WO2000055202A1 - A member of the frzb family, frazzled - Google Patents

Abstract

Bos taurus Frazzled polypeptides and polynucleotides and method for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for screening for compounds which either agonize or antagonize Bos taurus Frazzled. Such compounds are expected to be useful in treatment of human diseases, including, but not limited to: chronic and acute inflammation, arthritis, rheumatoid arthritis, osteoarthritis, septicimia, autoimmune diseases (e.g., inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stoke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (e.g., osteoporosis), cancer (e.g., lymphoproliferative disorders), atherosclerosis, Alzheimer's disease, retinitis pigmentosa, macular degeneration, and any other degenerative eye diseases.

Description

A Member of the Frzb Family, Frazzled

Cross-Reference to Related Application

This application claims the benefit of priority of U S Provisional Application No. 60/125,038, filed on March 18, 1999, the entire disclosure of which is incorporated herein by reference Field of the Invention

This mvention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in identifying compounds that may be agonists and/or antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides.

Background of the Invention The drug discovery process is currently undergoing a fundamental revolution as it embraces

'functional genomics,' that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on 'positional cloning'. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position. Functional genomics relies heavily on the vaπous tools of biomformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and characteπze further genes and their related polypeptides/protems, as targets for drug discovery

Accumulating evidence suggests that the secreted fπzzled-related proteins (SFRPs) may control morphogenesis by binding Wnts proteins extracellularly (Wang, et al , Cell, 88.757-766

(1997), Leyns, et al , Cell, 88:747-756 (1997)), thus antagonizing their ability to signal through the plasma membrane-bound frizzled receptors. Furthermore, it has been demonstrated that at least one of the SFRPs, termed FrzB-1, can interact with Wnt 5 A but does not appear to act as an antagonist (Lm, et al , Proc Natl Acad Set USA, 94:11196-111200 (1997)), suggesting that SFRPs may also act as chaperones for the Wnts. A prominent feature of both the fπzzled receptors and their soluble counterparts is the presence of a cysteme-πch domain (CRD) which is implicated in Wnt ligand binding (Bhanot, et al , Nature, 382.225-230 (1996))

In addition to their role in morphogenesis, recent data suggest that certain SFRP homologs, such as the SARPs (secreted apoptosis-related proteins) have the ability to either sensitize cells to apoptosis or inhibit the apoptotic response (Melkonyan, et al , Proc Natl Acad Set USA,

94.13636- 13641 (1997)) In this regard, there is mounting evidence to suggest that the SFRPs may play a role m the development of the retma m the eye (Chang, et al , Hum Molec Genet ,&' 575-583 (1999)), and that the aberrant expression of these proteins and the induction of apoptosis may lead to the development of retinal disease.

Summary of the Invention

The present invention relates to Bos taurus Frazzled, in particular Bos taurus Frazzled polypeptides and Bos taurus Frazzled polynucleotides, recombmant mateπals and methods for their production. In another aspect, the invention relates to methods for identifying agonists and antagonists/inhibitors of the Bos taurus Frazzled gene This invention further relates to the generation of in vitro and in vivo comparison data relating to the polynucleotides and polypeptides m order to predict oral absorption and pharmacokmetics in man of compounds that either agonize or antagonize the biological activity of such polynucleotides or polypeptides. Such a comparison of data will enable the selection of drugs with optimal pharmacokmetics in man, i e., good oral bioavailabihty, blood-bram barrier penetration, plasma half life, and minimum drug interaction.

The present invention further relates to methods for creating transgemc animals, which overexpress or underexpress or have regulatable expression of a Frazzled gene and "knock-out" animals, preferably mice, in which an animal no longer expresses a Frazzled gene. Furthermore, this invention relates to transgemc and knock-out animals obtained by using these methods. Such animal models are expected to provide valuable insight into the potential pharmacological and toxicological effects in humans of compounds that are discovered by the aforementioned screening methods as well as other methods. An understanding of how a Bos taurus Frazzled gene functions m these animal models is expected to provide an insight into treating and preventing human diseases including, but not limited to chronic and acute inflammation, arthritis, rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, 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 (e g , lymphoproliferative disorders), atherosclerosis, Alzheimer's disease, retmitis pigmentosa, macular degeneration, and any other degenerative eye diseases, hereinafter referred to as "the Diseases," amongst others.

