WO1997014966A1 - Procede d'activation d'une nouvelle voie de regulation de ligand - Google Patents

Procede d'activation d'une nouvelle voie de regulation de ligand Download PDF

Info

Publication number
WO1997014966A1
WO1997014966A1 PCT/CA1996/000679 CA9600679W WO9714966A1 WO 1997014966 A1 WO1997014966 A1 WO 1997014966A1 CA 9600679 W CA9600679 W CA 9600679W WO 9714966 A1 WO9714966 A1 WO 9714966A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
receptor tyrosine
tyrosine kinase
eph
cell
Prior art date
Application number
PCT/CA1996/000679
Other languages
English (en)
Other versions
WO1997014966B1 (fr
Inventor
Anthony Pawson
Mark Henkemeyer
Original Assignee
Mount Sinai Hospital Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mount Sinai Hospital Corporation filed Critical Mount Sinai Hospital Corporation
Priority to CA 2234090 priority Critical patent/CA2234090A1/fr
Priority to JP9515370A priority patent/JPH11515105A/ja
Priority to EP96932414A priority patent/EP1019728A1/fr
Publication of WO1997014966A1 publication Critical patent/WO1997014966A1/fr
Publication of WO1997014966B1 publication Critical patent/WO1997014966B1/fr

Links

Classifications

    • 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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • the invention relates to a novel ligand regulatory pathway, to methods for identifying substances capable of activating the novel pathway, methods for assaying for agonists or antagonists of the novel pathway, and to methods and pharmaceutical compositions for affecting neuronal development and regeneration.
  • Embryonic development of multicellular organisms is a highly ordered process that requires coordination of individual cells. Every cell must decipher the numerous signals it receives and then properly execute commands in order to achieve the correct position and differentiated state in the animal. The extraordinar controls over cell growth, determination, migration and adhesion are mediated by molecules located on the plasma membrane surface.
  • a class of membrane associated molecules known to regulate cellular interactions are receptor tyrosine kinase proteins.
  • the evolutionary conservation of genes encoding receptor tyrosine kinase proteins and their targets has emphasized the importance of these proteins in intracellular communication, and has also provided model systems for genetic analysis of tyrosine kinase signalling pathways.
  • Eph subfamily is made up of at least thirteen related but unique gene sequences in higher vertebrates (Hirai et al., Science 238:1717-1720, 1987; Letwin et al., Oncogene 3:621-627, 1988; Lindberg et al., Mol. Cell. Biol. 10:6316-6324, 1990; Lhotak et al, Mol. Cell. Biol.
  • cell adhesion-like domains in this family of tyrosine kinases suggests that these proteins function in cell-cell interactions.
  • the other major families of proteins implicated in cell adhesion include the cadherins, selectins, integrins, and those of the immunoglobulin superfamily (reviewed by Hynes, R.O. and Landers, A.D., Cell 68, 303-322, 1992).
  • the extracelluar regions of cell adhesion molecules frequently contain peptide repeats, such as FN III motifs, epidermal growth factor (EGF) repeats, or Ig loops that may direct protein-protein interactions at the cell surface.
  • EGF epidermal growth factor
  • ectopic N-cadherin expression during gastrulation stage Xenopus embryos has been shown to interfere with segregation of the neural tube from the ectoderm (Derrick et al., Neuron 4:493-506, 1990; Fujimori et al., Development 110:97-104, 1990).
  • Uttle is known about how these adhesive interactions are regulated and how they function in cell signalling pathways during normal development.
  • a critical stage in the development of the nervous system is the projection of axons to their targets. Navigational decisions are made at the growth cones of the migrating axons.
  • a function in axonal pathfinding is evident for the Drosophila abl tyrosine kinase when abl mutations are combined with mutations in other genes including the neural cell adhesion molecule, fasciclin I (fas I, Elkins et ah, Cell 60:565-575, 1990) or disabled (dab, Gertler et al., Cell 58:103-113, 1989).
  • fasciclin I fasciclin I
  • abl tyrosine kinase is specifically localized to the axonal compartment of the embryonic Central Nervous System (CNS) (Gertler et al, Cell 58:103-113, 1989).
  • the present inventors have identified and characterized a novel ligand regulatory pathway that plays a crucial role in cell-cell interactions and axonogenesis in the development and regeneration of the nervous system.
