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WO1994009037A1 - NOUVELLE CLASSE DE RPTPases: LEURS DOMAINES STRUCTURAUX ET LIGANDS - Google Patents

NOUVELLE CLASSE DE RPTPases: LEURS DOMAINES STRUCTURAUX ET LIGANDS Download PDF

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WO1994009037A1
WO1994009037A1 PCT/US1993/009838 US9309838W WO9409037A1 WO 1994009037 A1 WO1994009037 A1 WO 1994009037A1 US 9309838 W US9309838 W US 9309838W WO 9409037 A1 WO9409037 A1 WO 9409037A1
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receptor
receptor protein
ligand
protein
molecules
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PCT/US1993/009838
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WO1994009037A9 (fr
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Joseph Schlessinger
Gilad Barnea
Martin H. Grumet
Richard U. Margolis
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New York University Medical Center
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Priority to EP93924934A priority Critical patent/EP0677063A4/fr
Priority to JP6510272A priority patent/JPH08502487A/ja
Priority to AU54433/94A priority patent/AU5443394A/en
Publication of WO1994009037A1 publication Critical patent/WO1994009037A1/fr
Publication of WO1994009037A9 publication Critical patent/WO1994009037A9/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4725—Proteoglycans, e.g. aggreccan
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a new class of receptor protein tyrosine phosphatase molecule, the family of ligands that binds this new class of receptor, and uses of such receptors and ligands.
  • the members of this new class of receptor protein tyrosine phosphatase molecule are proteoglycans and/or possess an extracellular carbonic anhydrase structural domain.
  • the characterization of one member of this new class, RPTP/5, is described in the working examples presented herein.
  • the ligands which bind the receptor protein tyrosine phosphatases of the invention are members of the cell adhesion molecule (CAM) family of extracellular molecules.
  • CAM cell adhesion molecule
  • Extracellular molecules As a means by which to receive stimuli from their immediate environment. These extracellular signals are essential for the correct regulation of such diverse cellular processes as differentiation, contractility, secretion, cell division, contact inhibition, and metabolism.
  • the extracellular molecules which can include, for example, hormones, growth factors, or neurotransmitters, act as ligands that bind specific cell surface receptors. The binding of these ligands to their receptors triggers a cascade of reactions that brings about both the ampli ⁇ fication of the original stimulus and the coordinate regulation of the separate cellular processes mentioned above.
  • a central feature of this process is the reversible phosphorylation of certain proteins.
  • the phosphorylation or dephosphorylation of amino acid residues triggers conformational changes in regulated proteins that alter their biological properties.
  • Proteins are phosphorylated by protein kinases and are dephosphorylated by protein phosphatases. Protein kinases and phosphatases are classified according to the amino acid residues they act on, with one class being serine-threonine kinases and phosphatases
  • the protein kinases and phospha ⁇ tases may be further defined as being receptors, i.e., the enzymes are an integral part of a transmembrane, ligand-binding molecule, or as non-receptors, meaning they respond to an extracellular molecule indirectly by being acted upon by a ligand-bound receptor.
  • Phosphorylation is a dynamic process involving competing phosphorylation and dephosphorylation reactions, and the level of phosphorylation at any given instant reflects the relative activities, at that instant, of the protein kinases and phosphatases that catalyze these reactions.
  • PTPases protein tyrosine phosphatases
  • the non-receptor class is composed of low molecular weight, cytosolic, soluble proteins. All known non-receptor PTPases contain a single conserved catalytic phosphatase domain of approximately 230 amino acid residues.
  • the receptor class is made up of high molecular weight, receptor-linked PTPases, termed RPTPases.
  • RPTPases Structurally resembling growth factor receptors, RPTPases consist of an extracellular, putative ligand- binding domain, a single transmembrane segment, and an intracellular catalytic domain (reviewed in Fischer et al. , 1991, Science 253:401-406) .
  • the intracellular segments of almost all RPTPases are very similar. These intracellular segments consist of two catalytic phosphatase domains of the type described above, separated by an approximately 58 amino acid residue segment.
  • This two domain motif is usually located approximately 78 to 95 amino acid residues from the transmembrane segment and is followed by a relatively short carboxy-terminal amino acid sequence.
  • the only known exception is the isoform HPTP/3 (Krueger, N.X. et al. , 1990, EMBO J. 9_:3241), which contains only one catalytic phosphatase domain.
  • RPTPase extracellular domains are highly divergent.
  • certain RPTPases possess a heavily glycosylated external domain and a conserved cysteine-rich region (Thomas, M.L. et al.. 1985, Cell 1:83; Thomas, M.L. et al. , 1987, Proc. Natl. Acad. Sci. USA 84 . :5360; Ralph, S.J. et al.. 1987, EMBO J. 6 :1251-1257) while others contain immunoglobulin G-like (Ig) domains linked to fibronectin type III repeats (Streuli, M. et al..
  • RPTPases contains only multiple fibronectin type III repeats (Krueger, N.X. et al.. 1990, EMBO J. 9:3241), while certain RPTPases have smaller external domains that contain several potential glycosylation sites (Jirik, F.R. et al. , 1990, FEBS Lett. 273:239) .
  • the ligands that regulate RPTPs have not been identified. It has been speculated that circulating extracellular factors are unlikely to bind to those receptors containing Ig and/or fibronectin Type III repeats and that interaction with other surface antigens, perhaps on other cells, is more likely to be the case with these receptors.
  • tyrosine-specific phosphatase genes are candidate recessive oncogenes or tumor suppressor genes.
  • the human RPTPase, RPTP7 has been shown to map to a chromosomal region, 3pl4-21, which is frequently deleted in renal cell and lung carcinomas (LaForgia, S. et al.. 1991, Proc. Natl. Acad. Sci. USA ⁇ 8 . :5036-5040) .
  • the present invention relates to a new class of receptor protein tyrosine phosphatase molecule, to the family of ligands that binds this new class of receptor, and to the uses of such receptors and ligands.