Description of the Invention

In a first aspect, the present invention relates to Bos taurus Frazzled polypeptides Such polypeptides include isolated polypeptides comprising an ammo acid sequence having at least a 95% identity, most preferably at least a 97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO.2. Such polypeptides include those comprising the ammo acid of SEQ ID NO:2 (a) an isolated polypeptide encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 1;

(b) an isolated polypeptide comprising a polypeptide sequence having at least a 95%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2; (c) an isolated polypeptide comprising the polypeptide sequence of SEQ ID NO:2;(d) an isolated polypeptide having at least a 95%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;

(e) the polypeptide sequence of SEQ ID NO:2; and

(f) variants and fragments thereof; and portions of such polypeptides m (a) to (e) that generally contain at least 30 ammo acids, more preferably at least 50 ammo acids, thereof.

Polypeptides of the present invention are believed to be members of the f zzled-related proteins family of polypeptides. They are, therefore, of interest, because numerous studies suggest that members of the frizzled family play key roles in 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 originally isolated from calf articular cartilage. Furthermore, it has been proposed that at sites of active bone and cartilage remodeling, exemplified by osteoarthritis 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-hke 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. Furthermore, the polypeptides of the present invention can be used to establish assays to predict oral absorbtion and pharmacokmetics m man and thus enhance compound and formulation design, among others. These properties, either alone or in the aggregate, are hereinafter referred to as "Bos taurus Frazzled activity" or "Bos taurus Frazzled polypeptide activity" or "biological activity of Frazzled." Preferably, a polypeptide of the present invention exhibits at least one biological activity of Bos taurus Frazzled

Polypeptides of the present mvention also include vaπants of the aforementioned polypeptides, including alleles and splice vaπants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative. Particularly preferred vaπants are those in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acids are inserted, substituted, or deleted, in any combination. Particularly preferred pπmers will have between 20 and 25 nucleotides. Preferred fragments of polypeptides of the present mvention include an isolated polypeptide comprising an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO 2, or an isolated polypeptide comprising an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO.2.

Also preferred are biologically active fragments which are those fragments that mediate activities of Frazzled, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigemc or lmmunogenic m an animal, especially in a human. Particularly preferred are fragments compπsmg receptors or domains of enzymes that confer a function essential for viability of Bos taurus or the ability to initiate, or maintain cause the Diseases in an individual, particularly a human.

Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these vaπants may be employed as intermediates for producing the full-length polypeptides of the invention. The polypeptides of the present invention may be in the form of a "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 that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance, multiple histidine residues, or an additional sequence for stability during recombmant production. The present invention also includes vaπants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characteπstics. Typical substitutions are among Ala, Nal, 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, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added in any combmation

Polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurπng 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. In a further aspect, the present invention relates to Bos taurus Frazzled polynucleotides. Such polynucleotides include isolated polynucleotides compπsing a nucleotide sequence encodmg a polypeptide having at least a 95% identity, to the ammo acid sequence of SEQ ID NO .2, over the entire length of SEQ ID NO 2 In this regard, polypeptides which have at least a 97% identity are highly preferred, while those with at least a 98-99% identity are more highly preferred, and those with at least a 99% identity are most highly preferred. Such polynucleotides include a polynucleotide compnsmg the nucleotide sequence contained in SEQ ID NO: 1 encoding the polypeptide of SEQ ID NO:2. Further polynucleotides of the present invention include isolated polynucleotides compnsmg a nucleotide sequence having at least a 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, over the entire coding region. In this regard, polynucleotides which have at least a 97% identity are highly prefeπed, while those with at least a 98-99% identity are more highly preferred, and those with at least a 99% identity are most highly preferred. Further polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence having at least a 95% identity, to SEQ ID NO: l over the entire length of SEQ ID NO.1. In this regard, polynucleotides which have at least a 97% identity are highly preferred, while those with at least a 98-99% identify are more highly preferred, and those with at least a 99% identity are most highly preferred. Such polynucleotides include a polynucleotide compnsmg the polynucleotide of SEQ ID NO: 1 , as well as the polynucleotide of SEQ ID NO: 1.

The mvention also provides polynucleotides which are complementary to all the above described polynucleotides.

The nucleotide sequence of SEQ ID NO:l shows homology with Homo sapiens fπzzled-related protein [frpHE]. The nucleotide sequence of SEQ ID NO:l is a cDNA sequence and compπses a polypeptide encoding sequence (nucleotide 1 to 1038) encoding a polypeptide of 346 ammo acids, the polypeptide of SEQ ID NO.2. The nucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence of SEQ ID NO:l or it may be a sequence other than SEQ ED NO: 1, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2. The polypeptide of SEQ ID NO:2 is structurally related to other proteins of the fπzzled-related proteins family, having homology and/or structural sirmlaπty with

Homo sapiens fπzzled-related protein (frpHE).

Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one Frazzled activity

Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library derived from mRNA in cells of Bos taurus bovme heart, spleen, lung and kidney, 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 hbranes or can be synthesized using well known and commercially available techniques. When polynucleotides of the present invention are used for the recombmant production of polypeptides of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence, or other fusion peptide portions. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the mvention, the marker sequence is a hexa-histidine 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 compπse non-coding 5 ' and 3 ' sequences, such as transcnbed, non-translated sequences, splicing and polyadenylation signals, πbosome binding sites and sequences that stabilize mRNA.

Further embodiments of the present invention include polynucleotides encoding polypeptide vanants that compnse the amino acid sequence of SEQ ID NO:2 and in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 1 to 1 or 1 ammo acid residues are substituted, deleted or added, in any combination. Particularly preferred probes will have between 30 and 50 nucleotides, but may have between 100 and 200 contiguous nucleotides of the polynucleotide of SEQ ID NO: 1

A preferred embodiment of the invention is a polynucleotide of consisting of or compnsmg nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 1038 set forth in SEQ ID NO:l of Table 1, both of which encode a Frazzled polypeptide The invention also includes a polynucleotide consisting of or compπsmg a polynucleotide of the formula.

X-(R₁)_mJR₂)-(R₃)_n-Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occuπence of Rj and

R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R₂ is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof. In the polynucleotide formula above, R₂ is oriented so that its 5' end nucleic acid residue is at the left, bound to Ri and its 3' end nucleic acid residue is at the right, bound to R3. Any stretch of nucleic acid residues denoted by either R and/or R₂, where m and or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Where, in a prefeπed embodiment, X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double- stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary In another prefeπed embodiment m and/or n is an integer between 1 and 1000. Other prefeπed embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500

Polynucleotides that are identical, or are substantially identical to a nucleotide sequence of SEQ ID NO.1 , may be used as hybridization probes for cDNA and genomic DNA or as pnmers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present mvention and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than Bos taurus) that have a high sequence identity to SEQ ID NO.1 Typically these nucleotide sequences are 95% identical to that of the referent. Prefeπed probes or pnmers will generally compnse at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides, and may even have at least 100 nucleotides. Particularly prefeπed pnmers will have between 20 and 25 nucleotides. A polynucleotide encoding a polypeptide of the present invention, mcludmg homologs and orthologs from a species other than Bos taurus, may be obtained by a process compnsmg the steps of screening an appropnate library under stnngent hybridization conditions with a labeled probe having the sequence of SEQ ID NO.1 or a fragment thereof, preferably of at least 15 nucleotides m length, and isolating full-length cDNA and genomic clones compnsmg said polynucleotide sequence. Such hybπdization techniques are well known to the skilled artisan. Prefeπed stringent hybndization conditions include overnight incubation at 42°C m a solution compnsmg: 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 0 lx SSC at about 65°C Thus, the present invention also includes isolated polynucleotides, preferably of at least 100 nucleotides in length, obtained by screening an appropriate library under stnngent hybndization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides

The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5' end of the cDNA. This is a consequence of reverse transcriptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remain attached to the template during the polymerization reaction), failing to complete a DNA copy of the mRNA template during 1st strand cDNA synthesis

There are several methods available and well known to those skilled m the art to obtain full- length cDNAs, or extend short cDNAs, for example, those based on the method of Rapid

Amplification of cDNA ends (RACE) (see, for example, Frohman, et al , Proc Natl Acad Sci , USA 85, 8998-9002, 1988) Recent modifications of the technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc ), for example, have significantly simplified the search for longer cDNAs In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence gated onto each end Nucleic acid amplification

(PCR) is then earned out to amplify the 'missing' 5' end of the cDNA using a combination of gene specific and adaptor specific ohgonucleotide pnmers The PCR reaction is then repeated using 'nested' primers, that is, pnmers designed to anneal withm the amplified product (typically an adaptor specific primer that anneals further 3' m the adaptor sequence and a gene specific pnmer that anneals further 5' m the known gene sequence) The products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' pnmer

Recombmant polypeptides of the present invention may be prepared by processes well known m the art from genetically engineered host cells compnsmg expression systems Accordingly, in a further aspect, the present invention relates to expression systems compnsmg a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombmant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs denved from the DNA constructs of the present invention

For recombmant 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 Sprmg Harbor, N Y ( 1989) Prefeπed methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection Representative examples of appropπate hosts include bactenal 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 Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells. A great vanety of expression systems can be used, for instance, chromosomal, episomal and virus-deπved systems, e g , vectors deπved from bactenal plasmids, from bacteπophage, 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, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids. The expression systems may compπse control regions that regulate as well as engender expression Generally, any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropπate nucleotide sequence may be mserted into an expression system by any of a vanety of well-known and routine techniques, such as, for example, those set forth in Sambrook, et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra).

