WO2000075280A2 - A member of the frzb family, frazzled - Google Patents

Abstract

FRAZZLED polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing FRAZZLED polypeptides and polynucleotides in the design of protocols for the treatment of chronic and acute inflammation, arthritis, osteoarthritis and other osteopenic conditions, Paget's disease, rheumatoid arthritis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g., lymphoproliferative disorders), atherosclerosis, and Alzheimer's disease, among others, and diagnostic assays for such conditions.

Description

A MEMBER OF THE FRZB FAMILY, FRAZZLED

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-m-part of U.S. Application Seπal No. 09/238,256, filed January 27, 1999, which is a continuation-m-part of U.S. Application Seπal No. 09/111,894, filed July 8, 1998, which is a contmuation-in-part of U.S. Application Seπal No. 08/978,981, filed November 26,

1997, which claims the benefit of U.S. Provisional Application No. 60/047,408, filed May 22, 1997, all of whose contents are incorporated herein by reference in their entireties.

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 FRZB family, hereinafter referred to as FRAZZLED. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION Recently, a number of studies have focused on the identification and characteπzation of proteins which control developmental patterning. These proteins are members of a large family (referred to as the frizzled family), exemplified by frizzled and smoothened [Moon, et al., Cell 88: 725- 728 (1997)]. Smoothened is a 7 transmembrane protein which associates with the 12 transmembrane protein, patched, to regulate signaling of the soluble agonist, indian hedgehog (Stone, et al., Science 384(14): 129-134 (1996)] . Indian hedgehog and parathyroid hormone-related peptide appear to regulate the differentiation of chondrocytes in mammalian systems [Nortkamp, et al. Science 273(2): 613-622 (1996)]. The control of the chondrocyte phenotype could be cπtically important in the maintenance of cartilage homeostasis in diseases involving both bone and cartilage including osteoarthπtis, osteoporosis and rheumatoid arthπtis. In addition to the plasma membrane-associated members of the frizzled family, a soluble frizzled-related protein subfamily has recently been descπbed. Members of this family, referred to as either Frzb, Fπtz, frezzled or sFRPs (soluble frizzled related proteins), appear to control signaling by binding frizzled agonists, extracellularly [Moon, et al., Cell 88: 725-728 (1997); Wang, et al, Cell 88: 757-766 (1997); Leyns, et al., Cell 88: 747-756 (1997)]. The first descπption of Frzb was from extracts of bovine articular cartilage [Hoang, et al., J. Biol. Chem. 271(42): 26131-26137 (1996)]. In that study, it was reported that Frzb was expressed m chondrocytes in both developing cartilage rudiments and at sites of long bone growth. These authors also descπbed the human Frzb homologue and reported that it is 94% identical to the bovine sequence. More recently, several sFRPs have been identified in the mouse [Rattner, et al., PNAS 94: 2859-2863 (1997)]. One member of this subfamily, sFRP-3 is 92% identical to bovine and human Frzb. When sFRP-3 was expressed as a construct containing a hydrophobic transmembrane domain, it had the ability to bind the frizzled agonist, wingless, confirming that the soluble mammalian sFRP-3 has the ability to bind frizzled agonists The numerous studies descπbed above suggest that members of the frizzled family play key roles m cartilage and bone morphogenesis. However, it is unclear what role, if any, these proteins play in the maintenance of adult bone and/or cartilage. Consistent with a potential role in mature tissues, Frzb was oπgmally isolated from calf articular cartilage. Furthermore, it has been proposed that at sites of active bone and cartilage remodeling, exemplified by osteoarthπtis and fracture callus healing [Hughes, et al., J. Bone Miner. Res. 10(4): 533-544 (1995)], there may be differentiation of hypertrophic chondrocytes into osteoblast-like bone forming cells. Aberrant control of this process may result in the subchondral bone sclerosis observed in osteoarthritis, which may lead to the development and progression of this disease. This invention descπbes a novel member of the human Frzb family.

This indicates that the FRZB family has an established, proven history as therapeutic targets. Clearly there is a need for identification and characteπzation of further members of FRZB family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, chronic and acute inflammation, arthπtis, osteoarthπtis and other osteopenic conditions, Paget's disease, rheumatoid arthπtis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, ADDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g., lymphopro ferative disorders), atherosclerosis, and Alzheimers disease.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to FRAZZLED polypeptides and recombinant mateπals and methods for their production. Another aspect of the invention relates to methods for usmg such

FRAZZLED polypeptides and polynucleotides. Such uses include the treatment of chronic and acute inflammation, arthπtis, osteoarthπtis and other osteopenic conditions, Paget's disease, rheumatoid arthπtis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g., lymphoprohferative disorders), atherosclerosis, and Alzheimers disease. 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 FRAZZLED imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with mappropπate FRAZZLED activity or levels.

DESCRIPTION OF THE INVENTION

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

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

"FRAZZLED activity or FRAZZLED polypeptide activity" or "biological activity of the FRAZZLED or FRAZZLED polypeptide" refers to the metabolic or physiologic function of said

FRAZZLED including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and lmmunogenic activities of said FRAZZLED.

"FRAZZLED 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, chimeπc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.

"Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original 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 herein.

"Polynucleotide" generally refers to any polyπbonucleotide or polydeoxπbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising 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 tπple-stranded regions comprising RNA or DNA or both RNA and DNA.

