WO2003033688A1

WO2003033688A1 - Regulation of human receptor tyrosine phosphatase

Info

Publication number: WO2003033688A1
Application number: PCT/EP2002/011473
Authority: WO
Inventors: Jiing-Ren Liou
Original assignee: Bayer Healthcare Ag
Priority date: 2001-10-16
Filing date: 2002-10-14
Publication date: 2003-04-24

Abstract

Reagents that regulate human receptor tyrosine phosphatase and reagents which bind to human receptor tyrosine phosphatase gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, diabetes, CNS disorders, obesity, COPD or cardiovascular disorders.

Description

REGULATION OF HUMAN RECEPTOR TYROSINE PHOSPHATASE

FIELD OF THE INVENTION

The invention relates to the regulation of human receptor tyrosine phosphatase.

BACKGROUND OF THE INVENTION

Phosphorylation of proteins is a fundamental mechanism for regulating diverse cellular processes. U.S. Patent 5,952,212. While the majority of protein phosphor- ^' ylation occurs at serine and threonine residues, phosphorylation at tyrosine residues is attracting a great deal of interest since the discovery that many oncogene products and growth factor receptors possess intrinsic protein tyrosme kinase activity. The importance of protein tyrosme phosphorylation in growth factor signal transduction, cell cycle progression and neoplastic transformation is now well established. Hunter et al, Ann. Rev. Biochem. 54, 987-30, 1985; Ullrich et al, Cell 61, 203-12, 1990; Nurse, Nature 344, 503-08, 1990; Cantley et al, Cell 64, 281-302, 1991.

Biochemical studies have shown that phosphorylation on tyrosme residues of a variety of cellular proteins is a dynamic process involving competing phosphorylation and dephosphorylation reactions. The regulation of prot-ein , tyrosine phosphorylation is mediated by the reciprocal actions of protein tyrosme kinases (PTKases or PTKS) and protein tyrosine phosphatases (PTPs). The tyrosme phosphorylation reactions are catalyzed by PTKs. Tyrosine phosphorylated proteins can be specifically dephosphorylated through the action of PTPs. The level of protein tyrosme phosphorylation of intracellular substances is determined by the balance of PTK and PTP activities. Hunter, Cell 58, 1013-16, 1989. REGULATION OF HUMAN RECEPTOR TYROSINE PHOSPHATASE Protein Tyrosine Kinases

The protein tyrosine kinases (PTKS; ATPφrotein-tyrosine O-phosphotransferase, EC 2.7.1.112) are a large family of proteins that includes many growth factor receptors and potential oncogenes. Hanks et al., Science 241, 42-52, 1988. Many PTKs have been linked to initial signals required for induction of the cell cycle. Weaver et al, Mol. Cell. Biol. 11(9), 4415-22, 1991. PTKs comprise a discrete family of enzymes having common ancestry with, but major differences from, serine/threonine-specific protein kinases. Hanks et al, supra. The mechanisms leading to changes in activity of PTKs are best understood in the case of receptor-type PTKs having a transmembrane topology. Ullrich et al., 1990, supra. The binding of specific ligands to the extracellular domain of members of receptor-type PTKs is thought to induce their oligomerization, leading to an increase in tyrosine kinase activity and activation of the signal transduction pathways. Ullrich et al, 1990, supra. Deregulation of kinase activity through mutation or overexpression is a well-established mechanism for cell transformation. Hunter et al, 1985; Ullrich et al, 1990.

Protein Tyrosine Phosphatases

The protein phosphatases are composed of at least two separate and distinct families: the protein serine/threonine phosphatases and the protein tyrosine phosphatases (PTPs; protein-tyrosine-phosphate phosphohydrolase, EC 3.13.48. Hunter, 1989. The PTPs are a family of proteins that have been classified into two subgroups. The first subgroup is made up of the low molecular weight, intracellular enzymes that contain a single conserved catalytic phosphatase domain. All known intracellular type PTPs contain a single conserved catalytic phosphatase domain. Examples of the first group of PTPs include (1) placental PTP IB (Charbonneau et al, Proc Natl. ^' Acad. Sci. USA 86, 5252-56, 1989; Chernoff et al, Proc. Natl. Acad. Sci. USA 87, 2735-89, 1990, (2) T-cell PTP (Cool et al, Proc. Natl. Acad. Sci. USA 86, 5257-61,

1989, (3) rat brain PTP (Guan et al, Proc. Natl. Acad. Sci. USA 87, 1501-02, 1990, (4) neuronal phosphatase (STEP) (Lombroso et al, Proc. Natl. Acad. Sci. USA 88, 7242-46, 1991, and (5) cytoplasmic phosphatases that contain a region of homology to cytoskeletal proteins (Gu et al, Proc. Natl. Acad. Sci. USA 88, 5867-871, 1991; Yang et al, Proc. Natl. Acad. Sci. USA 88, 5949-53, 1991.

The second subgroup is made up of the high molecular weight, receptor-linked PTPs, termed RPTPs. RPTPs consist of (a) an intracell lar catalytic region, (b) a single transmembrane segment, and (c) a putative ligand-binding extracellular domain. The structures and sizes of the putative ligand-binding extracellular "receptor" domains of RPTPs are quite divergent. In contrast, the intracellular catalytic regions of RPTPs are highly homologous. All RPTPs have two tandemly duplicated catalytic phosphatase homology domains, with the prominent exception of an RPTP termed HPTPβ, which has only one catalytic phosphatase domain. Tsai et al, J. Biol. Chem. 266, 0534-43, 1991.

Since the initial purification, sequencing, and cloning of a PTP, additional potential PTPs have been identified at a rapid pace. The number of different PTPs that have been identified is increasing steadily, leading to speculations that this family may be as large as the PTK family. Hunter, 1989.

Conserved amino acid sequences designated "consensus sequences" have been identified in the catalytic domains of known PTPs. Krueger et al, EMBO J. 9, 3241-52, 1990; Yi et al, Mol. Cell. Biol. 12, 836-46, 1992. Yi et al aligned the catalytic phosphatase domain sequences of the following PTPs: LCA, PTP1B, TCPTP, LAR, DLAR, and HPTPα, HPTPβ, and HPTPγ. This alignment includes the following consensus sequences (Yi et al, supra): l. K C X X Y W P 2. H C S X G X G RX G.

Krueger et al., aligned the catalytic phosphatase domain sequences of PTP1B, TCPTP, LAR, LCA, HPTPα, β, δ, ε and ζ, and DLAR and DPTP. This alignment includes the following consensus sequences (Krueger et al, supra): l. K C X X Y W P

2. H C S X G X G R X G.

It is becoming clear that dephosphorylation of tyrosme residues can by itself function as an important regulatory mechanism. Dephosphorylation of a C-terminal tyrosme residue has been shown to activate tyrosme kinase activity in the src family of tyrosine kinases. Hunter, Cell 49,. 1-A, 1987. Tyrosme dephosphorylation has been suggested to be an obligatory step in the mitotic activation of the maturation-promoting factor (MPF) kinase. Morla et al, Cell 58, 193-203, 1989. These observations point out the need in the art for understanding the mechanisms that regulate tyrosine phosphatase activity.

Because of the importance of these enzymes in a variety of cellular processes, there is a continuing need in the art to identify additional tyrosine phosphatases, which can be regulated to provide therapeutic effects.

BRIEF SUMMARY OF THE INVENTION

It is an object of the invention to provide reagents and methods of regulating a human receptor tyrosine phosphatase. This and other objects of the invention are provided by one or more of the embodiments described below.

One embodiment of the invention is a receptor tyrosme phosphatase polypeptide comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 87% identical to the amino acid^' sequence shown in SEQ ID NO: 2; and

the amino acid sequence shown in SEQ ID NO: 2. Yet another embodiment of the invention is a method of screening for agents hich decrease extracellular matrix degradation. A test compound is contacted with a receptor tyrosine phosphatase polypeptide comprising an amino acid sequence '^'selected from the group consisting of:

amino acid sequences which are at least about 87% identical to the amino acid sequence shown in SEQ ID NO: 2; and

the amino acid sequence shown in SEQ ID NO: 2.

Binding between the test compound and the receptor tyrosine phosphatase polypeptide is detected. A test compound which binds to the receptor tyrosme phosphatase polypeptide is thereby identified as a potential agent for decreasing extra- cellular matrix degradation. The agent can work by decreasing the activity of the receptor tyrosine phosphatase.

Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a poly- nucleotide encoding a receptor tyrosme phosphatase polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; and

the nucleotide sequence shown in SEQ ED NO: 1.

Binding of the test compound to the polynucleotide is detected. A test compound - which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the amount of the receptor tyrosine phosphatase through interacting with the receptor tyrosme phosphatase mRNA.

Another embodiment of the mvention is a method of screening for agents which regulate extracellular matrix degradation. A test compound is contacted with a receptor tyrosine phosphatase polypeptide comprising an amino acid sequence selected from the group consisting of:

the amino acid sequence shown in SEQ ID NO: 2.

A receptor tyrosine phosphatase activity of the polypeptide is detected. A test com- pound which increases receptor tyrosme phosphatase activity of the polypeptide relative to receptor tyrosine phosphatase activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation. A test compound which decreases receptor tyrosine phosphatase activity of the polypeptide relative to receptor tyrosine phosphatase activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.

Even another embodiment of the mvention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a receptor tyrosme phosphatase product of a polynucleotide which comprises a- nucleotide sequence selected from the group consisting of:

nucleotide sequences which are at least about 50%o identical to the nucleotide^" sequence shown in SEQ LD NO: 1; and

the nucleotide sequence shown in SEQ ID NO: 1. Binding of the test compound to the receptor tyrosine phosphatase product is detected. A test compound which binds to the receptor tyrosine phosphatase product is thereby identified as a potential agent for decreasing extracellular matrix degradation.

Still another embodiment of the mvention is a method of reducing extracellular matrix degradation. A cell is contacted with a reagent which specifically binds- to a polynucleotide encoding a receptor tyrosine phosphatase polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ LD NO: 1; and

the nucleotide sequence shown in SEQ ID NO: 1.

Receptor tyrosme phosphatase activity in the cell is thereby decreased.