  • the present inventors have determined that Eph subfamily receptor tyrosine kinases activate a ligand regulatory pathway in cells expressing ligands for the Eph subfamily receptor tyrosine kinases.
  • Activation of the Ugand regulatory pathway results in downstream activation of a series of regulatory pathways in the cells that control gene expression, cell division, cytoskeletal architecture, cell metabohsm. cell migration and ceU-cell interactions.
  • the ligand regulatory pathway may be activated by an Eph subfamily receptor tyrosine kinase lacking in an active catalytic kinase domain.
  • an Eph subfamily receptor tyrosine kinase is essential for formation of a commissure in the brain and that this essential function is independent of an intact catalytic kinase domain.
  • the direct demonstration of a vital function in neuronal development for an Eph subfamily receptor tyrosine kinase is unprecedented, as is the showing of a function for a receptor tyrosine kinase which is mediated by the extracellular domain, independently of the catalytic kinase domain of the receptor.
  • a protein having the extracellular, transmembrane and juxtamembrane domains of an Eph subfamily receptor tyrosine kinase can provide a signal to a cell expressing a Ugand for the receptor tyrosine kinases and thereby activate a ligand regulatory pathway in the cell expressing the Ugand.
  • the present invention provides a method of activating a ligand regulatory pathway in a ceU, comprising reacting an Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein, with a cell expressing a Ugand for an Eph subfamily receptor tyrosine kinase on the surface of the cell thereby activating the ligand regulatory pathway in the cell.
  • the protein or part of the protein is lacking in catalytic kinase activity.
  • the part of the protein comprises an extraceUular, transmembrane and juxtamembrane domain, or only an extracellular domain of an Eph subfamUy receptor tyrosine kinase, preferably Nuk.
  • the invention also provides a method for identifying a substance which is capable of binding to a Ugand for an Eph subfamily receptor tyrosine kinase and activating a ligand regulatory pathway in a cell, comprising reacting a cell expressing a ligand for an Eph subfamily receptor tyrosine kinase on the surface of the ceU, with at least one test substance, under conditions which permit the formation of substance-Ugand complexes, and assaying for substance-Ugand complexes, for free substance, for non-complexed Ugands, or for activation of the ligand.
  • Activation of the ligand may be assayed by measuring phosphorylation of the Ugand, or binding of SH2 domains to the ligand, or by assaying for a biological affect on the ceU, such as inhibition or stimulation of proliferation, differentiation or migration.
  • the substance is an Eph subfamily receptor tyrosine kinase protein, which is not the native receptor tyrosine kinase protein for the Ugand, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein.
  • the part of the protein comprises an extracellular, transmembrane and juxtamembrane domain.
  • the part of the protein comprises an extraceUular domain.
  • Another aspect of the invention provides a method for assaying a medium for an agonist or antagonist of a Ugand regulatory pathway in a cell which comprises providing a cell expressing a Ugand for an Eph subfamily receptor tyrosine kinase on the surface of the cell, reacting the ceU with an Eph subfamily receptor tyrosine kinase protein or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein, and a suspected agonist or antagonist, under conditions which permit the formation of ligand-receptor tyrosine kinase protein complexes on the cell surface, and assaying for ligand-receptor tyrosine kinase protein complexes, for free receptor tyrosine kinase protein, for non-complexed proteins, for activation of the receptor tyrosine kinase protein, or for activation of the Ugand.
  • activation of the ligand is assayed by measuring phosphorylation of the Ugand or binding of SH2 domains to the ligand or by assaying for a biological affect on the cell, such as inhibition or stimulation of proliferation, differentiation or migration.
  • the invention stiU further provides a method for affecting neuronal development or regeneration in a mammal comprising administering to a mammal an effective amount of a purified and isolated Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein.
  • the protein or part of the protein is lacking in a catalytic kinase domain.
  • the part of the protein comprises an extracellular, juxtamembrane or transmembrane domain.
  • the part of the protein comprises at least one of an extracellular, juxtamembrane and transmembrane domain, preferably an extracellular domain.
  • the invention provides a method for stimulating or inhibiting axonogenesis in a mammal comprising administering to a mammal an effective amount of a purified and isolated Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein.
  • the part of the protein comprises an extraceUular domain of an Eph subfamily receptor tyrosine kinase.
  • the protein or part of the protein is lacking in a catalytic kinase domain.
