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1. WO2000034473 - DOMAINE TRANSMEMBRANAIRE 7 ZSIG56

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Description

SEVEN TRANSMEMBRANE DOMAIN RECEPTOR ZSIG56

BACKGROUND OF THE INVENTION

The seven transmembrane domain receptors are a functionally diverse group encoded by a large gene superfamily. Two characteristic features of this receptor superfamily are the presence of seven helical transmembrane domains and a cytoplasmic domain, the latter of which is believed to be responsible for coupling the receptor to G proteins. This superfamily has been reviewed by Lameh et al., Pharm Res. 7 : 1213-1221, 1990; Hargrave, Curr. Opin. Struct. Biol. 1:575-581, 1991; and Probst et al., DNA and Cell Biol. 11:1-20, 1992.

These G protein-coupled receptors are intrinsic membrane proteins characterized by seven alpha-helical membrane-spanning domains, each of about 22-24 hydrophobic amino acid residues, linked by 3 extracellular and 3 intracellular loops, an N-terminal, extracellular domain and a C-terminal, cytoplasmic tail. The ligand binding site is found within the space enclosed by the transmembrane and/or extracellular domains. Binding of a ligand to the receptor results in a conformational change to the cytoplasmic face of the receptor. The receptor, in particular the cytoplasmic loops between transmembrane domains 5 and 6, and the cytoplasmic tail, then act as a catalyst to activate a signal-transducing heterotrimeric G protein that in turn, activates or inhibits one or more effector molecules (generally an enzyme or certain ion channels) that generate an intracellular second messenger.

When activated, the G protein releases its bound GDP and binds GTP resulting in the formation of a G protein-GTP complex which then associates with an effector molecule. Hydrolysis of GTP returns the G protein to its inactive state. Effector molecules include, adenylate cyclase, which stimulates or inhibits cAMP formation; phosphoinositidase C, which hydrolyses phosphatidyl-inositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and 1,2-diacylglycerol; cyclic nucleotide phosphodiesterases, which upregulate cyclic nucleotide-regulated ion channel activity; and hyperpolarizing potassium channels. G protein-coupled receptors are bound by a variety of ligands including, peptides, hormones, neurotransmitters, lipids, ions, small molecules and external sensory stimuli such as odorants and light.

G protein-coupled receptors have a wide variety of ligands including hormones, neurotransmitters, lipids, peptides and odorants. Many of these ligands are useful as therapeutics when combined with their G protein-coupled receptor. These include PTH, calcitonin, amylin, glucagon, GLP-1, histamine, dopamine and epinephrine, for example.

Hundreds of examples encompassing over 20 different sub-families of G protein-coupled receptors have so far been cloned, many of which can be grouped into categories depending on which effector molecule they predominantly control. Three large sub-families have been characterized, the rhodopsin family; the secretin family; and the glutamate family. Within these families are numerous smaller sub-groups including, receptors for gonadotropin, chemokines, platelet activating factors, thrombin, and vasopressins, for example.

Vasoactive intestinal polypeptide receptors have a wide range of activities. In the periphery they induce relaxation of smooth muscles, such as in intestine and blood vessels and trachea. They regulate secretion such as in stomach, pancreas, gall bladder and intestinal epithelium. They modulate cells of the immune system. Within the central nervous system they are involved in excitatory ana inhibitory functions. Other members of this sub-family such as secretin stimulate secretion of enzymes and ions in the pancreas and intestine. Growth hormone releasing factor regulates synthesis and release of growth hormone from the anterior pituitary.

The secretin family contains receptors with specificity for peptidic hormones including, secretin, calcitonm, parathyroid hormone, parathyroid hormone-related peptide, corticotropin-releasmg factor, vasoactive intestinal peptide, glucagon-like peptide 1, growth hormone-releasing hormone, glucagon, pituitary adenylate cyclase-activatmg polypeptide, and insect diuretic hormone. One member of the secretin family, leukocyte activation antigen CD97, is involved early in leukocyte activation, in particular, it is involved in adhesion and signal transduction. The receptor cDNA was found in T cell and peripheral blood mononuclear cell libraries. A strong correlation is seen between CD97 concentrations and inflammation in lymphoid tissue (Hamann et al., J. Immunol. 155 : 1942-50, 1995; Gray et al., J. Immunol. 157: 5438-47, 1996). CD97 is expressed at low levels on normal, resting lymphocytes, and upon cellular activation the surface expression of CD97 is up-regulated. It has been suggested that CD97 exists as two noncovalently associated subunits that are generated by processing of the propeptide and that the extracellular subunit exists in a stable form in fluids surrounding inflammatory sites. CD97 shares 40% homology in the extracellular domain with another member of the secretin family, EMR1. The N-terminal portion of these receptors is longer than other members of this family and contains EGF-like repeats (Baud et al., Genomics 26:334-44, 1995; Gray et al., ibid) . Other members of the G protein- coupled receptor family with differential expression m lymphocytes have also been found. In particular, ChemR1, a chemokme receptor was detected by Northern blots m T lymphoblastic cell lines Jurkat and MOLT-4, but not in the pre-B lymphoblastic cell line JM-1 and by PCR in unstimulated CD4+ and CD8+ T cells (Samson et al., Eur. J. Immunol. 26:3021-28, 1996). The IP10/Mig receptor, is expressed in IL-2-actιvated T lymphocytes, but is not detectable in resting T lymphocytes, B lymphocytes, monocytes and granulocytes (Loetscher et al., J. Exp. Med. 184:963-9, 1996).

The demonstrated in vivo and in vitro activities of these G protein-coupled receptor family members illustrate the enormous clinical potential of, and need for, other G protein-coupled receptors, G protein-coupled receptor ligands, agonists, and antagonists. The present invention addresses this need by providing a novel seven transmembrane domain receptor and related compositions and methods that should be apparent to those skilled in the art from the teachings herein.

SUMMARY OF THE INVENTION

Within one aspect the invention provides an isolated polynucleotide molecule that encodes a polypeptide selected from the group consisting of: a) a polypeptide comprising amino acid residues 1 to 693 of SEQ ID NO:2; b) a polypeptide comprising amino acid residues 26 to 693 of SEQ ID NO:2; c) a polypeptide comprising amino acid residues 109 to 693 of SEQ ID NO:2; d) a polypeptide comprising amino acid residues 182 to 693 of SEQ ID NO:2; e) a polypeptide comprising amino acid residues 282 to 693 of SEQ ID NO:2; f) a polypeptide comprising amino acid residues 288 to 693 of SEQ ID N0:2; g) a polypeptide comprising amino acid residues 372 to 693 of SEQ ID NO:2; h) degenerate polynucleotide sequence of a), b), c), d), e), f) or g); and i) polynucleotide sequence complementary to a), b), c), d), e), f), g) or h). Within one embodiment the polynucleotide is selected from the group consisting of: a) nucleotides 190-2268 of SEQ ID NO : 1 ; b) nucleotides 514-2268 of SEQ ID NO:1; c) nucleotides 733-2268 of SEQ ID NO:1; d) nucleotides 1033-2268 of SEQ ID NO:1; e) nucleotides 1051-2268 of SEQ ID NO:1; f) nucleotides 1293-2268 of SEQ ID NO : 1 ; g) nucleotides 265-2268 of SEQ ID NO:1; h) a degenerate polynucleotide sequence of a), b), c), d), e), f) or g); and i) a polynucleotide sequence complementary to a), b), c), d), e), f), g) or h).

Within another aspect the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a polynucleotide molecule as described herein; and a transcription terminator. Within a related embodiment the expression vector further comprises a secretory signal sequence operably linked to the polynucleotide molecule. Within another embodiment is provided a cultured cell into which has been introduced an expression vector described herein, wherein the cultured cell expresses the polypeptide encoded by the polynucleotide molecule. Within a further embodiment is provided a method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector described herein; whereby the cell expresses the polypeptide encoded by the polynucleotide segment; and recovering the expressed polypeptide.

The invention also provided an isolated polypeptide selected from the group consisting of: a) a polypeptide comprising amino acid residues 1 to 25 of SEQ ID NO:2; b) a polypeptide comprising amino acid residues 109 to 693 of SEQ ID NO:2; c) a polypeptide comprising amino acid residues 182 to 693 of SEQ ID NO: 2; d) a polypeptide comprising amino acid residues 282 to 693 of SEQ ID NO: 2; e) a polypeptide comprising amino acid residues 288 to 693 of SEQ ID NO:2; f) a polypeptide comprising amino acid residues 372 to 693 of SEQ ID NO:2; g) a polypeptide comprising amino acid residues 26 to 693 of SEQ ID NO:2; and h) a polypeptide molecule having the sequence of SEQ ID NO:2.

The invention also provides a polypeptide selected from the group consisting of: amino acid residues 26 to 106 of SEQ ID NO: 2, amino acid residues 26 to 181 of SEQ ID NO:2, amino acid residues 26 to 278 of SEQ ID NO : 2 , amino acid residues 26 to 285 of SEQ ID NO:2, amino acid residues 26 to 369 of SEQ ID NO:2, amino acid residues 109 to 179 of SEQ ID NO : 2 , amino acid residues 109 to 278 of SEQ ID NO:2, amino acid residues 109 to 285 of SEQ ID NO:2, amino acid residues 109 to 369 of SEQ ID NO: 2, amino acid residues 181 to 278 of SEQ ID NO: 2, amino acid residues 182 to 285 of SEQ ID NO:2, amino acid residues

182 to 369 of SEQ ID NO: 2, and amino acid residues 288 to

369 of SEQ ID NO: 2.