If a polypeptide of the present invention is to be expressed for use screening assays, it is generally prefeπed that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested pnor to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered m order to recover and purify the polypeptide If produced lntracellularly, the cells must first be lysed before the polypeptide is recovered.

Polypeptides of the present invention can be recovered and punfied from recombmant 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 puπfication. Well known techniques for refolding protems may be employed to regenerate active conformation when the polypeptide is denatured duπng isolation and/or punfication.

Bos taurus Frazzled gene products can be expressed in transgemc animals Animals of any species, including, but not limited to mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro-pigs, goats, and non-human pπmates, e g , baboons, monkeys, chimpanzees, may be used to generate Frazzled transgemc animals

This invention further relates to a method of producing transgemc animals, preferably Bos taurus, over-expressing Frazzled, which method may compπse the introduction of several copies of a segment compnsmg at least the polynucleotide sequence encoding SEQ ID NO:2 with a suitable promoter into the cells of a Bos taurus embryo, or the cells of another species, at an early stage.

This invention further relates to a method of producing transgemc animals, preferably Bos taurus, under-expressing or regulatably expressing Frazzled, which method may compnse the introduction of a weak promoter or a regulatable promoter (e.g. , an mducible or repressible promoter) respectively, expressibly linked to the polynucleotide sequence of SEQ ID NO:l into the cells of a Bos taurus embryo at an early stage.

This invention also relates to transgemc animals, characteπzed m that they are obtained by a method, as defined above Any technique known in the art may be used to introduce a Bos taurus Frazzled transgene into animals to produce a founder line of animals. Such techniques include, but are not limited to: pronuclear micromjection (U.S. Patent No. 4,873,191); retrovirus mediated gene transfer into germ lines (Nan der Putten, et al , Proc Natl. Acad. Sci , USA 82: 6148-6152 (1985); gene targeting in embryonic stem cells (Thompson, et al., Cell 56: 313-321 (1989); electropolation of embryos (Lo, Mol. CellBiol 3: 1803-1814 (1983); and sperm-mediated gene transfer (Lavitrano, et al, Cell 57: 717-723 (1989); etc.

For a review of such techniques, see Gordon, Intl. Rev. Cytol 115: 171-229 (1989).

A further aspect of the present invention involves gene targeting by homologous recombination m embryonic stem cells to produce a transgemc animal with a mutation in a Frazzled gene ("knock-out" mutation). In such so-called "knock-out" animals, there is inactivation of the Frazzled gene or altered gene expression, such that the animals are useful to study the function of the Frazzled gene, thus providing animals models of human disease, which are otherwise not readily available through spontaneous, chemical or iπadiation mutagenesis. Another aspect of the present invention involves the generation of so-called "knock-m" animals m which a portion of a wild-type gene is fused to the cDΝA of a heterologous gene. This invention further relates to a method of producing "knock-out" animals, preferably mice, no longer expressing Frazzled. By using standard cloning techniques, a Bos taurus Frazzled cDΝA (SEQ ID NO: 1) can be used as a probe to screen suitable hbraπes to obtain the munne Frazzled genomic DNA clone. Using the munne genomic clone, the method used to create a knockout mouse is charactenzed in that: a suitable mutation is produced in the polynucleotide sequence of the munne Frazzled genomic clone, which inhibits the expression of a gene encoding munne Frazzled, or inhibits the activity of the gene product; said modified munne Frazzled polynucleotide is introduced into a homologous segment of munne genomic DNA, combined with an appropnate marker, so as to obtain a labeled sequence compnsmg said modified munne genomic DNA, said modified munne genomic DNA compπsmg the modified polynucleotide is transfected into embryonic stem cells and coπectly targeted events selected in vitro; then said stem cells are remjected into a mouse embryo; then said embryo is implanted into a female recipient and brought to term as a chimera which transmits said mutation through the germlme; and homozygous recombmant mice are obtained at the F2 generation which are recognizable by the presence of the marker