The invention thus provides a human receptor tyrosine phosphatase that can be used to identify test compounds that may act, for example, as activators or inhibitors at the enzyme's active site. Human receptor tyrosine phosphatase and fragments thereof also are useful in raising specific antibodies that can block the enzyme and effectively reduce its activity.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the DNA-sequence encoding a receptor tyrosme phosphatase Polypeptide (SEQ ID NO:l). Fig. 2 shows the amino acid sequence deduced from the DNA- sequence of Fig.1 (SEQ ID NO:2). Fig. 3 shows the amino acid sequence of the protein identified by trembl|AF063249|AF063249_l (SEQ ID NO:3). Fig. 4 shows the amino acid sequence of a receptor tyrosine phosphatase Polypeptide (SEQ LD NO:4). Fig. 5 shows the amino acid sequence of a receptor tyrosme phosphatase Polypeptide (SEQ ID NO:5). Fig. 6 shows the DNA-sequence encoding a receptor tyrosine phosphatase Polypeptide (SEQ ID NO:6). Fig. 7 shows the DNA-sequence encoding a receptor tyrosme phosphatase Polypeptide (SEQ TD NO:7):

Fig. 8 shows the DNA-sequence encoding a receptor tyrosine phosphatase Polypeptide (SEQ TD NO: 8). Fig. 9 shows the BLASTP - alignment of 492_Protein (SEQ

TD NO:2) against trembl|AF063249|AF063249_l (SEQ LD NO:3). This hit is scoring at: 0.0 (expectation value); Alignment length (overlap): 2301 ; Identities :

86 % ; Scoring matrix : BLOSUM62 (used to infer consensus pattern) ; Database searched : nrdb_l_. Fig. 10 shows the BLASTP - alignment of 492_Protein (SEQ ID NO:2) against pdb|lLAR|lLAR-A larfragment: phosphatase. This hit is scoring at: 3e-60 (expectation value); Alignment length (overlap): 308; Identities: 40

% ; Scoring matrix : BLOSUM62 (used to infer consensus pattern) ; Database searched : nrdb_l_. Fig. 11 shows the BLASTP - alternative alignment of

492_Protein (SEQ LD NO:2) against pdb|lLAR|lLAR-

A lar fragment: phosphatase. This hit is scoring at: 3e-

44 (expectation value); Alignment length (overlap): 245;

Identities: 40 %; Scoring matrix: BLOSUM62 (used to infer consensus pattern); Database searched: nrdb_l_. Fig. 12 shows the HMMPFAM - alignment of 492_Protein

(SEQ ID NO:2) against pfam|hmm|Yjphosphatase

Protein-tyrosine phosphatase. This hit is scoring at:

374.0; Scoring matrix: BLOSUM62 (used to infer consensus pattern).

Fig. 13 shows the ATP_GTP_A region, transmembrane segments, and fibronectin type III domain region prediction. ATP_GTP_A region from residue 1621 to 1629. Source: [prosite]; Tyrosine specific protein phosphatases signature; -Consensus pattern: [LINMF]-

H-C-x(2)-G-x(3)-[STC]-[STAGP]-x-[LINMFY] [C is the active site residue]; ATP/GTP -binding site motif A (P-loop); -Consensus pattern: [AG]-x(4)-G-K-[ST]; High scoring transmembrane segments: From 1906 to 1928: length= 23, score=69.00 ** 1906 IILSNTLCIL

SΠLLGTALF AFAL: 5(21.7%); A: 3(13.0%); I:

6(26.1%); Fibronectin type III domain region.

Fig. 14 shows the Genewise output: use rat "glomerular' mesangial cell receptor protein-tyrosine phosphatase precursor" (AF063249) (SEQ LD ΝO:3) as a template on human genomic DNA (NT_019546; chrl2:8890000188900001-32923-88900001269821); Score 3940.84 bits over entire alignment; Scores as bits over a synchronous coding model. Fig. 15 shows the Genewise output: use partial protein

(492_BAC_p, below) of rat "glomerular mesangial cell receptor protein-tyrosine phosphatase precursor" (AF063249) (SEQ ID NO:3) as a template on human 12q BAC RPll-288D9 (gi|15011617]gb|AC083812.24). DET AILED DESCRIPTION OF THE INVENTION

The invention relates to an isolated polynucleotide from the group consisting of:

a) a polynucleotide encoding a receptor tyrosme phosphatase polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 87% identical to the amino acid sequence shown in SEQ LD NO: 2; and the amino acid sequence shown in SEQ LD NO: 2. b) a polynucleotide comprising the sequence of SEQ ID NO: 1 ; c) a polynucleotide wliich hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a receptor tyrosine phosphatase polypeptide; d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a receptor tyrosine phosphatase polypeptide; and e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a receptor tyrosine phosphatase polypeptide.

Furthermore, it has been discovered by the present applicant that a novel receptor tyrosine phosphatase, particularly a human receptor tyrosme phosphatase, can be used in therapeutic methods to treat diabetes, a CNS disorder, COPD or a cardiovascular disorder. Human receptor tyrosme phosphatase comprises the amino acid sequence shown in SEQ ID NO:2. A coding sequence for human receptor tyrosine phosphatase is shown in SEQ LD NO:l. This sequence is located on^' chromosome 12. Related ESTs (AF169351; AR073855; AR073854; AR073853; AR073852; AR073851; AR073850) are expressed in colon and K562 CML cell line. Human receptor tyrosme phosphatase is 86% identical over 2301 amino acids to glomerular mesangial cell receptor protein-tyrosine phosphatase precursor (FIG. 1). Human receptor tyrosine phosphatase of the invention is expected to be useful for the same purposes as previously identified receptor tyrosine phosphatase enzymes.

Human receptor tyrosine phosphatase is believed to be useful in therapeutic methods to treat disorders such as diabetes, obesity, a CNS disorder, COPD, and cardiovascular disorders. Human receptor tyrosme phosphatase also can be used to screen for human receptor tyrosine phosphatase activators and inhibitors.

Polypeptides

Human receptor tyrosme phosphatase polypeptides according to the invention comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700,

725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625, 1650, 1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975, 2000, 2025, 2050, 2075, 2100, 2125, 2150, 2175, 2200, 2225, 2250, 2275 or 2299 contiguous amino acids selected from the amino acid sequence shown in SEQ LD NO:2 or a biologically active variant thereof, as defined below. A receptor tyrosine phosphatase polypeptide of the invention therefore can be a portion of a receptor tyrosine phosphatase protein, a full-length receptor tyrosine phosphatase protein, or a fusion protein comprising all or a portion of a receptor tyrosine phosphatase protein.

Biologically active variants

Human receptor tyrosme phosphatase polypeptide variants that are biologically active, e.g., retain an enzymatic activity, also are receptor tyrosme phosphatase polypeptides. Preferably, naturally or non-naturally occurring receptor tyrosme phosphatase polypeptide variants have amino acid sequences which are at least about 87, preferably about 90, 96, 96, 98, or 99% identical to the amino acid sequence shown in SEQ LD NO:2 or a fragment thereof. Percent identity between a putative receptor tyrosme phosphatase polypeptide variant and an amino acid sequence of SEQ ID NO:2 is determined using the Blast2 alignment program (Blosum62, Expect

10, standard genetic codes). .

Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an amino acid sequence.

They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a human receptor tyrosine phosphatase polypeptide can be found using computer programs well known in the art, such as DNASTAR software.

The invention additionally, encompasses receptor tyrosme phosphatase polypeptides that are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications can be carried out by known techniques including, but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, N8 protease, ΝaBH₄, acetylation, ^' formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc. Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N- terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The receptor tyrosme phosphatase polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.

The invention also provides chemically modified derivatives of receptor tyrosine phosphatase polypeptides that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immuno- genicity (see U.S. Patent No. 4,179,337). The chemical moieties for derivitization can be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like. The polypeptides can be modified at random or predetermined positions within the molecule and can include one, two, three, or more attached chemical moieties.

Whether an amino acid change or a polypeptide modification results in a biologically active receptor tyrosme phosphatase polypeptide can readily be determined by assaying for enzymatic activity, as described for example, in Calvert-Evers & Hammond, Cell Biol. Int. 24, 559-68, 2000.

Fusion proteins

Fusion proteins are useful for generating antibodies against receptor tyrosme phosphatase polypeptide amino acid sequences and for use in various assay systems. ^' For example, fusion proteins can be used to identify proteins that interact with portions of a human receptor tyrosine phosphatase polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.

A human receptor tyrosine phosphatase polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625, 1650,

1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975, 2000, 2025, 2050, 2075, 2100, 2125, 2150, 2175, 2200, 2225, 2250, 2275 or 2299 contiguous amino acids of SEQ LD NO:2 or of a biologically active variant, such as those described above. The first polypeptide segment also can comprise full-length receptor tyrosme phosphatase protein.

The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include β-galactosidase, β- glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutmin (HA) tags, Myc tags, VSV- G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4

DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the receptor tyrosine phosphatase polypeptide-encoding sequence and the^' heterologous protein sequence, so that the receptor tyrosme phosphatase polypeptide can be cleaved and purified away from the heterologous moiety. A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA constract which comprises coding sequences selected from SEQ LD NO:l in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, Wl), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC;

Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA- KITS).

Identification of species homologs

Species homologs of human receptor tyrosine phosphatase polypeptide can be obtained using receptor tyrosine phosphatase polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of receptor tyrosine phosphatase polypeptide, and expressing the cDNAs as is known in the art.

Polynucleotides

A human receptor tyrosine phosphatase polynucleotide can be single- or double- stranded and comprises a coding sequence or the complement of a coding sequence for a receptor tyrosme phosphatase polypeptide. A coding sequence for human receptor tyrosine phosphatase is shown in SEQ LD NO:l.

Degenerate nucleotide sequences encoding human receptor tyrosine phosphatase polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to the nucleotide sequence shown in SEQ TD NO:l or its complement also are receptor tyrosine phosphatase polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of

-12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species homologs, and variants of receptor tyrosine phosphatase polynucleotides that encode biologically active receptor tyrosine phosphatase polypeptides also are receptor tyrosine phosphatase polynucleotides. Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ ID NO:l or its complement also are receptor tyrosme phosphatase polynucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides.

Identification of polynucleotide variants and homologs

Variants and homologs of the receptor tyrosine phosphatase polynucleotides described above also are receptor tyrosme phosphatase polynucleotides. Typically, homologous receptor tyrosine phosphatase polynucleotide sequences can be identi- fied by hybridization of candidate polynucleotides to known receptor tyrosine phosphatase polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions~2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50 °C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each homologous sequences can be identified which contain at most about

25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.