  • the members of this new class of receptor protein tyrosine phosphatase molecule are proteoglycans and/or possess an extracellular carbonic anhydrase structural domain.
  • RPTPjS The characterization of one such receptor molecule, RPTPjS, is described in the working examples presented herein.
  • the ligands which bind the receptor protein tyrosine phosphatases of the invention are members of the cell adhesion molecule (CAM) family of extracel- lular molecules.
  • CAM cell adhesion molecule
  • the discovery that CAMs bind receptor protein tyrosine phosphatases represents the first identification of a natural ligand for this type of receptor. Binding of two CAMs, namely N-CAM and Ng-CAM, to the receptor protein tyrosine phosphatases of the invention is demonstrated in the working examples presented herein.
  • the receptors and the receptor-binding ligands of the invention may be used to develop compounds and strategies for modulating cellular processes under the control of the receptor protein tyrosine phosphatases.
  • Such processes include, but are not limited to, normal cellular functions such as differentiation, metabolism, cell cycle control, and neuronal function; cellular behavior such as motility and contact inhibition, in addition to abnormal or potentially deleterious processes such as virus-receptor interactions, inflammation, cellular transformation to a cancerous state, and the development of Type 2, insulin Independent, diabetes mellitus.
  • cellular behavior such as motility and contact inhibition
  • abnormal or potentially deleterious processes such as virus-receptor interactions, inflammation, cellular transformation to a cancerous state, and the development of Type 2, insulin Independent, diabetes mellitus.
  • Compounds that may interfere with ligand binding are described and methods for identifying other potential ligands, such as CAM-type ligands, growth factors, or extracellular matrix components, are discussed.
  • FIG. 1 The amino acid sequence of RPTP3.
  • the protein sequence of RPTP/3 containing 2308 amino acids is indicated.
  • the hydrophobic signal peptide is underlined, the transmembrane peptide is underlined and the transmembrane peptide is. designated in bold- type.
  • the 21 potential N-glycosylation sites are indicated by the arrows.
  • the CAH-related domain and the two phosphatase domains, DI and DII, are indicated by the boxes.
  • FIG. 2 Chromosomal localization of human RPTP/S.
  • A Presence of the RPTP/3 gene in a panel of 17 rodent-human hybrids. A completely stippled box indicates that the hybrid named in the left column contains the chromosome indicated in the upper row; lower-right stippling indicates presence of the long arm (or part of the long arm, indicated by a smaller fraction of stippling) of the chromosome shown above the column; upper left stippling indicates presence of the short arm (or partial short arm) of the chromosome listed above the column; an open box indicates absence of the chromosome above the column; the column for chromosome 7 is boldly outlined and stippled to highlight correlation of presence of this chromosome with the presence of the RPTP/3 gene.
  • RPTP/3 maps to 7q31- q33. Chromosome in situ hybridization of a 1.8 kb RPTP3 cDNA to normal human metaphases confirmed local ⁇ ization of the gene to 7q and revealed a peak of grains centered over region 7q31.3 - 7q32 as illustrated on the right to the chromosome sketch. Each dot representing an autoradiographic grain.
  • FIG. 3 Analysis of the expression of RPTP3 in various urine tissues and cell lines.
  • A. Poly A+ RNA (1 ⁇ g per sample) from the various murine tissues indicated were loaded onto a 1.0% agarose/2.2M formaldehyde gel and probed with the per amplified murine DNA fragment, pBSMBDII (described in Materials and Methods, Section 6.1.4).
  • B. The blot in A. was stripped of probe and rehybridized with a 3 P labeled rat actin probe.
  • RNA gel 20 ⁇ g of total cellular RNA (lanes 1-5) and 1 ⁇ g of Poly A+ RNA (lane 6) isolated from the various glioblastoma and neuroblas- toma cell lines indicated were loaded onto on RNA gel and probed with a DNA fragment isolated from the human brain stem cDNA clone that begins with sequences just 5' of the transmembrane region and extends and includes all of the sequences in phosphatase domain I.
  • FIG. 4 Northern blots to identify alternative splicing of RPTP/S transcripts.
  • A A schematic diagram of the protein encoded by the full length RPTPjS cDNA compared to the putative protein encoded by the two independently isolated cDNA clones that carry an identical deletion of 258 bp in the extracellular region of the protein. The position of the deletion is indicated by the dotted line with the number of the amino acid that remains at both the 5' and 3 ' end of the deletion indicted. The location of the two probes used in Northern analysis (probes 1 and 2) are indicated. TM, transmembrane peptide; DI, phosphatase domain I and DII, phosphatase domain II.
  • B A schematic diagram of the protein encoded by the full length RPTPjS cDNA compared to the putative protein encoded by the two independently isolated cDNA clones that carry an identical deletion of 258 bp in the extracellular region of the protein. The position of the deletion is indicated by the dotted line
  • RNA (1 ⁇ g) isolated from the Lan 5 neuroblastoma cell line was separated on a RNA formaldehyde gel and probed with human probe 1 (PI) that contains 1.3 kb of sequences derived from the extreme 5' end of the cDNA clone and human probe 2 (P2) that contains 1.6 kb of sequences derived from the portion of the full length cDNA clone that is deleted in clones BS-dl4 and Cau- dll.
  • PI human probe 1
  • P2 human probe 2
  • FIG. 5 In situ hybridization analysis of RPTP/3 in developing and adult mouse brain.
  • A. A sagittal section through an embryonic day 20 (E20) mouse shows that RPTP/3 is preferentially expressed in the developing central nervous system. The highest level of expression is seen in the ventricular zone (VZ) .
  • B. A sagittal section through the adult mouse brain shows discrete bands of expression in the Purkinje cell of the cerebellum, the dentate gyrus (OG) , and the anterior horn of the lateral ventricle (AH) .
