to
the art, e.g., radioactively, fluorescently, enzymatically.
Therefore, the specificity of binding of compounds having affinity for
MC-3R is shown by measuring the affinity of the compounds for recombinant
cells
expressing the cloned receptor or for membranes from these cells. Expression
of the
cloned receptor and screening for compounds that bind to MC-3R or that inhibit
the
binding of a known, radiolabeled ligand of MC-3R to these cells, or membranes
prepared from these cells, provides an effective method for the rapid
selection of
compounds with high affinity for MC-3R. Such ligands need not necessarily be
radiolabeled but can also be nonisotopic compounds that can be used to
displace
bound radiolabeled compounds or that can be used as activators in functional
assays.
Compounds identified by the above method are likely to be agonists or
antagonists of
MC-3R and may be peptides, proteins, or non-proteinaceous organic molecules.
Compounds may modulate by increasing or attenuating the expression of DNA or
RNA encoding MC-3R, or by acting as an agonist or antagonist of the MC-3R
receptor protein. These compounds that modulate the expression of DNA or RNA
encoding MC-3R 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 test sample with the levels of expression or
fimction in
a standard sample. Kits containing MC-3R, antibodies to MC-3R, or modified MC-
3R may be prepared by known methods for such uses.
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Therefore, the present invention relates to methods of expressing
rhMC-3R in recombinant systems and of identifying agonists and antagonists of
rhMC-3R. When screening compounds in order to identify potential
pharmaceuticals
that specifically interact with a target receptor, it is necessary to ensure
that the
compounds identified are as specific as possible for the target receptor. To
do this, it
is necessary to screen the compounds against as wide an array as possible of
receptors
that are similar to the target receptor. Thus, in order to find compounds that
are
potential pharmaceuticals that interact with receptor A, it is necessary not
only to
ensure that the compounds interact with receptor A (the "plus target") and
produce
the desired pharmacological effect through receptor A, it is also necessary to
determine that the compounds do not interact with receptors B, C, D, etc. (the
"minus
targets"). In general, as part of a screening program, it is important to have
as many
minus targets as possible (see Hodgson, 1992, BiolTechnology 10:973-980).
Rhesus
MC-3R proteins and the DNA molecules encoding this receptor protein have the
additional utility in that they can be used as "minus targets" in screens
designed to
identify compounds that specifically interact with other G-protein coupled
receptors.
Due to homology to GPCRs, the rhMC-3R of this invention is believed to
function
similarly to GPCRs and have similar biological activity. They are useful in
understanding the biological and physiological effects in the rhesus to in
identify
melanocortin active process in primates, followed by human clinical trials.
More
notable, rhMC-3R agonists will be identified and evaluated for their effects
on food
intake, weight gain, and metabolic rate to identify novel-anti-obesity agents
that are
effective in primates. They may also be used to scan for rhesus monkey
melanocortin
agonists and antagonists; as in particular to test the specificity of
identified ligands.
To this end, the present invention relates in part to methods of
identifying a substance which modulates MC-3R receptor activity, which
involves:
(a) combining a test substance in the presence and absence of a
MC-3R receptor protein wherein said MC-3R receptor protein comprises the amino
acid sequence as set forth in SEQ 1D N0:2; and,
(b) measuring and comparing the effect of the test substance in the
presence and absence of the MC-3R receptor protein.
In addition, several specific embodiments are disclosed herein to show
the diverse type of screening or selection assay which the skilled artisan may
utilize in
tandem with an expression vector directing the expression of the MC-3R
receptor
protein. Methods for identifying agonists and antagonists of other receptors
are well
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known in the art and can be adapted to identify agonists and antagonists of MC-
3R.
Therefore, these embodiments are presented as examples and not as limitations.