Species homologs of the receptor tyrosine phosphatase polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of receptor tyrosine phosphatase polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T_m of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973). Variants of human receptor tyrosme phosphatase polynucleotides or receptor tyrosine phosphatase polynucleotides of other species can therefore be identified by hybridizing a putative homologous receptor tyrosine phosphatase polynucleotide with a polynucleotide having a nucleotide sequence of SEQ TD NO:l or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to receptor tyrosme phosphatase polynucleotides or their complements following stringent hybridization and/or wash conditions also are receptor tyrosine phosphatase polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.

Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20 °C below the calculated T_m of the hybrid under study. The T_m of a hybrid between a receptor tyrosine phosphatase polynucleotide having a nucleotide sequence shown in SEQ ID

NO:l or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and^' McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962): T_m = 81.5 °C - 16.6(log₁₀[Na⁺]) + 0.41(%G + C) - 0.63(%formamide) - 600/1), where / = the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% formamide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C. Highly stringent wash conditions include, for example, 0.2X SSC at 65 °C.

Preparation of polynucleotides

A human receptor tyrosine phosphatase polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated receptor tyrosme phosphatase polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise receptor tyrosme phos- phatase nucleotide sequences. Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.

Human receptor tyrosine phosphatase cDNA molecules can be made with standard molecular biology techniques, using receptor tyrosine phosphatase mRNA as a template. Human receptor tyrosine phosphatase cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using ^' either human genomic DNA or cDNA as a template. Alternatively, synthetic chemistry techniques can be used to synthesize receptor tyrosine phosphatase polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a human receptor tyrosine phosphatase polypeptide having, for example, an amino acid sequence shown in SEQ ID NO:2 or a biologically active variant thereof.

Extending polynucleotides

Both methods are well known in the art. Briefly, filters with bacterial colonies containing the library in pBluescript or bacterial lawns containing lambda plaques are denatured, and the DNA is fixed to the filters. The filters are hybridized with the labeled probe using hybridization conditions described by Davis et al, 1986. The partial sequences, cloned into lambda or pBluescript, can be used as positive controls to assess background binding and to adjust the hybridization and washing stringencies necessary for accurate clone identification. The resulting autoradio- grams are compared to duplicate plates of colonies or plaques; each exposed spot corresponds to a positive colony or plaque. The colonies or plaques are selected, expanded and the DNA is isolated from the colonies for further analysis and sequencing.

Positive cDNA clones are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the partial sequence and the other primer from the vector. Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size detennined from Northern blot Analysis.

Once one or more overlapping cDNA clones are identified, the complete sequence of the clones can be determined, for example after exonuclease III digestion (McCombie et al, Methods 3, 33-40, 1991). A series of deletion clones are generated, each of which is sequenced. The resulting overlapping sequences are assembled into ^'a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a highly accurate final sequence.

Various PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al, Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or .more, and to anneal to the target sequence at temperatures about 68-72 °C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment ^' of the DNA molecule before performing PCR. Another method which can be used to retrieve unknown sequences is that of Parker et al, Nucleic Acids Res. 19, 3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFLNDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in wliich an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample.

Obtaining Polynucleotides

Receptor tyrosine phosphatase polypeptides can be obtained, for example, by^' purification from human cells, by expression of receptor tyrosine phosphatase polynucleotides, or by direct chemical synthesis. Prote in purification

Receptor tyrosine phosphatase polypeptides can be purified from any human cell which expresses the receptor, including host cells which have been transfected with receptor tyrosine phosphatase polynucleotides. A purified receptor tyrosine phosphatase polypeptide is separated from other compounds that normally associate with the receptor tyrosme phosphatase polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.

Receptor tyrosine phosphatase polypeptide can be conveniently isolated as a complex with its associated G protein, as described in the specific examples, below. A preparation of purified receptor tyrosme phosphatase polypeptides is at least .80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS- polyacrylamide gel electrophoresis.

Expression of polynucleotides

To express a human receptor tyrosine phosphatase polynucleotide, the polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding receptor tyrosine phosphatase polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.

A variety of expression vector/host systems can be utilized to contain and express sequences encoding a human receptor tyrosine phosphatase polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transfonned with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMN; tobacco mosaic virus,

TMN) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translated regions of the vector — enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including^' constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the

BLUESCRLPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses • (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a human receptor tyrosme phosphatase polypeptide, vectors based on SN40 or EBN can be used with an appropriate selectable marker.

Bacterial and yeast expression systems

In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the receptor tyrosine phosphatase polypeptide. For example, when a large quantity of a human receptor tyrosine phosphatase polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRJPT vector, a sequence encoding the receptor tyrosine phosphatase polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced. pLΝ vectors (Van Heeke & Schuster, J Biol. Chem. 264, 5503-5509, 1989) or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage^' sites so that the cloned polypeptide of interest can be released from the GST moiety at will. In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) and Grant et al, Methods Enzymol. 153, 516-544, 1987.

Plant and insect expression systems

If plant expression vectors are used, the expression of sequences encoding receptor tyrosme phosphatase polypeptides can be driven by any of a number of promoters. For example, viral promoters such as the 35 S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al, EMBO J. 3, 1671-1680, 1984; Broglie et al, Science 224, 838-843, 1984; Winter et al, Results Probl. Cell Differ. 17, 85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).

An insect system also can be used to express a human receptor tyrosine phosphatase polypeptide. For example, in one such system Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. Sequences encoding receptor tyrosine phosphatase polypeptides can be- cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of receptor tyrosine phosphatase polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat^' protein. The recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which receptor tyrosine phosphatase polypeptides can be expressed (Engelhard et al, Proc. Nat. Acad. Sci. 91, 3224-3227, 1994). Mammalian expression systems

A number of viral-based expression systems can be used to express receptor tyrosine phosphatase polypeptideis in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding receptor tyrosine phosphatase polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing a human receptor tyrosine phosphatase polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984). If desired, transcription enhancers, such as the Rous sarcoma virus (RSN) enhancer, can be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DΝA than can be contained and expressed in a plasmid. HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).

Specific initiation signals also can be used to achieve more efficient translation of sequences encoding receptor tyrosme phosphatase polypeptides. Such signals include the ATG imtiation codon and adjacent sequences. In cases where sequences encoding a human receptor tyrosine phosphatase polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG imtiation codon) should be provided. The initiation codon should be in the conect reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al, Results Probl. Cell Differ. 20, 125-162, 1994).

Host cells

A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed receptor tyrosme phosphatase polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipi- dation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate conect insertion, folding and/or function. Different host cells that have specific cellular machinery and characteristic mechamsms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 . University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the conect modification and processing of the foreign protein.

Stable expression is prefened for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express receptor tyrosme phosphatase polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced receptor tyrosme phosphatase sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, ^' R.I. Freshney, ed., 1986. Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al, Cell 11, 223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al, Cell 22, 817-23, 1980) genes that can be employed in tk^~ or aprf cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al, Proc. Natl. Acad. Sci. 77, 3567-70, 1980), npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al, J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Munay, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, wliich allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol. Biol. 55, 121-131, 1995).

Detecting expression

Although the presence of marker gene expression suggests that the receptor tyrosme phosphatase polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a human receptor tyrosine phosphatase polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode an receptor tyrosine phosphatase polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a receptor tyrosine phosphatase polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the receptor tyrosine phosphatase polynucleotide. Altematively, host cells which contain a human receptor tyrosine phosphatase polynucleotide and which express a human receptor tyrosine phosphatase polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques that include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding an receptor tyrosine phosphatase polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a human receptor tyrosine phosphatase polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a receptor tyrosine phosphatase polypeptide to detect transformants which contain an receptor tyrosine phosphatase polynucleotide.

A variety of protocols for detecting and measuring the expression of a human receptor tyrosine phosphatase polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a human receptor tyrosine phosphatase polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al, SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al, J. Exp. Med. 158, 1211-1216, 1983).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for^' producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding receptor tyrosine phosphatase polypeptides include oligo- labeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding a human receptor tyrosine phosphatase polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels . which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Expression and purification of polypeptides

Host cells transformed with nucleotide sequences encoding a human receptor tyrosine phosphatase polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode receptor tyrosine phos- phatase polypeptides can be designed to contain signal sequences which direct secretion of soluble receptor tyrosme phosphatase polypeptides tlirough a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane- bound receptor tyrosine phosphatase polypeptide.

As discussed above, other constructions can be used to join a sequence encoding a human receptor tyrosme phosphatase polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating^' peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor Xa or enterokmase (Invitrogen, San Diego, CA) between the purification domain and the receptor tyrosme phosphatase polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a human receptor tyrosme phosphatase polypeptide and 6 histidine residues preceding a thioredoxin or an enterokmase cleavage site. The histidine residues facilitate purification by LMAC (immobilized metal ion affimty chromatography, as described in Porath et al, Prot. Exp. Purifi 3, 263-281, 1992), while the enterokmase cleavage site provides a means for purifying the receptor tyrosine phosphatase polypeptide from the fusion protein. Vectors that contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 12, 441-453, 1993.

Chemical synthesis

Sequences encoding a human receptor tyrosine phosphatase polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Carut ers et al, Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980). Alternatively, a human receptor tyrosme phosphatase polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of receptor tyrosine phosphatase polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.

The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND

MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983). The composition of a synthetic receptor tyrosine phosphatase polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence, of the receptor tyrosine phosphatase polypeptide can be altered during direct synthesis and or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

As will be understood by those of skill in the art, it may be advantageous to produce receptor tyrosme phosphatase polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons prefened by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter receptor tyrosme phosphatase polypeptide- encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.

Antibodies

Any type of antibody known in the art can be generated to bind specifically to an epitope of a human receptor tyrosine phosphatase polypeptide. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')₂, and Fv, which are capable of binding an epitope of a human receptor tyrosine phosphatase polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve noncontiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.

An antibody which specifically binds to an epitope of a human receptor tyrosine phosphatase polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.

Typically, an antibody which specifically binds to a human receptor tyrosine phosphatase polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies which specifically bind to receptor tyrosine phosphatase polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a human receptor tyrosine phosphatase polypeptide from solution.

Human receptor tyrosine phosphatase polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a human receptor tyrosine phosphatase polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., alurninum hydroxide), and^' surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Gueriή) and Corynebacteήum parvum are especially useful.

Monoclonal antibodies which specifically bind to a human receptor tyrosme phosphatase polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al., Nature 256, 495-497, 1985; Kozbof et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl. Acad. Sci. 80, 2026-2030, 1983; Cole et al, Mol. Cell Biol. 62, 109-120,

1984).