Further provided is an antibody or antibody fragment that specifically binds to a polypeptide described herein. Within one embodiment the antibody is selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b); and d) human monoclonal antibody. Within another embodiment the antibody fragment is selected from the group consisting of F(ab'), F(ab), Fab', Fab, Fv, scFv, and minimal recognition unit. Within another embodiment is provided an anti-idiotype antibody that specifically binds to the antibody described above.

These and other aspects of the invention will become evident upon reference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a multiple sequence alignment between zsig56, human HE6 receptor (HSHE6, Osterhoff et al., DNA Cell Biol. 16:379-89, 1997) (SEQ ID NO: 3), human CD97 (HSU76764, Gray et al., J. Immunol. 157:5438-47, 1996) (SEQ ID NO : 4 ) and human EMR1 hormone receptor (EMR1, Baud et al., Genomics 26:334-44, 1995) (SEQ ID N0:5).

Figure 2 shows a Hopp/Woods hydrophilicity profile of the amino acid sequence shown in SEQ ID NO: 2. The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. These residues are indicated m the figure by lower case letters.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms:

The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.

Affinity tags include a poly-histidme tract, protein A

(Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,

Methods Enzymol. 198:3, 1991), glutathione S transferase

(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988), streptavidm binding peptide, or other antigenic epitope or binding domain. See, m general, Ford et al., Protein Expression and Purification 2: 95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).

The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism withm populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.

The terms "amino-termmal" and "carboxyl-termmal" are used herein to denote positions withm polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-termmal to a reference sequence withm a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carooxyl terminus of the complete polypeptide.

The term "complement/anti-complement pair" denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotm and avidm (or streptavidm) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <109 M-1.

The term "complements of a polynucleotide molecule" is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.

The term "contig" denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'-ATGGCTTAGCTT-3' are 5'-TAGCTTgagtct-3' and 3'-gtcgacTACCGA-5'.

The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term "expression vector" is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is m a form suitable for use withm genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).

An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or deri vatized forms.

The term "operably linked", when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.

The term "ortholog" denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.

A "polynucleotide" is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides withm a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will m general not exceed 20 nt in length.

A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides".

"Probes and/or primers" as used herein can be RNA or DNA. DNA can be either cDNA or genomic DNA.

Polynucleotide probes and primers are single or double-stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences or its complements. Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used. PCR primers are at least 5 nucleotides in length, preferably 15 or more nt, more preferably 20-30 nt. Short polynucleotides can be used when a small region of the gene is targeted for analysis. For gross analysis of genes, a polynucleotide probe may comprise an entire exon or more. Probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art.

The term "promoter" is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.

A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell. Membrane-bound receptors are characterized by a multi-peptide structure comprising an extracellular ligand-bmdmg domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule (s) in the cell. This interaction in turn leads to an alteration m the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of mositol lipids and hydrolysis of phospholipids. In general, receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietm receptor and IL-6 receptor).

The term "secretory signal sequence" denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.

The term "splice variant" is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to ±10%.

All references cited herein are incorporated by reference in their entirety.

The novel zsig56 polypeptide-encodmg polynucleotides of the present invention were initially identified by querying an EST database for secretory signal sequences characterized by an upstream methionine start site, a hydrophobic region of approximately 13 amino acids and a cleavage site, wherein cleavage occurs between the alanine and arginine amino acid residues, in an effort to select for secreted proteins. Polypeptides corresponding to ESTs meeting those search criteria were compared to known sequences to identify secreted proteins having homology to known ligands. Two EST sequences were discovered and predicted to encode a secreted protein. Full length sequencing thereof allowed the sequence to be characterized as encoding a seven transmembrane domain receptor.

The full length nucleotide sequence encoding zsig56 polypeptide is described in SEQ ID NO:1 was obtained from a retina library. The deduced amino acid sequence is described in SEQ ID NO : 2. Analysis of the DNA encoding a zsig56 polypeptide (SEQ ID NO:1) revealed an open reading frame encoding 693 amino acids (SEQ ID NO:2) comprising a signal peptide of 25 amino acid residues (residue 1 to residue 25 of SEQ ID NO:2, nucleotides 190-264 of SEQ ID NO:1) and a mature polypeptide of 668 amino acids (residue 26 to residue 693 of SEQ ID NO:2, nucleotides 265-2268 of SEQ ID NO:1). Those skilled m the art will recognize that predicted secretory signal sequence domain boundaries are approximations based on primary sequence content, and may vary slightly; however, such estimates are generally accurate to withm ±4 amino acid residues. The mature polypeptide includes an extracellular domain, (residue 26 to residue 401 of SEQ ID NO:2, nucleotides 265-1392 of SEQ ID NO:1). Also included are seven transmembrane domains: transmembrane domain 1 (residue 402 to 430 of SEQ ID NO: 2, nucleotides 266-1479 of SEQ ID NO:1), transmembrane domain 2 (residue 448 to residue 463 of SEQ ID NO: 2, nucleotides 1531-1578 of SEQ ID NO:1), transmembrane domain 3 (residue 479 to residue 496 of SEQ ID NO: 2, nucleotides 1626-1677 of SEQ ID NO:1), transmembrane domain 4 (residue 519 to residue 539 of SEQ ID NO:2, nucleotides 1744-1806 of SEQ ID NO:1), transmembrane domain 5 (residue 577 to residue 597 of SEQ ID NO:2, nucleotides 1918-1980 of SEQ ID NO:1), transmembrane domain 6 (residue 606 to residue 630 of SEQ ID NO:2, nucleotides 2005-2079 of SEQ ID NO:1) and transmembrane domain 7 (residue 637 to residue 661 of SEQ ID NO:2, nucleotides 2098-2172 of SEQ ID NO:1). There are three extracellular and three cytoplasmic loops between the transmembrane domains: extracellular loop 1 (residue 431 to residue 447 of SEQ ID NO:2, nucleotides 267-1478 of SEQ ID NO:1), extracellular loop 2 (residue 540 to residue 576 of SEQ ID NO: 2, nucleotides 1807-1917 of SEQ ID NO:1)) and extracellular loop 3 (residue 631 to residue 636 of SEQ ID NO:2, nucleotides 2080-2097 of SEQ ID NO:1) and cytoplasmic loop 1 (residue 431 to residue 447 of SEQ ID NO:2, 1480-1530 of SEQ ID NO:1), cytoplasmic loop 2 (residue 497 to residue 518 of SEQ ID NO:2, nucleotides 1678-1745 of SEQ ID NO:1) and cytoplasmic loop 3 (residue 598 to residue 605 of SEQ ID NO:2, nucleotides 2080-2097 of SEQ ID NO:1). A short cytoplasmic C-terminal tail (residue 662 to residue 693 of SEQ ID NO:2, nucleotides 2099-2268 of SEQ ID NO:1) completes the receptor. Those skilled in the art will recognize that these domain boundaries are approximate, and are based on alignments with known proteins and predictions of protein folding. These features indicate that the receptor encoded by the DNA sequence of SEQ ID NO: 2 is a member of the G-protem coupled receptor family.

Zsig56 is most closely related to the HE6 Tm7 receptor (Osterhoff et al., ibid.) and the EMR1 hormone receptor (Gaud et al., ibid.). The most conserved region between these proteins lies in the transmembrane domain and a short stretch of the extracellular domain immediately N-terminal to the transmembrane domain (amino acid residue 344 to amino acid residue 666 of SEQ ID NO:2). Withm this region the percent identity at the amino acid level between these receptors is:

Zsig56 (SEQ ID NO:2) and HE6 (SEQ ID NO : 3 , Osterhoff et al., ibid.) share additional homology withm the extracellular domain, from amino acid residue 32 to amino acid residue 344 of SEQ ID NO:2.

Figure 1 shows a multiple amino acid sequence alignment of zsig56 with HE6, EMR1 and CD97. Amino acid residues that appear to be conserved between these proteins withm the extracellular domain include amino acid residues 346, 349, 361, 365, 366, 377 and 379 of SEQ ID NO: 2. Withm the loops, conserved amino acid residues include amino acid residues 562-564 of SEQ ID NO:2. Conserved cysteme residues, residues 475 and 562 of SEQ ID NO: 2 may serve to from a disulfide bond between the first and second extracellular loops. Withm the hydrophobic regions of the transmembrane domains, hydrophilic and charged residues that are conserved among family members may be involved in ligand binding and/or signal transmission. Residues meeting these requirements include amino acid residues 410, 414, 483, 523, 525, 527, 576, 585, 614, 632, 641, 650 and 651 of SEQ ID NO:2. In addition to completely conserved hydrophilic residues withm the transmembrane domains, positions withm the transmembrane domains that are always hydrophilic with a subfamily may also be involved in ligand binding and signal transmission. Residues meeting these requirements include amino acid residues 408 and 457 of SEQ ID NO:2.