The term polynucleotide also includes 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, tπtylated bases and unusual bases such as inosine. A vaπety of modifications has been made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabohcally 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 o gonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly refeπed to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as proteins. Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids. "Polypeptides" include ammo acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chains and the ammo or carboxyl termini. It will be appreciated that the same type of modification may be present in 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 include acetylation, acylation, ADP-πbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide deπvative, covalent attachment of a hpid or pid derivative, covalent attachment of phosphotidyhnositol, 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 amino acids to proteins such as arg ylation, 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 in POSTTRANSLATIONAL COVALENT

MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al , "Analysis for protein modifications and nonprotem cofactors", Meth Enzymol (1990) 182.626- 646 and Rattan et al, "Protein Synthesis. Posttranslational Modifications and Aging", Ann NYAcad

"Vaπant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the vaπant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in ammo acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A vaπant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. A vaπant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurπng 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 the case may be, as determined by compaπng 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" can be readily calculated by known methods, including but not limited to those descπbed 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 Hemje, G., Academic Press, 1987; and Sequence Analysis Primer, Gπbskov, M. and Devereux, J., eds., M Stockton Press, New

York, 1991; and Caπllo, H., and Lipman, D., SIAM J. Applied Math , 48- 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity 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. 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 peptide comparisons (along with no penalty for end gaps). 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 Available as: The "gap" program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid compaπsons.

A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided in ( 1 ) and (2) below.

(1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO: l, wherem said polynucleotide sequence may be identical to the reference 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 withm the reference sequence, and wherem said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides m 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, 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 • is the symbol for the multiplication operator, 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 ED

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.

By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is it may be 100% identical, or it may include up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity. Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups with the reference sequence. The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of ammo acids in SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or: n_n < x_n - (x_n • y), wherem n_n is the number of amino acid alterations, x_n is the total number of ammo acids in SEQ ID NO:2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., • is the symbol for the multiplication operator, and wherem any non-mteger product of x_n and y is rounded down to the nearest integer pπor to subtracting it from x_n. (2) 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 polypeptide sequence may be identical to the reference sequence of SEQ ID NO: 2 or may include up to a certain integer number of ammo 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 wherem said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or m one or more contiguous groups withm the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO:2, or: n_a < x_a - (x_a • y), wherein n_a is the number of ammo acid alterations, x_a is the total number of ammo acids in SEQ ID

NO.2, 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 • is the symbol for the multiplication operator, 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. By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO.2, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity. Such alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- 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. The number of ammo acid alterations for a given % identity is determined by multiplying the total number of amino acids m SEQ ID NO:2 by the integer defining the percent identity divided by 100 and then 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 am o acid alterations, x_a is the total number of ammo acids in SEQ ID NO:2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and • is the symbol for the multiplication operator, and wherem 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 FRAZZLED polypeptides (or FRAZZLED proteins) . The FRAZZLED polypeptides include the polypeptide of SEQ ID NOS:2 and 4; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2; and polypeptides comprising the ammo 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 mcluded withm FRAZZLED polypeptides are polypeptides having the ammo acid sequence which have at least 80% identity to the polypeptide having the ammo 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 FRAZZLED polypeptide exhibit at least one biological activity of FRAZZLED.

The FRAZZLED 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 ammo acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidme residues, or an additional sequence for stability duπng recombinant production.

Fragments of the FRAZZLED 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 ammo acid sequence of the aforementioned FRAZZLED polypeptides. As with FRAZZLED polypeptides, fragments may be "free-standing," or compπsed withm 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 FRAZZLED polypeptide. In this context "about" mcludes 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 FRAZZLED polypeptides, except for deletion of a continuous seπes of residues that includes the amino terminus, or a continuous seπes of residues that includes the carboxyl terminus or deletion of two continuous seπes of residues, one including the ammo terminus and one including the carboxyl terminus. Also preferred are fragments characteπzed by structural or functional attπbutes such as fragments that compπse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet- formmg regions, turn and turn-forming regions, coil and coil-formmg 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 FRAZZLED 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 lmmunogemc m an animal, especially in a human.

Preferably, all of these polypeptide fragments retain the biological activity of the FRAZZLED, including antigenic activity. Among the most preferred fragment is that having the amino acid sequence of SEQ ID NO: 4. Vaπants of the defined sequence and fragments also form part of the present invention. Preferred vaπants are those that vary from the referents by conservative ammo acid substitutions — i.e., those that substitute a residue with another of like characteπstics. Typical such substitutions are among Ala, Val, Leu and lie; 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 vaπants in which several, 5-10, 1-5, or 1-2 ammo acids are substituted, deleted, or added in any combination.

The FRAZZLED polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occumng polypeptides, recombmantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for prepaπng such polypeptides are well understood in the art.

Polynucleotides of the Invention

Another aspect of the invention relates to FRAZZLED polynucleotides. FRAZZLED polynucleotides include isolated polynucleotides which encode the FRAZZLED polypeptides and fragments, and polynucleotides closely related thereto. More specifically, FRAZZLED polynucleotide of the invention include a polynucleotide compπsing the nucleotide sequence contained m SEQ ID

NO: 1 encoding a FRAZZLED polypeptide of SEQ ID NO: 2, and polynucleotides having the particular sequences of SEQ ED NOS: 1 and 3. FRAZZLED polynucleotides further include a polynucleotide compπsing a nucleotide sequence that has at least 80%) identity over its entire length to a nucleotide sequence encoding the FRAZZLED polypeptide of SEQ ID NO:2, and a polynucleotide comprising a nucleotide sequence that is at least 80% identical to that of SEQ D 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 FRAZZLED polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ED 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 FRAZZLED polynucleotides.