  • FIG. 6 Identification of endogenous RPTP ⁇ protein expression in Lan 5 cells. Immunoprecipi- tation of RPTPjS with normal rabbit serum (NRS, lane 1) and immune RPTP3 antiseru ( ⁇ PTP/3, lanes 2 and 3) from lysates of 35 S methionine-labeled Lan 5 cells that had been labeled in the absence (lanes 1 and 2) or presence of tunicamycin (lane 3) . Apparent molecular weight is approximately 300 kD in the absence, and 250 kD is the presence, of tunicamycin.
  • FIG. 7 Identification of a CAH-related domain in the extracellular region of RPTP/3.
  • the amino acid sequences that are boxed in black are those that are identical in all six isoforms of CAH.
  • the sequences that are boxed in the gray hatches are those that are identical between the CAH-related domains of RPTPS and RPTP ⁇ .
  • FIG. 8 Polyacryla ide gel of an immunoprecipita- tion, using 35 S-NaS0 4 -labeled cell lysates from 293 cells transfected with RPTP/3 DNA (Lane 1) or from control, 293 cells transfected with vector alone (Lane 2) . Antiserum used was directed against RPTP/3, as described in Section 6.1.5.
  • FIG. 9 Polyacrylamide gel of an immunoprecipita- tion, using 35 S-Met-labeled cell lysates from 293 cells transfected with RPTPjS DNA (Lane 1) or from control, 293 cells transfected with vector alone (Lane 2) . Antiserum used was directed against RPTP/3, as described in Section 6.1.5.
  • FIG. 10 Polyacrylamide gel of an immunoprecipi- tation, using 35 S-Met-labeled cell lysates from 293 cells transfected with RPTPjS DNA (Lanes 3 and 4) or from control, 293 cells transfected with vector alone (Lane 1 and 2) . Lanes 2 and 4 represent lysates that have been chondroitinase ABC-treated, while 1 and 3 are untreated lysates. Antiserum used was directed against RPTP/3, as described in Section 6.1.5.
  • FIG. 11 Effects of the proteoglycan 3F8 on aggregation of Ng-CAM-Covaspheres. Green-fluorescing Ng-CAM-Covaspheres after incubation for 2 hours at 25° (A) in the presence of 10 ⁇ g/ l of BSA. (B) 30 ⁇ g/ l 3F8 proteoglycan. Covaspheres were visualized using a Nikon Diaphot microscope equipped for fluorescence and were photographed using a N2000 camera.
  • FIG. 12 Inhibition of NG-CAM-Covasphere aggre ⁇ gation by 3F8.
  • FIG. 13 Inhibition of N-CAM-Covasphere aggre ⁇ gation by chondroitinase-treated 3F8 (circles) . The appearance of superthreshold aggregates of Covaspheres coated with N-CAM was measured after 2 hours.
  • FIG. 14 Comparison of the amino acid sequences of the carbonic anhydrase domains contained in rat 3F8 and human RPTPjS proteins. Top sequence represents the RPTP/3 sequence, bottom line the 3F8 sequence.
  • This invention involves a new class of receptor protein tyrosine phosphatase molecule whose members are proteoglycans and/or possess an extracellular carbonic anhydrase structural domain.
  • CAMs cell adhesion molecules
  • the receptor and the receptor-binding ligands of the invention may be used to develop compounds and strategies for modulating cellular processes under the control of the receptor protein tyrosine phosphatases.
  • Such processes include, but are not limited to, normal cellular functions such as differentiation, metabolism, cell cycle control, and neuronal function; cellular behaviors such as motility, contact inhibition, and signal transduction; in addition to abnormal or potentially deleterious processes such as virus-receptor interactions, inflam ⁇ mation, cellular transformation to a cancerous state, and the development of Type 2, insulin independent diabetes mellitus.
  • the RPTPases of the invention that are proteoglycans may be modified with macromolecules composed of glycosaminoglycan (GAG) chains (glycans) covalently bound to the RPTPase protein core.
  • GAG components may consist of such units as hexosamine (D- glucosamine (GlcN) or D-galactosamine (GalN) ) , and either hexuronic acid (HexA; D-glucuronic acid (GlcA) or L-iduronic acid (IdoA)) or galactose units (as in keratin sulfate) that are arranged in alternating, unbranched sequence, and carry sulfate substituents in various positions.
  • GAG components may consist of such units as hexosamine (D- glucosamine (GlcN) or D-galactosamine (GalN) ) , and either hexuronic acid (Hex
  • the glycan backbones of the RPTPase molecules may include, but are not limited to, a basic structure composed of (HexA-GalN) n , (HexA- GlcN) n , or (Gal-GlcN) n disaccharide units. While these structures connote the basic structure of the RPTPase modifications, such modifications may also contain marked heterogeneity within as well as between the individual polysaccharide chains. Such heterogeneity is an expected byproduct of the mechanism of GAG biosynthesis, and may include, but is not limited to differences in sulfate substitutions along the chain and epimerization of one unit to another (GlcA to IdoA, for example) .
  • At least one glycan chain must be attached to the protein core of each proteoglycan RPTPase.
  • Glycan chains may, but are not required to, be attached to the protein core at the serine (Ser) amino acid residue of the sequence, Ser-Gly-X-Gly, where Gly is a glycine amino acid residue and X is any amino acid residue.
  • the members of the RPTPase class of the invention may include an extracellular stretch of amino acids that shares similarity with the known carbonic anhydrase isoforms (Deutsch, H.F., 1987, Int. J. Biochem. 19_:101-113) . Such sequences need not have carbonic anhydrase enzymatic activity.
  • One or more complete or partial carbonic anhydrase motifs may be present on a single RPTPase molecule.
  • CAH region of similarity there may exist amino acid substitutions, as well as short amino acid deletions, and/or short amino acid additions that diverge from the known CAH isoforms.