To
this end, the present invention includes assays by which MC-3R modulators
(such as
agonists and antagonists) may be identified. Accordingly, the present
invention
includes a method for determining whether a substance is a potential agonist
or
antagonist of MC-3R that comprises:
(a) transfecting or transforming cells with an expression vector that
directs expression of MC-3R in the cells, resulting in test cells;
(b) allowing the test cells to grow for a time sufficient to allow
MC-3R to be expressed;
(c) exposing the cells to a labeled ligand of MC-3R in the presence
and in the absence of the substance;
(d) measuring the binding of the labeled ligand to MC-3R; where
if the amount of binding of the labeled ligand is less in the presence of the
substance
than in the absence of the substance, then the substance is a potential
agonist or
antagonist of MC-3R.
The conditions under which step (c) of the method is practiced are
conditions that are typically used in the art for the study of protein-ligand
interactions:
e.g., physiological pH; salt conditions such as those represented by such
commonly
used buffers as PBS or in tissue culture media; a temperature of about
4°C to about
55°C. The test cells may be harvested and resuspended in the presence
of the
substance and the labeled ligand. In a modification of the above-described
method,
step (c) is modified in that the cells are not harvested and resuspended but
rather the
radioactively labeled known agonist and the substance are contacted with the
cells
while the cells are attached to a substratum, e.g., tissue culture plates.
The present invention also includes a method for determining whether
a substance is capable of binding to MC-3R, i.e., whether the substance is a
potential
agonist or an antagonist of MC-3R, where the method comprises:
(a) transfecting or transforming cells with an expression vector that
directs the expression of MC-3R in the cells, resulting in test cells;
(b) exposing the test cells to the substance;
(c) measuring the amount of binding of the substance to MC-3R;
(d) comparing the amount of binding of the substance to MC-3R in
the test cells with the amount of binding of the substance to control cells
that have not
been transfected with MC-3R;
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wherein if the amount of binding of the substance is greater in the test
cells as compared to the control cells, the substance is capable of binding to
MC-3R.
Determining whether the substance is actually an agonist or antagonist can
then be
accomplished by the use of functional assays such as, e.g., the assay
involving the use
of promiscuous G-proteins described below.
The conditions under which step (b) of the method is practiced are
conditions that are typically used in the art for the study of protein-ligand
interactions:
e.g., physiological pH; salt conditions such as those represented by such
commonly
used buffers as PBS or in tissue culture media; a temperature of about
4°C to about
55°C. The test cells are harvested and resuspended in the presence of
the substance.
Chen et al. (1995, Analytical Biochemistry 226: 349-354) describe a
colorometric assay which utilizes a recombinant cell transfected with an
expression
vector encoding a G-protein coupled receptor with a second expression vector
containing a promoter with a cAMP responsive element fused to the LacZ gene.
Activity of the overexpressed G-protein coupled receptor is measured as the
expression and OD measurement of 13-Gal. Therefore, another aspect of this
portion
of the invention includes a non-radioactive method for determining whether a
substance is a potential agonist or antagonist of MC-3R that comprises:
(a) transfecting or transforming cells with an expression vector
encoding MC-3R, resulting in test cells;
(b) transfecting or transforming the test cells of step (a) with an
expression vector which comprises a cAMP-inducible promoter fused to a
colorometric gene such a LacZ;
(c) allowing the transfected cells to grow for a time sufficient to
allow MC-3R to be expressed;
(d) harvesting the transfected cells and resuspending the cells in
the presence of a known agonist of MC-3R and/or in both the presence and
absence of
the test compound;
(e) measuring the binding of the known agonist and test compound
to overexpressed MC-3R by a colorometric assay which measures expression off
the
cAMP-inducible promoter and comparing expression levels in the presence of the
known agonist as well as in the presence and absence of the unknown substance
so as
to determine whether the unknown substance acts as either a potential agonist
or
antagonist of MC-3R.
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Additional methods of identifying agonists or antagonists include but
are by no means limited to the following:
I. (a) transfecting or transforming cells with a first expression
vector which directs expression of MC-3R and a second expression vector which
directs the expression of a promiscuous G-protein, resulting in test cells;
(b) exposing the test cells to a substance that is a suspected
agonist of MC-3R;
(c) measuring the level of inositol phosphates in the cells;
where an increase in the level of inositol phosphates in the cells as compared
to the
level of inositol phosphates in the cells in the absence of the suspected
agonist
indicates that the substance is an agonist of MC-3R.