In addition, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al,

Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985). Monoclonal and other antibodies also can be "humanized" to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in

GB2188638B. Antibodies that specifically bind to a human receptor tyrosme phosphatase polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.

Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies that specifically bind to receptor tyrosme phosphatase polypeptides. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991). _.

Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al, 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15,

159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J Biol. Chem. 269, 199-206.

A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DΝA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Nerhaar et al, 1995, Int. J. Cancer 61, 497-501; Νicholls et al, 1993, J Immunol. Meth. 165, 81- 91).

Antibodies which specifically bind to receptor tyrosme phosphatase polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837,

1989; Winter etal, Nature 349, 293-299, 1991).

Other types of antibodies can be constructed and used therapeutically in methods of the mvention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immuno globulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.

Antibodies according to the mvention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a human receptor tyrosine phosphatase polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.

Antisense oligonucleotides

Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of receptor tyrosine phosphatase gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. ^' Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmaiin et al, Chem. Rev. 90, 543-583, 1990. Modifications of receptor tyrosine phosphatase gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the receptor tyrosine phosphatase gene. Oligonucleotides derived from the transcription imtiation site, e.g., between positions -10 and +10 from the start site, are prefened. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al, in Huber & Can, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco,

N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a human receptor tyrosme phosphatase polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, A, or 5 or more stretches of contiguous nucleotides which are precisely complementary to an receptor tyrosine phosphatase polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent receptor tyrosine phosphatase nucleotides, can provide sufficient targeting specificity for receptor tyrosine phosphatase mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least A, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular receptor tyrosme phosphatase polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting their ability to hybridize to a human receptor tyrosine phosphatase polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al, Chem. Rev.. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett. 215, 3539-3542, 1987.

Ribozymes

Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515,

1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of a human receptor tyrosine phosphatase polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the receptor tyrosine phosphatase polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to fhe target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).

Specific ribozyme cleavage sites within a human receptor tyrosine phosphatase RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides conesponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate receptor tyrosme phosphatase RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribo- nuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA constract. Mechanical methods, such as microinj ection, liposome-mediated fransfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing

DNA construct into cells in which it is desired to decrease receptor tyrosine phosphatase expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the constract can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

As taught in Haseloff et al, U.S. Patent 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

Differentially expressed genes

Described herein are methods for the identification of genes whose products interact with human receptor tyrosine phosphatase. Such genes may represent genes that are differentially expressed in disorders including, but not limited to, diabetes, CNS disorders, COPD, and cardiovascular disorders. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human receptor tyrosine phosphatase gene or gene product may itself be tested for differential expression.

The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.

To identify differentially expressed genes total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from conesponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et^' al, ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.

Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subfractive hybridization (Hedrick et al, Nature 308, 149-53; Lee et al, Proc Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and, preferably, differential display (Liang & Pardee, Science 257, 967-71, 1992; U.S. Patent 5,262,311).

The differential expression information may itself suggest relevant methods for the treatment of disorders involving the human receptor tyrosine phosphatase. For example, treatment may include a modulation of expression of the differentially ex- pressed genes and/or the gene encoding the human receptor tyrosine phosphatase.

The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human receptor tyrosme phosphatase gene or gene product are up-regulated or down-regulated.

Screening methods

The invention provides assays for screening test compounds that bind to or modulate the activity of a human receptor tyrosine phosphatase polypeptide or a human receptor tyrosine phosphatase polynucleotide. A test compound preferably binds to a human receptor tyrosine phosphatase polypeptide or polynucleotide. More preferably, a test compound decreases or increases enzymatic activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound. Test compounds

Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The com- pounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al, J. Med. Chem. 37, 2678, 1994; Cho et al, Science 261, 1303, 1993; Carell et al, Angew. Chem. Int. Ed. Engl.

33, 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al, J. Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Patent 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. U.S.A.

89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; Devlin, Science 249, 404-406, 1990); Cwirla et al, Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Patent 5,223,409). High through-put screening

Test compounds can be screened for the ability to bind to receptor tyrosme phosphatase polypeptides or polynucleotides or to affect receptor tyrosine phosphatase activity or receptor tyrosme phosphatase gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.

Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by

Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as. dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.

Another example of a free format assay is described by Chelsky, "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in

Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying^' combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UN-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change. Yet another example is described by Salmon et al., Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.

Another high throughput screening method is described in Beutel et al, U.S. Patent 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.

Binding assays

For binding assays, the test compound is preferably a small molecule that binds to and occupies, for example, the active site of the receptor tyrosme phosphatase polypeptide, such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.

In binding assays, either the test compound or the receptor tyrosine phosphatase polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound that is bound to the receptor tyrosme phosphatase polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

Alternatively, binding of a test compound to a human receptor tyrosine phosphatase polypeptide can be determined without labeling either of the interactants. For example, a microphysiometer can be used to detect binding of a test compound with a human receptor tyrosine phosphatase polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a human receptor tyrosine phosphatase polypeptide

(McCormell et al, Science 257, 1906-1912, 1992).

Determining the ability of a test compound to bind to a human receptor tyrosme phosphatase polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal.

Chem. 63, 2338-2345, 1991, and Szabo et al, Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In yet another aspect of the invention, a human receptor tyrosine phosphatase polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268, 12046-12054, 1993; Bartel et al, BioTechniques 14, 920-924,

1993; Iwahuchi et al, Oncogene 8, 1693-1696, 1993; and Brent W094/10300), to identify other proteins which bind to or interact with the receptor tyrosine phosphatase polypeptide and modulate its activity.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct, polynucleotide encoding a human receptor tyrosine phosphatase polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein

("prey" or "sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor.

Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein that interacts with the receptor tyrosme phosphatase polypeptide. .

It may be desirable to immobilize either the receptor tyrosine phosphatase polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the receptor tyrosine phosphatase polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an anay, so that the location of individual test compounds can be tracked. Binding of a test compound to a human receptor tyrosine phosphatase polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

In one embodiment, the receptor tyrosine phosphatase polypeptide is a fusion protein comprising a domain that allows the receptor tyrosine phosphatase polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed receptor tyrosine phosphatase polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a human receptor tyrosine phosphatase polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated receptor tyrosine phosphatase polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N- hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to a receptor tyrosme phosphatase polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the receptor tyrosine phosphatase polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.

Methods for detecting such complexes, in addition to those described above for the

GST-immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to the receptor tyrosine phosphatase polypeptide or test compound, enzyme-linked assays wliich rely on detecting an activity of the^' receptor tyrosine phosphatase polypeptide, and SDS gel electrophoresis under non- reducing conditions . Screening for test compounds which bind to a human receptor tyrosine phosphatase polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a receptor tyrosine phosphatase polypeptide or polynucleotide can be used in a cell-based assay system. A receptor tyrosine phosphatase polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a receptor tyrosme phosphatase polypeptide or polynucleotide is determined as described above.

Enzymatic activity

Test compounds can be tested for the ability to increase or decrease the enzymatic activity of a human receptor tyrosine phosphatase polypeptide. Enzymatic activity can be measured, for example, as described in Calvert-Evers & Hammond, Cell Biol. Int. 24, 559-68, 2000.

Enzyme assays can be carried out after contacting either a purified receptor tyrosine phosphatase polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound that decreases enzymatic activity of a human receptor tyrosine phosphatase polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing receptor tyrosine phosphatase activity. A test compound which increases enzymatic activity of a human receptor tyrosme phosphatase polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human receptor tyrosine phosphatase activity.

Gene expression

In another embodiment, test compounds that increase or decrease receptor tyrosme phosphatase gene expression are identified. A receptor tyrosme phosphatase polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the receptor tyrosine phosphatase polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.

The level of receptor tyrosine phosphatase mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of a human receptor tyrosine phosphatase polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a human receptor tyrosme phosphatase polypeptide.

Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell that expresses a human receptor tyrosme phosphatase polynucleotide can be used in a cell-based assay system. The receptor tyrosine phosphatase polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used. Pharmaceutical compositions

The invention also provides pharmaceutical compositions that can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the in- vention can comprise, for example, a human receptor tyrosine phosphatase polypeptide, receptor tyrosine phosphatase polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a receptor tyrosine phosphatase polypeptide, or mimetics, activators, or inhibitors of a human receptor tyrosine phosphatase polypeptide activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drags or hormones.

In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the mvention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, infra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, shinies, suspensions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, marmitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxyprop- ylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pynolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpynolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as

Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.^' Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present mvention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactio, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the conesponding free base forms. In other cases, the prefened preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co.,

Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.

Therapeutic indications and methods

Human receptor tyrosme phosphatase can be regulated to treat diabetes, CNS^' disorders, COPD, and cardiovascular disorders. Diabetes mellitus is a common metabolic disorder characterized by an abnormal elevation in blood glucose, alterations in lipids and abnormalities (complications) in the cardiovascular system, eye, kidney and nervous system. Diabetes is divided into two separate diseases: type 1 diabetes (juvenile onset), which results from a loss of cells which make and secrete insulin, and type 2 diabetes (adult onset), which is caused by a defect in insulin secretion and a defect in insulin action.

Type 1 diabetes is initiated by an autoimmune reaction that attacks the insulin secreting cells (beta cells) in the pancreatic islets. Agents that prevent this reaction from occurring or that stop the reaction before destruction of the beta cells has been accomplished are potential therapies for this disease. Other agents that induce beta cell proliferation and regeneration also are potential therapies.

Type II diabetes is the most common of the two diabetic conditions (6% of the population). The defect in insulin secretion is an important cause of the diabetic condition and results from an inability of the beta cell to properly detect and respond to rises in blood glucose levels with insulin release. Therapies that increase the response by the beta cell to glucose would offer an important new treatment for this disease.

The defect in insulin action in Type II diabetic subjects is another target for therapeutic intervention. Agents that increase the activity of the insulin receptor in muscle, liver, and fat will cause a decrease in blood glucose and a normalization of plasma lipids. The receptor activity can be increased by agents that directly stimulate the receptor or that increase the intracellular signals from the receptor. Other therapies can directly activate the cellular end process, i.e. glucose transport or various enzyme systems, to generate an insulin-like effect and therefore a produce beneficial outcome. Because overweight subjects have a greater susceptibility to Type LI diabetes, any agent that reduces body weight is a possible therapy. Both Type I and Type diabetes can be treated with agents that mimic insulin action or that treat diabetic complications by reducing blood glucose levels. Likewise, agents that reduces new blood vessel growth can be used to treat the eye complications that develop in both diseases.