  • Such divergent sequences are acceptable as long as the overall amino acid sequence similarity to CAH remains at least about 25% and/or the tertiary structure or the domain remains similar to that of CAH.
  • RPTP/3 the characterization of one member, of this new class of RPTPase molecule.
  • RPTP/3 not only contains a CAH-like domain but is also a proteoglycan.
  • the molecules that act as the preferred ligands for the receptors of the invention are cell adhesion molecules (CAMs) .
  • CAMs cell adhesion molecules
  • Such molecules include, but are not limited to, any member of the classes of Ca 2+ -indepen- dent CAMs, cadherins, which are Ca 2+ -dependent CAMs, and integrins, which are Ca 2+ - or Mg 2+ -dependent CAMs.
  • Ca 2+ -independent CAMs include such molecules as the.N- CAM family, Ng-CAM, LI, Jl, Fasciclin III, and MAG molecules.
  • the cadherins include such molecules as N- cadherin, E-cadherin, P-cadherin, L-CAM, B-cadherin, and T-cadherin.
  • N- cadherin E-cadherin
  • P-cadherin P-cadherin
  • L-CAM L-CAM
  • B-cadherin B-cadherin
  • T-cadherin T-cadherin.
  • receptor phosphatases themselves may function as cell adhesion molecules because some of them contain motifs such as IgG-like or fibronectin Type III repeats typical of CAMs.
  • motifs such as IgG-like or fibronectin Type III repeats typical of CAMs.
  • PTPases with IgG and fibronectin motifs may also under go homotypic interactions. It is of note, however, that IgG-like and fibronectin motifs are found in many surface receptors and proteins which do not undergo homotypic interations.
  • CAMs act as ligands for the RPTPase*.molecules of this invention, which contain no IgG-like and fibronectin Type III motifs.
  • a ligand/receptor interaction does, in fact, occur between the RPTPase class of molecule disclosed in this invention and CAMs, where no interaction has previously been predicted to occur.
  • the ligands of the invention may be transmembrane proteins, glycosylphosphatidylino ⁇ itol-linked membrane proteins, or secreted proteins.
  • the molecules that constitute the ligands of this invention may contain one or more peptide domains, including, but not limited to, one or more Ig (immunoglobulin) domains (Williams, A.F., 1987, Immunol. Today 8 . :298-303), one or more fibronectin type III domains (Hynes, R.O. , 1990, Fibronectins, Springer-Verlag, New York), and/or one or more ectodomains (Takeichi, M. , 1991, Science 251:1451-1455) .
  • Ig domains may share characteristics with both immunoglobulin constant and variable regions. Such characteristics may include pairs of cysteine residues, spaced approximately 60 amino acids apart, that form disulfide bonds with each other. Molecules may exhibit one or amino acid repeats of the sequence DRE, DXNDN, DXD, DVNE, DXE, and/or DPD. If the molecules are transmembrane proteins, such sequences should be present in the extracellular portion of the molecule.
  • the RPTPase molecules of this invention may be proteoglycans
  • several other non-CAM-like ligands may exist.
  • extracellular matrix molecules as vitronectin, fibronectin, and laminin have been known to bind to the GAGs of certain proteoglycans.
  • growth factors such as fibro- blast growth factors, and Schwann cell growth factor, have also been demonstrated to have affinity for proteoglycan GAG chains. Therefore, molecules including, but not limited to extracellular matrix molecules and growth factors are potential ligands for the RPTPase class of molecule presented in this invention.
  • RPTPase LIGANDS Depending on the individual molecule, some RPTPase molecules may become activated upon ligand binding, and others may become inactivated (the activity referred to here being the RPTPases' phospha ⁇ tase activity) . Ligand binding to RPTPase molecules may affect a variety of cellular processes.
  • Such processes include, but are not limited to, normal cellular functions such as differentiation, metabo- lism, cell cycle control, and neuronal function; cellular behavior, such as motility and contact inhibition; in addition to abnormal or potentially deleterious processes such as virus-receptor interac- tions, inflammation, cellular transformation to a cancerous state, and the development of Type 2, insulin independent diabetes mellitus.
  • RPTPase/CAM binding may exert an effect on the above-mentioned processes within the RPTPase-exhibiting cell.
  • CAMs are often cell surface proteins, RPTPase/CAM binding may elicit an effect on the CAM-exhibiting cell.
  • RPTPases may contribute to the control of such cellular processes by exerting an effect directly on the CAM ligand itself, via, for example, a CAM phosphorylation/ dephosphorylation reaction.
  • the receptors and the receptor-binding ligands of the invention may be used as drugs that can modulate the cellular processes under the control of the RPTPases.
  • methods are presented below for the identification of compounds that affect RPTPase activity, and such compounds may also be used as drugs that can modulate one or more of the cellular processes mentioned above.
  • the receptors or their ligands may be used directly to modulate processes such as those mentioned above.
  • soluble RPTPases may be adminis ⁇ tered, using techniques well known to those skilled in the art, that could act to compete with endogenous transmembrane receptor molecules for available ligands, thus reducing or inhibiting ligand binding to endogenous RPTPases.
  • the effect of such a procedure would be to activate, reduce or block the signal normally transduced into the cell (either the RPTPase- exhibiting cell, or the CAM-exhibiting cell) via ligand binding to transmembrane RPTPase.
  • the RPTPases used here may include the entire molecule or, alternatively, only the RPTPase extracellular domain, or a part of the RPTPase extracellular domain thereof.
  • ligands may be administered, again, using techniques well known to those in the art. Such administration would lead to a greater than normal number of transmembrane RPTPases being bound by ligand, potentially causing an amplification of the ligand-bound state within cells exhibiting RPTPases.
  • the administered ligand may be composed of a modified form of said ligand such that receptor binding may still occur, but the normal result of such binding (receptor activation or inactivation, as the case may be) does not occur.