II. (a) transfecting or transforming cells with a first expression
vector of claim 3 which directs expression of MC-3R and a second expression
vector
which directs the expression of a promiscuous G-protein, resulting in test
cells;
(b) exposing the test cells to a substance that is an agonist
of MC-3R;
(c) subsequently or concurrently to step (b), exposing the
test cells to a substance that is a suspected antagonist of MC-3R;
(d) measuring the level of inositol phosphates in the cells;
where a decrease in the level of inositol phosphates in the cells in the
presence of the
suspected antagonist as compared to the level of inositol phosphates in the
cells in the
absence of the suspected antagonist indicates that the substance is an
antagonist of
MC-3R.
III. the method of II wherein the first and second expression
vectors of step (a) are replaced with a single expression vector which
expresses a
chimeric MC-3R protein fused at its C-terminus to a promiscuous G-protein.
The above-described methods can be modified in that, rather than
exposing the test cells to the substance, membranes can be prepared from the
test cells
and those membranes can be exposed to the substance. Such a modification
utilizing
membranes rather than cells is well known in the art and is described in,
e.g., Hess et
al., 1992, Biochem. Biophys. Res. Comm. 184:260-268. Accordingly, another
embodiment of the present invention includes a method for determining whether
a
substance binds and/or is a potential agonist or antagonist of MC-3R wherein
membrane preparations from the test cells are utilized in place of the test
cells. Such
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methods comprise the following and may utilized the physiological conditions
as
noted above:
(a) transfecting or transforming cells with an expression vector that
directs the expression of MC-3R in the cells, resulting in test cells;
(b) preparing membranes containing MC-3R from the test cells and
exposing the membranes to a ligand of MC-3R under conditions such that the
ligand
binds to the MC-3R in the membranes;
(c) subsequently or concurrently to step (b), exposing the
membranes from the test cells to a substance;
I 0 (d) measuring the amount of binding of the ligand to the MC-3R in
the membranes in the presence and the absence of the substance;
(e) comparing the amount of binding of the Iigand to MC-3R in the
membranes in the presence and the absence of the substance where a decrease in
the
amount of binding of the ligand to MC-3R in the membranes in the presence of
the
15 substance indicates that the substance is capable of binding to MC-3R.
The present invention also relates to a method for determining whether
a substance is capable of binding to MC-3R comprising:
(a) transfecting or transforming cells with an expression vector that
directs the expression of MC-3R in the cells, resulting in test cells;
20 (b) preparing membranes containing MC-3R from the test cells and
exposing the membranes from the test cells to the substance;
(c) measuring the amount of binding of the substance to the MC-
3R in the membranes from the test cells;
(d) comparing the amount of binding of the substance to MC-3R in
25 the membranes from the test cells with the amount of binding of the
substance to
membranes from control cells that have not been transfected with MC-3R, where
if
the amount of binding of the substance to MC-3R in the membranes from the test
cells is greater than the amount of binding of the substance to the membranes
from
the control cells, then the substance is capable of binding to MC-3R.
30 A preferred embodiment of the present invention is determining
various ligand binding affinities using 1251-labeled NDP-a,-MSH as the labeled
ligand in the presence of varying concentration of unlabeled ligands. The
activation
of the second messenger pathway may be determined by measuring the
intracellular
cAMP elicited by agonist at various concentration.
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The present invention also relates to polyclonal and monoclonal
antibodies raised in response to either the rhesus form of MC-3R, or a
biologically
active fragment thereof. Polyclonal or monoclonal antibodies may be raised
against
rhesus MC-3R or a synthetic peptide (usually from about 9 to about 25 amino
acids in
length) from a portion of rhesus MC-3R as disclosed in SEQ ID N0:2.