FRAZZLED of the invention is structurally related to other proteins of the FRZB family, as shown by the results of sequencing the cDNA encoding human FRAZZLED. The cDNA sequence of SEQ ED NO: 1 contains an open reading frame (nucleotide number 171 to 1208) encodmg a polypeptide of 346 amino acids of SEQ ED NO:2. The ammo acid sequence of Table 1 (SEQ ED NO:2) has about 51.1% identity (using FASTA) in 319 amino acid residues with mouse sFRP-3 (A. Ratner et al., Proc. Natl. Acad. Sci. U.S.A. 94, 2859-2863, 1997) The nucleotide sequence of Table 1 (SEQ ED NO:l) has about 81.6% identity (using FAST A) in 582 nucleotide residues with mouse sFRP-4 (A. Ratner et al., Proc. Natl. Acad. Sci. U.S.A. 94, 2859- 2863, 1997). Thus, FRAZZLED polypeptides and polynucleotides of the present invention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art.

Table l^a

CGCGGCCGGACCCCGCGGCCCCGCTTTGCTGCCGACTGGAGTTTGGGGGAAGA AACTCTCCTGCGCCCCAGAGGATTTCTTCCTCGGCGAAGGGACAGCGAAAGAT GAGGGTGGCAGGAAGAGAAGGGCGCTTTCTGTCTGCCGGGGTCGCAGCGCGAG AGGGCAGTGCCATGTTCCTCTCCATCCTAGTGGCGCTGTGCCTGTGGCTGCAC CTGGCGCTGGGCGTGCGCGGCGCGCCCTGCGAGGCGGTGCGCATCCCTATGTG CCGGCACATGCCCTGGAACATCACGCGGATGCCCAACCACCTGCACCACAGCA CGCAGGAGAACGCCATCCTGGCCATCGAGCAGTACGAGGAGCTGGTGGACGTG AACTGCAGCGCCGTGCTGCGCTTCTTCCTCTGTGCCATGTACGCGCCCATTTG CACCCTGGAGTTCCTGCACGACCCTATCAAGCCGTGCAAGTCGGTGTGCCAAC GCGCGCGCGACGACTGCGAGCCCCTCATGAAGATGTACAACCACAGCTGGCCC GAAAGCCTGGCCTGCGACGAGCTGCCTGTCTATGACCGTGGCGTGTGCATCTC GCCTGAAGCCATCGTCACGGACCTCCCGGAGGATGTTAAGTGGATAGACATCA CACCAGACATGATGGTACAGGAAAGGCCTCTTGATGTTGACTGTAAACGCCTA AGCCCCGATCGGTGCAAGTGTAAAAAGGTGAAGCCAACTTTGGCAACATATCT CAGCAAAAACTACAGCTATGTTATTCATGCCAAAATAAAAGCTGTGCAGAGGA GTGGCTGCAATGAGGTCACAACGGTGGTGGATGTAAAAGAGATCTTCAAGTCC TCATCACCCATCCCTCGAACTCAAGTCCCGCTCATTACAAATTCTTCTTGCCA GTGTCCACACATCCTGCCCCATCAAGATGTTCTCATCATGTGTTACGAGTGGC GCTCAAGGATGATGCTTCTTGAAAATTGCTTAGTTGAAAAATGGAGAGATCAG CTTAGTAAAAGATCCATACAGTGGGAAGAGAGGCTGCAGGAACAGCGGAGAAC AGTTCAGGACAAGAAGAAAACAGCCGGGCGCACCAGTCGTAGTAATCCCCCCA AACCAAAGGGAAAGACTCCTGCTCCCAAACCAGCCAGTCCCAAGAAGAACATT AAAACTAGGAGTGCCCAGAAGAGAACAAACCCGAAAAGAGTGTGAGCTAACTA GTTTCCAAAGCGGAGACTTCCGACTTCCTTACAGGATGAGGCTGGGCATTGCC TGGGACAGCCTATGTAAGGCCATGTGCCCCTTGCCCTAACAACTCACTGCAGT GCTCTTCATAGACACATCTTGCAGCATTTTTCTTAAGGCTATGCTTCAGTTTT TCTTTGTAAGCCATCACAAGCCATAGTGGTAGGTTTGCCCTTTGGTACAGAAG GTGAGTTAAAGCTGGTGGAAAAGGCTTATTGCATTGCATTCAGAGTAACCTGT GTGCATACTCTAGAAGAGTAGGGAAAATAATGCTTGTTACAATTCGACCTAAT ATGTGCATTGTAAAATAAATGCCATATTTCAAACAAAACACGTAATTTTTTTA CAGTATGTTTTATTACCTTTTGATATCTGTTGTTGCAATGTTAGTGATGTTTT AAAATGTGATCGAAAATATAATGCTTCTAAGAAAGGAACAGTAGTGGAATGAA TGTCTAAAAGATCTTTATGTGTTTATGGTCTGCAGAAGGATTTTTGTGATGAA AGGGGATTTTTTGAAAAA