  • the invention also relates to a pharmaceutical composition which comprises a purified and isolated Eph subfamily receptor tyrosine kinase protein or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein for affecting neuronal development or regeneration and a pharmaceutically acceptable carrier, diluent or excipient.
  • the part of the protein may comprise an extraceUular domain of an Eph subfamily receptor tyrosine kinase, and the protein or part of the protein may be lacking in a catalytic kinase domain.
  • Figure 1 shows the amino acid sequences of members of the Eph subfamily of receptor tyrosine kinases, dots indicate spaces introduced in order to optimize alignment, conserved cysteine residues are marked with asterisks and, arrows indicate the boundaries of the catalytic kinase domain;
  • Figure 2 shows the nucleotide sequence encoding the Nuk tyrosine kinase protein as shown in SEQ ID NO: 1;
  • Figure 3 shows the amino acid sequence of Nuk tyrosine kinase protein as shown in SEQ ID NO:2 and a schematic diagram of the regions of the Nuk receptor tyrosine kinase protein;
  • Figure 4 shows a recombinant DNA molecule having a Nuk 7 null mutation obtained by deletion of exon 2, corresponding to codons 29 to 50 as shown in SEQ ID NO: 1;
  • Figure 5 shows a recombinant DNA molecule encoding the Nuk 2 mutation in the ATP binding region of the kinase domain of Nuk protein, and a lac Z reporter gene;
  • Figure 6A is a photomicrograph showing a transverse section taken through the brain of heterozygous Nuk J /+ mice across the anterior of the frontal lobes;
  • Figure 6B is a photomicrograph showing a transverse section taken through the brain of homozygous Nuk J Wuk 1 mice across the anterior of the frontal lobes
  • Figure 6C is a photomicrograph showing a transverse section taken through the brain of homozygous Nuk 7 /Nuk 7 mice across the anterior of the frontal lobes;
  • Figure 7 A is a photomicrograph of a horizontal section taken through the brain of a Nuk 7 /+ mouse across the anterior of the frontal lobes, showing the medial tract of the anterior commissure;
  • Figure 7B is a photomicrograph of a horizontal section taken through the brain of a homozygous Nuk 7 /Nuk 7 mouse across the anterior of the frontal lobes, showing the absence of the medial tract of the anterior commissure;
  • Figure 8 shows horizontal sections taken through the brains of Nuk 7 /Nuk 7 (bottom) and Nuk 7 /+ (top) mice injected in one frontal lobe with a fluorescent dye, fast blue;
  • Figure 9 is a diagram illustrating the fast blue tracing of the temporal lobe
  • Figure 10 is a diagram illustrating the axon pathways affected in Nuk/Sek4 double homozygotes
  • Figure 11 shows an alignment of the amino acid sequences of ligands of the Eph subfamily of receptor tyrosine kinase proteins, amino acids identical in at least five out of nine proteins are shown in inverse type, the cysteine residues common to aU nine proteins are marked by asterisks;
  • Figure 12 is a diagram showing membrane anchored Ugands for Eph subfamily receptor tyrosine kinase proteins.
  • Figure 13 is a diagram showing a potential signalling role for Lerks.
  • DETAILED DESCRIPTION OF THE INVENTION As hereinbefore mentioned, the present inventors have identified and characterized a novel ligand regulatory pathway that plays a crucial role in cell-cell interactions and axonogenesis in the development and regeneration of the nervous system. The present inventors have determined that Eph subfamily receptor tyrosine kinases activate a Ugand regulatory pathway in ceUs expressing ligands for the Eph subfamily receptor tyrosine kinases.
  • Nuk Eph subfamUy receptor tyrosine kinase
  • Nuk was found to be essential for formation of at least one commissure in the brain, the medial tract of the anterior commissure. In nuU mice, lacking in Nuk expression the medial tract was found not to form. In Nuk 2 /Nuk 2 mice, expressing a fusion protein comprising the Nuk protein extracellular domain and ⁇ -galactosidase, the medial tract of the anterior commissure formed and was of a normal appearance. Therefore, the extraceUular domain of Nuk protein is required for formation of the medial tract of the anterior commissure. Nuk protein did not appear to be expressed in the medial tract of the anterior commissure, but expression was detected ventrally underlying the commissure.