Monospecific
antibodies to rhesus MC-3R are purified from mammalian antisera containing
antibodies reactive against rhesus MC-3R or are prepared as monoclonal
antibodies
reactive with rhesus MC-3R using the technique of Kohler and Milstein ( 1975,
Nature 256: 495-497). Monospecific antibody as used herein is defined as a
single
antibody species or multiple antibody species with homogenous binding
characteristics for rhesus MC-3R. 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 rhesus MC-3R, as described above. Rhesus MC-3R-specific
antibodies are raised by immunizing animals such as mice, rats, guinea pigs,
rabbits,
goats, horses and the like, with an appropriate concentration of rhesus MC-3R
protein
or a synthetic peptide generated from a portion of rhesus MC-3R with or
without an
immune adjuvant.
Preimmune serum is collected prior to the first immunization. Each
animal receives between about 0.1 pg and about 1000 ~g of rhesus MC-3R protein
associated with an acceptable immune adjuvant. Such acceptable adjuvants
include,
but are not limited to, Freund's complete, Freund's incomplete, alum-
precipitate,
water in oil emulsion containing Corynebacterium parvum and tRNA. The initial
immunization consists of rhesus MC-3R 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 deternune 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 rhesus MC-3R 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 7 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 rhesus MC-3R are
prepared by immunizing inbred mice, preferably Balb/c, with rhesus MC-3R
protein.
The mice are immunized by the IF' or SC route with about 1 ~g to about 100 pg,
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preferably about 10 pg, of rhesus MC-3R 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 pg of rhesus MC-3R 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 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 rhesus MC-3R
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 vivo by injection of pristine
primed Balb/c mice, approximately 0.5 ml per mouse, with about 2 x 106 to
about 6 x
106 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-rhesus MC-3R 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.
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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 rhesus MC-3R 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 rhesus MC-3R peptide fragments, or full-length rhesus MC-3R.
Rhesus MC-3R antibody affinity columns are made, for example, by
adding the antibodies to Affigel-10 (Biorad), a gel support which is pre-
activated with
N-hydroxysuccinimide esters such that the antibodies form covalent linkages
with the
agarose gel bead support. The antibodies are then coupled to the gei 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 rhesus MC-3R or
rhesus
MC-3R protein fragments are slowly passed through the column. The column is
then
washed with phosphate buffered saline until the optical density (A2gp) falls
to
background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6). The
purified rhesus MC-3R protein is then dialyzed against phosphate buffered
saline.
The assays described above can be carried out with cells that have
been transiently or stably transfected or stably transformed with rhMC-3R.
Transfection is meant to include any method known in the art for introducing
rhMC-
3R into the test cells. For example, transfection includes calcium phosphate
or
calcium chloride mediated transfection, lipofection, infection with a
retroviral
construct containing rhMC-3R, and electroporation. Transfonmation is meant to
encompass a genetic change to the target cell resulting from an incorporation
of DNA.
Where binding of the substance or agonist to rhMC-3R is measured,
such binding can be measured by employing a labeled substance or agonist. The
substance or agonist can be labeled in any convenient manner known to the art,
e.g.,
radioactively, fluorescently, enzymatically.
The DNA molecules, RNA molecules, recombinant protein and
antibodies of the present invention may be used to screen and measure levels
of
rhesus MC-3R. The recombinant proteins, DNA molecules, RNA molecules and
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antibodies lend themselves to the formulation of kits suitable for the
detection and
typing of rhesus MC-3R. 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 MC-3R or anti-MC-3R antibodies suitable
for
detecting rhesus MC-3R. 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
rhesus MC-3R may be formulated according to known methods such as by the
admixture of a pharmaceutically acceptable carrier. Examples of such carriers
and
methods of formulation may be found in Remington's Pharmaceutical Sciences. To
form a pharmaceutically acceptable composition suitable for effective
administration,
such compositions will contain an effective amount of the protein, DNA, RNA,
modified rhesus MC-3R, or either MC-3R agonists or antagonists including
tyrosine
kinase activators or inhibitors.
Therapeutic or diagnostic compositions of the invention are
administered to an individual in amounts sufficient to treat or diagnose
disorders.