Central and peripheral nervous system disorders also can be treated, such as primary and secondary disorders after brain injury, disorders of mood, anxiety disorders, disorders of thought and volition, disorders of sleep and wakefulness, diseases of the motor unit, such as neurogenic and myopathic disorders, neurodegenerative disorders such as Alzheimer's and Parkinson's disease, and processes of peripheral and chronic pain.

Pain that is associated with CNS disorders also can be treated by regulating the activity of human receptor tyrosine phosphatase. Pain which can be treated includes that associated with central nervous system disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemonhage, vascular malformation). Non-central neuropathic pain includes that associated with post mastectomy pain, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HIN/ALDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia. Pain associated with cancer and cancer treatment also can be treated, as can headache pain (for example, migraine with aura, migraine without aura, and other migraine disorders), episodic and chronic tension-type headache, tension-type like headache, cluster headache, and chronic^' paroxysmal hemicrania. Central and peripheral nervous system disorders also can be treated, such as primary and secondary disorders after brain injury, disorders of mood, anxiety disorders, disorders of thought and volition, disorders of sleep and wakefulness, diseases of the motor unit, such as neurogenic and myopathic disorders, neurodegenerative disorders such as Alzheimer's and Parkinson's disease, and processes of peripheral and chronic pain.

Pain that is associated with CNS disorders also can be treated by regulating the activity of human receptor tyrosine phosphatase. Pain which can be treated mcludes that associated with central nervous system disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemonhage, vascular malformation). Non-central neuropathic pain includes that associated with post mastectomy pain, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HIN/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia. Pain associated with cancer and cancer treatment also can be treated, as can headache pain (for example, migraine with aura, migraine without aura, and other migraine disorders), episodic and chronic tension-type headache, tension-type like headache, cluster headache, and chronic paroxysmal hemicrania.

Cardiovascular diseases include the following disorders of the heart and the vascular system: congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular anhythmias, hypertensive vascular diseases,^' and peripheral vascular diseases. Heart failure is defined as a pathophysiologic state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failure, such as high-output and low-output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause.

Myocardial infarction (MI) is generally caused by an abrupt decrease in coronary blood flow that follows a thrombotic occlusion of a coronary artery previously nanowed by arteriosclerosis. MI prophylaxis (primary and secondary prevention) is included, as well as the acute treatment of MI and the prevention of complications.

Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which inadequate to meet the myocardial requirement for oxygen. This group of diseases mcludes stable angina, unstable angina, and asymptomatic ischemia.

Anhythmias include all forms of atrial and ventricular tachyarrhythmias (atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexcitation syndrome, ventricular tachycardia, ventricular flutter, and ventricular fibrillation), as well as bradycardic forms of anhythmias.

Vascular diseases include primary as well as all kinds of secondary arterial hypertension (renal, endocrine, neurogenic, others). The disclosed gene and its product may be used as drag targets for the treatment of hypertension as well as for the prevention of all complications.

Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue^' oxygen demand. It mcludes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon, and venous disorders. This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an anti- sense nucleic acid molecule, a specific antibody, ribozyme, or a human receptor tyrosme phosphatase polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

A reagent which affects receptor tyrosine phosphatase activity can be administered to a human cell, either in vitro or in vivo, to reduce receptor tyrosine phosphatase activity. The reagent preferably binds to an expression product of a human receptor tyrosme phosphatase gene. If the expression product is a protein, the reagent is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.

In one embodiment, the reagent is delivered using a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin. A liposome useful in the present mvention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the fransfection efficiency of a liposome is about 0.5 μg of DNA per 16 nmole of liposome delivered to about 10 cells, more preferably about 1.0 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome de- livered to about 10 cells. Preferably, a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.

Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More prefened liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyeth- ylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.

Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Patent 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from, about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes.

In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al, J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al, J. Biol. Chem. 266, 338-42 (1991).

Determination of a therapeutically effective dose

The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases enzymatic activity relative to the enzymatic activity which occurs in the absence of the therapeutically effective dose.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to detennine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeutically effective in 50%) of the population) and LD₅o (the dose lethal to 50%ι of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅o/ED₅o.

Pharmaceutical compositions that exhibit large therapeutic indices are prefened. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of admimstration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drag combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, fransfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated fransfection.

Effective in vivo dosages of an antibody are in the range of about 5 μg to about

50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about

100 μg of DNA. If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.

Preferably, a reagent reduces expression of a human receptor tyrosme phosphatase gene or the activity of a receptor tyrosine phosphatase polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a human receptor tyrosine phosphatase gene or the activity of a human receptor tyrosme phosphatase polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to receptor tyrosine phosphatase-specific mRNA, quantitative RT-PCR, immunologic detection of a human receptor tyrosine phosphatase polypeptide, or measurement of enzymatic activity.

In any of the embodiments described above, any of the pharmaceutical compositions ^• of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, accordmg to conventional pharmaceutical principles. The combination of therapeutic agents can act syner- gistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. Diagnostic methods

Human receptor tyrosme phosphatase also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding receptor tyrosine phosphatase in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.

Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.

Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions accordmg to their specific melting or partial melting temperatures (see, e.g., Myers et al, Science 230, 1242, 1985). Sequence changes at specific locations can also be^' revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85,

4397-4401, 1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

Altered levels of receptor tyrosme phosphatase also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.

All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present mvention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the mvention.

EXAMPLE 1

Detection of receptor tyrosine phosphatase activity

The polynucleotide of SEQ LD NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-receptor tyrosme phosphatase polypeptide obtained is transfected into human embryonic kidney 293 cells. To prepare membrane fractions, the cells are sonicated in a hypotonic lysis buffer (25 mM Tris-HCl, pH 7.5/25 mM sucrose/0.1 mM EDTA/5 mM MgC12/5 mM DTT/1 mM phenylmethylsulfonyl fTuoride/0.5 μg/ml leupeptin/1 μg/ml aprotinin), nuclei are removed by low-speed centrifugation, and membrane fractions are obtained by centrifugation at 100,000 x g for 60 min at 4°C. The resulting pellets are suspended^' by sonication in lysis buffer, brought to a concentration of 2 mg/ml, and used to measure receptor tyrosine phosphatase activity.. A nonradioactive protein-tyrosine phosphatase assay kit (Roce Molecular Biochemicals) is used accordmg to the manufacturer's instractions. Signals on Western blots are detected by chemi- luminescence (ECL, Amersham Pharmacia Biotech. It is shown that the polypeptide of SEQ ID NO: 2 has a receptor tyrosine phosphatase activity.

EXAMPLE 2

Expression of recombinant human receptor tyrosine phosphatase

The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human receptor tyrosine phosphatase polypeptides in yeast. The receptor tyrosme phosphatase-encoding DNA sequence is derived from SEQ LD NO:l. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pPICZ B with the conesponding restriction enzymes the modified DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in Pichia pastoris, driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast.

The yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea. The bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San

Diego, CA) according to manufacturer's instractions. Purified human receptor tyrosine phosphatase polypeptide is obtained. EXAMPLE 3

Identification of test compounds that bind to receptor tyrosine phosphatase polypeptides

Purified receptor tyrosme phosphatase polypeptides comprising a glutathione-S- transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. Human receptor tyrosme phosphatase polypeptides comprise the amino acid sequence shown in SEQ TD NO:2. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Confrol samples are incubated in the absence of a test compound.

The buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a human receptor tyrosme phosphatase polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in wliich a test compound is not incubated is identified as a compound which binds to a human receptor tyrosine phosphatase polypeptide.

EXAMPLE 4

Identification of a test compound which decreases receptor tyrosine phosphatase gene expression

A test compound is admimstered to a culture of human cells transfected with a receptor tyrosine phosphatase expression construct and incubated at 37 °C for 10 to

45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control. RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a ³²P-labeled receptor tyrosme phosphatase-specific probe at 65 ° C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ TD NO:l. A test compound that decreases the receptor tyrosine phosphatase-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of receptor tyrosme phosphatase gene expression.

EXAMPLE 5

Identification of a test compound which decreases receptor tyrosine phosphatase activity

A test compound is administered to a culture of human cells transfected with a receptor tyrosme phosphatase expression construct and incubated at 37 °C for 10 to 45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control. Enzymatic activity is measured using the method of Calvert-Evers & Hammond, Cell Biol. Int. 24, 559-68, 2000.

A test compound which decreases the enzymatic activity of the receptor tyrosine phosphatase relative to the enzymatic activity in the absence of the test compound is identified as an inhibitor of receptor tyrosine phosphatase activity.

EXAMPLE 6

Tissue-specific expression of receptor tyrosine phosphatase

The qualitative expression pattern of receptor tyrosme phosphatase in various tissues is determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR).

Quantitative expression profiling.

To demonstrate that receptor tyrosine phosphatase is involved in the disease process of diabetes, the following whole body panel is screened to show predominant or relatively high expression: subcutaneous and mesenteric adipose tissue, adrenal gland, bone manow, brain, colon, fetal brain, heart, hypothalamus, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, small intestine, spleen, stomach, testis, thymus, thyroid, trachea, and uterus. Human islet cells and an islet cell library also are tested. As a final step, the expression of receptor tyrosme phosphatase in cells derived from normal individuals with the expression of cells derived from diabetic individuals is compared.

To demonstrate that receptor tyrosine phosphatase is involved in CNS disorders, the following tissues are screened: fetal and adult brain, muscle, heart, lung, kidney, liver, thymus, testis, colon, placenta, trachea, pancreas, kidney, gastric mucosa, colon, liver, cerebellum, skin, cortex (Alzheimer's and normal), hypothalamus, cortex, amygdala, cerebellum, hippocampus, choroid, plexus, thalamus, and spinal cord.

To demonstrate that receptor tyrosme phosphatase is involved in the disease process of COPD, the initial expression panel consists of RNA samples from respiratory tissues and inflammatory cells relevant to COPD: lung (adult and fetal), trachea, freshly isolated alveolar type II cells, cultured human bronchial epithelial cells, cultured small airway epithelial cells, cultured bronchial sooth muscle cells, cultured H441 cells (Clara-like), freshly isolated neutrophils and monocytes, and cultured monocytes (macrophage-like). Body map profiling also is carried out, using total RNA panels purchased from Clontech. The tissues are adrenal gland, bone manow, brain, colon, heart, kidney, liver, lung, mammary gland, pancreas, prostate, salivary gland, skeletal muscle, small intestine, spleen, stomach, testis, thymus, trachea, thyroid, and uterus.