  • Nuk protein Ligands of Nuk protein are thought to be expressed in the medial tract of the commissure. Nuk protein also appears to play an important role in the formation of the habenular interpeduncle tract in the brain. Complete formation of the habenular interpeduncle tract was shown to require expression of at least two members of the Eph subfamily of receptor tyrosine kinase proteins and appeared to require expression of Nuk protein having a catalytic kinase domain.
  • the invention relates to a method of activating a Ugand regulatory pathway in a ceU, comprising reacting an Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein with a cell expressing a ligand for an Eph subfamUy receptor tyrosine kinase on the surface of the ceU thereby activating the Ugand regulatory pathway in the ceU.
  • Ugand regulatory pathway used herein refers to the interactions of an Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein with a cell expressing a ligand for an Eph subfamUy receptor tyrosine kinase on the surface of the ceU thereby activating the Ugand regulatory pathway in the ceU.
  • Ugand regulatory pathway used herein refers to the interactions of an Eph sub
  • Eph subfamily receptor tyrosine kinase protein with a cell surface ligand for an Eph subfamUy receptor tyrosine kinase protein to form a Ugand receptor tyrosine kinase protein eomplex thereby activating a series of downstream regulatory pathways in the Ugand expressing cell that affect the cell, for example by controlling gene expression, ceU division, cytoskeletal architecture, cell metabolism, migration, cell-cell interactions and spatial positioning.
  • downstream regulatory pathways are the GAP/Ras pathway, the pathway that regulates the breakdown of the polyphosphoinositides through phosphoUpase C (PLC) and the Src/ tyrosine kinase and Ras pathways.
  • Eph subfamily receptor tyrosine kinase proteins refers to proteins of the Eph subfamily which are characterised as encoding a structurally related cysteine rich extracellular domain containing a single immunoglobulin (Ig)-like loop near the N-terminus and two fibronectin in (FN UI) repeats adjacent to the plasma membrane. The structure of the extracellular region is thought to determine Ugand binding specificity. The intracellular regions contain the juxtamembrane and the catalytic kinase domain. Receptor mediated signal transduction is initiated in the receptor expressing cell by ligand binding to the extracellular domain, which facilitates dimerization of the receptor and autophosphorylation.
  • Ig immunoglobulin
  • FN UI fibronectin in
  • Eph family members Over a dozen members of the Eph subfamily have been identified (van der Geer et al., 1994, Annu. Rev. Cell Biol.. 10:251-237). Examples of Eph family members include mouse Nuk and its homologs Hek5, Cek5 in chickens (Pasquale, Cell Regulation 2:523-534, 1991), Sek3 in mice, and Erk in humans; Eek (Chan and Watt, Oncogene 6:1057-1061 1991); rat Elk and its homologs including Cek6a in chickens and xEK (Lhotak et al., 1991, Mol. CeU. Biol.
  • Sek has been shown to be segmentally expressed in specific rhombomeres of the mouse hindbrain (Nieto et al, Development 116:1137-1150, 1992).
  • Other members of the family include Eck (Lindberg and Hunter, 1990, Mol. Cell Biol.
  • Eph subfamily receptor tyrosine kinases, or parts thereof, which bind to transmembrane Ugands are used in the present invention.
  • preferred Eph subfamily receptor tyrosine kinases, or parts thereof, used in the present invention include mouse Nuk and its homologs Hek5, Cek5 in chickens, and Erk; rat Elk and its homologs including Cek ⁇ a in chickens and xEK; human Hek2 and its homologs including Sek4 in mice and CeklO in chickens; and human Htk and its homologs including Mykl in mice.
  • the cartoon in Figure 3 shows the location of the various domains of Nuk protein. FoUowing a 26 amino acid hydrophobic signal peptide, the Nuk protein extraceUuar domain is composed of an Ig-like domain and two FN III repeats. The Nuk protein extracelluar domain also contains 20 cysteines whose position is conserved in the Eph fa ⁇ ly (Lhotak et al., Mol. Cell. Biol. 11:2496-2502, 1991). A hydrophobic transmembrane domain divides the Nuk protein into approximately two halves, a 548 amino acid extraceUuar region and a 419 amino acid cytoplasmic region which contains a tyrosine kinase catalytic domain.