The effective amount may vary according to a variety of factors such as 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
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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,
S syrups and emulsions, or by injection. Likewise, they may also be
administered in
intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical
with or
without occlusion, or intramuscular form, all using forms well known to those
of
ordinary skill in the pharmaceutical arts.
Advantageously, compounds of the present invention may be
administered in a single daily dose, or the total daily dosage may be
administered in
divided doses of two, three or four times daily. Furthermore, compounds for
the
present invention can be administered in intranasal form via topical use of
suitable
intranasal vehicles, or via transdermal routes, using those forms 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.
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EXAMPLE 1
Isolation of the rhesus MC-3R gene
A rhesus monkey (M. mulatta) genomic DNA library (Clontech) was
screened with a random primed probe using human MC-3R (Gantz, et al., 1993,
J. Biol. Chem. 268: 8246-8250) as template and random hexamer oligonucleotides
as
primer (Molecular Cloning by Maniatis, Fritsch and Sambrook, p. 131, Cold
Spring
Harbor Laboratories, 1982). One positive lambda phage clone was identified.
The
phage clone was digested with BstXI and HindIII to generate a 1.6 kb fragment
encoding the rhesus MC-3R C-terminal sequence and 3' UTR. Another digestion of
the lambda clone with BamHI generated a 5 kb fragment encoding the full length
rhesus MC-3R with a XbaI site at 109 nucleotides up-stream of the ATG codon
and a
HindIII site at 592 nucleotides down-stream of the stop codon. The XbaI and
HindIII
fragment from the lambda phage clone was subcloned into pcDNA3.neo for
expression studies.
EXAMPLE 2
Stable expression of rhesus MC-3R
The receptor plasmid was transfected into CHO dhfr- cells using
lipofectamin (GIBCO). Transfected cells were selected by 6418 for at least 2-3
weeks before binding and functional assays were performed. To determine the
apparent binding affinity of peptides, transfected cells in 12- well plate
were washed
once with PBS and binding was done in HBSS with 25 mM Hepes, 0.5% NaN3, 0.1%
BSA, and protease inhibitor cocktail (4 ug/ml leupeptin, 40 ug/ml bacitracin,
S ug/ml
aprotinin, 10 uM phospharamidon and 100 uM AEBSF). IZSI-NDP-a-MSH
(Amersham) at a concentration of 0.1 nM was used and non-specific binding was
determined in the presence of 1 uM unlabeled NDP-a-MSH. After incubation at
room temperature for 3 hours, wells were washed 3 times by PBS, lysed in 0.5
ml of
0.05% SDS and counted by a gamma counter. Alternatively, the same results were
obtained using cell suspensions followed by GF/C filter filtration {Huang, et
al., 1994,
Biochemistry 33: 3007-3013).
To determine the functional activation of receptors, stably transfected
CHO cells were washed once with PBS without Ca /Mgr, dissociated by non-
enzymatic dissociation medium (Specialty Media, Inc.) at 37°C for 10
min.
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Dissociated cells were resuspended in EBSS buffer containing 25 mM Hepes, S mM
MgCl2, 0.1 % BSA and 50 mM IBMX and incubated with agonist at room temperature
for 45 min. The agonist activation was terminated by boiling for 4 min.
Intracellular
cyclic AMP concentration was measured by the Amersham SPA assay system
(Huang, et al., 1997, J. Receptor Signal Transduc. Res. 17:599-607).
EXAMPLE 3
Pharmacological Properties of Rhesus MC-3R
Human MC-3R were obtained from Dr. Ira Gantz (Gantz, et al., 1993,
J. Biol. Chem. 268: 8246-8250), subcloned into the pIItESlneo expression
vector
(Clontech), and stably transfected into CHO cells. The human MC-3R is used as
a
reference for the pharmacological properties of the rhesus MC-3R. Single
clones
were used in all pharmacological studies as described above. The functional
properties of rhMC-3R were determined by heterologous expression in CHO cells.