Vaπous methods for producing mutations in non-human animals are contemplated and well known in the art. In a prefeπed method, a mutation is generated in a munne Frazzled allele by the introduction of a DNA construct compnsmg DNA of a gene encoding munne Frazzled, which munne gene contains the mutation. The mutation is targeted to the allele by way of the DNA construct. The DNA of the gene encodmg munne Frazzled compnsed in the construct may be foreign to the species of which the recipient is a member, may be native to the species and foreign only to the individual recipient, may be a construct compnsed of synthetic or natural genetic components, or a mixture of these. The mutation may constitute an insertion, deletion, substitution, or combination thereof. The DNA construct can be introduced into cells by, for example, calcium-phosphate DNA co-precipitation It is prefeπed that a mutation be introduced into cells using electroporation, micromjection, virus infection, hgand-DNA conjugation, virus-hgand-DNA conjugation, or lrposomes

Another embodiment of the instant invention relates to "knock-out" animals, preferably mice, obtained by a method of producing recombmant mice as defined above, among others

Another aspect of this invention provides for in vitro Frazzled "knock-outs", i e , tissue cultures Animals of any species, including, but not limited to mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro-pigs, goats, and non-human pnmates, e g , baboons, monkeys, chimpanzees, may be used to generate in vitro Frazzled "knock-outs" Methods for "knocking out" genes in vitro are descπbed in Galh-Tahadoros, et al , Journal of Immunological Methods 181" 1-15 (1995)

Transgemc, "knock-m", and "knock-out" animals, as defined above, are a particularly advantageous model, from a physiological point of view, for studying fπzzled-related proteins Such animals will be valuable tools to study the functions of a Frazzled gene. Moreover, such animal models are expected to provide information about potential toxicological effects m humans of any compounds discovered by an aforementioned screening method, among others An understanding of how a Bos taurus Frazzled gene functions in these animal models is expected to provide an msight into treating and preventing human diseases including, but not limited to. chronic and acute inflammation, arthritis, rheumatoid arthntis, osteoarthntis, septicemia, autoimmune diseases (e g , inflammatory bowel disease, psonasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases

(eg , osteoporosis), cancer (e g., lymphoprohferative disorders), atherosclerosis, Alzheimer's disease, retmitis pigmentosa, macular degeneration, and any other degenerative eye diseases.

Polypeptides of the present invention are responsible for many biological functions, including many disease states, m particular the Diseases mentioned herein. It is, therefore, an aspect of the invention to devise screening methods to identify compounds that stimulate (agonists) or that inhibit

(antagonists) the function of the polypeptide, such as agonists, antagonists and inhibitors. Accordingly, m a further aspect, the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function of the polypeptide. In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for the Diseases mentioned herein mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures Such agonists and antagonists so-identified may be natural or modified substrates, gands, receptors, enzymes, etc., as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Co gan, et al , CURRENT PROTOCOLS IN IMMUNOLOGY 1(2)- Chapter 5 (1991)) The screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes beanng the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound. Alternatively, a screening method may involve measuring or, qualitatively or quantitatively, detecting the competition of binding of a candidate compound to the polypeptide with a labeled competitor (e g , agonist or antagonist). Further, screening methods may test whether the candidate compound results in a signal generated by an agonist or antagonist of the polypeptide, using detection systems appropriate to cells bearing the polypeptide. Antagonists are generally assayed in the presence of a known agonist and an effect on activation by the agonist by the presence of the candidate compound is observed. Further, screening methods may simply comprise the steps of mixing a candidate compound with a solution comprising a polypeptide of the present mvention, to form a mixture, measuring Bos taurus Frazzled activity in the mixture, and comparing a Bos taurus Frazzled activity of the mixture to a control mixture which contains no candidate compound.

Polypeptides of the present invention may be employed in conventional low capacity screening methods and also high-throughput screening (HTS) formats. Such HTS formats include not only the well-established use of 96- and, more recently, 384-well microtiter plates but also emerging methods such as the nanowell method described by Schullek, et al , Anal Biochem., 246, 20-29, (1997).

Fusion proteins, such as those made from Fc portion and Bos taurus Frazzled polypeptide, as herein described, can also be used for high-throughput screening assays to identify antagonists of antagonists of the polypeptide of the present mvention (see D. Bennett, et al, J Mol Recognition, 8:52-58 (1995); and K Johanson, et al , J Bwl Chem., 270(16):9459-9471 (1995)).