  • a ligand with such a design would act in much the same way that administra ⁇ tion of soluble RPTPase would, in that both procedures would have the final effect of reducing the number of functionally bound RPTPase transmembrane molecules, therefore lowering or blocking the normal extracel- lular signal being transduced into the RPTPase- exhibiting cell via normal ligand binding to transmembrane RPTPase.
  • the effect on a CAM ligand- exhibiting cell would also be the same in that an overall lower number of endogenous CAM ligands would be bound, therefore lowering or blocking the effect of RPTPase binding on such CAM-exhibiting cells.
  • agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in "Remington's
  • Suitable routes may include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcu- taneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injec ⁇ tions, just to name a few.
  • the agents of the invention may be formulated in aqueous solu- tions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • RPTPases and/or their ligands may also be used to screen for additional molecules that can act to modu ⁇ late the activity of cellular processes such as those described above.
  • compounds that bind to RPTPase molecules may be identified.
  • One method that may be pursued in the isolation of such RPTPase- binding molecules would include the attachment of RPTPase molecules to a solid matrix, such as agarose or plastic beads, microtiter wells, or petri dishes, and the subsequent incubation of attached RPTPase molecules in the presence of a potential RPTPase- binding compound or compounds. After incubation, unbound compounds are washed away, and the RPTP-bound compounds are recovered.
  • Bound molecules could be eluted from the RPTPase molecules by, for example, competing them away from the RPTPase molecules with the addition of excess ligand.
  • the effect of a compound on the phosphatase activity of RPTPase molecules can also be determined.
  • Such a compound may, for example, be one isolated using a procedure such as the binding technique described above.
  • One method that may be utilized for determining the effects of a compound on RPTPase phosphatase activity would involve exposing such a compound to a preparation of cultured cells that express the RPTPase of the invention, and subsequently measuring the phosphatase activity of the culture.
  • the compound of interest may be introduced to the cells, for example, by addition of the compound to the tissue culture medium.
  • the phosphatase activity of the cells within the tissue culture preparation may be determined by measuring the level of cellular phospho- tyrosine within the culture, using method that are well known in the art (Honegger et al. , 1987, Cell 51:199-209; Margolis et al. , 1989, Cell 57:1101-1107) .
  • RPTPases may be incorporated into apparatuses including but not limited to affinity columns such that large numbers of molecules may be screened quickly by being applied to said apparatuses. Those molecules with an affinity for RPTPases will be bound. Such binding will also bring about a partial purifica ⁇ tion of the molecules of interest.
  • the bound molecules should be eluted off the above described apparatuses, for example by competing them away from the RPTPases with excess ligand, and the process should be repeated until the molecule of interest is purified to the extent necessary.
  • RPTPj3 human receptor protein tyrosine phosphatase molecule
  • a cDNA clone containing a portion of the coding sequences for RPTP/3 was isolated after screening a ⁇ gtll human infant brain stem cDNA library under conditions of reduced stringency with a nick trans ⁇ lated LCA probe that included both phosphatase domains (Kaplan, R. et al. , 1990, Proc. Natl. Acad. Sci. USA 82:7000-7004). Since the 5' end of this gene was not present in the original clone, the library was rescreened with a DNA fragment that was generated from the 5'end of the original clone.
  • the probe was labeled with 32 P dCTP utilizing the random prime method (USB) and hybridization was performed under moderately stringent conditions at 42°C in a buffer containing 50% formamide, 5XSSC, 20mM Tris-CL pH 7.6, IX Denhardt's solution, 0.1% SDS and 100 ⁇ g/ml of sheared and denatured salmon sperm DNA. After hybridization, phage filters were washed three times for 20 min at 50°C in a buffer containing O.lXSSC/0.1% SDS and then were processed for autoradiography. The brainstem library was rescreened a total of three times in order to isolate overlapping cDNA clones that contained the entire coding sequence for RPTPjS.
  • USB random prime method
  • cDNA inserts from positive recombinant plaque- purified were subcloned into the plasmid vector, Blue Script (Stratagene) , and sequenced by the dideoxy chain termination method using the Sequenase Version 2.0 Kit (USB) . 6.1.2 CHROMOSOMAL LOCALIZATION Isolation, propagation and characterization of parental and somatic cell hybrids used in this study have been described (Durst, M. et al.. 1987, Proc. Natl. Acad. Sci USA 84.:1070-1074) . Presence of specific human chromosomes or regions of chromosomes has been confirmed by DNA hybridization using probes for genes assigned to specific chromosome regions.
  • Fig. 2A depicts diagrammatically the chromosomes or partial chromosomes retained in most of the hybrids used.
  • Chromosomal in situ hybridization was performed as described previously (Cannizzano, L.A. et al. , 1991, Cancer Res. 5L:3818-3820) .
  • Slides containing metaphase chromosomes from normal male (46 XY) peripheral blood lymphocytes were aged at 4°C for 7-10 days and pretreated with ribonuclease A (Sigma) for 1 hour at 37°C.
  • the chromosomal DNA was denatured in a hybridization mixture containing 50% formamide, 2X SSC and 10% dextran sulfate (pH 7.0). Hybridization was carried out at 37°C overnight.
  • oligonucleotides in conserved phosphatase domain II were synthesized according to the nucleotide sequence of human RPTPj3. These oligos, in conjunction with phage DNA from a mouse brain cDNA library that was purchased from Clonetech (Palo Alto, CA) , were used in the polymerase chain reaction with Taq polymerase (Perkin-Elmer) to amplify homologous mouse RPTP/3 sequences. The amplified product was purified and cloned into the Blue Script plasmid vector (Stratagene, La Jolla, CA) . Homology was confirmed by DNA sequence analysis as described above. This subcloned fragment will be referred to as pBSMBDII.