Mock transfected CHO cells did not exhibit any binding of [~2sI]NDP-a-MSH or
any
cAMP response to a-MSH. When the rhMC-3R was transfected into CHO cells,
specific binding of [i2sl]NDP-a-MSH was detected, and this specific binding
can be
inhibited by various peptides (Figure 3A). Agonist-elicited accumulation of
cAMP
can also be detected in transfected cells (Figure 3B). Most peptides bind to
or
activate the rhesus MC-3R or human MC-3R with similar potency (within 10-
fold).
Shu-9119 does not activate MC-3R, and its binding affinity is significantly
more
potent at the rhesus MC-3R than at the human MC-3R (Table 1). Of all agonists
tested, only MT-II is a partial agonist for both rhesus and human MC-3R. Shu-
9119
is shown in this Example Section to be an antagonist for both the rhesus and
human
MC-3R. Rhesus MC-3R is shown to be pharmacologically similar to human MC-3R
with respect to all tested agonists.
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Table 1
Ligands IC50, nM EC50, nM Maximal response (%)
rhMC-3R hMC-3R rhMC-3R hMC-3R rhMC-3R
hMC-3R
NDP 0.510.1(2)0.910.2(4)0.110.05(3)0.810.2(2)100 100
a-MSH 1115(2) 46112(3) 110.2(3) 413(4) 100 100
y2-MSH 356144(2)1O5f33(2)311(3) Sf3(4) 100 92
MTII St2(2) 51(2) 0.710.3(3)211(4) 79 61
Shu9119 O.1f0.01(3)2f1(3) - - 0 0
ACTH1-24 61(2) 2211(1) 110.5(3) 110.4(4)100 100
The present invention is not to be limited in scope by the specif c
embodiments described herein. Indeed, various modifications of the invention
in
addition to those described herein will become apparent to those skilled in
the art
fibm the foregoing description. Such modifications are intended to fall within
the
scope of the appended claims.
Various publications are cited herein, the disclosures of which are
incorporated by reference in their entireties.
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SEQUENCE LISTING
<110> Merck & Co., Inc.
<120> DNA MOLECULES ENCODING THE MELANOCORTIN
3 RECEPTOR PROTEIN FROM RHESUS MONKEY
<130> 20189 PCT
<150> 60/107,725
<151> 1998-11-09
<160> 2
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 1909
<212> DNA
<213> rhesus monkey (Macaca mulatta)
<400>
1
tctatctatctatctctccctctctggagaaactaaagtctagactggacagcatccaca60
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ccgatgctgcctaacctctctgagcaccctgcagcccctcctgccagcaaccggagcggc240
agtgggttctgtgagcaggtcttcatcaagccggaggtcttcctggctctgggcatcgtc300
agtctgatggaaaacatcctggtgatcctggctgtggtcaggaatggcaacctgcactct360
cccatgtacttcttcctgtgcagcctggctgcagccgacatgctggtgagcctgtccaac420
tccctggagaccatcatgatcgccgtgatcaacagcgactccctgaccttggaggaccag480
tttatccagcacatggataatatcttcgactctatgatttgcatctccctggtggcctcc540
atctgcaacctcctggccattgccatcgacaggtacgtcaccatcttctatgcccttcgg600
taccacagcatcatgacagtgaggaaagccctcaccttgatcggggtcatctgggtctgc660
tgcggcatctgcggcgtgatgttcatcatctactccgagagcaagatggtcatcgtgtgt720
ctcatcaccatgttcttcgccatggtgctcctcatgggcaccctatatatccacatgttc780