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

Table 2^C

MFLSI VALCL HLALGVRGAPCEAVRIPMCRHMP NITR PNH HHSTQENAILAIEQYEELVDVNC SAV RFFLCAMYAPICT EFLHDPIKPCKSVCQRARDDCEP MK YNHSWPESLACDELPVYDRGVCIS PEAIVTD PEDVKWIDITPDMMVQERPLDVDCKRLSPDRCKCKKVKPTLATYLSKNYSYVIHAKIKAVQ RSGCNEVTTWDVKEIFKSSSPIPRTQVPLITNSSCQCPHI PHQDVLIMCYEWRΞRMM LENC VEK RDQ SKRSIQWEER QEQRRTVQDKKKTAGRTSRSNPPKPKGKTPAPKPASPKKNIKTRΞAQKRTNPKR V

An ammo acid sequence of a human FRAZZLED (SEQ ED NO: 2). One polynucleotide of the present invention encoding FRAZZLED may be obtained using standard cloning and screening, from a cDNA library deπved from mRNA in cells of human osteoblasts using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252: 1651- 1656; Adams, M.D. et al, Nature, (1992) 555:632-634; 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 hbraπes or can be synthesized using well known and commercially available techniques.

The nucleotide sequence encoding FRAZZLED polypeptide of SEQ ED NO:2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 171 to 1208 of SEQ ED 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 ED NO:2.

When the polynucleotides of the invention are used for the recombinant production of FRAZZLED 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 puπfication of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidme peptide, as provided m the pQE vector (Qiagen, Inc.) and descπbed in Gentz et al , 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 transcπbed, non-translated sequences, splicing and polyadenylation signals, πbosome binding sites and sequences that stabilize mRNA.

Further preferred embodiments are polynucleotides encoding FRAZZLED vaπants compπsing the amino acid sequence of FRAZZLED polypeptide of Table 2 (SEQ ED NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acid residues are substituted, deleted or added, m any combination.

Among the preferred polynucleotides of the present invention is contained in Table 3 (SEQ ED NO: 3) encoding the amino acid sequence of Table 4 (SEQ ID NO: 4).