  • RNA was prepared with the Strategene RNA isolation kit. Poly A + RNA was further selected utilizing oligo dT cellulose chromatography (Stratagene) . For Northern analysis, the RNA was separated on a 1.0% agarose/2.2 M formaldehyde gel and transferred to a Nytran membrane (Schleicher and Schuell) by capillary action. The membrane was prehy- bridized and hybridized in 0.5 M sodium phosphate pH 7.2, 7% SDS, ImM EDTA, 100 ⁇ g/ml salmon sperm DNA and then washed in 40mM sodium phosphate ph 7.2, 1% SDS, 1 mM EDTA at 65°C.
  • RNA isolated from various mouse tissues a 32 P-labeled probe was made utilizing pBSMBDII as template in the random prime labeling reaction (USB) .
  • the human glioblastoma and neuroblastoma RNA blots were probed with labeled restriction fragments isolated from different parts of the human RPTP/3 cDNA clones.
  • ANTIBODIES A peptide derived from the carboxy-terminal 15 amino acids of human RPTP/3 was synthesized and coupled to Keyhole limpet hemocyanin according to previously published procedures (Harlow, E. and Lane, D. , 1988, in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 77-88) . This was used as immunogen to inoculate two rabbits to produce polyclonal antisera against RPTPjS. Anti-EGF receptor immunoprecipitates were performed with RK2 antibody which recognizes the glycosylated and nonglycosylated forms of the EGF receptor (Kris, R.M. et al.. 1985, Cell 4O: 619-625).
  • DMEM Dulbecco's modified Eagles medium
  • FBS fetal bovine serum
  • tunicamycin involved incubating the cultures with 10 ⁇ g/ml tunicamycin (Sigma) for 1 hour prior to 35 S methionine labeling. Treated and untreated cells were washed twice with methionine free DMEM and then labeled for 4 hours with 0.15 mCi/ml 32 S methionine (purchased from New England Nuclear) in DMEM minus methionine containing 1% dialyzed FBS.
  • Lysates from 35 S-NaS0 4 - labeled cultures were immunoprecipitated without preclearing, with anti-RPTP/3 antiserum for 2 hours at 4°C.
  • the immunocomplexes were then precipitated with Protein A Sepharose (Sigma) for 45 min at 4°C and washed 10 times with RIPA buffer (20mM Tris-Cl ph 7.6, 300 mM NaCl, 2mM EDTA, 1.0% Triton X-100, 1.0% sodium deoxycholate and 0.1% SDS).
  • the immunoprecipitated material was analyzed on SDS-polyacrylamide gels (7.5% for 35 S-Methionine, 5% for 35 S-NaS0 4 ) and then fluorographed.
  • the oligonucleotide was labeled with [ ⁇ - 35 S] dATP (NEN Dupont) using terminal deoxynucleotidyltransferase (Boerhinger Mannheim) and purified using Sephadex G25 quick spin columns (Boerhinger Mannheim) .
  • the specific activity of the labeled probes was between 5 X 10 8 - 1 x 10 9 cpm/ ⁇ g.
  • Prehybridizations and hybridizations were carried out in a buffer containing 50% deionized formamide, 4X SSC, IX Denhardt's, 500 ⁇ g/ml denatured salmon sperm DNA, 250 ⁇ g/ml yeast tRNA and 10% dextran sulfate.
  • the tissue was incubated for 12 h at 45°C in hybridization solution containing the labeled probe (1 x 10° cpm/section) and 10 mM dithiothreitol (DTT) .
  • Controls for specificity were performed on adjacent sections by diluting the labeled oligonucleotides with a 30 fold concentration of the appropriate unlabeled oligonucleotide and by hybridization with a sense probe.
  • the sections were washed in 2 changes of 2X SSC at room temperature for 1 h, IX SSC at 55°C for 30 min., 0.5X SSC at 55°C for 30 min, 0.5X SSC at room temperature for 15 min and dehydrated in 60%, 80%, and 100% ethanol. After air drying, the sections were exposed to X-ray film for 5-10d.
  • RPTP/3 belongs to the high molecular weight, transmembrane class of tyrosine phosphatases and is encoded by 2308 amino acids.
  • the protein contains a signal peptide (underlined in FIG.
  • the 8.8 and 6.4 kb transcripts were identical in size to the two transcripts observed in RNA isolated from mouse brain tissue (FIG. 3A) .
  • the deleted clones maintained the same extreme 5' and 3' ends of the RPTPj3 gene in addition to the sequences encoding the transmembrane peptide and the two phosphatase domains.
  • a transcript corresponding to the deleted clone would be approximately 2.6 kb smaller than the transcript corresponding to the undeleted, full-length clone.
  • FIG. 3C there is a transcript of 6.4 kb that is approximately 2.4 kb smaller than the largest transcript which is 8.8 kb in length.
  • Northern blot hybridization analysis was performed utilizing RNA isolated from the Lan 5 cell line.
  • Duplicate blots were made from this RNA and hybridized with two distinct probes.
  • One probe (probe 1) was derived from sequences in the 5' end of RPTPj3 that are present in the deleted and full length cDNA clones.
  • the other probe (probe 2) encompasses the sequences that are no longer present in the deleted cDNA clones.
  • the location of probes A and B in the full length RPTPj3 cDNA is shown in FIG. 4A.
  • Comparison of the Northern analysis with the two probes revealed that probe 1 hybridized to the three distinct transcript (FIG. 4B, PI) whereas probe 2 hybridized to the 7.5 and 8.8 kb transcripts but failed to hybridize to the 6.4 kb transcript (FIG. 4B, P2) .
  • RPTP/3 In order to obtain further insight into the expression of RPTP/3, an in situ hybridization analysis was performed to look for the expression of RPTP/3 in the developing mouse embryo. Studies were also performed to determine whether RPTP/S gene expression is diffuse or restricted to specific regions of the adult brain. The results of this analysis confirm that RPTP/3 is preferentially expressed in the central nervous system. In the day 20 mouse embryo (E20) , high level of expression was observed in the ventricular zone of the brain (FIG 5A.) and in the spinal cord. A similar pattern of expression, with variable levels of intensity, has been seen from embryonic day 13 to postnatal day 7.