The Bos Taurus 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 Bos taurus Frazzled mRNA and protein m cells For example, an ELISA may be constructed for measuπng secreted or cell associated levels of Bos taurus Frazzled protein using monoclonal and polyclonal antibodies by standard methods known m the art, and this can be used to discover agents (i.e. antagonists or agonists) which may inhibit or enhance the production of Bos taurus Frazzled from suitably manipulated cells or tissues The Bos taurus Frazzled protein may be used to identify membrane bound or soluble hgand or receptors through standard hgand/receptor binding techniques known in the art. These include, but are not limited to, hgand binding and crosslinkmg assays in which the Bos taurus Frazzled is labeled with a radioactive isotope (e.g. ¹²⁵I), chemically modified (e.g. biotmylated), 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 punfication and cloning of the receptor, these binding assays can be used to identify agonists and antagonists of Bos taurus Frazzled which compete with the binding of Bos taurus Frazzled to its receptors or hgands. The above binding assays can be used to identify cell which respond biologically to Bos taurus Frazzled. Cells which respond to Bos taurus Frazzled may show changes m mtracellular signal transduction pathways and in gene expression. These changes can be used m screens for agonists or antagonists which mimic or inhibit the action of Bos taurus Frazzled respectively

Examples of potential polypeptide antagonists include antibodies or, in some cases, ohgopeptides or proteins that are closely related to hgands, substrates, receptors, enzymes, etc , as the case may be, of a Frazzled polypeptide, eg , a fragment of a hgand, substrate, receptor, enzyme, etc. ; or small molecules which bind to a Frazzled polypeptide but do not elicit a response, so that an activity of a Frazzled polypeptide is prevented Thus, m another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, inhibitors, hgands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which compounds comprise a member selected from the group consisting of: (a) a polypeptide of the present invention,

(b) a recombmant cell expressing a polypeptide of the present invention; or

(c) a cell membrane expressing a polypeptide of the present invention, which polypeptide is preferably that of SEQ ID NO:2.

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

It will also be readily appreciated by the skilled artisan that a polypeptide of the present mvention may also be used m a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by:

(a) determining in the first instance the three-dimensional structure of the polypeptide; (b) deducing the three-dimensional structure for the likely reactive or binding sιte(s) of an agonist, antagonist or inhibitor;

(c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding or reactive site, and

(d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitors It will be further appreciated that this will normally be an iterative process

In an alternative prefeπed embodiment, the present invention relates to the use of Bos taurus Frazzled polypeptides, polynucleotides, and recombmant materials thereof in selection screens to identify compounds which are neither agonists nor antagonist/inhibitors of Bos taurus Frazzled. The data from such a selection screen is expected to provide in vitro and in vivo comparisons and to predict oral absorption, pharmacokmetics in humans. The ability to make such a comparison of data will enhance formulation design through the identification of compounds with optimal development characteπstics, i e , high oral bioavailabihty, UID (once a day) dosmg, reduced drug interactions, reduced variability, and reduced food effects, among others

The following definitions are provided to facilitate understanding of certain terms used frequently herein " Allele" refers to one or more alternative forms of a gene occurnng at a given locus in the genome.

"Fragment" of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. "Fragment" of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ ID NO: 1.

"Fusion protein" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. In one example, EP-A-0 464 discloses fusion proteins comprising various portions of constant region of immunoglobulm molecules together with another human protein or part thereof In many cases, employing an immunoglobulm Fc region as a part of a fusion protein is advantageous for use m therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e g., EP-A 0232 262] On the other hand, for some uses, it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected, and purified.

"Homolog" is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or similarity between the two sequences as hereinbefore defined. Falling withm this genenc term are the terms, "ortholog", and "paralog". "Ortholog" refers to polynucleotides/genes or polypeptide that are homologs via speciation, that is closely related and assumed to have commend descent based on structural and functional considerations. "Paralog" refers to polynucleotides/genes or polypeptide that are homologs via gene duplication, for instance, duplicated vanants within a genome.

"Identity" reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences In general, identity refers to an exact nucleotide to nucleotide or ammo acid to ammo acid coπespondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared. For sequences where there is not an exact coπespondence, a "% identity" may be determined. In general, the two sequences to be compared are aligned to give a maximum coπelation between the sequences This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment A % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length

"Similarity" is a further, more sophisticated measure of the relationship between two polypeptide sequences In general, "similarity" means a comparison between the ammo acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact coπespondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact coπespondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated 'score' from which the "% similaπty" of the two sequences can then be determined.