The extracellular domain of zsig56 including several potential proteolytic cleavage sites, residues 107-108 (lys-arg) of SEQ ID NO:2, residues 180-181 (lys-arg) of SEQ ID NO:2, residues 281-282 (arg-arg) of SEQ ID NO:2, residues 286-287 (lys-arg) of SEQ ID NO : 2 and residues 370-71 (arg-arg) of SEQ ID NO:2. Additionally there is an arg-gly-his-arg, residues 26-29 of SEQ ID NO:2 and a lys-gly-arg-arg site at residues 195-198 of SEQ ID NO:2 and an arg-arg site at residues 198-199 of SEQ ID NO:2. The extracellular domain of zsig56 may be cleaved or processed into an active form through the action of prohormone convertases. The processed polypeptides are often amidated at the 5' end through the action of an amidatmg enzyme, such as peptidyl-glycyl, α-amidatmg monooxygenase (PAM).

The most prevalent cleavage or processing site is a dibasic amino acid prohormone convertase site. Since there are only a few dibasic amino acid combinations, including KK (lys-lys), RR (arg-arg), RK (arg-lys) and KR

(lys-arg), non-dibasic cleavage and processing sites have also been observed. An example of a non-dibasic site is that found in gastrm (NR). Such cleavage or processing sites may be incorporated into zsig56 polypeptides, fragments or fusion proteins. Therefore the present invention also provides post-translationally modified polypeptides or polypeptide fragments having the amino acid sequence from amino acid residue:


Cleavage of the extracellular domain of zsig56 at amino acid residue 281 of SEQ ID NO:2 would leave an extracellular domain of about 120 amino acid residues which is in the range of G protem-coupled receptors such as glucagon among others.

The extracellular domain of the zsig56 polypeptide of SEQ ID NO:2 also contains seven potential N-lmked glycosylation sites based on the motif N-X-T/S were N is asparagme, X is any amino acid residue and T/S represents either the residue threonme or serme, (residues 39-41, 148-150, 171-173, 234-236, 303-305, 324-326 and 341-343 of SEQ ID NO:2). Also there are eight cysteme residues (residues 35, 91, 121, 177, 346, 366, 377 and 379 of SEQ ID NO:2. The extracellular domain also contains 22 prolme residues (residues 50, 52, 70, 72, 76, 82, 84, 133, 134, 146, 152, 164, 165, 193, 200, 204, 242, 270, 339, 346, 353 and 358 of SEQ ID NO:2) suggesting that the molecule adopts a rather extended conformation in solution. O-linked glycosylation is also predicted as the extracellular domain contains over 63 serme or threonme residues. In particular, the region between residues 351 and 400 of SEQ ID NO:2 is especially rich in serme and threonme residues (24%).

Thirty-eight percent of the amino acid residues withm the cytoplasmic domain of the zsig56 polypeptide of SEQ ID NO : 2 are either serme or threonme which may be subject to phosphorylation. Also withm the this domain are three sub-regions (residues 660-662, 678-689 and 690-692 of SEQ ID NO:2) of sequence which are potential identity to the substrate for protein kmase C (PKC) as represented by the motif S/T-X-K/R, where X is any amino acid residue and S/T is either a serme (S) or a threonme (T) residue and K/R is either a lysme (K) or an argmine (R) residue. The presence of consensus sites for PCK-dependent phosphorylation suggests that zsig56 plays a role m cell signaling. There are no consensus sites for cAMP dependent protein kmase activity.

The present invention further provides polynucleotide molecules, including DNA and RNA molecules, encoding zsig56 proteins. The polynucleotides of the present invention include tne sense strand; the anti-sense strand; and the DNA as double-stranded, having both the sense and anti-sense strand annealed together by their respective hydrogen bonds. A representative DNA sequence encoding a zsig56 protein is set forth in SEQ ID NO:1. DNA sequences encoding other zsig56 proteins can be readily generated by those of ordinary skill in the art based on the genetic code. Counterpart RNA sequences can be generated by substitution of U for T.

Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO:6 is a degenerate DNA sequence that encompasses all DNAs that encode the zsig56 polypeptide of SEQ ID NO:2. Those skilled m the art will recognize that the degenerate sequence of SEQ ID NO: 6 also provides all RNA sequences encoding SEQ ID N0:2, by substituting U for T. Thus, zsig56 polypeptide-encodmg polynucleotides comprising nucleotide 1 to nucleotide 2079 of SEQ ID NO: 6 and their respective RNA equivalents are contemplated by the present invention. Table 1 sets forth the one-letter codes used withm SEQ ID NO: 6 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide (s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.

TABLE 1


The degenerate codons used in SEQ ID NO: 6, encompassing all possible codons for a given amino acid, are set forth in Table 2.

One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serme (WSN) can, in some circumstances, encode argmme (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucme. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO : 2. Variant sequences can be readily tested for functionality as described herein.

One of ordinary skill in the art will also appreciate that different species can exhibit "preferential codon usage." In general, see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr. Biol. 6:315-24, 1996; Wam-Hobson, et al., Gene 13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J. Mol . Biol. 158: 573-97, 1982. As used herein, the term "preferential codon usage" or "preferential codons" is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2). For example, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular specie0 can be introduced into the polynucleotides of the present invention by a variety of methods known m the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient withm a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO: 6 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.

The conserved amino acids or sequences of amino acids withm a domain of zsig56 can be used as a tool to identify new family members. For instance, reverse transcription-polymerase chain reaction (RT-PCR) can be used to amplify sequences encoding the conserved residues or region from RNA obtained from a variety of tissue sources or cell lines. In particular, highly degenerate primers designed from the zsig56 sequences are useful for this purpose.

Analysis of the tissue distribution of the mRNA corresponding to this novel DNA by Northern blot and Dot blot analysis showed strong expression m kidney and thyroid and weaker but obvious expression in spinal cord, placenta, trachea, heart, lung, PBLs, brain, pancreas, prostate, stomach, spleen, testis and colon. A transcript of about 4.0 kb was observed m all tissues.

Withm preferred embodiments of the invention, isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:1, or to a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.

As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zsig56 RNA. Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include kidney and thyroid. Total RNA can be prepared using guanidmium isothiocyanate extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)+ RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972). Complementary DNA (cDNA) is prepared from poly (A)+ RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding zsig56 polypeptides are then identified and isolated by, for example, hybridization or PCR.

A full-length clone encoding zsig56 can be obtained by conventional cloning procedures. Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic mtron. Methods for preparing cDNA and genomic clones are well known and withm the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library.

Expression libraries can be probed with antibodies to zsig56, receptor fragments, or other specific binding partners.

The polynucleotides of the present invention can also be synthesized using automated equipment. The current method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 bp) is technically straightforward and can be accomplished by synthesizing tne complementary strands and then annealing them. For the production of longer genes (>300 bp), however, special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. Gene synthesis methods are well known in the art. See, for example, Glick and

Pasternak, Molecular Biotechnology, Principles &

Applications of Recombmant DNA, ASM Press, Washington, D.C., 1994; Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984; and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990.

The present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are zsig56 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human zsig56 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zsig56 as disclosed herein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A zsig56-encodmg cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human zsig56 sequence disclosed herein. Withm an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zsig56 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.

Those skilled in the art will recognize that the sequence disclosed in SEQ ID NO:1 represents a single allele of human zsig56 and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of tne DNA sequence shown in SEQ ID NO:1, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are withm the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO: 2. cDNAs generated from alternatively spliced mRNAs , which retain the prrperties of the zsig56 polypeptide are included withm the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.

The present invention also provides isolated zsig56 polypeptides that are substantially homologous to the polypeptides of SEQ ID NO:2 and their orthologs. The term "substantially homologous" is used herein to denote polypeptides having at least 80% sequence identity to the sequences shown in SEQ ID NO:2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO: 2 or its orthologs.) Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Hemkoff and Hemkoff, Proc. Natl. Acad. Sci. USA 89: 10915-9, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Hemkoff and Hemkoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as:

Total number of identical matches

______________________________________ ________x 100

[length of the longer sequence plus the

number of gaps introduced into the longer sequence in order to align the two sequences] Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above.

Variant zsig56 polypeptides or substantially homologous zsig56 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-termmal extensions, such as an amino-terminal methionme residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zsig56 polypeptide and the affinity tag. Preferred such sites include thrombm cleavage sites and factor Xa cleavage sites.


The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline, cιs-4-hydroxyproline, trans-4-hydroxyproline, W-methyl-glycme, allo-threonme, methylthreonine, hydroxyethyl-cysteme, hydroxyethylhomocysteme, nitroglutamine, homo-glutamine, pipecolic acid, thiazolidme carboxylic acid, dehydroprol ine, 3- and 4-methylprolme, 3,3-dιmethyl-prolme, tert-leucme, norvalme, 2-azaphenylalanme, 3-azaphenylalanme, 4-azapnenylaln anin e, and 4-fluorophenyl- alanme. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vi tro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylatmg tRNA are known m the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E . coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymoi. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out m Xenopus oocytes by micromjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Withm a third method, E . coli cells are cultured m the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acιd(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanme). The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vi tro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for zsig56 amino acid residues.

Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanme-scannmg mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88: 4498-502, 1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699-708, 1996. Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. Amino acid residues that might be considered essential in the zsig56 polypeptide are cysteme residues at amino acid residues 475 and 562 of SEQ ID NO: 2; the charged transmembrane domain residues 410, 414, 483, 523, 525, 527, 576, 585, 641, 650 and 651 of SEQ ID NO:2, and the conserved hydrophilic residues 408 and 457 of SEQ ID NO:2. Other residues of interest include potential proteolytic cleavage sites at residues 107-108, 180-181, 281-282, 286-287, 370-71, 26-29, 195-198 and 198-199 of SEQ ID NO:2. Additionally there are N-glycosylation sites predicted at residues 39-41, 148-150, 171-173, 234-236, 303-305, 324-326 and 341-343 of SEQ ID NO:2). Other resiαues that may be invariant include eight cysteme residues (residues 35, 91, 121, 177, 346, 366, 377 and 379 of SEQ ID NO:2 and 22 prolme residues (residues 50, 52, 70, 72, 76, 82, 84, 133, 134, 146, 152, 164, 165, 193, 200, 204, 242, 270, 339, 346, 353 and 358 of SEQ ID NO:2). Additionally, the region between residues 351 and 400 of SEQ ID NO:2 is especially rich in serme and threonme residues. Withm the cytoplasmic domain are three sub-regions (residues 660-662, 678-689 and 690-692 of SEQ ID NO:2) of sequence identity to the substrate for protein kmase C. A hydrophobicity profile of SEQ ID NO:2 is shown in Figure 2. Those skilled m the art will recognize that this hydrophobicity profile will be taken into account when designing alterations in the amino acid sequence of a zsig56 polypeptide, so as not to disrupt the overall profile.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions m a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Variants of the disclosed zsig56 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vi tro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.

Mutagenesis methods as disclosed herein can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides m host cells. Mutagenized DNA molecules that encode active polypeptides (e.g., ligand binding receptors) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

Using the methods discussed herein, one of ordinary skill in the art can identify and/or prepare a variety of polypeptide fragments or variants of SEQ ID NO:2 or that retain the ligand binding properties of the wild-type zsig56 protein. Such polypeptides may include additional amino acids from an extracellular ligand-binding domain of a G protem-coupled receptor as well as part or all of the transmembrane and cytoplasmic domains; affinity tags; and the like.

For any zsig56 polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.

The zsig56 polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, can be produced m genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher euKaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a zsig56 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, withm an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that withm certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design withm the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.

To direct a zsig56 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of zsig56, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo . The secretory signal sequence is operably linked to the zsig56 DNA sequence, i.e., the two sequences are joined m the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830).

Alternatively, the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway. The present invention provides for such fusion polypeptides. A signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid residues 1-25 of SEQ ID NO: 2 is be operably linked to another polypeptide using methods known in the art and disclosed herein. The secretory signal sequence contained m the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway. Such constructs have numerous applications known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein. Such fusions may be used in vivo or in vi tro to direct peptides through the secretory pathway.

Cultured mammalian cells are suitable hosts withm the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 5:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and viral vectors (Miller and Rosman, BioTechmques 7:980-90' 1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production of recombmant polypeptides m cultured mammalian cells is disclosed, for example, by Levmson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Rmgold, U.S. Patent No. 4,656,134. Suitable cultureα mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-Kl; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionem genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycm. Selection is carried out m the presence of a neomycm-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification". Amplification is carried out by culturmg transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g. hygromycm resistance, multi-drug resistance, puromycm acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Smkar et al., J. Biosci. (Bangalore; 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarmo et al., U.S.

Patent No. 5,162,222 and WIPO publication WO 94/06463. Methods for making recombmant proteins in baculovirus systems are known in the art and commercially available from such sources as Life Technologies, Rockville, MD and Invitrogen Inc., San Diego, CA. See for example King and Possee, The Baculovirus Expression System: A Laboratory Guide, London, Chapman & Hall; O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; Richardson, C. D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995; Luckow et al., J Virol 67:4566-79, 1993; Hill-Pernns and Possee, J. Gen. Virol. 71:971-6, 1990; Bonnmg et al., J. Gen. Virol. 75 : 1551- 6 , 1994; Chazenbalk and Rapoport, J. Biol. Chem. 270: 1543-9, 1995; and Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombmant DNA, ASM Press, Washington, D.C., 1994.

Fungal cells, including yeast cells, can also be used withm the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pas toπs , and Pichia methanolica . Methods for transforming S . cerevisiae cells with exogenous DNA and producing recombmant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kmgsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha , Schizosaccnaromyces pombe, Kl uyveromyces la ctis , Kl uyveromyces fragil is , Ustilago maydis , Pichia pastoris , Pichia methanolica , Pichia guillermondn and Candida mal tosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergill us cells may be utilized according to the methods of McKmght et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Summo et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533.

The use of Pichia methanolica as host for the production of recombmant proteins is disclosed Raymond, U.S. Patent No. 5,716,808, Raymond, U.S. Patent No. 5,736,383, Raymond et al., Yeast 14: 11-23, 1998, and in international publication Nos. WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.

Prokaryotic host cells, including strains of the bacteria Escheπchia , Bacill us and other genera are also useful host cells withm the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.). When expressing a zsig56 polypeptide in bacteria such as E . coli , the polypeptide may be retained m the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guamdme isothiocyanate or urea. The denatured polypeptide can then be refolded and dimeπzed by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space m a soluble and functional form by disrupting the cells (by, for example, somcation or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known m the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency m an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell. P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto™ Peptone (Difco Laboratories, Detroit, MI), 1% Bacto™ yeast extract (Difco Laboratories), 0.004% adenme and 0.006% L-leucine).

Moreover, using methods described in the art, polypeptide fusions, or hybrid zsig56 proteins, are constructed using regions or domains of the inventive zsig56 in combination with those of other G protem-coupled receptor family proteins, or heterologous proteins (Sledziewski et al. US Patent No. 5,576,210; Kobilka et al., Science 240:13106, 1988; Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opm. Biology, 5:511-5, 1994, and references therein). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.

Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain(s) conferring a biological function may be swapped between zsig56 of the present invention with the functionally equivalent domain(ss) from another G protein-coupled receptor family member. Such domains include, but are not limited to, the secretory signal sequence, extracellular domain, transmembrane domains, extracellular and cytoplasmic loops, the cytoplasmic C-termmal tail, or combinations of such domains. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known G protem-coupled receptor family proteins, depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.

It is preferred to purify the polypeptides and polypeptide fragments of the present invention to ≥80% purity, more preferably to ≥90% purity, even more preferably ≥95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.

Expressed recombmant zsig56 polypeptides (or zsig56 fusion polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include deπvatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred. Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to oe used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccmimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodnmide coupling chemistries. These and other solid media are well known and widely used m the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated by exploitation of their properties. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidme-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3 :: 1-7 , 1985). Histidme-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelatmg agents. Other methods of purification include purification of glycosylated proteins by lectm affinity chromatography and ion exchange chromatograpny (Methods in Enzymol., Vol. 182, "Guide to Protein Purification", Deutscher, (ed.), Acad. Press, San Diego, 1990, pp. 529-39). Withm additional emoodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., maltose-binding protein, FLAG tag, Glu-Glu tag) may be constructed to facilitate purification.

Zsig56 polypeptides or fragments thereof may also be prepared through chemical synthesis. Zsig56 polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionme amino acid residue. Polypeptides, especially polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. The polypeptides are preferably prepared by solid phase peptide synthesis, for example as described by Merπfield, J. Am. Chem. Soc. 85:2149, 1963.

Zsig56 polypeptides can also be used to identify inhibitors (antagonists) of its activity. This would include molecules capable of binding zsig56, but which do not stimulate, or reduce the stimulation of, a response pathway withm the cell. Such zsig56 antagonists would include antibodies; oligonucleotides which bind either to the zsig56 polypeptide or to its ligand; natural or synthetic analogs of zsig56 polypeptides which retain the ability to bind the ligand but do not result m either ligand or receptor signaling. Such analogs could be peptides or peptide-like compounds. As such, zsig56 antagonists would be useful as therapeutics for treating certain disorders where blocking signal from either a zsig56 ligand or receptor would be beneficial.

In particular, such zsig56 antagonists are generally identified by their ability to bind to the zsig56 receptor, and thereby reduce the stimulation of a response pathway withm the cell. To identify zsig56 antagonists, test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of zsig56. A variety of assays designed to measure receptor binding or the stimulation/inhibition of receptor-dependent cellular responses are known m the art. For example, zsig56-responsιve cell lines can be transfected with a reporter gene construct that is responsive to a zsig56-stιmulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a zsig56-DNA response element operably linked to a gene encoding an assayable protein, such as luciferase. DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE) insulin response element (IRE) (Nasπn et al., Proc. Natl. Acad. Sci. USA 82:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563-72, 1989). Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener, Molec. Endocrmol. 4 (8):1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 56:335-44; 1989. Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zsig56 on the target cells as evidenced by a decrease in zsig56 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block zsig56 binding to cell-surface receptors, as well as compounds that block processes m the cellular pathway subsequent to receptor-ligand binding. In the alternative, compounds or other samples can be tested for direct blocking of ligand binding to the zsig56 receptor by using a zsig56 ligand tagged with a detectable label (e.g., 125I, biotm, horseradish peroxidase, FITC, and the like). Withm assays of this type, the ability of a test sample to inhibit the binding of labeled ligand to the receptor is indicative of inhibitory activity, which can be confirmed through secondary assays. Receptors used withm binding assays may be cellular receptors or isolated, immobilized receptors.