  • Nuk is most highly related to the full length amino acid sequence of human Hek5 and also to chicken Cek5 (96% identity; Pasquale, Cell Regulation 2:523-534, 1991) and to short PCR products of mRNA from rats (Tyro 5; Lai and Lemke, Neuron 6:691-704, 1991) and humans (Erk; Chan and Watt, Oncogene 6:1057-1061 1991).
  • the close identity between Nuk and CekS suggest they represent the mammaUan and avian orthologs of the same progenitor gene.
  • the absence of fuU length cDNAs for Tyro 5 and Erk precludes the determination of whether these sequences correspond to the same or a closely related but different gene.
  • Eph subfamily receptor tyrosine kinase protein for use in activating a Ugand regulatory pathway may be an isoform or a part of the protein having at least 20 contiguous amino acids of the protein.
  • An isoform contains the same number and kinds of amino acids as the protein, but the isoform has a different molecular structure.
  • the isoforms contemplated for use in the methods of the invention are isoforms having the same functional properties as the Eph subfamily receptor tyrosine kinase proteins.
  • the part of the protein having at least 20 contiguous amino acids comprises an Eph subfamUy tyrosine kinase protein, preferably Nuk, lacking a catalytic kinase domain.
  • the part of the protein containing at least one of the extraceUular domain, the transmembrane domain and the juxtamembrane domain or parts thereof, preferably, the extraceUular domain is used in the methods herein.
  • the extracellular domain is characterised by a cysteine rich region, whose position is conserved in the extracellular domain of Eph family members an immunoglobulin-like domain near the amino terminus (Ig-Uke), and two fibronectin type IU repeats (FN III).
  • ExtraceUular domains of Eph subfamily receptor tyrosine kinase proteins may be identified based on the above-noted features and based on a comparison of the amino acid sequences of the extraceUular domains of known Eph subfamily receptor tyrosine kinase proteins.
  • the extracellular domain may be generaUy defined as the region extraceUular to the transmembrane domain, which is indicated in bold underline in Figure 1.
  • the protein may also be a protein having substantial sequence identity with the sequence of an Eph subfamily receptor tyrosine kinase protein.
  • sequence having substantial identity means those amino acid sequences having sUght or inconsequential sequence variations from the sequence of an Eph subfamily receptor tyrosine kinase protein. The variations may be attributable to local mutations or structural modifications. Suitable proteins may have over 95%, preferably over 97%, most preferably over 99% identity with an Eph subfamUy receptor tyrosine kinase protein.
  • An Eph subfamUy receptor tyrosine kinase or part thereof may be selected for use in the present invention based on the nature of the Ugand which is targeted or selected. The selection of a particular Ugand and complementary Eph subfamUy receptor tyrosine kinase in the method of the invention will aUow for the identification of specific substances that affect a Ugand regulatory pathway.
  • An Eph subfamily receptor tyrosine kinase or part thereof may be prepared from
  • Eph subfamily receptor tyrosine kinase proteins isolated from cells which are known to express the proteins.
  • the protein or part of the protein may be prepared using recombinant DNA methods known in the art.
  • nucleic acid molecules having a sequence which codes for an Eph subfamUy receptor tyrosine kinase protein, or a part of the protein may be prepared and incorporated in a known manner into an appropriate expression vector which ensures good expression of the protein or part thereof.
  • Possible expression vectors include but are not Umited to cosmids, plasmids, or modified viruses, so long as the vector is compatible with the host ceU used.
  • Suitable transcription and translation elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropriate transcription and translation elements is dependent on the host ceU chosen, and may be readUy accompUshed by one of ordinary skill in the art. Examples of such elements include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host ceU chosen and the vector employed, other genetic elements, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. It wiU also be appreciated that the necessary transcriptional and translation elements may be supplied by the native receptor tyrosine kinase protein and/ or its flanking regions.
  • the recombinant molecules may also contain a reporter gene which facilitates the selection of host ceUs transformed or transfected with a recombinant molecule.
  • reporter genes are genes encoding a protein such as ⁇ -galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG.
  • the reporter gene is lac Z . Transcription of the reporter gene is monitored by changes in the concentration of the reporter protein such as ⁇ -galactosidase, chloramphenicol acetyltransferase, or firefly luciferase.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, eiectroporation etc.
  • Methods for transforming transfecting, etc. host ceUs to express foreign DNA are weU known in the art (see, e.g., Itakura et al., U.S. Patent No. 4,704,362; Hinnen et al., PNAS USA 75:1929-1933, 1978; Murray et al., U.S. Patent No. 4,801,542; Upshall et al., U.S. Patent No. 4,935,349; Hagen et al., U.S. Patent No. 4,784,950; Axel et al., U.S. Patent No.