Methods for comparing the identity and similarity of two or more sequences are well known in the art. Thus for instance, programs available m the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J., et al, Nucleic Acids Res, 12, 387-395, 1984, available from Genetics Computer Group, Madison, Wisconsin, USA), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % similanty between two polypeptide sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman (J Mol Biol , 147:195-197, 1981, Advances m Applied Mathematics, 2, 482- 489, 1981) and finds the best single region of similarity between two sequences. BESTFIT is more suited to comparing two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer. In comparison,

GAP aligns two sequences, finding a "maximum similarity", according to the algoπthm of Neddleman and Wunsch (J Mo I Biol , 48, 443-453, 1970). GAP is more suited to companng sequences that are approximately the same length and an alignment is expected over the entire length. Preferably, the parameters "Gap Weight" and "Length Weight" used in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively.

Preferably, % identities and similanties are determined when the two sequences being compared are optimally aligned

Other programs for determining identity and/or similarity between sequences are also known in the art, for instance the BLAST family of programs (Altschul S.F., et al , J Mol Biol , 215, 403-410, 1990, Altschul S.F., et al , Nucleic Acids Res , 25:389-3402, 1997, available from the

National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA and accessible through the home page of the NCBI at www.ncbi.nlm.mh.gov) and FASTA (Pearson W R, Methods in Enzymology, 183 63-99 (1990), Pearson W R and Lipman D.J., Proc Nat Acad Sci USA, 85. 2444-2448 (1988) (available as part of the Wisconsin Sequence Analysis Package) Preferably, the BLOSUM62 ammo acid substitution matnx (Hemkoff S. and Henikoff J G.,

Proc Nat Acad Sci USA, 89: 10915-10919 (1992)) is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into ammo acid sequences before comparison. Preferably, the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a polynucleotide or a polypeptide sequence of the present invention, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore described. Alternatively, for instance, for the purposes of interpreting the scope of a claim including mention of a "% identity" to a reference polynucleotide, a polynucleotide sequence having, for example, at least 95% identity to a reference polynucleotide sequence is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference sequence. Such point mutations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion. These point mutations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides m the reference sequence or in one or more contiguous groups withm the reference sequence. In other words, to obtain a polynucleotide sequence having at least 95% identity to a reference polynucleotide sequence, up to 5% of the nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore descnbed. The same applies mutatis mutandis for other % identities such as 96%, 97%, 98%, 99% and 100%.

For the purposes of interpreting the scope of a claim including mention of a "% identity" to a reference polypeptide, a polypeptide sequence having, for example, at least 95% identity to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include up to five point mutations per each 100 ammo acids of the reference sequence. Such point mutations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion. These point mutations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups withm the reference sequence. In other words, to obtain a sequence polypeptide sequence having at least 95% identity to a reference polypeptide sequence, up to 5% of the ammo acids of the m the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore descnbed. The same applies mutatis mutandis for other % identities such as 96%, 97%, 98%, 99%, and 100%.

A prefeπed 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 95, 97 or 100% identity to the reference sequence of SEQ ID NO: 1 , wherein 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 m 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.1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO: 1, or:

ⁿn ≤ ^xn - (^xn • ). wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides m SEQ ID NO: l, y is 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-mteger product of x_n and y is rounded down to the nearest integer pnor 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.

(2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 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 amino acid alterations as compared to the reference sequence, wherein said 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 amino- 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 in 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 amino 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 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 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-mteger product of x_a and y is rounded down to the nearest integer pnor to subtracting it from x_a.

"Isolated" means altered "by the hand of man" from its natural state, i e., if it occurs in nature, it has been changed or removed from its ongmal environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated ' but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is "isolated", as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombmant method is "isolated" even if it is still present in said organism, which organism may be living or non-living. "Knock-m" refers to the fusion of a portion of a wild-type gene to the cDNA of a heterologous gene

"Knock-out" refers to partial or complete suppression of the expression of a protein encoded by an endogenous DNA sequence in a cell The "knock-out" can be affected by targeted deletion of the whole or part of a gene encoding a protein, m an embryonic stem cell. As a result, the deletion may prevent or reduce the expression of the protein in any cell in the whole animal in which it is normally expressed.

"Splice Nanant" as used herein refers to cDΝA molecules produced from RΝA molecules initially transcπbed from the same genomic DΝA sequence but which have undergone alternative RΝA splicing. Alternative RΝA splicing occurs when a primary RΝA transcnpt undergoes splicing, generally for the removal of mtrons, which results in the production of more than one mR A molecule each of that may encode different ammo acid sequences. The term splice variant also refers to the proteins encoded by the above cDΝA molecules.