  • the level of expression is much lower in the adult brain, and is discretely localized to the Purkinje cell layer of the cerebellum, the dentate gyrus, and the anterior horn of lateral ventricle (FIG. 5B) .
  • the addition of a 30 fold excess of unlabeled oligonucleotide completely blocked the labeling in all of these areas indicating that this probe is hybridizing to mRNA in a sequence specific manner.
  • Results from the Northern blot' and in situ hybridization analyses demonstrate that RPTPjS has a restricted tissue specificity to specific regions in the central nervous system and therefore may play an important role in the development of the nervous system.
  • RPTPjS transcripts were identified in the Lan 5 neuroblastoma cell line, these cells were subse ⁇ quently used to detect endogenous protein expression.
  • Cell lysates prepared from cultures labeled with 35 S- methionine for 4 hours were immunoprecipitated with normal rabbit serum or anti-RPTPj3 antiserum (FIG. 6) .
  • a protein with apparent weight of approximately 300 kd was recognized by the immune but not by the normal rabbit serum (lanes 1 and 2) .
  • tunicamycin was added to the cells during the 35 S-methionine labeling period.
  • the effects of tunicamycin treatment on RPTPjS mobility was compared to the cell line were drug's ability to inhibit the glycosylation of the EGF receptor, which is also expressed in this cell line.
  • Untreated cell lysates and lysates prepared from cells treated with tunicamycin were immunoprecipitated with an antibody (RK2) that recognizes the 170 kd glycosylated form and the 135 kd nonglycosylated form of the EGF receptor.
  • RK2 an antibody that recognizes the 170 kd glycosylated form and the 135 kd nonglycosylated form of the EGF receptor.
  • the molecular weight of the protein detected in FIG. 6, lane 3, is approximately 250 kD a value consistent with that of the core protein whose predicted molecular weight as deduced from the amino acid sequence is approximately 254 kd.
  • FIG. 7A Alignment of the CAH-related domains of RPTPjS and RPTP ⁇ with the six known isoforms of CAH is shown in FIG. 7A.
  • FIG. 7A Alignment of the CAH-related domains of RPTPjS and RPTP ⁇ with the six known isoforms of CAH is shown in FIG. 7A.
  • RPTPases, ⁇ and ⁇ may represent a new subgroup of tyrosine phosphatases that will be charac ⁇ terized by the presence of CAH-related sequences in their extracellular domains.
  • RPTPj3 exhibits the characteristics of a proteoglycan. Specifically, it is shown that the RPTPjS protein is covalently modified with high molecular weight, sulfate-containing moieties, and that such moieties are sensitive to chondroitinase ABC treatment.
  • lane 5 which contains the control lysate, exhibits no such material.
  • 6.2.8.3 CHONDROITINASE TREATMENT 293 cells transfected with RPTP/3 DNA as well as control 293 cells transfected with vector alone were 35 S-methionine labeled. Lysates were immunoprecipitated using an anti-RPTPjS antiserum and then chondroitinase ABC treated for 1 hour. The gel illustrated in FIG. 10 shows the results of one such immuno ⁇ precipitation. Lane 3 and 4 contain non-treated and treated RPTP/3-transfected lysates, respectively.
  • Ng-CAM and N-CAM were purified from 14-d embryonic chicken brains by immunoaffinity chromato- graphy using specific monoclonal antibodies (Grumet, M. and Edelman, G.M. , 1988, J. Cell Biol. 106:487- 503) . Analysis of the proteins on SDS/PAGE showed that Ng-CAM consisted of a major component of 135 kDa and lesser amounts of the 200 kDa and 80 kDa species as described (Grumet, M.
  • 3F8 proteoglycan was then isolated by immunoaffinity chromatography, using monoclonal antibodies coupled to CNBr-activated Sepharose 4B (Rauch, U. et al. , 1991, J. Biol. Chem. 266:14785-14801) .
  • Analysis of the proteins on SDS-PAGE following chondroitinase-treat- ment showed that the core glycoprotein obtained by chondroitinase treatment of the 3F8 proteoglycan from either early postnatal or adult brain migrated on SDS- PAGE as a single bad at 400 kDa (Rauch, U. et al.. 1991, J. Biol. Chem. 266:14785-14801) .
  • proteoglycans were digested for 45-60 min at 37°C with protease-free chondroitinase ABC (Seikagaku America Inc., Rockville, MD) in 100 mM Tris-HCl buffer (pH 8.0 at 37°C) containing 30 mM sodium acetate. A ratio of 1.5 mM chondroitinase/ ⁇ g proteoglycan protein was used for the 3F8 proteoglycan. Completeness of digestion was confirmed by SDS-PAGE, which demonstrated that the large native proteoglycan which did not enter the separating gel was converted to discrete core glycoprotein bands after enzyme treatment (Rauch, U. et al. , 1991, J. Biol. Chem. 266:14785-14801) .
  • Polyclonal rabbit antibodies raised against chicken Ng-CAM were prepared as previously described (Grumet, M. et al. , 1984, Proc. Natl. Acad. Sci USA 1:267-271).
  • proteoglycans To test the sensitivity of proteoglycans to proteolysis, solutions containing 0.1 mg/ml proteoglycan were treated with 10 ⁇ g/ml of trypsin for 1 h at 37°C and the reaction was terminated by addition of 20 ⁇ g/ml of soybean trypsin inhibitor.
  • the 3F8 proteoglycan inhibited aggregation of Ng- CAM-Covaspheres at 30 ⁇ g/ml (FIG. 11) . It is unlikely that the proteoglycans inhibited Covasphere aggregation by a trivial mechanism such as proteolysis of Ng-CAM because it was found that incubation of the 3F8 proteoglycan with Ng-CAM for 1 h at 37°C had no effect of the molecular sizes of the Ng-CAM components when resolved by SDS-PAGE. To compare the effects of different proteoglycans we measured the appearance of superthreshold aggre ⁇ gates of Covaspheres using a Coulter Counter to detect aggregates larger than a given size. The aggregation of Ng-CAM-Covaspheres was inhibited in a concentra- tion-dependent manner by the 3F8 proteoglycan (FIG. 12).
  • the inhibitory effect of 3F8 proteoglycan on the aggregation of Ng-CAM- and N-CAM-coated beads were maximal at approximately 10 ⁇ g/ml.
  • the amount of proteoglycan in solution was 0.6 ⁇ g and the amount of Ng-CAM on the Covaspheres was approximately 0.3 ⁇ g (see Materials and Methods, Section 7.1.2), suggesting that the brain proteoglycan can perturb homophilic Ng-CAM binding at approximately stoichio- metric levels with Ng-CAM.

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Abstract

L'invention se rapporte à une nouvelle classe de molécules de tyrosine phosphatase récepteur protéique (RPTPase), à la famille de ligands qui lie cette nouvelle classe de récepteurs et à l'utilisation de tels récepteurs et ligands.
PCT/US1993/009838 1992-10-15 1993-10-14 NOUVELLE CLASSE DE RPTPases: LEURS DOMAINES STRUCTURAUX ET LIGANDS WO1994009037A1 (fr)

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EP93924934A EP0677063A4 (fr) 1992-10-15 1993-10-14 NOUVELLE CLASSE DE RPTPases: LEURS DOMAINES STRUCTURAUX ET LIGANDS.
JP6510272A JPH08502487A (ja) 1992-10-15 1993-10-14 新規なクラスのｒｐｔｐアーゼ：それらの構造ドメイン及びリガンド
AU54433/94A AU5443394A (en) 1992-10-15 1993-10-14 A new class of rptpases: their structural domains and ligands

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996037776A1 (fr) * 1995-05-26 1996-11-28 Sugen, Inc. Ligands fonctionnels de la contactine molecule de reconnaissance des cellules axonales
EP0684989A4 (fr) * 1993-02-10 1998-06-03 Univ New York NOUVELLE PHOSPHOTYROSINE PHOSPHATASE--g(b) DE TYPE RECEPTEUR.
WO1999050385A3 (fr) * 1998-03-30 1999-11-18 Harvard College Regulation de la synthese de glycosaminoglycane, techniques apparentees et reactifs connexes
US6682905B1 (en) 1990-07-11 2004-01-27 New York University Receptor-type phosphotyrosine phosphatase-alpha
US7108994B2 (en) 1990-07-11 2006-09-19 New York University Receptor-type phosphotyrosine phosphatase-alpha
WO2019222547A1 (fr) * 2018-05-17 2019-11-21 The Board Of Trustees Of The Leland Stanford Junior University Inhibition de récepteur par recrutement de phosphatases

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WO1992001050A1 (fr) * 1990-07-11 1992-01-23 New York University Nouvelle phosphotyrosine phosphatase de type recepteur

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WO1992001050A1 (fr) * 1990-07-11 1992-01-23 New York University Nouvelle phosphotyrosine phosphatase de type recepteur

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International Journal of Biochemistry, Volume 19, Number 2, issued 1987, H.F. DEUTSCH, "Carbonic Anhydrases", pages 101-113, see entire document. *
Journal of Experimental Medicine, Volume 168, issued November 1988, M. STREULI et al., "A New Member of the Immunoglobulin Superfamily that has a Cytoplasmic Region Homologous to the Leukocyte Common Antigen", pages 1523-1530, see entire document. *
Proceedings of the National Academy of Sciences, USA, Volume 86, issued November 1989, M. STREULI et al., "A Family of Receptor-Linked Protein Tyrosine Phosphatases in Humans and Drosophila", pages 8698-8702, see entire document. *
Proceedings of the National Academy of Sciences, USA, Volume 87, issued September 1990, R. KAPLAN et al., "Cloning of Three Human Tyrosine Phosphatases Reveals a Multigene Family of Receptor-Linked Protein-Tyrosine-Phosphatases Expressed in Brain", pages 7000-7004, see entire document. *
Science, Volume 251, issued 22 March 1991, M. TAKEICHI, "Cadherin Cell Adhesion Receptors as a Morphogenetic Regulator", pages 1451-1455, see entire document. *
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See also references of EP0677063A4 *
The EMBO Journal, Volume 9, Number 10, issued 1990, N.X. KRUEGER et al., "Structural Diversity and Evolution of Human Receptor-Like Protein Tyrosine Phosphatases", pages 3241-3252, see entire document. *
The Journal of Biological Chemistry, Volume 266, Number 22, issued 05 August 1991, U. RAUCH et al., "Isolation and Characterization of Developmentally Regulated Chondroitin/Keratin Sulfate Proteoglycans of Brain Identifed with Monoclonal Antibodies", pages 14785-14801, see entire document. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6682905B1 (en) 1990-07-11 2004-01-27 New York University Receptor-type phosphotyrosine phosphatase-alpha
US7108994B2 (en) 1990-07-11 2006-09-19 New York University Receptor-type phosphotyrosine phosphatase-alpha
EP0684989A4 (fr) * 1993-02-10 1998-06-03 Univ New York NOUVELLE PHOSPHOTYROSINE PHOSPHATASE--g(b) DE TYPE RECEPTEUR.
WO1996037776A1 (fr) * 1995-05-26 1996-11-28 Sugen, Inc. Ligands fonctionnels de la contactine molecule de reconnaissance des cellules axonales
WO1999050385A3 (fr) * 1998-03-30 1999-11-18 Harvard College Regulation de la synthese de glycosaminoglycane, techniques apparentees et reactifs connexes
WO2019222547A1 (fr) * 2018-05-17 2019-11-21 The Board Of Trustees Of The Leland Stanford Junior University Inhibition de récepteur par recrutement de phosphatases

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