  • Suitable host cells include a wide variety of prokaryotic and eukaryotic host ceUs, including bacterial, mammalian, yeast or other fungi, viral, plant, or insect ceUs.
  • Eph subfamily receptor tyrosine kinase protein or parts thereof may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).
  • Conjugates of the protein, or parts thereof, with other molecules, such as proteins or polypeptides may be prepared and used in the methods described herein. This may be accompUshed, for example, by the synthesis of N-terminal or C-terminal fusion proteins.
  • fusion proteins may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of an Eph subfamily receptor tyrosine kinase protein or parts thereof, and the sequence of a selected protein or marker protein with a desired biological function.
  • the resultant fusion proteins contain Eph subfamily receptor tyrosine kinase protein or a part thereof fused to the selected protein or marker protein as described herein.
  • proteins which may be used to prepare fusion proteins include immunoglobulins and parts thereof such as the constant region of immunoglobulin ⁇ l, and lymphokines such as gamma interferon, tumor necrosis factor, IL-1, IL-2.IL-3, 11-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, GM-CSF, CSF-1 and G-CSF.
  • Sequences which encode the above-described proteins may generaUy be obtained from a variety of sources, including for example, depositories which contain plasmids encoding sequences including the American Type Culture Collection (ATCC, RockvUle Maryland), and the British Biotechnology Limited (Cowley, Oxford England).
  • Examples of such plasmids include BBG 12 (containing the GM-CSF gene coding for the mature protein of 127 amino acids), BBG 6 (which contains sequences encoding gamma interferon), ATCC No. 39656 (which contains sequences encoding TNF), ATCC No. 20663 (which contains sequences encoding alpha interferon,) ATCC Nos. 31902 and 39517 (which contains sequences encoding beta interferon), ATCC No. 67024 (which contains a sequence which encodes Interleukin-lfi), ATCC Nos. 39405, 39452, 39516, 39626 and 39673 (which contains sequences encoding Interleukin-2), ATCC Nos.
  • BBG 12 containing the GM-CSF gene coding for the mature protein of 127 amino acids
  • BBG 6 which contains sequences encoding gamma interferon
  • ATCC No. 39656 which contains sequences encoding TNF
  • Eph subfamUy receptor tyrosine kinase protein, isoforms or parts thereof, used in the method of the invention may be insolubilized.
  • the receptor protein or part thereof, preferably the extracellular domain may be bound to a suitable carrier.
  • suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, sUk, etc.
  • the carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc.
  • the insolubiUzed receptor tyrosine kinase protein may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
  • the receptor tyrosine kinase protein or parts thereof may also be expressed on the surface of a ceU using the methods described herein.
  • Ligands for Eph subfamUy receptor tyrosine kinases may be identified based on homology with known Ugands and based on their interaction with the extraceUular domain of Eph subfamUy receptor tyrosine kinases. At least seven Ugands for Eph subfamily receptor tyrosine kinases have been identified, aU of which are membrane anchored via either a GPI linkage or transmembrane domain (see Figure 12), including B61 (Holzmann et al., 1990, Mol. Cell Biol.
  • ELF-1 Chozlosky et al., 1995 Oncogne 10:299-306
  • LERK-4 Kozlosky et al, 1994, supra
  • ELF-1 AL-1 /RAGS (GPI- anchored, Drescher, et al, 1995, Cell, 82:359-370)
  • LERK-4 HTKL/ELF-2/Lerk5, LERK- 2/CEK5-L/ELK-L (Tessier-Lavigne, M., 1995, supra).
  • Ligands of Eph subfamily receptor tyrosine kinases show significant homology with each other.
  • An aUgnment of the amino acid sequences of ligands of Eph subfamily receptor tyrosine kinases are shown in Figure 11 (excerpted from Drescher, et al., 1995, supra.
  • Ligands for the Eph subfamily receptor tyrosine kinases are known to show promiscuous interactions with different Eph subfamily receptors (BrambUla et al., 1995, EMBO J. 14:3116-3126).
  • the Ugands are ligands which are membrane anchored via a transmembrane domain.