ctcttcgccaggctccacgtccagcgcatcgcagtgctgccccctgctggcgtggtggcc840
ccacagcagcactcctgcatgaagggggctgtcaccatcactatcctgctgggtgttttc900
atcttctgctgggcgcctttcttcctccacctggtcctcatcatcacctgccccaccaat960
ccctactgcatctgctacacggcccatttcaacacctacctggttctcatcatgtgcaac1020
tccgtcatcgaccccctcatctacgccttccgcagcctggagctgcgcaacacgttcaag1080
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catccagccaagagacaaaaacaacgctcagacgggacgtaaaagggtgttaggagctgg1200
aactgtgcttggcttcgtctgtaagctcgtggccctttgcagacgggacacggcgtagga1260
tgggctgtctgtgaggatctgtgtgtgggtaagtcagtttgatctagcacatagcctgga1320
agaatcaggcaaagcagccctgagtgtcatctgtgttcattgctaggcacccagggtttg1380
tggcccctgcctgcttattggctttgtaccagtaactgtgcttcaagccaaccagaccgg1440
agggctctcgtgagcagaaagagtgcttagacttccggcaagcatcctggctcacagcgg1500
ccacctcctgaccactaccgggagagctttgcacatattctgtgggagattgagtgaagc1560
cctgaaaacaatgtgatatttgctgctcccttccagaacttacatctgtgccagcctccc1620
cgaacccctgcacagagacatgacccccttctccctgtgccgttgtcatggttgttatta1680
ttgttggagttttgttcgttaaaatctaagcttgggcccgaacaaaaactcatctcagaa1740
gaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttaaacggtctcc1800
agcttaagtttaaaccgctgatcagcctcgactgtgccttctagttgccagccatctgtt1860
gtttgcccctcccccgtgccttccttgacctggaaggtgccactcccac 1909

CA 02349950 2001-05-08
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<210> 2
<211> 323
<212> PRT
<213> rhesus monkey (Macaca mulatta)
<400> 2
Met Asn Ser Ser Cys Cys Leu Ser Ser Val Ser Pro Met Leu Pro Asn
1 5 10 15
Leu Ser Glu His Pro Ala Ala Pro Pro Ala Ser Asn Arg Ser Gly Ser
20 25 30
Gly Phe Cys Glu Gln Val Phe Ile Lys Pro Glu Val Phe Leu Ala Leu
35 40 45
Gly Ile Val Ser Leu Met Glu Asn Ile Leu Val Ile Leu Ala Val Val
50 55 60
Arg Asn Gly Asn Leu His Ser Pro Met Tyr Phe Phe Leu Cys Ser Leu
65 70 75 80
Ala Ala Ala Asp Met Leu Val Ser Leu Ser Asn Ser Leu Glu Thr Ile
85 90 95
Met IIe Ala Val Ile Asn Ser Asp Ser Leu Thr Leu Glu Asp Gln Phe
100 105 110
Ile Gln His Met Asp Asn Ile Phe Asp Ser Met Ile Cys Ile Ser Leu
115 120 125
Val Ala Ser Ile Cys Asn Leu Leu Ala Ile Ala Ile Asp Arg Tyr Val
130 135 140
Thr Ile Phe Tyr Ala Leu Arg Tyr His Ser Ile Met Thr Val Arg Lys
145 150 155 160
Ala Leu Thr Leu Ile Gly Val Ile Trp Val Cys Cys Gly Ile Cys Gly
165 170 175
Val Met Phe Ile Ile Tyr Ser Glu Ser Lys Met Val Ile Val Cys Leu
180 185 190
Ile Thr Met Phe Phe Ala Met Val Leu Leu Met Gly Thr Leu Tyr Ile
195 200 205
His Met Phe Leu Phe Ala Arg Leu His Val Gln Arg Ile Ala Val Leu
210 215 220
Pro Pro Ala Gly VaI Val Ala Pro Gln Gln His Ser Cys Met Lys Gly
225 230 235 240
Ala Val Thr Ile Thr Ile Leu Leu Gly Val Phe Ile Phe Cys Trp Ala
245 250 255
Pro Phe Phe Leu His Leu Val Leu Ile Ile Thr Cys Pro Thr Asn Pro
260 265 270
Tyr Cys Ile Cys Tyr Thr Ala His Phe Asn Thr Tyr Leu Val Leu Ile
275 280 285
Met Cys Asn Ser Val Ile Asp Pro Leu Ile Tyr Ala Phe Arg Ser Leu
290 295 300
Glu Leu Arg Asn Thr Phe Lys Glu Ile Leu Cys Gly Cys Asn Ser Met
305 310 315 320
Asn Leu Gly
-2-