Table 3^C

CGCGGAGTCC GGGACTGGAG CTGCCCGGGC GGGTTCGCGC CCCGAAGGCT

51 GAGAGCTGGC GCTGCTCGTG CCCTGTGTGC CAGACGGCGG AGCTCCGCGG

101 CCGGACCCCG CGGCCCCGCT TTGCTGCCGA CTGGAGTTTG GGGGAAGAAA 151 CTCTCCTGCG CCCCAGAGGA TTTCTTCCTC GGCGAAGGGA CAGCGAAAGA

201 TGAGGGTGGC AGGAAGAGAA GGGCGCTTTC TGTCTGCCGG GGTCGCAGCG

251 CGAGANGGCA GTGCCATGTT CCTCTCCATC CTAGTGGCGC TGTGCCTGTG

301 GCTGTCACCT GGGGCTGGGC GTGTCGCGGC GCCCCTGACG AGGTCGGTGC

351 GCATCCCTAT GTGCCGGCAC ATGCCCTGGA ACATCACGCG GATGCCCAAC

401 CACCTGCACC ACAGCACGCA GGAGAACGCC ATCCTGGCCA TCGAGCAGTA

451 CGAGGAGCTG GTGGACGTGA ACTGCAGCGC CGTGCTGCGC TTCTTCCTCT

501 GTGCCATGTA CGCGCCCATT TGCACCCTGG AGTTCCTGCA CGACCCTATC

551 AAGCCGTGCA AGTCGGTGTG CCAACGCGCG CGCGACGACT GCGAGCCCCT

501 CATGAAGATG TACAACCACA GCTGGCCCGA AAGCCTGGCC TGCGACGAGC

651 TGCCTGTCTA TGACCGTGGC GTGTGCATCT CGCCTGAAGC CATCGTCACG

701 GACCTCCCGG AGGATGTTAA GTGGATAGAC ATCACACCAG ACATGATGGT

751 ACAGGAAAGG CCTCTTGATG TTGACTGTAA ACGCCTAAGC CCCGATCGGT

801 GCAAGTGTAA AAAGGTGAAG CCAACTTTGG CAACATATCT CAGCAAAAAC

851 TACAGCTATG TTATTCATGC CAAAATAAAA GCTGTGCAGA GGAGTGGCTG

901 CAATGAGGTC ACAACGGTGG TGGATGTAAA AGAGATCTTC AAGTCCTCAT

951 CACCCATCCC TCGAACTCAA GTCCCGCTCA TTACAAATTC TTCTTGCCAG

1001 TGTCCACACA TCCTGCCCCA TCAAGATGTT CTCATCATGT GTTACGAGTG

1051 GCGCTCAAGG ATGATGCTTC TTGAAAATTG CTTAGTTGAA AAATGGAGAG

1101 ATCAGCTTAG TAAAAGATCC ATACAGTGGG AAGAGAGGCT GCAGGAACAG

1151 CGGAGAACAG TTCAGGACAA GAAGAAAACA GCCGGGCGCA CCAGTCGTAG

1201 TAATCCCCCC AAACCAAAGG GAAAGACTCC TGCTCCCAAA CCAGCCAGTC 1251 CCAAGAAGAA CATTAAAACT AGGGGTCGAC CCACGCGTCC GAAGAGAACA

1301 AACCCGAAAA GAGTGTGAGC TAACTAGTTT CCAAAGCGGA GACTTCCGAC

1351 TTCCTTACAG GATGAGGCTG GGCATTGCCT GGGACAGCCT ATGTAAGGCC

1401 ATGTGCCCCT TGCCCTAACA ACTCACTGCA GTGCTCTTCA TAGACACATC

1451 TTGCAGCATT TTTCTTAAGG CTATGCTTCA GTTTTTCTTT GTAAGCCATC

1501 ACAAGCCATA GTGGTAGGTT TGCCCTTTGG TACAGAAGGT GAGTTAAAGC

1551 TGGTGGAAAA GGCTTATTGC ATTGCATTCA GAGTAACCTG TGTGCATACT

1601 CTAGAAGAGT AGGGAAAATA ATGCTTGTTA CAATTCGACC TAATATGTGC

1651 ATTGTAAAAT AAATGCCATA TTTCAAACAA AACACGTAAT TTTTTTACAG

1701 TATGTTTATT ACCTTTTGAT ATCTGTTGTT GCAATGTTAG TGATGTTTAA

1751 AATGTGATCG AAAATATAAT GCTTCTAAGA AGGAACAGTA GTGGGAATGA

1801 ATGTCTAAAA GATCTTTATG TGTTTATGGT CTGCCAGAAG GATTTTTGTG

1851 ATGAAAGGGG ATTTTTTGAA AAATCTAGGG GAAGTAGCCA TATGGGAAAA

1901 TTATNATGTG TCTTTTTTAC ATGGACTTCC AGCTCCGTTT TTTGGCTNGG

1951 AAACTCTNAA AACCAAANT

A partial nucleotide sequence of a human FRAZZLED (SEQ ID NO: 3).

Table 4°

1 MRVAGREGRF LSAGVAARXG SAMFLSI VA LCLWLSPGAG RVAAPLTRSV

51 RIPMCRHMPW NITRMPNHLH HSTQENAILA IEQYEELVDV NCSAVLRFFL

101 CAMYAPICTL EFLHDPIKPC KSVCQRARDD CEPLMKMYNH SWPESLACDE

151 LPVYDRGVCI SPEAIVTDLP EDVKWIDITP DMMVQERPLD VDCKRLSPDR

201 CKCKKVKPTL ATYLSK YSY VIHAKIKAVQ RSGCNEVTTV VDVKEIFKSS

251 SPIPRTQVPL ITNSSCQCPH ILPHQDVLIM CYEWRSRMML LENCLVEKWR

301 DQLSKRSIQ EERLQEQRRT VQDKKKTAGR TSRSNPPKPK GKTPAPKP S

351 PKKNIKTRGR PTRPKRTNPK RV

^d A partial ammo acid sequence of a human FRAZZLED (SEQ ID NO: 4).

The present invention further relates to polynucleotides that hybπdize to the herein above- descπbed sequences. In this regard, the present invention especially relates to polynucleotides which hybπdize under stπngent conditions to the here above-descπbed polynucleotides. As herein used, the term "stπngent conditions" means hybπdization will occur only if there is at least 80%, and preferably at least 90%>, and more preferably at least 95%, yet even more preferably 91-99% identity between the sequences. Polynucleotides of the invention, which are identical or sufficiently identical to a nucleotide sequence contained m SEQ ED NO: 1 or a fragment thereof (including that of SEQ ED NO:3), may be used as hybπdization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding FRAZZLED 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 similaπty to the FRAZZLED gene. Such hybπdization 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 compπse 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 FRAZZLED polypeptide, including homologs and orthologs from species other than human, compπses the steps of screening an appropπate library under stingent hybπdization conditions with a labeled probe having the SEQ ED NO: 1 or a fragment thereof (including that of SEQ ED NO: 3), and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybπdization techniques are well known to those of skill in the art. Thus in another aspect, FRAZZLED polynucleotides of the present invention further include a nucleotide sequence compπsing a nucleotide sequence that hybπdize under stπngent condition to a nucleotide sequence having SEQ ED NO: 1 or a fragment thereof (including that of SEQ ED NO:3). Also included with FRAZZLED polypeptides are polypeptide compπsing amino acid sequence encoded by nucleotide sequence obtained by the above hybπdization condition. Stπngent hybπdization conditions are as defined above or, alternatively, conditions under overnight incubation at

42°C m a solution compπsing: 50% formamide, 5xSSC (150mM NaCl, 15mM tπsodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65°C.

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

Vectors, Host Cells, Expression

The present invention also relates to vectors which compπse a polynucleotide or polynucleotides of the present mvention, 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 deπved from the DNA constructs of the present mvention.

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 descπbed 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 Spπng Harbor Laboratory Press, Cold Spπng Harbor, NN. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, canonic pid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropπate hosts include bacteπal cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtihs cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophύa S2 and Spodoptera Sf cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells. A great vaπety of expression systems can be used. Such systems include, among others, chromosomal, episomal and virus-deπved systems, e.g., vectors deπved from bacteπal plasmids, from bacteπophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SN40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors deπved from combinations thereof, such as those deπved from plasmid and bacteπophage 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 appropπate nucleotide sequence may be inserted mto an expression system by any of a vaπety of well-known and routine techniques, such as, for example, those set forth m Sambrook et al, MOLECULAR CLONING, A LABORATORY

MANUAL (supra).

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the peπplasmic space or mto the extracellular environment, appropπate 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 FRAZZLED 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 pπor to use in the screening assay If FRAZZLED polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced mtracellularly, the cells must first be lysed before the polypeptide is recovered.