"Transgemc animal" refers to an animal to which exogenous DΝA has been introduced while the animal is still in its embryonic stage In most cases, the transgemc approach aims at specific modifications of the genome, e g , by introducing whole transcπptional units into the genome, or by up- or down-regulating pre-existing cellular genes. The targeted character of certain of these procedures sets transgemc technologies apart from expenmental methods in which random mutations are confeπed to the germlme, such as administration of chemical mutagens or treatment with ionizing solution. "Polynucleotide" generally refers to any polyπbonucleotide or polydeoxnbonucleotide, which may be unmodified RΝA or DΝA or modified RΝA or DΝA "Polynucleotides" include, without limitation, single- and double-stranded DΝA, DΝA that is a mixture of single- and double- stranded regions, single- and double-stranded RΝA, and RΝA 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 tnple-stranded regions comprising RNA or DNA or both RNA and DNA The term "polynucleotide" also includes DNAs or RNAs comprising one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tntylated bases and unusual bases such as inosine. A vanety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found m nature, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often refeπed to as ohgonucleotides.

"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 oligomers, and to longer chains, generally refeπed to as proteins. Polypeptides may comprise ammo acids other than the 20 gene-encoded amino acids "Polypeptides" include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described m basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may 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 to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may comprise 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 post-translation 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 derivative, covalent attachment of a lipid or hpid derivative, covalent attachment of phosphotidylmositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-lmks, formation of cysteme, 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 proteins such as argmylation, and ubiquitmation (see, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H Freeman and Company, New York, 1993; Wold, F., Post-translational 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 protein modifications and nonprotem cofactors", Meth Enzymol (1990) 182 626- 646 and Rattan, et al , "Protein Synthesis Post-translational Modifications and Aging", Ann NY Acad Sci (1992) 663 48-62) "Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties A typical vaπant 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 ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical vaπant 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 vanant 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 variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc vanant, 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 All publications including, but not limited to, patents and patent applications, cited in this specification or to which this patent application claims pπoπty, 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 selected from the group consisting of: (1) an isolated polynucleotide compπsmg a nucleotide sequence encoding a polypeptide having at least a 95% identity to the ammo acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2; (n) an isolated polynucleotide compnsmg a nucleotide sequence having at least a 95% identity over its entire length to a nucleotide sequence encoding the polypeptide of SEQ ID NO:2; (in) an isolated polynucleotide compnsmg a nucleotide sequence having at least a 95% identity to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1 ; (IV) an isolated polynucleotide compnsmg a nucleotide sequence encoding the polypeptide of SEQ ID NO:2; (v) an isolated polynucleotide that is the polynucleotide of SEQ ID NO: 1 ; and (vi) an isolated polynucleotide with a nucleotide sequence of at least 100 nucleotides in length obtained by screening an appropnate library under stnngent hybndization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof of at least 15 nucleotides; or a nucleotide sequence complementary to said isolated polynucleotide. 2. An isolated polypeptide selected from the group consisting of: (1) an isolated polypeptide having at least a 95% identity to the amino acid sequence of SEQ ID NO.

2 over the entire length of SEQ ID NO:2, (n) an isolated polypeptide comprising the ammo acid sequence of SEQ ID NO:2; and (in) an isolated polypeptide that is the ammo acid sequence of SEQ ID NO:2.

3. A method for screening to identify compounds that stimulate or that inhibit a function or level of the polypeptide of Claim 2, compnsmg a method selected from the group consisting of: (a) measuring or, quantitatively or qualitatively, detecting the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound; (b) measuring the competition of the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof m the presence of a labeled competitor, (c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes bearing the polypeptide; (d) mixing a candidate compound with a solution comprising a polypeptide of Claim 2, to form a mixture, measuring activity of the polypeptide in the mixture, and comparing the activity of the mixture to a to a control mixture which contains no candidate compound; and (e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells.

4. An agonist or an antagonist of the polypeptide of Claim 2.

5. An agonist or an antagonist of the Bos taurus Frazzled identified by the method of Claim 3.

6. An expression system compnsmg a polynucleotide capable of producing a polypeptide of Claim 2 when said expression system is present in a compatible host cell.

7. A process for producing a recombmant host cell comprising the step of introducing the expression vector of Claim 6 into a cell, such that the host cell, under appropnate culture conditions, produces said polypeptide.

8. A recombmant host cell produced by the process of Claim 7.

9. A membrane of a recombmant host cell of Claim 8 expressing said polypeptide.

10. A process for producing a polypeptide comprising cultuπng a host cell of Claim 9 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture.