WO1999046290A1 - A human angiotensin ii/vasopressin receptor (aii/avp) like gene (cbdakd01) - Google Patents

Abstract

CBDAKD01 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CBDAKD01 polypeptides and polynucleotides in the design of protocols for the treatment of hypertension, heart disease, cancer, and kidney disease, among others, and diagnostic assays for such conditions.

Description

A Human Angiotensin II/Vasopressin receptor (AII/AVP) Like Gene (CBDAKDOl)

FIELD OF INVENTION

This invention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to the Angiotensin and/or Vasopressin Receptor family, hereinafter referred to as CBDAKDOl. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

Rat angiotensin/vasopressin receptor (AII/AVP) is a dual receptor which can bind to both angiotensin II and vasopressin. This indicates that the Angiotensin and/or Vasopressin Receptor family has an established, proven history as therapeutic targets. Clearly there is a need for identification and characterization of further members of the Angiotensin and/or Vasopressin Receptor family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, hypertension, heart disease, cancer, and kidney disease.

SUMMARY OF THE INVENTION In one aspect, the invention relates to CBDAKDOl polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such CBDAKDOl polypeptides and polynucleotides. Such uses include the treatment of hypertension, heart disease, cancer, and kidney disease, among others. In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with CBDAKDOl imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate CBDAKDOl activity or levels.

DESCRIPTION OF THE INVENTION Definitions

The following definitions are provided to facilitate understanding of certain terms used frequently herein.

"CBDAKDOl" refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or an allelic variant thereof.

1 "CBDAKDOl activity or CBDAKDOl polypeptide activity" or ' biological activity of the CBDAKDOl or CBDAKDOl polypeptide" refers to the metabolic or physiologic function of said CBDAKDOl including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and immunogenic activities of said CBDAKDO 1 "CBDAKDOl gene" refers to a polynucleotide having the nucleotide sequence set forth in SEQ

ID NO 1 or allelic vaπants thereof and/or their complements

"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimenc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobu n expression library "Isolated" means altered "by the hand of man" from the natural state If an "isolated" composition or substance occurs m nature, it has been changed or removed from its oπginal environment, or both For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herem "Polynucleotide" generally refers to any polyπbonucleotide or polydeoxπbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotides" include, without limitation smgle- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybπd molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions In addition, "polynucleotide" refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA The term polynucleotide also mcludes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases mclude, for example, tntylated bases and unusual bases such as inosme A vanety of modifications has been made to DNA and RNA, thus, "polynucleotide" embraces chemically, enzymatically or metabo cally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteπstic of viruses and cells "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides

"Polypeptide" refers to any peptide or protein compnsmg two or more amino acids jomed to each other by peptide bonds or modified peptide bonds, l e , peptide isosteres "Polypeptide" refers to both short chains, commonly referred to as peptides, o gopeptides or ohgomers, and to longer chams, generally referred to as proteins Polypeptides may contain ammo acids other than the 20 gene-encoded amino acids "Polypeptides" mclude amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art

2 Such modifications are well described in basic texts and m more detailed monographs, as well as in a voluminous research literature Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini It will be appreciated that the same type of modification may be present m the same or varying degrees at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods Modifications mclude acetylation, acylation, ADP-πbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a hpid or hpid denvative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to protems such as arginylation, and ubiquitmation See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York, 1993 and Wold, F , Posttranslational Protein Modifications Perspectives and Prospects, pgs 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York, 1983, Seifter et al , "Analysis for protem modifications and nonprotein cofactors", Meth Enzy ol (1990) 182 626-646 and Rattan et al , "Protein Synthesis Posttranslational Modifications and Aging", Ann NYAcadSci (1992) 663 48-62

"Variant" as the term is used herem, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical vanant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below A typical vanant of a polypeptide differs m ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in many regions, identical A variant and reference polypeptide may differ m ammo acid sequence by one or more substitutions, additions, deletions in any combmation A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code A vanant of a polynucleotide or polypeptide may be a naturally occurring

3 such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed. , Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds. , Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology , von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al. , NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al. , J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992) Gap Penalty: 12 Gap Length Penalty: 4

A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps). Preferred parameters for polynucleotide comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = + 10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3

A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for polynucleotide comparisons.

Preferred polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polynucleotide reference sequence of SEQ ID NO:l, wherein said reference sequence may be identical to the sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO:l by the numerical percent of the respective percent identity and subtracting that product from said total number of nucleotides in SEQ ID NO:l, or:

n_n < x_n - (x_n • y),

wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO:l, and y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and wherein any non-integer product of x_n and y is rounded down to the nearest integer prior to subtracting it from x_n. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations. Preferred polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, wherein said reference sequence may be identical to the sequence of SEQ ID NO: 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the numerical percent of the respective percent identity and subtracting that product from said total number of amino acids in SEQ ID NO:2, or:

n_a < x_a - (x_a « y),

wherein n_a is the number of amino acid alterations, x_a is the total number of amino acids in SEQ ID NO:2, and y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90% , 0.95 for 95 %, 0.97 for 97% or 1.00 for 100% , and wherein any non-integer product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a.

Polypeptides of the Invention

In one aspect, the present invention relates to CBDAKDOl polypeptides (or CBDAKDOl proteins). The CBDAKDOl polypeptides include the polypeptide of SEQ ID NO:2; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2; and polypeptides comprising the amino acid sequence which have at least 80% identity to that of SEQ ID NO:2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2. Furthermore, those with at least 97-99% are highly preferred. Also included within CBDAKDOl polypeptides are polypeptides having the amino acid sequence which have at least 80% identity to the polypeptide having the amino acid sequence of SEQ ID NO:2 over its entire length, and still more preferably at least 90% identity, and still more preferably at least 95% identity to SEQ ID NO:2. Furthermore, those with at least 97-99% are highly preferred. Preferably CBDAKDOl polypeptide exhibit at least one biological activity of CBDAKDOl. The CBDAKDOl polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.

Fragments of the CBDAKDOl polypeptides are also included in the invention. A fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned CBDAKDOl polypeptides. As with CBDAKDOl polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of CBDAKDOl polypeptide. In this context "about" includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.

Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of CBDAKDO 1 polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl teirninus. Also preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Other preferred fragments are biologically active fragments. Biologically active fragments are those that mediate CBDAKDOl activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human.

Preferably, all of these polypeptide fragments retain the biological activity of the CBDAKDOl, including antigenic activity. Variants of the defined sequence and fragments also form part of the present invention. Preferred variants are those that vary from the referents by conservative amino acid substitutions — i.e., those that substitute a residue with another of like characteristics. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination. The CBDAKDOl polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally C -curring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

Polynucleotides of the Invention

Another aspect of the invention relates to CBDAKDOl polynucleotides. CBDAKDOl polynucleotides include isolated polynucleotides which encode the CBDAKDOl polypeptides and fragments, and polynucleotides closely related thereto. More specifically, CBDAKDOl polynucleotide of the invention include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding a

CBDAKDOl polypeptide of SEQ ID NO: 2, and polynucleotide having the particular sequence of SEQ ID NO: 1. CBDAKDOl polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBDAKDOl polypeptide of SEQ ID NO:2, and a polynucleotide comprising a nucleotide sequence that is at least 80% identical to of SEQ ID NO: 1 over its entire length. In this regard, polynucleotides at least 90% identical are particularly preferred, and those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred. Also included under CBDAKDOl polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO: 1 to hybridize under conditions useable for amplification or for use as a probe or marker. The invention also provides polynucleotides which are complementary to such CBDAKDOl polynucleotides.

CBDAKDOl of the invention is structurally related to other proteins of the Angiotensin and/or Vasopressin Receptor family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO: 1) encoding human CBDAKDO 1. The cDNA sequence of SEQ ID NO: 1 contains an open reading frame (nucleotide number 47 to 1588) encoding a polypeptide of 514 amino acids of SEQ ID NO:2. The amino acid sequence of Table 2 (SEQ ID NO:2) has about 30% identity (using FASTA) in 485 amino acid residues with rat angioptensin/vasopressin receptor (AII/AVP) (N.Ruiz-Opazo,et al.,Nature,Med.l:1074-1081,1995). The nucleotide sequence of Table 1 (SEQ ID NO:l) has about 57.9% identity (using FASTA) in 489 nucleotide residues with rat angioptensin/vasopressin receptor (AII/AVP) (N.Ruiz-Opazo,et al.,Nature,Med.l:1074-1081,1995). Thus, CBDAKDOl polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art. Table 1'