Quantitative expression profiling is performed by the form of quantitative PCR analysis called "kinetic analysis" firstly described in Higuchi et al, BioTechnology^' 10, 413-17, 1992, and Higuchi et al, BioTechnology 11, 1026-30, 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies. If the amplification is performed in the presence of an internally quenched fluorescent oligonucleotide (TaqMan probe) complementary to_. the target sequence, the probe is cleaved by the 5 '-3' endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al, Proc. Natl. Acad. Sci.

U.S.A. 88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al, Genome Res. 6, 986-94, 1996, and Gibson et al, Genome Res. 6, 995-1001, 1996).

The amplification of an endogenous control can be performed to standardize the amount of sample RNA added to a reaction. In this kind of experiment, the confrol of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous confrol can be independently quantified in the same tube if probes labeled with different dyes are used.

All "real time PCR" measurements of fluorescence are made in the ABI Prism 7700.

RNA extraction and cDNA preparation. Total RNA from the tissues listed above are used for expression quantification. RNAs labeled "from autopsy" were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) accordmg to the manufacturer's protocol.

Fifty μg of each RNA were treated with DNase I for 1 hour at 37°C in the following reaction mix: 0.2 U/μl RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/μl RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; lOmM MgCl₂; 50 mM NaCl; and 1 mM DTT. After incubation, RNA is extracted once with 1 volume of phenolxhloroform so- amyl alcohol (24:24:1) and once with chloroform, and precipitated with 1/10 volume of 3 M sodium acetate, pH5.2, and 2 volumes of ethanol.

Fifty μg of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, TX). After resuspension and spectrophoto- metric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is 200ng/μL. Reverse transcription is carried out with 2.5μM of random hexamer primers.

TaqMan quantitative analysis. Specific primers and probe are designed accordmg to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5' end FAM (6-carboxy-fluorescein) and at the 3' end with TAMRA (6-carboxy-tetra- methyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.

Total cDNA content is normalized with the simultaneous quantification (multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).

The assay reaction mix is as follows: IX final TaqMan Universal PCR Master Mix (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from 20X stock); 300 nM forward primer; 900 nM reverse primer; 200 nM probe; 10 ng cDNA; and water to 25 μl.

Each of the following steps are carried out once: pre PCR, 2 minutes at 50° C, and 10 minutes at 95°C. The following steps are carried out 40 times: denaturation, 15 seconds at 95 °C, annealing/extension, 1 minute at 60°C. The experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA). At the end of the run, fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user's manual in order to achieve better background subtraction as well as signal linearity with the starting target quantity.

EXAMPLE 7

Diabetes: In vivo testing of compounds/target validation

Glucose Production

Over-production of glucose by the liver, due to an enhanced rate of gluconeogenesis, is the major cause of fasting hyperglycemia in diabetes. Overnight fasted normal rats or mice have elevated rates of gluconeogenesis as do sfreptozotocin-induced diabetic rats or mice fed ad libitum. Rats are made diabetic with a single intravenous injection of 40 mg/kg of sfreptozotocin while C57BL/KsJ mice are given 40-60 mg/kg i.p. for 5 consecutive days. Blood glucose is measured from tail-tip blood and then compounds are administered via different routes (p.o., i.p., i.v., s.c). Blood is collected at various times thereafter and glucose measured. Alternatively, compounds are admimstered for several days, then the animals are fasted overnight, blood is collected and plasma glucose measured. Compounds that inhibit glucose production will decrease plasma glucose levels compared to the vehicle-treated control group.

Insulin Sensitivity

Both ob/ob and db/db mice as well as diabetic Zucker rats are hyperglycemic,^' hypermsulmemic and insulin resistant. The animals are pre-bled, their glucose levels measured, and then they are grouped so that the mean glucose level is the same for each group. Compounds are administered daily either q.d. or b.i.d. by different routes (p.o., i.p., s.c.) for 7-28 days. Blood is collected at various times and plasma glucose and insulin levels determined. Compounds that improve insulin sensitivity in these models will decrease both plasma glucose and insulin levels when compared to the vehicle-treated control group.

Insulin Secretion

Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the administration of a glucose load. When measuring insulin levels, compounds are admimstered by different routes (p.o., i.p., s.c. or i.v.) to overnight fasted normal rats or mice. At the appropriate time an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later. Plasma insulin levels are determined. Compounds that enhance insulin secretion will increase plasma insulin levels compared to animals given only glucose. When measuring glucose disappearance, animals are bled at the appropriate time after compound admimstration, then given either an oral or intraperitoneal glucose load (lg/kg), bled again after 15, 30, 60 and 90 minutes and plasma glucose levels determined. Compounds that increase insulin levels will decrease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose.

Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the administration of a glucose load. When measuring insulin levels, test compounds which regulate receptor tyrosine phosphatase are administered by different routes

(p.o., i.p., s.c, or i.v.) to overnight fasted normal rats or mice. At the appropriate time an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later. Plasma insulin levels are determined. Test compounds that enhance insulin^' secretion will increase plasma insulin levels compared to ammals given only glucose. When measuring glucose disappearance, ammals are bled at the appropriate time after compound admimstration, then given either an oral or intraperitoneal glucose load (lg/kg), bled again after 15, 30, 60, and 90 minutes and plasma glucose levels determined. Test compounds that increase insulin levels will decrease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose.

Glucose Production

Over-production of glucose by the liver, due to an enhanced rate of gluconeogenesis, is the major cause of fasting hyperglycemia in diabetes. Overnight fasted normal rats or mice have elevated rates of gluconeogenesis as do streptozotocin-induced diabetic rats or mice fed ad libitum. Rats are made diabetic with a single intravenous injection of 40 mg/kg of sfreptozotocin while C57BL/KsJ mice are given 40- 60 mg/kg i.p. for 5 consecutive days. Blood glucose is measured from tail-tip blood and then compounds are administered via different routes (p.o., i.p., i.v., s.c). Blood is collected at various times thereafter and glucose measured. Alternatively, compounds are administered for several days, then the animals are fasted overnight, blood is collected and plasma glucose measured. Compounds that inhibit glucose production will decrease plasma glucose levels compared to the vehicle-treated control group.

Insulin Sensitivity

Both ob/ob and db/db mice as well as diabetic Zucker rats are hyperglycemic, hyperinsulinemic and insulin resistant. The animals are pre-bled, their glucose levels measured, and then they are grouped so that the mean glucose level is the same for each group. Compounds are administered daily either q.d. or b.i.d. by different routes (p.o., i.p., s.c.) for 7-28 days. Blood is collected at various times and plasma glucose and insulin levels determined. Compounds that improve insulin sensitivity in^' these models will decrease both plasma glucose and insulin levels when compared to the vehicle-treated control group. Insulin Secretion

Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the admimstration of a glucose load. When measuring insulin levels, compounds are admimstered by different routes (p.o., i.p., s.c. or i.v.) to overnight fasted normal rats or mice. At the appropriate time an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later. Plasma insulin levels are determined. Compounds that enhance insulin secretion will increase plasma insulin levels compared to animals given only glucose. When measuring glucose disappearance, animals are bled at the appropriate time after compound administration, then given either an oral or intraperitoneal glucose load (lg/kg), bled again after 15, 30, 60 and 90 minutes and plasma glucose levels determined. Compounds that increase insulin levels will decrease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose.

EXAMPLE 8

In vivo testing of compounds/target validation

Pain

Acute pain. Acute pain is measured on a hot plate mainly in rats. Two variants of hot plate testing are used: In the classical variant animals are put on a hot surface (52 to 56 °C) and the latency time is measured until the animals show nocifensive behavior, such as stepping or foot licking. The other variant is an increasing temperature hot plate where the experimental animals are put on a surface of neutral temperature. Subsequently this surface is slowly but constantly heated until the animals begin to^' lick a hind paw. The temperature which is reached when hind paw licking begins is a measure for pain threshold. Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t, i.c.v., s.c, intradermal, transdermal) prior to pain testing.

Persistent pain. Persistent pain is measured with the formalin or capsaicm test, mainly in rats. A solution of 1 to 5% formalin or 10 to 100 μg capsaicin is injected into one hind paw of the experimental animal. After formalin or capsaicin application the animals show nocifensive reactions like flinching, licking and biting of the affected paw. The number of nocifensive reactions within a time frame of up to 90 minutes is a measure for- intensity of pain.

Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to formalin or capsaicin administration.

Neuropathic pain. Neuropathic pain is induced by different variants of unilateral sciatic nerve injury mainly in rats. The operation is performed under anesthesia. The first variant of sciatic nerve injury is produced by placing loosely constrictive ligatures around the common sciatic nerve. The second variant is the tight ligation of about the half of the diameter of the common sciatic nerve. In the next variant, a group of models is used in which tight ligations or transections are made of either the L5 and L6 spinal nerves, or the L% spinal nerve only. The fourth variant involves an axotomy of two of the tliree terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact whereas the last variant comprises the axotomy of only the tibial branch leaving the sural and common nerves uninjured. Control animals are treated with a sham operation.

Postoperatively, the nerve injured animals develop a chronic mechanical allodynia,^' cold allodynioa, as well as a thermal hyperalgesia. Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC

Inc-Life Science Instruments, Woodland Hills, SA, USA; Electronic von Frey System, Somedic Sales AB, Hδrby, Sweden). Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy), or by means of a cold plate of 5 to 10 °C where the nocifensive reactions of the affected hind paw are counted as a measure of pain intensity. A further test for cold induced pain is the counting of nocifensive reactions, or duration of nocifensive responses after plantar administration of acetone to the affected hind limb. Chronic pain in general is assessed by registering the circadanian rhythms in activity (Surjo and Arndt, Universitat zu Kδln, Cologne, Germany), and by scoring differences in gait (foot print patterns; FOOTPRINTS program, Klapdor et al., 1997. A low cost method to analyze footprint patterns. J. Neurosci. Methods 75, 49-54).

Compounds are tested against sham operated and vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to pain testing.

Inflammatory Pain. Inflammatory pain is induced mainly in rats by injection of 0.75 mg canageenan or complete Freund's adjuvant into one hind paw. The animals develop an edema with mechanical allodynia as well as thermal hyperalgesia. Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc-Life Science Instruments, Woodland Hills, SA,

USA). Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy, Paw thermal stimulator, G. Ozaki, University of California, USA). For edema measurement two methods are being used. In the first method, the animals are sacrificed and the affected hindpaws sectioned and weighed. The second method comprises differences in paw volume by measuring water displacement in a plethysmometer (Ugo Basile, Comerio, Italy).