The zsig56 receptor polypeptides of the present invention can also be used in assay systems to identify ligands and agonists. Such assay systems permit rapid identification of substances having selective activity on cells expressing the zsig56 receptor. Zsig56 is a member of the G protem-coupled receptor family. Such receptors are known to transduce signal via activation of adenylate cyclase, leading to elevation of cellular cAMP levels (Lm et al., Science 254:1022-24, 1991). Assay system exploiting the receptor's ability to elevate cAMP levels can be used as a way to detect molecules that are able to stimulate the zsig56 receptor and initiate signal transduction.

Receptor activation can be detected by: (1) measurement of adenylate cyclase activity (Salomon et al., Anal. Biochem. 58:541-48, 1974; Alvarez and Daniels, Anal . Biochem. 187:989103, 1990); (2) measurement of change in intracellular cAMP levels using conventional radioimmunoassay methods (Sterner et al., J. Biol. Chem. 247 : 1106-13, 1972; Harper and Brooker, J. Cyc. Nucl. Res. 2:207-18, 1975); or (3) through use of a cAMP scintillation proximity assay (SPA) method (Amersham Corp., Arlington Heights, IL).

An alternative assay system involves selection of substances that are able to induce expression of a cyclic AMP response element (CRE)-luciferase reporter gene, as a consequence of elevated cAMP levels, in cells expressing a zsig56 receptor, but not m cells lacking zsig56 receptor expression. Other G protem-coupled receptors, such as the glucagon receptor or the calcitonm receptor, that transduces signal through adenylate cyclase-mediated elevation of cAMP can be used as positive controls.

This CRE-luciferase assay measures the end result of a multi-step signal transduction pathway triggered when a zsig56 receptor ligand or agonist stimulates the Gs-coupled zsig56 receptor. The complexity of this pathway provides multiple mechanisms for induction of luciferase transcription at points that are downstream of the zsig56 receptor, and therefore may not be zsig56 receptor-specific (e.g., forskolm's direct activation of adenylate cyclase). Any response triggered by nonspecific mducers is eliminated by counter screening using receptor-negative cell lines.

An assay system that uses a ligand-bmdmg receptor (or an antibody, one member of a complement/ anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed. Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234 : 554-63, 1993. A receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film withm the flow cell. A test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change m the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.

This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.

Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimetric assays (Cunningham et al., Science 253: 545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

Zsig56 polypeptides can also be used for purification of zsig56 receptor ligands. The polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use. Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, Nhydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting medium will generally be configured in the form of a column, and fluids containing ligand are passed through the column one or more times to allow ligand to bind to the receptor polypeptide. The ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HCl ), or pH to disrupt ligand-receptor binding.

The invention also provides anti-zsig56 antibodies. Antibodies to zsig56 can be obtained, for example, using as an antigen the product of a zsig56 expression vector, or zsig56 isolated from a natural source. Particularly useful anti-zsig56 antibodies "bind specifically" with zsig56. Antibodies are considered to be specifically binding if the antibodies bind to a zsig56 polypeptide, peptide or epitope with a binding affinity

(Ka) of 106 M-1 or greater, preferably 10 M or greater, more preferably 10 8 M-1 or greater, and most preferably 10 9 M-1 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, ibid.). Suitable antibodies include antibodies that bind with zsig56 in particular domains.

Antι-zsig56 antibodies can be produced using antigenic zsig56 epitope-bearmg peptides and polypeptides. Antigenic epitope-bearmg peptides and polypeptides of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within SEQ ID NO:2. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that bind with zsig56. It is desirable that the amino acid sequence of the epitope-bearmg peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues, while hydrophobic residues are preferably avoided). The hydrophobicity plot provided in Figure 2 provides such information. Using the plot, antigenic regions can be selected such as those found in the fragments, amino acid residue 435-440, 26-31, 253-258, 278-283 and 276-281 of SEQ ID NO:2. Moreover, amino acid sequences containing prolme residues may be also be desirable for antibody production.

Polyclonal antibodies to recombmant zsig56 protein or to zsig56 isolated from natural sources can be prepared using methods well-known to those of skill m the art. See, for example, Green et al., "Production of Polyclonal Antisera," in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al., "Expression of foreign proteins in E . coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995). The immunogenicity of a zsig56 polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zsig56 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like," such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such as horses, cows, dogs, chicken, rats, mice, rabbits, hamsters, guinea pigs, goats, or sheep, an anti-zsig56 antibody of the present invention may also be derived from a subhuman primate antibody. General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., international patent publication No. WO 91/11465, and in Losman et al., Int. J. Cancer 46:310, 1990. Antibodies can also be raised in transgenic animals such as transgenic sheep, cows, goats or pigs, and can also be expressed in yeast and fungi in modified forms as will as in mammalian and insect cells.

Alternatively, monoclonal anti-zsig56 antibodies can be generated. Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al., Nature 256: 495 (1975), Coligan et al. (eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991), Picksley et al., "Production of monoclonal antibodies against proteins expressed in E . coli ," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising a zsig56 gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybπdomas, selecting positive clones which produce antibodies to the antigen, culturmg the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.

In addition, an antι-zsig56 antibody of the present invention may be derived from a human monoclonal antibody. Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies m response to antigenic challenge. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al., Nature Genet. 7:13, 1994, Lonberg et al., Nature 368:856, 1994, and Taylor et al., Int. Immun. 6:579, 1994.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protem-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Barnes et al., "Purification of Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments of anti-zsig56 antibodies. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. As an illustration, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages. As an alternative, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. patent No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys. 89:230, 1960, Porter, Biochem. J. 23;11 9, 1959, Edelman et al., in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and by Coligan, ibid.

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

For example, Fv fragments comprise an association of VH and VL chains. This association can be noncovalent, as described by Inbar et al., Proc. Natl. Acad. Sci. USA 69:2659, 1972. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as gluteraldehyde (see, for example, Sandhu, Crit. Rev. Biotech. 12:437, 1992).

The Fv fragments may comprise VH and VL chains which are connected by a peptide linker. These singlechain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E . coli . The recombmant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are described, for example, by Whitlow et al., Methods: A Companion to Methods in Enzymology 2 : 91 , 1991, also see, Bird et al., Science 242:423, 1988, Ladner et al., U.S. Patent No. 4,946,778, Pack et al., Bio/Technology 11:1271, 1993, and Sandhu, supra .

As an illustration, a scFV can be obtained by exposing lymphocytes to zsig56 polypeptide in vitro, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zsig56 protein or peptide). Genes encoding polypeptides having potential zsig56 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli . Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., U.S. Patent No. 5,223,409, Ladner et al., U.S. Patent No. 4,946,778, Ladner et al., U.S. Patent No. 5,403,484, Ladner et al., U.S. Patent No. 5,571,698, and Kay et al., Phage Display of Peptides and Proteins (Academic Press, Inc. 1996)) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA), and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide display libraries can be screened using the zsig56 sequences disclosed herein to identify proteins which bind to zsig56.

Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producmg cells (see, for example, Larrick et al . , Methods: A Companion to Methods in Enzymology 2: 106 , 1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al.

(eds.), page 166 (Cambridge University Press 1995), and

Ward et al., "Genetic Manipulation and Expression of

Antibodies," in Monoclonal Antibodies: Principles and

Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an antι-zsig56 antibody may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse lmmunoglobulm into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the murme counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murme constant regions. General techniques for cloning murine lmmunoglobulm variable domains are described, for example, by Orlandi et al., Proc. Nat. Acad. Sci. USA 86:3833, 1989. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:522, 1986, Carter et al., Proc. Nat. Acad. Sci. USA 89:4285, 1992, Sandhu, Crit. Rev. Biotech. 12:437, 1992, Singer et al., J. Immun. 150:2844, 1993, Sudhir (ed.), Antibody Engineering Protocols (Humana Press, Inc. 1995), Kelley, "Engineering Therapeutic Antibodies," m Protein Engineering: Principles and Practice, Cleland et al. (eds.), pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S. Patent No. 5, 693,762 (1997).

Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with antι-zsig56 antibodies or antibody fragments, using standard techniques. See, for example, Green et al., "Production of Polyclonal Antisera," in Methods In Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan, ibid. at pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotype antibodies can be prepared using antι-zsig56 antibodies or antibody fragments as lmmunogens with the techniques, described above. As another alternative, humanized anti-idiotype antibodies or subhuman primate anti-idiotype antibodies can be prepared using the above-described techniques.

Methods for producing anti-idiotype antibodies are described, for example, by Irie, U.S. Patent No.

5,208,146, Greene, et. al., U.S. Patent No. 5,637,677, and

Varthakavi and Mmocha, J. Gen. Virol. 77:1875, 1996.

Genes encoding polypeptides having potential zsig56 polypeptide binding domains, "binding proteins", can be obtained by screening random or directed peptide libraries displayed on phage (phage display) or on bacteria, such as E . coli . Nucleotide sequences encoding the polypeptides can be obtained m a number of ways, such as through random mutagenesis and random polynucleotide synthesis. Alternatively, constrained phage display libraries can also be produced. These peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such peptide display libraries are known in the art (Ladner et al., US Patent NO. 5,223,409; Ladner et al., US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and Ladner et al., US Patent NO. 5,571,698) and peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Peptide display libraries can be screened using the zsig56 sequences disclosed herein to identify proteins which bind to zsig56. These "binding proteins" which interact with zsig56 polypeptides can be used essentially like an antibody.

A variety of assays known to those skilled in the art can be utilized to detect antibodies and/or binding proteins which specifically bind to zsig56 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zsig56 protein or polypeptide.

Antibodies and binding proteins to zsig56 may be used for tagging cells that express zsig56; for isolating zsig56 by affinity purification; for diagnostic assays for determining circulating levels of zsig56 polypeptides; for detecting or quantitatmg soluble zsig56 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zsig56 polypeptide modulation of spermatogenesis or like activity m vi tro and m vivo . Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemilummescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotm-avidm or other complement/anti-complement pairs as intermediates. Moreover, antibodies to zsig56 or fragments thereof may be used in vi tro to detect denatured zsig56 or fragments thereof in assays, for example, Western Blots or other assays known in the art.

Antibodies or polypeptides herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For instance, polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance). More specifically, zsig56 polypeptides or antι-zsig56 antibodies, or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti-complementary molecule.

The present invention also provides methods for studying known or identifying new prohormone convertases, or endoproteases, enzymes which process prohormones and protein precursors. Precursor proteins are cleaved or processed into active form through the action of prohormone convertases (endoproteases). The most prevalent cleavage or processing site is a dibasic amino acid prohormone convertase site. There are only a few dibasic amino acid combinations, including lys-lys, arg-arg, arg-lys and lys-arg. Non-dibasic cleavage and processing sites have also been observed, for example, Asn-Arg is a non-dibasic site found in gastrm. Zsig56 polypeptides may be processed by prohormone convertases into an active from. Known prohormone convertases include, but are not limited to, prohormone convertase 3 (PC3), prohormone convertase 2 (PC2), furm, or similar convertases of the furm family such as prohormone convertase 4 (PC4) and PACE4.

Prohormone convertases sometimes exhibit tissue specificity. As a result, zsig56 polypeptides, which are expressed at high levels in kidney and/or thyroid, for example, are likely to be processed by prohormone convertases exhibiting such tissue specificity. In such methods of the present invention, known or suspected prohormone convertases (enzyme) are incubated with zsig56 polypeptides or fragments (substrate) or cells expressing zsig56 to produce multiple fragments resulting from complete and incomplete cleavage including a 71, 81, 82, 98, 104, 170, 177, 188, 208, 260, 261, 262, 322, 344, 406, 412 and 512 amino acid residue fragments. The enzyme and substrate are incubated together or co-expressed in a test cell for a time sufficient to achieve cleavage/processing of the zsig56 polypeptide or fragment or fusion thereof. Detection and/or quantification of cleavage products follows, using procedures that are known in the art. For example, enzyme kinetics techniques, measuring the rate of cleavage, can be used to study or identify prohormone convertases capable of cleaving zsig56 polypeptides, fragments or fusion proteins of the present invention.

Nucleic acid molecules disclosed herein can be used to detect the presence of a zsig56 gene or gene fragment in a biological sample. Such probe molecules include double-stranded nucleic acid molecules comprising the nucleotide sequences of SEQ ID NOs: 1 or 3, or fragments thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequences of SEQ ID NOs: 1 or 3, or a fragment thereof. Probe molecules may be DNA, RNA, oligonucleotides, and the like.

As an illustration, suitable probes include nucleic acid molecules that bind with a portion of a zsig56 domain or motif, such as the zsig56 extracellular domain (nucleotides 265-1392 of SEQ ID NO:1), extracellular or intracellular loops, or fragments thereof (nucleotides 267-1478, 1807-1917, 2080-2097, 1480-1530, 1678-1745, 1981-2004 of SEQ ID NO:1 or fragments thereof), transmembrane domains (266-1479, 1531-1578, 1626-1677, 1744-1806, 1918-1980, 2005-2079 and 2098-2172 of SEQ ID NO:1 or fragments thereof) or cytoplasmic domain (2099-2268 of SEQ ID NO:1 or fragments thereof). Other probes include nucleotide sequences encoding the proteolytic fragments described herein.

In a basic assay, a single-stranded probe molecule is incubated with RNA, isolated from a biological sample, under conditions of temperature and ionic strength that promote base pairing between the probe and target zsig56 RNA species. After separating unbound probe from hybridized molecules, the amount of hybrids is detected.

Well-established hybridization methods of RNA detection include northern analysis and dot/slot blot hybridization (see, for example, Ausubel ibid, and Wu et al. (eds.), "Analysis of Gene Expression at the RNA Level," in Methods in Gene Biotechnology, pages 225-239 (CRC Press, Inc. 1997)). Nucleic acid probes can be detectably labeled with radioisotopes such as 32P or 35S . Alternatively, zsig56 RNA can be detected with a nonradioactive hybridization method (see, for example, Isaac (ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes, Humana Press, Inc., 1993). Typically, nonradioactive detection is achieved by enzymatic conversion of chromogenic or chemiluminescent substrates. Illustrative nonradioactive moieties include biotin, fluorescein, and digoxigenin.

Zsig56 oligonucleotide probes are also useful for in vivo diagnosis. As an illustration, 18F-labeled oligonucleotides can be administered to a subject and visualized by positron emission tomography (Tavitian et al., Nature Medicine 4:467, 1998).

Numerous diagnostic procedures take advantage of the polymerase chain reaction (PCR) to increase sensitivity of detection methods. Standard techniques for performing PCR are well-known (see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols; Current Methods and Applications (Humana Press, Inc. 1993), Cotter

(ed.), Molecular Diagnosis of Cancer (Humana Press, Inc.

1996), Hanausek and Walaszek (eds.), Tumor Marker

Protocols (Humana Press, Inc. 1998), Lo (ed.), Clinical

Applications of PCR (Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

PCR primers can be designed to amplify a sequence encoding a particular zsig56 domain or motif, such as the zsig56 cysteine motif (encoded by about nucleotide 325-459 of SEQ

ID NO:1 or nucleotides 280-414 of SEQ ID NO:15).

One variation of PCR for diagnostic assays is reverse transcriptase-PCR (RT-PCR). In the RT-PCR technique, RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with zsig56 primers (see, for example, Wu et al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR," in Methods in Gene Biotechnology, CRC Press, Inc., pages 15-28, 1997). PCR is then performed and the products are analyzed using standard techniques.

As an illustration, RNA is isolated from biological sample using, for example, the guamdinium-thiocyanate cell lysis procedure described herein. Alternatively, a solid-phase technique can be used to isolate mRNA from a cell lysate. A reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short homopolymers of dT, or zsig56 anti-sense oligomers. Oligo-dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences. Zsig56 sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically at least 5 bases in length.

PCR amplification products can be detected using a variety of approaches. For example, PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining. Alternatively, fractionated PCR products can be transferred to a membrane, hybridized with a detectably-labeled zsig56 probe, and examined by autoradiography. Additional alternative approaches include the use of digoxigenin-labeled deoxyπbonucleic acid triphosphates to provide chemiluminescence detection, and the C-TRAK colorimetric assay.

Another approach is real time quantitative PCR

(Perkm-Elmer Cetus, Norwalk, Ct.). A fluorogenic probe, consisting of an oligonucleotide with both a reporter and a quencher dye attached, anneals specifically between the forward and reverse primers. Using the 5' endonuclease activity of Taq DNA polymerase, the reporter dye is separated from the quencher dye and a sequence-specific signal is generated that increases as amplification increases. The fluorescence intensity can be continuously monitored and quantified during the PCR reaction.

Another approach for detection of zsig56 expression is cycling probe technology (CPT), in which a single-stranded DNA target binds with an excess of DNA-RNA-DNA chimeπc probe to form a complex, the RNA portion is cleaved with RNase H, and the presence of cleaved chimeric probe is detected (see, for example, Beggs et al., J. Clin. Microbiol. 34:2985, 1996 and Bekkaoui et al., Biotechmques 20:240, 1996). Alternative methods for detection of zsig56 sequences can utilize approaches such as nucleic acid sequence-based amplification (NASBA), cooperative amplification of templates by cross-hybridization (CATCH), and the ligase chain reaction (LCR)

(see, for example, Marshall et al., U.S. Patent No.

5,686,272 (1997), Dyer et al., J. Virol. Methods 60:161,

1996; Ehπcht et al., Eur. J. Biochem. 243:358, 1997 and

Chadwick et al., J. Virol. Methods 70 : 59 , 1998). Other standard methods are known to those of skill in the art.

Zsig56 probes and primers can also be used to detect and to localize zsig56 gene expression m tissue samples. Methods for such m si tu hybridization are well-known to those of skill m the art (see, for example, Choo (ed.), In Situ Hybridization Protocols, Humana Press, Inc., 1994; Wu et al . (eds.), "Analysis of Cellular DNA or Abundance of mRNA by Radioactive In Si tu Hybridization (RISH)," in Methods m Gene Biotechnology, CRC Press, Inc., pages 259-278, 1997 and Wu et al. (eds.), "Localization of DNA or Abundance of mRNA by Fluorescence In Si tu Hybridization (RISH)," in Methods in Gene Biotechnology, CRC Press, Inc., pages 279-89, 1997).

Various additional diagnostic approaches are well-known to those of skill in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics Humana Press, Inc., 1991; Coleman and Tsongalis, Molecular Diagnostics, Humana Press, Inc., 1996 and Elles, Molecular Diagnosis of Genetic Diseases, Humana Press, Inc., 1996).

Probes and primers generated from the sequences disclosed herein can be used to map the zsig56 gene to a particular chromosome. Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250:245-50, 1990). Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels. Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL). These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence-tagged sites (STSs), and other nonpolymorphic and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers. The precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.

Sequence tagged sites (STSs) can also be used independently for chromosomal localization. An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome. An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based solely on DNA sequence they can be completely described withm an electronic database, for example, Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm. mh.gov), and can be searched with a gene sequence of interest for the mapping data contained withm these short genomic landmark STS sequences.

The present invention also contemplates use of such chromosomal localization for diagnostic applications. Briefly, the zsig56 gene, a probe comprising zsig56 DNA or RNA or a subsequence thereof, can be used to determine if the zsig56 gene is present on an identified chromosome or if a mutation has occurred. Detectable chromosomal aberrations at the zsig56 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).

In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient. Genetic samples for use withm the present invention include genomic DNA, cDNA, and RNA. The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:1 or SEQ ID NO: 6, the complement of SEQ ID NO:1 or SEQ ID NO: 6, or an RNA equivalent thereof. Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applications 1;5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108 :255-65, 1995). Ribonuclease protection assays (see, e.g., Ausubel et al., ibid., ch. 4) comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase. Hybridized regions of the RNA are protected from digestion. Withm PCR assays, a patient's genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations m the patient. Another PCR-based technique that can be employed is single strand conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications 1:34-8, 1991).

Tissue distribution of zsig56 mRNA, together with the known functions of related G protem-coupled receptors suggests a variety of functions for the zsig56 receptor. Expression of zsig56 mRNA in kidney suggests that the receptor regulates ion excretion. The polypeptides, nucleic acid, agonists (including the natural ligand, substrate, cofactor, etc.) and/or antagonists of the present invention may be used in treatment of disorders associated with changes in ion or electrolyte homeostasis, particularly disorders caused by, or resulting in, changes in calcium, phosphate, magnesium, zinc and copper levels. The molecules of the present invention may used to modulate electrolyte imbalances or to treat or prevent development of pathological conditions in such diverse tissue as bone, heart, kidney, pancreas and the vascular system. In particular, certain bone diseases, hypertension, renal failure, gout, congestive heart failure, hyperthyroidism, hyperparathyroidism, certain carcinomas, sarcoidosis, pancreatitis and drug-induced disorders that result in elevated levels of serum calcium, known as hypercalcemia, may be amenable to such diagnosis, treatment or prevention.

Secretin and parathyroid hormone (PTH) have reciprocal actions on Ca2+ and phosphate secretion by the kidney that produce long term changes in bone formation (Guyton, Textbook of Medical Physiology, 7 ed. 1986). PTH and parathyroid hormone-related protein (PTHRP) stimulate osteoclasts to resorb bone and are believed to provide the primary pathogenic mechanism for hypercalcemia. Also, excessive gastrointestinal absorption of calcium can lead to hypercalcemia and has been correlated with pancreatitis.

Depressed levels of serum calcium, or hypocalcemia, can lead to increased resorption of calcium from bone. Increased bone resorption can result in osteoporosis and Paget's disease. Molecules of the present invention can be used to identify and, in some cases treat, diseases where calcium regulation is abnormal.

To verify these capabilities in zsig56 polypeptides, polynucleotides, agonists or antagonists of the present invention, the molecules of the present invention are evaluated for regulating ion homeostasis in such disorders as bone resorption, according to procedures known in the art. Well established animal models are available to test in vivo efficacy of zsig56 polypeptides, ligands, antibodies, agonists or antagonists. For example, the hypocalcemic rat or mouse model can be used to determine the effect of zsig56 polypeptides, polynucleotides, agonists or antagonists on serum calcium, and the ovariectomized rat or mouse can be used as a model system for osteoporosis. Bone changes seen in these models and in humans during the early stages of estrogen deficiency are qualitatively similar. Calcitonin has been shown to be an effective agent for the prevention of bone loss in ovariectomized women and rats (Mazzuoli et al., Calcif. Tissue Int. 47:209-14, 1990; Wronski et al., Endocrinology 129: 2246-50, 1991). High dose estrogen has been shown to inhibit bone resorption and to stimulate bone formation in an ovariectomized mouse model (Bain et al., J. Bone Miner. Res. 8:435-42, 1993). If desired, zsig56 polypeptide performance in this regard can be compared to calcitonin and estrogen or the like.

The efficacy of zsig56 polypeptides, polynucleotides agonists or antagonists in hypertension is tested in vivo utilizing spontaneously hypertensive rats (SHR) for systemic hypertension (Marche et al., Clin. Exp. Pharmacol. Physiol. Suppl. 1:S114_6' 1995).

Assays measuring in vivo effects of synthetic zsig56 polypeptides, polynucleotides, agonists or antagonists include a Left Ventricular Hypertrophy model (Feldman et al., Circ. Res. 73:184-92, 1993), which measures remodeling and repair after congestive heart failure and chronic pressure overload.

Expression of zsig56 mRNA in thyroid supports a role for this receptor in therapeutic regimes to treat hypo and hyperthyroidism and weight disorders.

Many of the regulatory peptides that are important in maintaining nutritional homeostasis are found in the gastrointestinal environment and signal through G protein-coupled receptors. For a review of gut peptides see, Mulvihill et al., in Basic and Clinical Endocrinology, pp. 551-70, 4th edition Greenspan and Baxter editors, Appleton & Lange: Norwalk, Connecticut, 1994. Many of these gut peptides are synthesized as inactive precursor molecules that require multiple peptide cleavages to be activated. Exemplary peptides regulated by multiple cleavages and post-translational modifications include VIP, gastrin, secretin, motilin, glucagon and galanin. These gut hormones use several different mechanisms of action, endocrine, neurocrine and paracrine, either exclusively or in combination.

In view of the tissue distribution observed for zsig56 in colon and stomach, zsig56 polypeptides, agonists or antagonists would be useful for promoting stimulation of gastrointestinal (GI) cell contractility and GI motility, modulation of nutrient uptake and/or secretion of digestive enzymes and acids in vivo and in vitro . For example, agonist compounds are useful as components of defined cell culture media and regulate the uptake of nutrients, and thus are useful in specifically promoting the growth and/or development of gastrointestinal cells such as G cells, enterochromaffin cells and the epithelial mucosa of the stomach, duodenum, proximal jejunum, antrum and fundus.

To verify these capabilities in zsig56 polypeptides, polynucleotides, agonists or antagonists of the present invention, the molecules of the present invention are evaluated for their ability to regulate gastrointestinal motility and secretion of digestive enzymes and acids, according to procedures known in the art. The activity of molecules of the present invention can be measured using a variety of assays that measure stimulation of gastrointestinal cell contractility, modulation of nutrient uptake and/or secretion of digestive enzymes. Of particular interest are changes m contractility of smooth muscle cells. For example, the contractile response of segments of mammalian duodenum or other gastrointestinal smooth muscles tissue (Depoortere et al., J. Gastrointestinal Motility 1:150-9, 1989). An exemplary m vivo assay uses an ultrasonic micrometer to measure the dimensional changes radially between commissures and longiturdmally to the plane of the valve base (Hansen et al., Society of Thoracic Surgeons 60: S384-90, 1995).

Gastric motility is generally measured m the clinical setting as the time required for gastric emptylng and subsequent transit time through the gastrointestinal tract. Gastric emptylng scans are well known to those skilled in the art, and briefly, comprise use of an oral contrast agent, such as barium, or a radiolabeled meal. Solids and liquids can be measured independently. A test food or liquid is radiolabeled with an isotope (e.g. 99mTc) , and after mgestion or administration, transit time through the gastrointestinal tract and gastric emptylng are measured by visualization using gamma cameras (Meyer et al., Am. J. Dig. Pis. 21:296, 1976; Collins et al., Gut 24:1117, 1983; Maughan et al., Diabet. Med. 13 9 Supp. 5:S6-10, 1996 and Horowitz et al., Arch. Intern. Med. 145: 1467-72, 1985). These studies may be performed before and after the administration of a zsig56 polypeptide, polynucleotide, antagonist or agonist to quantify the efficacy as a therapeutic.

The many gut peptides have also been associated with neurological and CNS functions, "gut-brain" peptides. For example, neuropeptide Y (NPY), a peptide with receptors in both the bram and the gut has been shown to stimulate appetite when administered to the central nervous system (Gehlert, Life Sciences 55(6): 551-562, 1994). Motilm lmmunoreactivity has been identified m different regions of the bram, particularly the cerebellum, and in the pituitary (Gasparim et al., Hum. Genetics 94 (6): 671-674, 1994). Motilm has been found to coexist with neurotransmitter -aminobutyric acid in cerebellum (Chan-Patay, Proc. Sym. 50th Anniv. Meet. Br. Pharmalog. Soc.:1-24, 1982). Physiological studies have provided some evidence that motilm has an affect on feeding behavior (Rosenfield et al., Phys. Behav. 39(6): 735-736, 1987), bladder control, pituitary growth hormone release. Other gut-brain peptides, such as CCK, enkephalm, VIP and secretin have been shown to be involved in control of blood pressure, heart rate, behavior, and pam modulation, in addition to be active in the digestive system. Zsig56 was found to be expressed in various bram tissues. Therefore, a zsig56 polypeptide, polynucleotide, agonist or antagonist could be expected to have some neurological association.

Zsig56 also has significant homology with the receptor for alpha-latrotoxm, a potent toxin from black widow spiders (Krasnoperov et al., Neuron 18: 925-37, 1997 and Lelianova et al., J. Biol. Chem. 272:21504-08, 1997).

Alpha-latrotoxin stimulates neurotransmitter release from pre-synaptic nerve terminals. Expression of zsig56 in brain and spinal cord suggests this receptor plays a role in regulating neurotransmission. The molecules of the present invention would be of use in therapeutic application for treating a variety of nervous system disorders such as neurodegenerative diseases including multiple sclerosis, Alzheimer's disease and Parkinson's disease; schizophrenia; manic depression and repair of nerve tissue following damage due to strokes, brain damage caused by head injuries and paralysis caused by spinal injuries.

The alpha-latrotoxin receptor has been shown to regulate insulin release in pancreatic islets stimulated with alpha-latrotoxin (Lang et al., EMBO J. 17:648-57, 1998). Zsig56 mRNA has been detected in pancreas. To verify this capability in zsig56 polypeptides, polynucleotides, agonists or antagonists of the present invention, the molecules of the present invention are evaluated according to procedures known in the art. For example, see Lang et al., ibid.

Zsig56 also shares homology with the receptor for neuropeptide corticotropin releasing hormone. The molecules of the present invention would be useful in regulating the pituitary-hypothalamic-adrenal axis. Such molecules would be useful in therapeutic applications for treating stress induced disorders. This would include high blood pressure, any conditions associated with caticholamines and adrenal steroids such as certain forms of heart failure, immune depression, periodontal disease, see for example, Genco et al., Ann. Periodontol. 3;288-302, 1998; Guidi et al., Gerontology 44:247-61, 1998 and Theoreil et al., J. Occup. Health Psychol. 1:9-26, 1996.

The homology of zsig56 to the lymphocyte activation marker CD97 (Hamann et al., J. Immunol. 155:1942-50, 1995, Hamann et al., Genomics 32:144-7, 1996) suggests that zsig56 ligands, agonists or antagonists would be useful therapeutics for treatment of inflammatory disorders such as rheumatoid arthritis.

To verify the presence of this capability in zsig56 ligands, agonists or antagonists, such ligands, agonists or antagonists are evaluated with respect to their ability to inhibit acute inflammation. Such methods are known m the art, m particular, zsig56 ligands, agonists or antagonists can be tested for anti-mflammatory activity m the carrageenan-mduced rat footpad edema model (Winter et al., J. Pharmac. Exp. Ther. 141:369-76, 1963 and Miele et al., Nature 335:726-30,

1988). Other models include the endotoxm-mduced uveitis

(EIU) model (Chan et al., Arch. Ophthalmol. 109:278-81,

1991), Oxazolone-mduced inflammation model (Lloret and

Moreno, Biochem. Pharmocol. 44:1437, 1992), croton oil-induced inflammation model, PMA-mduced inflammation model

(Miele et al., ibid.), and dextran-mduced edema assay for anti-mflammatory agents (Ialenti et al., Agents Actions

29:48-9, 1990 and Rosa and Willoughby, J. Pharm. Pharmac.

23:297-8, 1971). Efficacy for treating diseases such as rheumatoid arthritis can be evaluated using indicators which would include a reduction in inflammation and relief of pam or stiffness, and m animal models indications would be derived from macroscopic inspection of joints and change in swelling of hind paws. If desired, zsig56 ligands, agonists or antagonists performance in this regard can be compared to other anti-mflammatory agents, m particular, dexamethasone, Enbrel, non-steroid anti-mflammatory drugs such as aspirin. In addition, zsig56 ligands, agonists or antagonists may be evaluated in combination with one or more anti-inflammatory agents to identify synergistic effects.

Zsig56 is also expressed in tissues associated with reproduction, testis, prostate and placenta. Hormones associated with the reproductive cascade such as gonadotropin-releasing hormone, follicle-stimulating hormone and luteinizing hormone stimulate G coupled-protein receptors and regulate spermatogenesis and testicular androgen production through the brain-testis axis. Zsig56 polypeptides and agonists would be useful for treating male and female reproductive disorders. Zsig56 polypeptides, agonists and antagonists can be used as therapeutics for male fertility by stimulation of spermatogenesis, independently or in conjunction with other gonadotropins or sex steroids such as testosterone.

In vivo assays for evaluating the effect of zsig56 polypeptides, agonists and antagonists on testes are well known in the art. For example, compounds can be injected intraperitoneally for a specific time duration. After the treatment period, animals are sacrificed and testes removed and weighed. Testicles are homogenized and sperm head counts are made (Meistrich et al., Exp. Cell Res. 99:72-78, 1976). Other activities, for example, chemotaxic activity that may be associated with proteins of the present invention can be analyzed. For example, late stage factors in spermatogenesis may be involved in egg-sperm interactions and sperm motility. Activities, such as enhancing viability of cryopreserved sperm, stimulating the acrosome reaction, enhancing sperm motility and enhancing egg-sperm interactions may be associated with the proteins of the present invention. Assays evaluating such activities are known (Rosenberger, J. Androl. 11:89-96, 1990; Fuchs, Zentralbl Gynakol 11:117-120, 1993; Neurwinger et al., Andrologia 22:335-9, 1990; Harris et al., Human Reprod. 3:856-60, 1988; and Jockenhovel, Andrologia 22: 171-178, 1990; Lessing et al., Fertil. Steril. 44:406-9 (1985); Zaneveld, In Male Infertility Chapter 11, Comhaire Ed., Chapman & Hall, London 1996). These activities are expected to result in enhanced fertility and successful reproduction.

Prostate adenocarcinoma is one of the most frequent cancers diagnosed in American men. It afflicts men over the age of 50 and intervention is dependent upon the stage to which the disease has progressed at the time of diagnosis. Consequently, markers and therapeutics are sought for prostate cancer, also, agents capable of reversing or blocking metastasis or capable of depressing elevated levels of prostate-cancer associated polypeptides are also sought for additional avenues of therapeutic intervention. Association of zsig56 with prostate suggests the molecules of the present invention would be useful as markers or in therapeutic regimes for treating prostate cancer.

Proteins of the present invention would also be useful as therapeutics for regulating pregnancy, gestation and birth.

The invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1

Extension of EST Sequence

The novel zsig56 polypeptide-encoding polynucleotides of the present invention were initially identified by querying an EST database for secretory signal sequences characterized by an upstream methionine start site, a hydrophobic region of approximately 13 amino acids and a cleavage site m an effort to select for secreted proteins. Polypeptides corresponding to ESTs meeting those search criteria were compared to known sequences to identify secreted proteins having homology to known ligands. An two ESTs were discovered and predicted to encode a seven transmembrane domain receptor. The full length cDNA sequence was derived from a retina library. Using an Invitrogen S.N.A.P.™ Mimprep kit (Invitrogen, Corp., San Diego, CA) according to manufacturer's instructions a 5 ml overnight culture in LB + 50 μg/ml ampicillm of a clone from the retina library were prepared. The template were sequenced on an ABIPRISM ™ model 377 DNA sequencer (Perkm-Elmer Cetus, Norwalk, Ct.) using the ABI PRISM™ Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkm-Elmer Corp.) according to manufacturer's instructions. The sequence was verified by sequence analysis carried out in a Hybaid OmniGene Temperature Cycling System (National Labnet Co., Woodbridge, NY). SEQUENCHER™ 3.0 sequence analysis software (Gene Codes Corporation, Ann Arbor, MI) was used for data analysis. The resulting 3710 bp sequence of zsig56 is disclosed m SEQ ID NO:1.

Example 2

Tissue Distribution

Human Multiple Tissue Northern Blots (MTN I, MTN II and MTN III; Clontech) were probed to determine the tissue distribution of human zsig56 expression. An approximately 750 bp probe encompassing the 5' end of the zsig56 polypeptide was derived by restriction digest and radioactively labeled using the MULTIPRIME DNA labeling kit (Amersham, Arlington Heights, IL) according to the manufacturer's instructions. The probe was purified using a NUCTRAP push column (Stratagene). EXPRESSHYB (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 65°C using 1.0 x 10 cpm/ml of labeled probe. The blots were then washed 4 times in 2X SSC and 0.1% SDS at 25°C, followed by 2 washes in 0.1X SSC and 0.1% SDS at 56°C. A 4.0 kb transcript was detected corresponding to zsig56 in kidney and thyroid. Signals of less intensity were also detected in spinal cord, placenta, trachea, heart, lung, PBLs, brain, pancreas, prostate, stomach, spleen, testis and colon.

A RNA Master Dot Blot (Clontech) that contained RNAs from various tissues that were normalized to 8 housekeeping genes was also probed and hybridized as described above. The expression pattern matched that of the Northern blot.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.