1 CAGGGCAGCC TTCAGTCTGA TTCAGGAGAA CGAGGTCCTC TTCACCATGT

51 GCTTCATCCC CCTGGTCTGC TGGATCGTGT GCACTGGACT GAAACAGCAG

101 ATGGAGAGTG GCAAGAGCCT TGCCCAGACA TCCAAGACCT CCACCGCGGT

151 GTACGTCTTC TTCCTTTCCA GTTTGCTGCA GCCCCGGGGA GGGAGCCAGG

201 AGCACGGCCT CTGCGCCCAC CTCTGGGGGC TCTGCTCTTT GGCTGCAGAT

251 GGAATCTGGA ACCAGAAAAT CCTGTTTGAA GAGTCCGACC TCAGGAATCA

301 TGGACTGCAG AAGGCGGATG TGTCTGCTTT CCTGAGGATG AACCTGTTCC

351 AAAAGGAAGT GGACTGCGAG AAGTTCTACA GCTTCATCCA CATGACTTTC

401 CAGGAGTTCT TTGCCGCCAT GTACTACCTG CTGGAAGAGG AAAAGGAAGG

451 AAGGACGAAC GTTCCAGGGA GTCGTTTGAA GCTTCCCAGC CGAGACGTGA

501 CAGTCCTTCT GGAAAACTAT GGCAAATTCG AAAAGGGGTA TTTGATTTTT

551 GTTGTACGTT TCCTCTTTGG CCTGGTAAAC CAGGAGAGGA CCTCCTACTT

601 GGAGAAGAAA TTAAGTTGCA TGATCTCTCA GCAAATCAGG CTGGAGCTGC

651 TGAAATGGAT TGAAGTGAAA GCCAAAGCTA AAAAGCTGCA TGATCAGCCC

701 AGCCAGCTGG AATTGTTCTA CTGTTTGTAC GAGATGCAGG AGGAGGACTT

751 CGTGCAAAGG GCCATGGACT ATTTCCCCAA GATTGAGATC AATCTCTCCA

801 CCAGAATGGA CCACATGGTT TCCTCCTTTT GCATTGAGAA CTGTCATCGG

851 GTGGAGTCAC TGTCCCTGGG GTTTCTCCAT AACATGCCCA AGGAGGAAGA

901 GGAGGAGGAA AAGGAAGGCC GACACCTTGA TATGGTGCAG TGTGTCCTCC

951 CAAGCTCCTC TCATGCTGCC TGTTCTCATG GGTTGGGGCG CTGTGGCCTC

1001 TCCCATGAGT GCTGCTTCGA CATCTCCTTG GTCCTCAGCA GCAACCAGAA 1051 GCTGGTGGAG CTGGACCTGA GTGACAACGC CCTCGGTGAC TTCGGAATCA

1101 GACTTCTGTG TGTGGGACTG AAGCACCTGT TGTGCAATCT GAAGAAGCTC

1151 TGGTTGGTGA ATTCTGCCTT ACGTCAGTCT GTTGTTCAGC TTTGTCCTCG

1201 GTACTCAGCA CTAATCAGAA TCTCACGCAC CTTTACTGCG AGGCAACACT

1251 CTCGGAGACA AGGGATCAAA CTACTCTGTG AGGGACTCTT GCACCCCGAC

1301 TGCAAGCTTC AGGTGTTGGA ATTAGACAAC TGCAACCTCA CGTCACACTG

1351 CTGCTGGGAT CTTTCCACAC TTCTGACCTC CAGCCAGAGC CTGCGAAAGC

1401 TGAGCCTGGG CAACAATGAC CTGGGCGACC TGGGGGTCAT GATGTTCTGT

1451 GAAGTGCTGA AACAGCAGAG CTGCCTCCTG CAGAACCTGG GGTTGTCTGA

1501 AATGTATTTC AATTATGAGA CAAAAAGTGC GTTAGAAACA CTTCAAGAAG

1551 AAAAGCCTGA GCTGACCGTC GTCTTTGAGC CTTCTTGGTA GGAGTGGAAA

1601 CGGGGCTGCC AGACGCCAGT GTTCTCCGGT CCCTCCAGCT GGGGGCCCTC

1651 AGGTGGAGAG AGCTGCGATC CATCCAGGCC AAGACCACAG CTCTGTGATC

1701 CTTCCGGTGG AGTGTCGGAG AAGAGAGCTT GCCGACGATG CCTTCCTGTG

1751 CAGAGCTTGG GCATCTCCTT TACGCCAGGG TGAGGAAGAC ACCAGGACAA

1801 TGACAGCATC GGGTGTTGTT GTCATCACAG CGCCTCAGTT AGAGGATGTT

1851 CCTCTGGTGA CCTCATGTAA TTAGCTCATT CAATAAAGCA CTTTCTTTAT

1901 TTTTCTCTTC TCTGTCTAAC CTTCTTTTTC CTATCTTTTT TTCTTCTTTG

1951 TTCTGTTTAC TTTTGCTCAT ATCATCATTC CCGCTATCTT TCTATTAACT

2001 GACCATAACA CAGAACTAGT TGACTATATA TTATGTTGAA ATTTTATGGC

2051 AGCTATTTAT TTATTTAAAT TTTTTGTAAT AGTTTTGTTT TCTAATAAGA

10 2101 AAAATCCATG CTTTTTGTAG CTGGTTGAAA ATTCAGGAAT ATGTAAAACT

2151 TTTTGGTATT TAATTAAATT GATTCCTTTT CTTAATTTTA AAAAAAAAAA

2201 AAAAAAAAAA AAAAAAAA

A nucleotide sequence of a human CBDAKDOl (SEQ ID NO: 1).

Table 2^b

1 MCFIPLVC I VCTGLKQQME SGKS AQTSK TSTAVYVFFL SSLLQPRGGS

51 QEHG CAHLW GLCSLAADGI NQKILFEES DLRNHGLQKA DVSAFLRMNL

101 FQKEVDCEKF YSFIHMTFQE FFAAMYYLLE EEKEGRTNVP GSRLKLPSRD

151 VTVLLENYGK FEKGYLIFW RFLFGLVNQE RTSYLEKKLS CMISQQIRLE

201 LLK IEVKAK AKKLHDQPSQ LELFYCLYEM QEEDFVQRAM DYFPKIEINL

251 STRMDHMVSS FCIENCHRVE SLSLGFLHNM PKEEEEEEKE GRHLDMVQCV

301 LPSSSHAACS HGLGRCGLSH ECCFDISLVL SSNQKLVELD LSDNA GDFG

351 IRLLCVGLKH LLCNI-KKLWL VNSALRQSW QLCPRYSALI RISRTFTARQ

401 HSRRQGIKLL CEGLLHPDCK LQVLE DNCN LTSHCC DLS TLLTSSQSLR

451 KLSLGNNDLG DLGVMMFCEV LKQQSCLLQN LGLSEMYFNY ETKSALETLQ

501 EEKPELTWF EPSW

" An amino acid sequence of a human CBDAKDOl (SEQ ID NO: 2).

One polynucleotide of the present invention encoding CBDAKDOl may be obtained using standard cloning and screening, from a cDNA library derived from mRNA in cells of human cord blood using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252: 1651-1656; Adams, M.D. et al., Nature, (1992) 355:632-634; Adams, M.D., et al., Nature (1995) 377 Supp:3-174). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.

11 The nucleotide sequence encoding CBDAKDOl polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 47 to 1588 of SEQ ID NO:l), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2. When the polynucleotides of the invention are used for the recombinant production of

CBDAKDO 1 polypeptide, the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself; the coding sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz etal, Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA. Further preferred embodiments are polynucleotides encoding CBDAKDO 1 variants comprise the amino acid sequence CBDAKDOl polypeptide of Table 2 (SEQ ED NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combination.

The present invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97-99% identity between the sequences.

Polynucleotides of the invention, which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding CBDAKDO 1 polypeptide and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to the CBDAKDO 1 gene. Such hybridization techniques are known to those of skill in the art. Typically these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent. The probes generally will comprise at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 30 and 50 nucleotides.

In one embodiment, to obtain a polynucleotide encoding CBDAKDOl polypeptide, including homologs and orthologs from species other than human, comprises the steps of screening an appropriate library under stingent hybridization conditions with a labeled probe having the SEQ ID NO: 1 or a fragment

12 thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Thus in another aspect, CBDAKDOl polynucleotides of the present invention further include a nucleotide sequence comprising a nucleotide sequence that hybridize under stringent condition to a nucleotide sequence having SEQ ID NO: 1 or a fragment thereof. Also included with CBDAKDOl polypeptides are polypeptide comprising amino acid sequence encoded by nucleotide sequence obtained by the above hybridization condition. Such hybria-ization tec--niques -ue weU -mown to mose ofski-l -nthe art. Stringent hybridization conditions are as defined above or, alternatively, conditions under overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNN followed by washing the filters in 0. lx SSC at about 65°C.

The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.

Vectors, Host Cells, Expression The present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY ( 1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium phosphate t-ransfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic Upid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

A great variety of expression systems can be used. Such systems include, among others, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses,

13 fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.

If the CBDAKDOl polypeptide is to be expressed for use in screening assays, generally, it is preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If CBDAKDOl polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered.

CBDAKDOl polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.

Diagnostic Assays

This invention also relates to the use of CBDAKDOl polynucleotides for use as diagnostic reagents. Detection of a mutated form of CBDAKDOl gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under- expression, over-expression or altered expression of CBDAKDO 1. Individuals carrying mutations in the CBDAKDOl gene may be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product

14 in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled CBDAKDOl nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al., Science (1985) 230: 1242. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method. See Cotton et al. , Proc Natl Acad Sci USA ( 1 85) 85 : 4397-4401. In another embodiment, an array of oligonucleotides probes comprising CBDAKDOl nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M.Chee et al., Science, Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or deterrnining a susceptibility to hypertension, heart disease, cancer, and kidney disease, through detection of mutation in the CBDAKDOl gene by the methods described.

In addition, hypertension, heart disease, cancer, and kidney disease, can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of CBDAKDOl polypeptide or CBDAKDOl mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as an CBDAKDOl polypeptide, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Thus in another aspect, the present invention relates to a diagonostic kit for a disease or suspectability to a disease, particularly hypertension, heart disease, cancer, and kidney disease, which comprises:

(a) a CBDAKDOl polynucleotide, preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof ; (b) a nucleotide sequence complementary to that of (a);

(c) a CBDAKDOl polypeptide, preferably the polypeptide of SEQ ID NO: 2, or a fragment thereof; or

(d) an antibody to a CBDAKDOl polypeptide, preferably to the polypeptide of SEQ ID NO: 2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.

15 Chromosome Assays

The nucleotide sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.

Antibodies

The polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the CBDAKDOl polypeptides. The term "immunospecific" means that the antibodies have substantiall greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.

Antibodies generated against the CBDAKDO 1 polypeptides can be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al, MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms including other mammals, may be used to express humanized antibodies .

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.

Antibodies against CBDAKDOl polypeptides may also be employed to treat hypertension, heart disease, cancer, and kidney disease, among others.

16 Vaccines

Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with CBDAKDOl polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from hypertension, heart disease, cancer, and kidney disease, among others. Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering CBDAKDOl polypeptide via a vector directing expression of CBDAKDOl polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.

Further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a CBDAKDOl polypeptide wherein the composition comprises a CBDAKDOl polypeptide or CBDAKDOl gene. The vaccine formulation may further comprise a suitable carrier. Since CBDAKDOl polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

Screening Assays

The CBDAKDOl polypeptide of the present invention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the CBDAKDOl polypeptide of the present invention. Thus, polypeptides of the invention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide of the present invention; or may be structural

17 or functional mimetics of the polypeptide of the present invention. See Coligan et al , Current Protocols in Immunology l(2):Chapter 5 (1991).

CBDAKDOl polypeptides -ire responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate CBDAKDOl polypeptide on the one hand and which can inhibit the function of CBDAKDO 1 polypeptide on the other hand. In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as hypertension, heart disease, cancer, and kidney disease. Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as hypertension, heart disease, cancer, and kidney disease.

In general, such screening procedures may involve using appropriate cells which express the CBDAKDO 1 polypeptide or respond to CBDAKDO 1 polypeptide of the present invention. Such cells include cells from mammals, yeast, Drosophila or E. coli. Cells which express the CBDAKDOl polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBDAKDOl polypeptide are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response. The ability of the cells which were contacted with the candidate compounds is compared with the same cells which were not contacted for CBDAKDO 1 activity.

The assays may simply test binding of a candidate compound wherein adherence to the cells bearing the CBDAKDOl polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the CBDAKDOl polypeptide, using detection systems appropriate to the cells bearing the CBDAKDOl polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.

Further, the assays may simply comprise the steps of mixing a candidate compound with a solution containing a CBDAKDOl polypeptide to form a mixture, measuring CBDAKDOl activity in the mixture, and comparing the CBDAKDOl activity of the mixture to a standard.

The CBDAKDOl cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of CBDAKDOl mRNA and protein in cells. For example, an ELISA may be constructed for measuring secreted or cell associated levels of CBDAKDOl protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of CBDAKDOl (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues. The CBDAKDOl protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to,

18 ligand binding and crosslinking assays in which the CBDAKDOl is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. In addition to being used for purification and cloning of the receptor, these binding assays can be used to identify agonists and antagonists of CBDAKDOl which compete with the binding of CBDAKDOl to its receptors, if any. Standard methods for conducting screening assays are well understood in the art.

Examples of potential CBDAKDOl polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the CBDAKDOl polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or small molecules which bind to the polypetide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.

Thus in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for CBDAKDOl polypeptides; or compounds which decrease or enhance the production of CBDAKDOl polypeptides, which comprises:

(a) a CBDAKDOl polypeptide, preferably that of SEQ ID NO:2;

(b) a recombinant cell expressing a CBDAKDOl polypeptide, preferably that of SEQ ID NO:2;

(c) a cell membrane expressing a CBDAKDOl polypeptide; preferably that of SEQ ID NO: 2; or (d) antibody to a CBDAKDOl polypeptide, preferably that of SEQ ID NO: 2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.

Prophylactic and Therapeutic Methods

This invention provides methods of treating abnormal conditions such as, hypertension, heart disease, cancer, and kidney disease, related to both an excess of and insufficient amounts of CBDAKDO 1 polypeptide activity.

If the activity of CBDAKDOl polypeptide is in excess, several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the CBDAKDO 1 polypeptide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by inhibiting a second signal, and thereby alleviating the abnormal condition. In another approach, soluble forms of CBDAKDOl polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous CBDAKDOl polypeptide may be administered. Typical embodiments of such competitors comprise fragments of the CBDAKDOl polypeptide.

19 In still another approach, expression of the gene encoding endogenous CBDAKDOl polypeptide can be inhibited using expression blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988). Alternatively, oligonucleotides which form triple helices with the gene can be supplied. See, for example, Lee et al, Nucleic Acids Res (1979) 6:3073; Cooney et al, Science (1988) 241:456; Dervan et al, Science (1991) 251:1360. These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo.

For treating abnormal conditions related to an under-expression of CBDAKDO 1 and its activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound which activates CBDAKDOl polypeptide, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of CBDAKDOl by the relevant cells in the subject. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be aclrninistered to a subject for engineering cells in vivo and expression of the polypeptide in vivo. For overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996). Another approach is to administer a therapeutic amount of CBDAKDOl polypeptides in combination with a suitable pharmaceutical carrier.

Formulation and Administration

Peptides, such as the soluble form of CBDAKDOl polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier. Such formulations comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Formulation should suit the mode of administration, and is well within the skill of the art. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.

20 Polypeptides and other compounds of the present mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds

Preferred forms of systemic administration of the pharmaceutical compositions mclude injection, typically by intravenous injection Other injection routes, such as subcutaneous, intramuscular, or -ntrapentoneal, can be used Alternative means for systemic administration mclude transmucosal and transdermal administration usmg penetrants such as bile salts or fusidic acids or other detergents In addition, if properly formulated in entenc or encapsulated formulations, oral administration may also be possible Administration of these compounds may also be topical and or localized, in the form of salves, pastes, gels and the like The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner Suitable dosages, however, are in the range of 0 1-100 μg/kg of subject Wide vanations m the needed dosage, however, are to be expected in view of the vanety of compounds available and the differing efficiencies of vanous routes of administration For example, oral administration would be expected to require higher dosages than administration by intravenous mjechon Variations m these dosage levels can be adjusted usmg standard empincal routmes for opt-mization, as is well understood m the art

Polypeptides used m treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as descnbed above Thus, for example, cells from a subject may be engmeered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector The cells -ire then introduced mto the subject

All publications, mcludmg but not limited to patents and patent applications, cited m this specification are herein incorporated by reference as if each individual publication were specifically and individually mdicated to be incorporated by reference herein as though fully set forth

21 SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: ZHANG, QING-HUA SHEN, YU MAO, MAO GU, BAI-WEI

(ii) TITLE OF THE INVENTION: A Human Angiotensin Il/Vasopressin receptor (AII/AVP) Like Gene (CBDAKDOl)

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: RATNER & PRESTIA

(B) STREET: P.O. BOX 980 (C) CITY: VALLEY FORGE

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19482

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: TO BE ASSIGNED

(B) FILING DATE:

(C) CLASSIFICATION: UNKNOWN

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: PRESTIA, PAUL F

22 (B) REGISTRATION NUMBER: 23,031

(C) REFERENCE/DOCKET NUMBER: GP-70382

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 610-407-0700

(B) TELEFAX: 610-407-0701

(C) TELEX: 846169

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2218 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CAGGGCAGCC TTCAGTCTGA TTCAGGAGAA CGAGGTCCTC TTCACCATGT GCTTCATCCC 60

CCTGGTCTGC TGGATCGTGT GCACTGGACT GAAACAGCAG ATGGAGAGTG GCAAGAGCCT 120

TGCCCAGACA TCCAAGACCT CCACCGCGGT GTACGTCTTC TTCCTTTCCA GTTTGCTGCA 180 GCCCCGGGGA GGGAGCCAGG AGCACGGCCT CTGCGCCCAC CTCTGGGGGC TCTGCTCTTT 240

GGCTGCAGAT GGAATCTGGA ACCAGAAAAT CCTGTTTGAA GAGTCCGACC TCAGGAATCA 300

TGGACTGCAG AAGGCGGATG TGTCTGCTTT CCTGAGGATG AACCTGTTCC AAAAGGAAGT 360

GGACTGCGAG AAGTTCTACA GCTTCATCCA CATGACTTTC CAGGAGTTCT TTGCCGCCAT 420

GTACTACCTG CTGGAAGAGG AAAAGGAAGG AAGGACGAAC GTTCCAGGGA GTCGTTTGAA 480 GCTTCCCAGC CGAGACGTGA CAGTCCTTCT GGAAAACTAT GGCAAATTCG AAAAGGGGTA 540

TTTGATTTTT GTTGTACGTT TCCTCTTTGG CCTGGTAAAC CAGGAGAGGA CCTCCTACTT 600

GGAGAAGAAA TTAAGTTGCA TGATCTCTCA GCAAATCAGG CTGGAGCTGC TGAAATGGAT 660

TGAAGTGAAA GCCAAAGCTA AAAAGCTGCA TGATCAGCCC AGCCAGCTGG AATTGTTCTA 720

CTGTTTGTAC GAGATGCAGG AGGAGGACTT CGTGCAAAGG GCCATGGACT ATTTCCCCAA 780 GATTGAGATC AATCTCTCCA CCAGAATGGA CCACATGGTT TCCTCCTTTT GCATTGAGAA 840

CTGTCATCGG GTGGAGTCAC TGTCCCTGGG GTTTCTCCAT AACATGCCCA AGGAGGAAGA 900

GGAGGAGGAA AAGGAAGGCC GACACCTTGA TATGGTGCAG TGTGTCCTCC CAAGCTCCTC 960

TCATGCTGCC TGTTCTCATG GGTTGGGGCG CTGTGGCCTC TCCCATGAGT GCTGCTTCGA 1020

CATCTCCTTG GTCCTCAGCA GCAACCAGAA GCTGGTGGAG CTGGACCTGA GTGACAACGC 1080 CCTCGGTGAC TTCGGAATCA GACTTCTGTG TGTGGGACTG AAGCACCTGT TGTGCAATCT 1140

GAAGAAGCTC TGGTTGGTGA ATTCTGCCTT ACGTCAGTCT GTTGTTCAGC TTTGTCCTCG 1200

GTACTCAGCA CTAATCAGAA TCTCACGCAC CTTTACTGCG AGGCAACACT CTCGGAGACA 1260

AGGGATCAAA CTACTCTGTG AGGGACTCTT GCACCCCGAC TGCAAGCTTC AGGTGTTGGA 1320

23 ATTAGACAAC TGCAACCTCA CGTCACACTG CTGCTGGGAT CTTTCCACAC TTCTGACCTC 1380

CAGCCAGAGC CTGCGAAAGC TGAGCCTGGG CAACAATGAC CTGGGCGACC TGGGGGTCAT 1440

GATGTTCTGT GAAGTGCTGA AACAGCAGAG CTGCCTCCTG CAGAACCTGG GGTTGTCTGA 1500

AATGTATTTC AATTATGAGA CAAAAAGTGC GTTAGAAACA CTTCAAGAAG AAAAGCCTGA 1560 GCTGACCGTC GTCTTTGAGC CTTCTTGGTA GGAGTGGAAA CGGGGCTGCC AGACGCCAGT 1620

GTTCTCCGGT CCCTCCAGCT GGGGGCCCTC AGGTGGAGAG AGCTGCGATC CATCCAGGCC 1680

AAGACCACAG CTCTGTGATC CTTCCGGTGG AGTGTCGGAG AAGAGAGCTT GCCGACGATG 1740

CCTTCCTGTG CAGAGCTTGG GCATCTCCTT TACGCCAGGG TGAGGAAGAC ACCAGGACAA 1800

TGACAGCATC GGGTGTTGTT GTCATCACAG CGCCTCAGTT AGAGGATGTT CCTCTGGTGA 1860 CCTCATGTAA TTAGCTCATT CAATAAAGCA CTTTCTTTAT TTTTCTCTTC TCTGTCTAAC 1920

CTTCTTTTTC CTATCTTTTT TTCTTCTTTG TTCTGTTTAC TTTTGCTCAT ATCATCATTC 1980

CCGCTATCTT TCTATTAACT GACCATAACA CAGAACTAGT TGACTATATA TTATGTTGAA 2040

ATTTTATGGC AGCTATTTAT TTATTTAAAT TTTTTGTAAT AGTTTTGTTT TCTAATAAGA 2100

AAAATCCATG CTTTTTGTAG CTGGTTGAAA ATTCAGGAAT ATGTAAAACT TTTTGGTATT 2160 TAATTAAATT GATTCCTTTT CTTAATTTTA AAAAAAAAAA AAAAAAAAAA AAAAAAAA 2218

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 514 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Cys Phe lie Pro Leu Val Cys Trp lie Val Cys Thr Gly Leu Lys 1 5 10 15

Gin Gin Met Glu Ser Gly Lys Ser Leu Ala Gin Thr Ser Lys Thr Ser

20 25 30

Thr Ala Val Tyr Val Phe Phe Leu Ser Ser Leu Leu Gin Pro Arg Gly 35 40 45 Gly Ser Gin Glu His Gly Leu Cys Ala His Leu Trp Gly Leu Cys Ser

50 55 60

Leu Ala Ala Asp Gly lie Trp Asn Gin Lys lie Leu Phe Glu Glu Ser 65 70 75 80

Asp Leu Arg Asn His Gly Leu Gin Lys Ala Asp Val Ser Ala Phe Leu 85 90 95

Arg Met Asn Leu Phe Gin Lys Glu Val Asp Cys Glu Lys Phe Tyr Ser

100 105 110

Phe lie His Met Thr Phe Gin Glu Phe Phe Ala Ala Met Tyr Tyr Leu

24 115 120 125

Leu Glu Glu Glu Lys Glu Gly Arg Thr Asn Val Pro Gly Ser Arg Leu

130 135 140

Lys Leu Pro Ser Arg Asp Val Thr Val Leu Leu Glu Asn Tyr Gly Lys 145 150 155 160

Phe Glu Lys Gly Tyr Leu lie Phe Val Val Arg Phe Leu Phe Gly Leu

165 170 175

Val Asn Gin Glu Arg Thr Ser Tyr Leu Glu Lys Lys Leu Ser Cys Met 180 185 190 lie Ser Gin Gin lie Arg Leu Glu Leu Leu Lys Trp lie Glu Val Lys 195 200 205

Ala Lys Ala Lys Lys Leu His Asp Gin Pro Ser Gin Leu Glu Leu Phe

210 215 220

Tyr Cys Leu Tyr Glu Met Gin Glu Glu Asp Phe Val Gin Arg Ala Met 225 230 235 240

Asp Tyr Phe Pro Lys lie Glu lie Asn Leu Ser Thr Arg Met Asp His

245 250 255

Met Val Ser Ser Phe Cys lie Glu Asn Cys His Arg Val Glu Ser Leu 260 265 270 Ser Leu Gly Phe Leu His Asn Met Pro Lys Glu Glu Glu Glu Glu Glu 275 280 285

Lys Glu Gly Arg His Leu Asp Met Val Gin Cys Val Leu Pro Ser Ser

290 295 300

Ser His Ala Ala Cys Ser His Gly Leu Gly Arg Cys Gly Leu Ser His 305 310 315 320

Glu Cys Cys Phe Asp lie Ser Leu Val Leu Ser Ser Asn Gin Lys Leu

325 330 335

Val Glu Leu Asp Leu Ser Asp Asn Ala Leu Gly Asp Phe Gly lie Arg 340 345 350 Leu Leu Cys Val Gly Leu Lys His Leu Leu Cys Asn Leu Lys Lys Leu 355 360 365

Trp Leu Val Asn Ser Ala Leu Arg Gin Ser Val Val Gin Leu Cys Pro

370 375 380

Arg Tyr Ser Ala Leu lie Arg lie Ser Arg Thr Phe Thr Ala Arg Gin 385 390 395 400

His Ser Arg Arg Gin Gly lie Lys Leu Leu Cys Glu Gly Leu Leu His

405 410 415

Pro Asp Cys Lys Leu Gin Val Leu Glu Leu Asp Asn Cys Asn Leu Thr 420 425 430 Ser His Cys Cys Trp Asp Leu Ser Thr Leu Leu Thr Ser Ser Gin Ser 435 440 445

Leu Arg Lys Leu Ser Leu Gly Asn Asn Asp Leu Gly Asp Leu Gly Val 450 455 460

25 Met Met Phe Cys Glu Val Leu Lys Gin Gin Ser Cys Leu Leu Gin Asn 465 470 475 480

Leu Gly Leu Ser Glu Met Tyr Phe Asn Tyr Glu Thr Lys Ser Ala Leu

485 490 495

Glu Thr Leu Gin Glu Glu Lys Pro Glu Leu Thr Val Val Phe Glu Pro

500 505 510

Ser Trp

26

Claims

What is claimed is:

1. An isolated polynucleotide comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBDAKDO 1 polypeptide of SEQ ID NO:2; or a nucleotide sequence complementary to said isolated polynucleotide.

2. The polynucleotide of claim 1 wherein said polynucleotide comprises the nucleotide sequence contained in SEQ ID NO:l encoding the CBDAKDOl polypeptide of SEQ ID N02.

3. The polynucleotide of claim 1 wherein said polynucleotide comprises a nucleotide sequence that is at least 80% identical to that of SEQ ID NO: 1 over its entire length.

4. The polynucleotide of claim 3 which is polynucleotide of SEQ ID NO: 1.

5. The polynucleotide of claim 1 which is DNA or RNA.

6. A DNA or RNA molecule comprising an expression system, wherein said expression system is capable of producing a CBDAKDO 1 polypeptide comprising an amino acid sequence, which has at least 80% identity with the polypeptide of SEQ ID NO:2 when said expression system is present in a compatible host cell.

7. A host cell comprising the expression system of claim 6.

8. A process for producing a CBDAKDOl polypeptide comprising culturing a host of claim 7 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture.

9. A process for producing a cell which produces a CBDAKDOl polypeptide thereof comprising transforming or transfecting a host cell with the expression system of claim 6 such that the host cell, under appropriate culture conditions, produces a CBDAKDOl polypeptide.

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10. A CBDAKDOl polypeptide comprising an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID N0:2 over its entire length.

1 1. The polypeptide of claim 10 which comprises the amino acid sequence of SEQ ID NO:2.

12. An antibody immunospecific for the CBDAKDOl polypeptide of claim 10.

13. A method for the treatment of a subject in need of enhanced activity or expression of CBDAKDOl polypeptide of claim 10 comprising:

(a) administering to the subject a therapeutically effective amount of an agonist to said polypeptide; and/or

(b) providing to the subject an isolated polynucleotide comprising a nucleotide sequence that has at least 80% identity to a nucleotide sequence encoding the CBDAKDOl polypeptide of SEQ ID NO:2 over its entire length; or a nucleotide sequence complementary to said nucleotide sequence in a form so as to effect production of said polypeptide activity in vivo.

14. A method for the treatment of a subject having need to inhibit activity or expression of CBDAKDOl polypeptide of claim 10 comprising: (a) administering to the subject a therapeutically effective amount of an antagonist to said polypeptide; and/or

(b) administering to the subject a nucleic acid molecule that inhibits the expression of the nucleotide sequence encoding said polypeptide; and/or

(c) administering to the subject a therapeutically effective amount of a polypeptide that competes with said polypeptide for its ligand, substrate , or receptor.

15. A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of CBDAKDOl polypeptide of claim 10 in a subject comprising:

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said CBDAKDOl polypeptide in the genome of said subject; and/or

(b) analyzing for the presence or amount of the CBDAKDOl polypeptide expression in a sample derived from said subject.

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16. A method for identifying compounds which inhibit (antagonize) or agonize the CBDAKDOl polypeptide of claim 10 which comprises:

(a) contacting a candidate compound with cells which express the CBDAKDOl polypeptide (or cell membrane expressing CBDAKDOl polypeptide) or respond to CBDAKDOl polypeptide; and

(b) observing the binding, or stimulation or inhibition of a functional response; or comparing the ability of the cells (or cell membrane) which were contacted with the candidate compounds with the same cells which were not contacted for CBDAKDOl polypeptide activity.

17. An agonist identified by the method of claim 16.

18. An antagonist identified by the method of claim 16.

19. A recombinant host cell produced by a method of Claim 9 or a membrane thereof expressing a CBDAKDOl polypeptide.

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