Compounds are tested against uninflamed as well as vehicle treated control groups.^' Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to pain testing. Diabetic neuropathic pain. Rats treated with a single intraperitoneal injection of 50 to 80 mg/kg sfreptozotocin develop a profound hyperglycemia and mechanical allodynia within 1 to 3 weeks. Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc. -Life Science Instruments, Woodland Hills, SA, USA).

Compounds are tested against diabetic and non-diabetic vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to pain testing.

Parkinson's disease

6-Hydroxydopamine (6-OH-DA) Lesion.

Degeneration of the dopaminergic nigrostriatal and striatopallidal pathways is the central pathological event in Parkinson's disease. This disorder has been mimicked experimentally in rats using single/sequential unilateral stereotaxic injections of 6-OH-DA into the medium forebrain bundle (MFB).

Male Wistar rats (Harlan Winkelmann, Gennany), weighing 200±250 g at the beginning of the experiment, are used. The rats are maintained in a temperature- and humidity-controlled environment under a 12 h light/dark cycle with free access to food and water when not in experimental sessions. The following in vivo protocols are approved by the governmental authorities. All efforts are made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques.

Animals are administered pargyline on the day of surgery (Sigma, St. Louis, MO, USA; 50 mg/kg i.p.) in order to inhibit metabolism of 6-OHDA by monoamine oxidase and desmethylimipramine HCl (Sigma; 25 mg/kg i.p.) in order to prevent uptake of 6-OHDA by noradrenergic terminals. Thirty minutes later the rats are anesthetized with sodium pentobarbital (50 mg/kg) and placed in a stereotaxic frame. In order to lesion the DA nigrostriatal pathway 4 μl of 0.01% ascorbic acid-saline containing 8 μg of 6-OHDA HBr (Sigma) are injected into the left medial fore-brain bundle at a rate of 1 μl/min (2.4 mm anterior, 1.49 mm lateral, -2.7 mm ventral to

Bregma and the skull surface). The needle is left in place an additional 5 min to allow diffusion to occur.

Stepping Test. Forelimb akinesia is assessed three weeks following lesion placement using a modified stepping test protocol. In brief, the animals are held by the experimenter with one hand fixing the hindlimbs and slightly raising the hind part above the surface. One paw is touching the table, and is then moved slowly sideways

(5 s for 1 m), first in the forehand and then in the backhand direction. The number of adjusting steps is counted for both paws in the backhand and forehand direction of movement. The sequence of testing is right paw forehand and backhand adjusting stepping, followed by left paw forehand and backhand directions. The test is repeated three times on three consecutive days, after an initial training period of three days prior to the first testing. Forehand adjusted stepping reveals no consistent differences between lesioned and healthy control animals. Analysis is therefore restricted to backhand adjusted stepping.

Balance Test. Balance adjustments following postural challenge are also measured during the stepping test sessions. The rats are held in the same position as described in the stepping test and, instead of being moved sideways, tilted by the experimenter towards the side of the paw touching the table. This maneuver results in loss of balance and the ability of the rats to regain balance by forelimb movements is scored on a scale ranging from 0 to 3. Score 0 is given for a normal forelimb placement. When the forelimb movement is delayed but recovery of postural balance detected, score 1 is given. Score 2 represents a clear, yet insufficient, forelimb reaction, as evidenced by muscle contraction, but lack of success in recovering balance, and score

3 is given for no reaction of movement. The test is repeated three times a day on each side for three consecutive days after an initial training period of three days prior to the first testing.

Staircase Test (Paw Reaching). A modified version of the staircase test is used for evaluation of paw reaching behavior three weeks following primary and secondary lesion placement. Plexiglass test boxes with a central platform and a removable staircase on each side are used. The apparatus is designed such that only the paw on the same side at each staircase can be used, thus providing a measure of independent forelimb use. For each test the animals are left in the test boxes for 15 min. The double staircase is filled with 7 x 3 chow pellets (Precision food pellets, formula: P, purified rodent diet, size 45 mg; Sandown Scientific) on each side. After each test the number of pellets eaten (successfully retrieved pellets) and the number of pellets taken (touched but dropped) for each paw and the success rate (pellets eaten/pellets taken) are counted separately. After three days of food deprivation (12 g per animal per day) the animals are tested for 11 days. Full analysis is conducted only for the last five days.

MPTP treatment. The neurotoxin l-methyl-4-phenyl-l,2,3,6- tetrahydro-pyridine (MPTP) causes degeneration of mesencephalic dopaminergic (DAergic) neurons in rodents, non-human primates, and humans and, in so doing, reproduces many of the symptoms of Parkinson's disease. MPTP leads to a marked decrease in the levels of dopamine and its metabolites, and in the number of dopaminergic terminals in the striatum as well as severe loss of the tyrosine hydroxylase (TH)-immunoreactive cell bodies in the substantia nigra, pars compacta.

In order to obtain severe and long-lasting lesions, and to reduce mortality, animals receive single injections of MPTP, and are then tested for severity of lesion 7-10 days later. Successive MPTP injections are admimstered on days 1, 2 and 3. Animals^' receive application of 4 mg/kg MPTP hydrochloride (Sigma) in saline once daily. All injections are intraperitoneal (i.p.) and the MPTP stock solution is frozen between injections. Animals are decapitated on day 11. Immunohistology. At the completion of behavioral experiments, all animals are anaesthetized with 3 ml thiopental (1 g/40 ml i.p., Tyrol Pharma). The mice are perfused transcardially with 0.01 M PBS (pH 7.4) for 2 min, followed by 4% paraformaldehyde (Merck) in PBS for 15 min. The brains are removed and placed in

4% paraformaldehyde for 24 h at 4 °C. For dehydration they are then transfened to a 20% sucrose (Merck) solution in 0.1 M PBS at 4 °C until they sink. The brains are frozen in methylbutan at -20 °C for 2 min and stored at -70 °C. Using a sledge microtome (mod. 3800-Frigocut, Leica), 25 μm sections are taken from the genu of the corpus callosum (AP 1.7 mm) to the hippocampus (AP 21.8 mm) and from AP

24.16 to AP 26.72. Forty-six sections are cut and stored in assorters in 0.25 M Tris buffer (pH 7.4) for immunohistochemistry.

A series of sections is processed for free-floating tyrosme hydroxylase (TH) im- munohistochemistry. Following three rinses in 0.1 M PBS, endogenous peroxidase activity is quenched for 10 min in 0.3% H₂O₂ ±PBS. After rinsing in PBS, sections are preincubated in 10% normal bovine serum (Sigma) for 5 min as blocking agent and transfened to either primary anti-rat TH rabbit antiserum (dilution 1 :2000).

Following overnight incubation at room temperature, sections for TH immuno- reactivity are rinsed in PBS (2 xlO min) and incubated in biotinylated anti-rabbit immunoglobulin G raised in goat (dilution 1:200) (Vector) for 90 min, rinsed repeatedly and transfened to Vectastain ABC (Vector) solution for 1 h. 3,.3' -Diaminobenzidine tetrahydrochloride (DAB; Sigma) in 0.1 M PBS, supplemented with 0.005%) H₂O₂ , serves as chromogen in the subsequent visualization reaction.

Sections are mounted on to gelatin-coated slides, left to dry overnight, counter-stained with hematoxylin dehydrated in ascending alcohol concentrations and cleared in butylacetate. Coverslips are mounted on entellan.

Rotarod Test. We use a modification of the procedure described by Rozas and

Labandeira-Garcia (1997), with a CR-1 Rotamex system (Columbus Instruments, Columbus, OH) comprising an IBM-compatible personal computer, a CIO-24 data acquisition card, a control unit, and a four-lane rotarod. unit. The rotarod unit consists of a rotating spindle (diameter 7.3 cm) and individual compartments for each mouse. The system software allows preprogramming of session protocols with varying rotational speeds (0-80 rpm). Infrared beams are used to detect when a mouse has fallen onto the base grid beneath the rotarod. The system logs the fall as the end of the experiment for that mouse, and the total time on the rotarod, as well as the time of the fall and all the set-up parameters, are recorded. The system also allows a weak cunent to be passed through the base grid, to aid training.

Dementia

The object recognition task. The object recognition task has been designed to assess the effects of experimental manipulations on the cognitive performance of rodents. A rat is placed in an open field, in which two identical objects are present. The rats inspects both objects during the first trial of the object recognition task. In a second trial, after a retention interval of for example 24 hours, one of the two objects used in the first trial, the 'familiar' object, and a novel object are placed in the open field. The inspection time at each of the objects is registered. The basic measures in the OR task is the time spent by a rat exploring the two object the second trial. Good retention is reflected by higher exploration times towards the novel than the 'familiar' object.

Administration of the putative cognition enhancer prior to the first trial pre- dominantly allows assessment, of the effects on acquisition, and eventually on consolidation processes. Administration of the testing compound after the first trial allows to assess the effects on consolidation processes, whereas administration before the second trial allows to measure effects on retrieval processes.

77ze passive avoidance task. The passive avoidance task assesses memory performance in rats and mice. The inhibitory avoidance apparatus consists of a two-compartment box with a light compartment and a dark compartment. The two compartments are separated by a guillotine door that can be operated by the experimenter. A threshold of 2 cm I separates the two compartments when the guillotine door is raised. When the door is open, the illumination in the dark compartment is about 2 lux. The light intensity is about 500 lux at the center of the floor of the light compartment.

Two habituation sessions, one shock session, and a retention session are given, separated by inter-session intervals of 24 hours. In the habituation sessions and the retention session the rat is allowed to explore the apparatus for 300 sec. The rat is placed in the light compartment, facing the wall opposite to the guillotine door. After an accommodation period of 15 sec. the guillotine door is opened so that all parts of the apparatus can be visited freely. Rats normally avoid brightly lit areas and will enter the dark compartment within a few seconds.

In the shock session the guillotine door between the compartments is lowered as soon as the rat has entered the dark compartment with its four paws, and a scrambled 1 mA footshock is admimstered for 2 sec. The rat is removed from the apparatus and put back into its home cage. The procedure during the retention session is identical to that of the habituation sessions.