FRAZZLED polypeptides can be recovered and puπfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, amon 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 puπfication. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured duπng isolation and or puπfication. Diagnostic Assays

This invention also relates to the use of FRAZZLED polynucleotides for use as diagnostic reagents. Detection of a mutated form of FRAZZLED 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 FRAZZLED. Individuals carrying mutations in the FRAZZLED gene may be detected at the DNA level by a vaπety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, uπne, saliva, tissue biopsy or autopsy mateπal. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques pπor 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 in compaπson to the normal genotype. Point mutations can be identified by hybπdizmg amplified DNA to labeled FRAZZLED 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 denatuπng 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 ( 1985) 85 : 4397-4401. In another embodiment, an array of o gonucleotides probes compπsing FRAZZLED 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 vaπety of questions m molecular genetics including gene expression, genetic linkage, and genetic vaπabihty. (See for example: M.Chee et al, Science, Vol 274, pp 610-613 (1996)). The diagnostic assays offer a process for diagnosing or determining a susceptibility to chronic and acute inflammation, arthπtis, osteoarthπtis and other osteopenic conditions, Paget's disease, rheumatoid arthπtis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, bram injury, AIDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g., lymphoprohferative disorders), atherosclerosis, and Alzheimers disease, through detection of mutation in the FRAZZLED gene by the methods descπbed.

In addition, chronic and acute inflammation, arthπtis, osteoarthπtis and other osteopenic conditions, Paget's disease, rheumatoid arthπtis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g , lymphoprohferative disorders), atherosclerosis, and Alzheimers disease, can be diagnosed by methods comprising determining from a sample deπved from a subject an abnormally decreased or increased level of FRAZZLED polypeptide or FRAZZLED mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known m 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 FRAZZLED polypeptide, in a sample deπved from a host are well-known to those of skill in the art.

Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, lmmunocytochemistry and ELISA assays.

Thus in another aspect, the present invention relates to a diagonostic kit for a disease or suspectabihty to a disease, particularly chronic and acute inflammation, arthπtis, osteoarthπtis and other osteopenic conditions, Paget's disease, rheumatoid arthπtis, septicemia, autoimmune diseases

(e.g., inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, ADDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g., lymphoprohferative disorders), atherosclerosis, and Alzheimers disease, which comprises:

(a) a FRAZZLED polynucleotide, preferably the nucleotide sequence of SEQ ED NO: 1, or a fragment thereof ;

(b) a nucleotide sequence complementary to that of (a); (c) a FRAZZLED polypeptide, preferably the polypeptide of SEQ ED NO: 2, or a fragment thereof; or

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

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

The nucleotide sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybπdize 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, Mendehan Inheπtance 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 (coinheπtance 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.

The gene coding for FRAZZLED has been localized to the 15q21-23 locus. 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 lmmunospecific for the FRAZZLED polypeptides. The term "lmmunospecific" means that the antibodies have substantiall greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the pπor art. Antibodies generated against the FRAZZLED polypeptides can be obtained by admmisteπng the polypeptides or epitope-beaπng 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 lme cultures can be used. Examples include the hybπdoma technique (Kohler, G. and Milstem, C, Nature (1975) 256:495-497), the tπoma technique, the human B-cell hybπdoma technique (Kozbor et al , Immunology Today (1983) 4:72) and the EBN-hybπdoma 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-descπbed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to puπfy the polypeptides by affinity chromatography.

Antibodies against FRAZZLED polypeptides may also be employed to treat chronic and acute inflammation, arthπtis, osteoarthπtis and other osteopenic conditions, Paget's disease, rheumatoid arthπtis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g., lymphoprohferative disorders), atherosclerosis, and Alzheimers disease, among others.

Vaccines

Another aspect of the mvention relates to a method for inducing an immunological response m a mammal which compπses inoculating the mammal with FRAZZLED polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from chronic and acute inflammation, arthπtis, osteoarthπtis and other osteopenic conditions, Paget's disease, rheumatoid arthπtis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AEDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g., lymphoprohferative disorders), atherosclerosis, and Alzheimers disease, among others. Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering FRAZZLED polypeptide via a vector directing expression of FRAZZLED 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 m that mammal to a FRAZZLED polypeptide wherem the composition compπses a FRAZZLED polypeptide or FRAZZLED gene. The vaccine formulation may further comprise a suitable carrier.

Since FRAZZLED polypeptide may be broken down m the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, mtradermal etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous steπle injection solutions which may contain anti-oxidants, buffers, bacteπostats and solutes which render the formulation mstonic with the blood of the recipient; and aqueous and non-aqueous steπle suspensions which may include suspending agents or thickening agents. The formulations may be presented m unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiπng only the addition of the steπle 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 FRAZZLED 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 FRAZZLED 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 hbraπes, and natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors, etc., as the case may be, of the polypeptide of the present invention; or may be structural or functional mimetics of the polypeptide of the present invention. See Co gan et al, Current Protocols in Immunology l(2):Chapter 5 (1991).

FRAZZLED polypeptides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate FRAZZLED polypeptide on the one hand and which can inhibit the function of FRAZZLED polypeptide on the other hand. In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as chronic and acute inflammation, arthπtis, osteoarthπtis and other osteopenic conditions, Paget's disease, rheumatoid arthπtis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases

(e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g., lymphoprohferative disorders), atherosclerosis, and Alzheimers disease.

Antagonists may be employed for a vaπety of therapeutic and prophylactic purposes for such conditions as chronic and acute inflammation, arthπtis, osteoarthπtis and other osteopenic conditions, Paget's disease, rheumatoid arthπtis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, bram injury, AEDS, metabolic and other bone diseases (e.g., osteoporosis), cancer mcludmg bone and cartilage cancers and related tumors (e.g., lymphoprohferative disorders), atherosclerosis, and Alzheimers disease In general, such screening procedures may involve using appropπate cells which express the FRAZZLED polypeptide or respond to FRAZZLED polypeptide of the present invention. Such cells include cells from mammals, yeast, Drosophila or E coll. Cells which express the FRAZZLED polypeptide (or cell membrane containing the expressed polypeptide) or respond to FRAZZLED 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 FRAZZLED activity.

The FRAZZLED 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 FRAZZLED mRNA and protein in cells. For example, an enzyme linked immunosorbent assay (ELISA) may be constructed for measuring secreted or cell associated levels of FRAZZLED protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents (i.e. antagonists or agonists) which may inhibit or enhance the production of FRAZZLED from suitably manipulated cells or tissues. The FRAZZLED protein may be used to identify membrane bound or soluble hgand or receptors through standard ligand/receptor binding techniques known in the art. These include, but are not limited to, gand binding and crosshnkmg assays m which the FRAZZLED is labeled with a radioactive isotope (e.g., 1251), chemically modified (e.g., biotinylated), or fused to a peptide sequence suitable for detection or puπfication, 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 puπfication and cloning of the receptor, these binding assays can be used to identify agonists and antagonists of FRAZZLED which compete with the binding of FRAZZLED to its receptors or ligands.

The above binding assays can be used to identify cells which respond biologically to FRAZZLED. Cells which respond to FRAZZLED may show changes in mtracellular signal transduction pathways and in gene expression. These changes can be used in screens for agonists or antagonists which mimic or inhibit the action of FRAZZLED, respectively.

The assays may simply test binding of a candidate compound wherein adherence to the cells beaπng the FRAZZLED 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 FRAZZLED polypeptide, using detection systems appropriate to the cells beaπng the FRAZZLED polypeptide. Inhibitors of activation are generally assayed m 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 FRAZZLED polypeptide to form a mixture, measuπng FRAZZLED activity in the mixture, and comparing the FRAZZLED activity of the mixture to a standard.

The FRAZZLED 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 FRAZZLED mRNA and protein in cells. For example, an ELISA may be constructed for measuring secreted or cell associated levels of FRAZZLED 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 FRAZZLED (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.

The FRAZZLED 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, hgand binding and crosshnkmg assays in which the FRAZZLED is labeled with a radioactive isotope (e.g., 1251), chemically modified (e.g., 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 puπfication and clonmg of the receptor, these binding assays can be used to identify agonists and antagonists of FRAZZLED which compete with the binding of FRAZZLED to its receptors, if any. Standard methods for conducting screening assays are well understood m the art.

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

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

(a) a FRAZZLED polypeptide, preferably that of SEQ ED NO:2,

(b) a recombinant cell expressing a FRAZZLED polypeptide, preferably that of SEQ ED NO:2; (c) a cell membrane expressing a FRAZZLED polypeptide; preferably that of SEQ ED NO: 2; or

(d) antibody to a FRAZZLED polypeptide, preferably that of SEQ ID NO: 2.

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

Prophylactic and Therapeutic Methods This invention provides methods of treating abnormal conditions such as, chronic and acute inflammation, arthπtis, osteoarthπtis and other osteopenic conditions, Paget's disease, rheumatoid arthπtis, septicemia, autoimmune diseases (e.g., inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, bram injury, AEDS, metabolic and other bone diseases (e.g., osteoporosis), cancer including bone and cartilage cancers and related tumors (e.g., lymphoprohferative disorders), atherosclerosis, and Alzheimers disease, related to both an excess of and insufficient amounts of FRAZZLED polypeptide activity.

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

In another approach, soluble forms of FRAZZLED polypeptides still capable of binding the hgand in competition with endogenous FRAZZLED polypeptide may be administered. Typical embodiments of such competitors comprise fragments of the FRAZZLED polypeptide. In still another approach, expression of the gene encoding endogenous FRAZZLED 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 Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988). Alternatively, ohgonucleotides 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 α/., Science (1991) 251:1360. These o gomers can be administered per se or the relevant ohgomers can be expressed in vivo. For treating abnormal conditions related to an under-expression of FRAZZLED and its activity, several approaches are also available. One approach compπses admimstenng to a subject a therapeutically effective amount of a compound which activates FRAZZLED polypeptide, i.e., an agonist as descπbed above, combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of FRAZZLED by the relevant cells in the subject. For example, a polynucleotide of the invention may be engineered for expression m 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 administered to a subject for eng eenng 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 FRAZZLED polypeptides in combination with a suitable pharmaceutical earner.

Formulation and Administration

Peptides, such as the soluble form of FRAZZLED polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical earner. Such formulations compnse a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excφient. Such earners 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 withm the skill of the art. The invention further relates to pharmaceutical packs and kits compπsing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.

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

Preferred forms of systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or mtrapeπtoneal, can be used. Alternative means for systemic administration include transmucosal and transdermal administration using 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, m 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 m view of the vaπety of compounds available and the diffeπng efficiencies of vaπous routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Naπations in these dosage levels can be adjusted using standard empincal routines for optimization, as is well understood m the art.

Polypeptides used in treatment can also be generated endogenously in the subject, m treatment modalities often referred to as "gene therapy" as descπbed above. Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DΝA or RΝA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.

Example 1 A partial clone encoding FRAZZLED (EST # 2105409) was identified through a search of a commercial EST database using the ammo acid sequence of a previously identified member of the Fnzzled family, Frzb. This clone was then fully sequenced and the full length sequence of this clone shared 73.8% identity with human Frzb (Hoang, et al., J. Biol. Chem. 271(42): 26131-26137 (1996)]. The clone encoding FRAZZLED was found in an osteoblast cell library. This gene is also expressed in chondrosarcoma, osteosarcoma, osteoclastoma, synovial fibroblasts, hodgkin's lymphoma, ovary, uterus, fetal lung, adipose and pancreatic tumor. Two ESTs corresponding to this gene from Soares ΝhHMPu SI cDΝA hbranes are found in the public EST database.

Example 2- Tissue distribution of FRAZZLED gene expression

Northern blot analysis was earned out to examine the expression of FRAZZLED mRNA expression m human tissues. A human multiple cell and multiple tissue northern blot (Clonetech

Laboratoπes, Inc.) were hybπdized with the entire nucleotide sequence of FRAZZLED cDNA labeled with ³²P using the redipπme DNA labeling system™ (Amersham Life Sciences), according to manufacturer's mstructiuons. Hybndization and washes were earned out according to manufacturer's mstructiuons and the blot was exposed to film at -70°C for 72 hours. FRAZZLED was expressed at high levels in ovary, testes and spleen. It was also expressed moderately m prostate, small intestine, colon, skeletal muscle and heart and at much lower levels in thymus, placenta, lung, kidney and pancreas. The in situ hybridization results are as follows: FRAZZLED in situ hybridization

^*+/- = weak positive, + = positive, ++ = strong positive

Example 3 - Raising of antibodies

Anti- peptide antibodies were raised to unique sequences in FRAZZLED and their selectivity for FRAZZLED was confirmed using either peptides (by ELISA) or baculovirus- expressed whole protein (by Western blotting). The peptide sequences used to raise polyclonal antibodies are as follows:

1. N-Gln-Glu-Gln-Arg-Arg-Thr-Val-Gln-Asp-Lys-Lys-Lys-Thr-Ala-C (SEQ ID NO:5) (QEQRRTVQDKKKTA - amino acids 292-305)

2. N-Lys-Asn-Ile-Lys-Thr-Arg-Ser-Ala-Gln-Lys-Arg-Thr-Asn-Pro-C (SEQ ID NO:6) (KNIKTRSAQKRTNP - amino acids- 330-343) Preincubation of the anti-FRAZZLED antibody with the appropriate peptide blocked the reactivity in both these systems.

The studies with FRAZZLED suggest that it is expressed in bone and cartilage -related tissues and may play a role in chondrocyte and osteoblast function. Recent data on a rat FRAZZLED homologue (92.5% identity with the human protein) shows that it is upregulated in rat tissues (mammary gland, ovary, and prostate) during physiological apoptosis. Interestingly,

FRAZZLED also appears to be highly expressed in these tissues, and in chondrocytes associated with fissuring in OA cartilage. Therefore, it may play a similar role in human cartilage and/or bone. All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

Claims

What is claimed is:

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

2. The polynucleotide of claim 1 wherem said polynucleotide comprises the nucleotide sequence contained in SEQ ED NO:l encoding the FRAZZLED polypeptide of SEQ ED NO2.

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

4. The polynucleotide of claim 3 which is polynucleotide of SEQ ED 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 FRAZZLED polypeptide compπsing an amino acid sequence, which has at least 80% identity with the polypeptide of SEQ ED 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 FRAZZLED polypeptide comprising cultuπng 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 FRAZZLED polypeptide thereof comprising transforming or transfectmg a host cell with the expression system of claim 6 such that the host cell, under appropπate culture conditions, produces a FRAZZLED polypeptide.

10. A FRAZZLED polypeptide comprising an ammo acid sequence which is at least 80%) identical to the ammo acid sequence of SEQ ED NO:2 over its entire length.

11. The polypeptide of claim 10 which comprises the ammo acid sequence of SEQ ID

NO.2

12. An antibody lmmunospecific for the FRAZZLED polypeptide of claim 10.

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

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

(b) providing to the subject an isolated polynucleotide compπsing a nucleotide sequence that has at least 80% identity to a nucleotide sequence encoding the FRAZZLED polypeptide of SEQ ED 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 FRAZZLED polypeptide of claim 10 comprising: (a) administeπng to the subject a therapeutically effective amount of an antagonist to said polypeptide; and/or

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

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

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

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said FRAZZLED polypeptide in the genome of said subject; and/or (b) analyzing for the presence or amount of the FRAZZLED polypeptide expression in a sample deπved from said subject.

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

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

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

20. A polypeptide comprising the ammo acid sequence set forth in SEQ ED NO:5.

21. A polypeptide comprising the ammo acid sequence set forth m SEQ ED NO:6.

22. An antibody to the polypeptide of claim 20.

23. An antibody to the polypeptide of claim 21.