The step-through latency, that is the first latency of entering the dark compartment (in sec.) during the retention session is an index of the memory performance of the animal; the longer the latency to enter the dark compartment, the better the retention is. A testing compound in given half an hour before the shock session, together with

1 mg*kg^_1 scopolamine. Scopolamine impairs the memory performance during the retention session 24 hours later. If the test compound increases the enter latency compared with the scopolamine-treated controls, is likely to possess cognition^' enhancing potential. The Morris water escape task. The Morris water escape task measures spatial orientation learning in rodents. It is a test system that has extensively been used to investigate the effects of putative therapeutic on the cognitive functions of rats and mice. The performance of an animal is assessed in a circular water tank with an escape platform that is submerged about 1 cm below the surface of the water. The escape platform is not visible for an animal swimming in the water tank. Abundant extra-maze cues are provided by the furniture in the room, including desks, computer equipment, a second water tank, the presence of tiie experimenter, and by a radio on a shelf that is playing softly.

The animals receive four trials during five daily acquisition sessions. A trial is started by placing an animal into the pool, facing the wall of the tank. Each of four starting positions in the quadrants north, east, south, and west is used once in a series of four trials; their order is randomized. The escape platform is always in the same position. A trial is terminated as soon as the animal had climbs onto the escape platform or when 90 seconds have elapsed, whichever event occurs first. The animal is allowed to stay on the platform for 30 seconds. Then it is taken from the platform and the next trial is started. If an animal did not find the platform within 90 seconds it is put on the platfonn by the experimenter and is allowed to stay there for 30 seconds. After the fourth trial of the fifth daily session, an additional trial is given as a probe trial: the platform is removed, and the time the animal spends in the four quadrants is measured for 30 or 60 seconds. In the probe trial, all animals start from the same start position, opposite to the quadrant where the escape platform had been positioned during acquisition.

Four different measures are taken to evaluate the performance of an animal during acquisition training: escape latency, traveled distance, distance to platform, and swimming speed. The following measures are evaluated for the probe trial: time (s)^' in quadrants and traveled distance (cm) in the four quadrants. The probe trial provides additional information about how well an animal learned the position of the escape platform. If an animal spends more time and swims a longer distance in the quadrant where the platform had been positioned during the acquisition sessions than in any other quadrant, one concludes that the platform position has been learned well.

In order to assess the effects of putative cognition enhancing compounds, rats or mice with specific brain lesions which impair cognitive functions, or animals treated with compounds such as scopolamine or MK-801, which interfere with normal learning, or aged animals which suffer from cognitive deficits, are used.

The T-maze spontaneous alternation task. The T-maze spontaneous alternation task (TeMCAT) assesses the spatial memory performance in mice. The start arm and the two goal arms of the T-maze are provided with guillotine doors which can be operated manually by the experimenter. A mouse is put into the start arm at the beginning of training. The guillotine door is closed. In the first trial, the 'forced trial', either the left or right goal arm is blocked by lowering the guillotine door. After the mouse has been released from the start arm, it will negotiate the maze, eventually enter the open goal arm, and return to the start position, where it will be confined for 5 seconds, by lowering the guillotine door. Then, the animal can choose freely between the left and right goal arm (all guillotine-doors opened) during 14 'free choice' trials. As soon a the mouse has entered one goal arm, the other one is closed. The mouse eventually returns to the start arm and is free to visit whichever go alarm it wants after having been confined to the start arm for 5 seconds. After completion of 14 free choice trials in one session, the animal is removed from the maze. During training, the animal is never handled.

The percent alternations out of 14 trials is calculated. This percentage and the total time needed to complete the first forced trial and the subsequent 14 free choice trials (in s) is analyzed. Cognitive deficits are usually induced by an injection of scopolamine, 30 min before the start of the training session. Scopolamine reduced the^' per-cent alternations to chance level, or below. A cognition enhancer, which is always administered before the training session, will at least partially, antagonize the scopolamine-induced reduction in the spontaneous alternation rate. EXAMPLE 9

Identification of test compound efficacy in a COPD animal model

Guinea pigs are exposed on a single occasion to tobacco smoke for 50 minutes. Animals are sacrificed between 10 minutes and 24 hour following the end of the exposure and their lungs placed in RNAlater™. The lung tissue is homogenised ,and total RNA was extracted using a Qiagens RNeasy™ Maxi kit. Molecular Probes RiboGreen™ RNA quantitation method is used to quantify the amount of RNA in each sample.

Total RNA is reverse transcribed, and the resultant cDNA is used in a real-time polymerase chain reaction (PCR). The cDNA is added to a solution containing the sense and anti-sense primers and the 6-carboxy-tetramethyl-rhodamine labeled probe of the receptor tyrosine phosphatase gene. Cyclophilin is used as the housekeeping gene. The expression of the receptor tyrosme phosphatase gene is measured using the TaqMan real-time PCR system that generates an amplification curve for each sample. From this curve a threshold cycle value is calculated: the fractional cycle number at which the amount of amplified target reaches a fixed threshold. A sample containing many copies of the receptor tyrosme phosphatase gene will reach this threshold earlier than a sample containing fewer copies. The threshold is set at 0.2, and the threshold cycle C-j is calculated from the amplification curve. The Cx value for the receptor tyrosme phosphatase gene is normalized using the CT; value for the house- keeping gene.

Expression of the receptor tyrosme phosphatase gene is increased by at least 3-fold between 10 minutes and 3 hours post tobacco smoke exposure compared to air^' exposed control animals. Test compounds are evaluated as follows. Animals are pre-treated with a test compound between 5 minutes and 1 hour prior to the tobacco smoke exposure and they are then sacrificed up to 3 hours after the tobacco smoke exposure has been completed. Control animals are pre-treated with the vehicle of the test compound via the route of administration chosen for the test compound. A test compound that reduces the tobacco smoke induced upregulation of receptor tyrosine phosphatase gene relative to the expression seen in vehicle treated tobacco smoke exposed animals is identified as an inhibitor of receptor tyrosine phosphatase gene expression.

REFERENCES

1. Wright MB, Hugo C, Seifert R, Disteche CM, Bowen-Pope DF. Proliferating and migrating mesangial cells responding to injury express a novel receptor protein- tyrosine phosphatase in experimental mesangial proliferative glomerulonephritis. J Biol Chem 1998 Sep ll;273(37):23929-37.

2. Zelivianski S, Dean J, Madhavan D, Lin FF, Lin MF. Expression of receptor protein tyrosine phosphatase alpha mRNA in human prostate cancer cell lines. Mol Cell Biochem 2000 May;208(l-2):ll-8.

3. Fauman EB, Saper MA. Stracture and function of the protein tyrosine phosphatases. Trends Biochem Sci 1996 Nov;21(l l):413-7.

4. Neel BG, Tonks NK. Protein tyrosine phosphatases in signal transduction. Curr Opin Cell Biol 1997 Apr;9(2): 193-204.

5. Calvert-Evers JL, Hammond KD. Temporal variations in protein tyrosme phosphatase activity during cell proliferation and differentiation. Cell Biol Int 2000;24(8):559-68.

Claims

1. An isolated polynucleotide being selected from the group consisting of: a. a polynucleotide encoding a receptor tyrosine phosphatase polypeptide comprising an amino acid sequence selected form the group consisting of: i. amino acid sequences which are at least about 87% identical to the amino acid sequence shown in SEQ TD NO: 2; and ii. the amino acid sequence shown in SEQ TD NO: 2. b. a polynucleotide comprising the sequence of SEQ ID NO: 1 ; c. a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a receptor tyrosine phosphatase polypeptide; d. a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a receptor tyrosme phosphatase polypeptide; and e. a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a receptor tyrosine phosphatase polypeptide.

2. An expression vector containing any polynucleotide of claim 1.

3. A host cell containing the expression vector of claim 2.

4. A substantially purified receptor tyrosine phosphatase polypeptide encoded by a polynucleotide of claim 1.

5. A method for producing a receptor tyrosine phosphatase polypeptide, wherein the method comprises the following steps: a. culturing the host cell of claim 3 under conditions suitable for the expression of the receptor tyrosine phosphatase polypeptide; and b. recovering the receptor tyrosine phosphatase polypeptide from the host cell culture.

6. A method for detection of a polynucleotide encoding a receptor tyrosine phosphatase polypeptide in a biological sample comprising the following steps: a. hybridizing any polynucleotide of claim 1 to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and b. detecting said hybridization complex.

7. The method of claim 6, wherein before hybridization, the nucleic acid material of the biological sample is amplified.

8. A method for the detection of a polynucleotide of claim 1 or a receptor tyrosine phosphatase polypeptide of claim 4 comprising the steps of: a. contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the receptor tyrosine phosphatase polypeptide and b. detecting the interaction

9. A diagnostic kit for conducting the method of any one of claims 6 to 8.

10. A method of screening for agents which decrease the activity of a receptor tyrosine phosphatase, comprising the steps of: a. contacting a test compound with any receptor tyrosme phosphatase polypeptide encoded by any polynucleotide of claiml ; b. detecting binding of the test compound to the receptor tyrosme phosphatase polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a receptor tyrosine phosphatase.

11. A method of screening for agents which regulate the activity of a receptor tyrosme phosphatase, comprising the steps of: a. contacting a test compound with a receptor tyrosine phosphatase polypeptide encoded by any polynucleotide of claim 1; and b. detecting a receptor tyrosme phosphatase activity of the polypeptide, wherein a test compound which increases the receptor tyrosme phosphatase activity is identified as a potential therapeutic agent for increasing the activity of the receptor tyrosme phosphatase, and wherein a test compound which decreases the receptor tyrosme phosphatase activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the receptor tyrosine phos- phatase.

12. A method of screening for agents which decrease the activity of a receptor tyrosine phosphatase, comprising the step of:

contacting a test compound with any polynucleotide of claim 1 and detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of receptor tyrosine phosphatase.

13. A method of reducing the activity of receptor tyrosme phosphatase, comprising the step of:

contacting a cell with a reagent which specifically binds to any^' polynucleotide of claim 1 or any receptor tyrosine phosphatase polypeptide of claim 4, whereby the activity of receptor tyrosine phosphatase is reduced.

14. A reagent that modulates the activity of a receptor tyrosine phosphatase polypeptide or a polynucleotide wherein said reagent is identified by the method of any of the claim 10 to 12.

15. A pharmaceutical composition, comprising:

the expression vector of claim 2 or the reagent of claim 14 and a pharmaceutically acceptable carrier.

16. Use of the expression vector of claim 2 or the reagent of claim 14 in the preparation of a medicament for modulating the activity of a receptor tyrosine phosphatase in a disease.

17. Use of claim 16 wherein the disease is diabetes, a CNS disorder, obesity, COPD or a cardiovascular disorder.