  • the selected ligands are Elk- L/LERK2/Efl-3/Cek5-L; hHtk-L/ELF-2/LERK5 (Tessier-Lavigne, M., 1995, CeU 82:345-348), and hElk-L3/EU-6. These ligands have highly conserved cytoplasmic reions with multiple potential sites for phosphorylation.
  • the amino acid sequences for hElk-L3, hHtk-L and hElk-L, and the extraceUular domains of the ligands can be found in GenBank (e.g. Accession Nos. L38734 (Htk) and L37361 (Efl-3)).
  • the Ugand should be expressed on the surface of the cell.
  • the ceU is one which expresses native ligand.
  • the invention also contemplates chimeric ceUs expressing a recombinant Ugand.
  • the invention also provides a method for identifying a substance which is capable of bmding to a Ugand for an Eph subfamUy receptor tyrosine kinase and activating a ligand regulatory pathway in a cell, comprising reacting a cell expressing a Ugand for an Eph subfamUy receptor tyrosine kinase with at least one substance which potentially can bind with the Ugand, under conditions which permit the formation of substance-Ugand complexes, and assaying for substance-Ugand complexes, for free substance, for non-complexed Ugands, or for activation of the ligand.
  • Activation of the ligand may be assayed by measuring phosphorylation of the Ugand, binding of SH2 domains to the Ugand, and where the Ugand is expressed on a ceU surface, by assaying for a biological affect on the ceU, such as inhibition or stimulation of proUferation, differentiation or migration.
  • SH2-domains of cytoplasmic signalling proteins are known to bind to phosphorylated receptor tyrosine kinase proteins.
  • the SH2 domains of p21 ras GTPase-activating protein (GAP), Src, and phosphoinositide-specific phosphoUpase C (PLC ⁇ ) may bind an Eph subfamily receptor tyrosine kinase protein.
  • SH2 domains of cytoplasmic signalling proteins may bind to phosphorylated Ugands to mediate the interactions of the phophorylated Ugand with signalling proteins of the downstream regulatory pathways in the ceU.
  • a signal transduction event in the Ugand expressing cell may be initiated. This could occur by activation of one or more cytoplasmic tyrosine kinases which would phosphorylate the intracellular domain of the Ugand, which would then lead to the binding of SH2 domain-containing proteins to the phosphorylated activated ligand.
  • a diagram of a potential signalling role for Lerks is shown in Figure 13.
  • the substance is an Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein.
  • the part of the protein comprises an extraceUular domain.
  • the substances is an Eph subfamUy receptor tyrosine kinase which is not the native receptor tyrosine kinase for the Ugand.
  • Conditions which permit the formation of substance-Ugand complexes may be selected having regard to factors such as the nature and amounts of the substance and the ligand.
  • the substance-Ugand complex, free substance or non-complexed Ugand may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel fUtration, fractionation,absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof.
  • antibody against the Ugand or the substance, or a labelled Ugand, or a labelled substance may be utilized.
  • Antibodies, receptor protein or substance may be labelled with a detectable substance as described above.
  • the substance used in the method of the invention may be insolubilized.
  • the receptor protein or substance may be bound to a suitable carrier.
  • suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl ceUulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
  • the carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc.
  • the insolubUized substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
  • the substance may also be expressed on the surface of a cell using the methods described herein. Where the substance is expressed on the surface of a cell the presence of a substance which can bind to and be activated by the receptor tyrosine kinase protein may be identified by assaying for activation of the substance or by assaying for a biological affect on the cell.
  • the above mentioned methods of the invention may be used to identify substances which bind with Ugands of the Eph subfamily of receptor tyrosine kinase proteins, thereby activating a ligand regulatory pathway in a cell, particularly those involved in neuronal development, axonal migration, pathfinding and regeneration. Identification and isolation of such substances will permit studies of the role of the substance in the developmental regulation of axonogenesis and neural regeneration, and permit the development of substances which affect these roles, such as functional or non-functional analogues of the extraceUular domain of an Eph subfamily receptor tyrosine kinase. It will be appreciated that such substances wiU be useful as pharmaceuticals to modulate axonogenesis, nerve ceU interactions and regeneration to treat conditions such as neurodegenerative diseases and cases of nerve injury.
  • Substances which bind to and activate the Ugand may be identified by assaying for protein tyrosine kinase activity i.e. by assaying for phosphorylation of the tyrosine residues of the Ugand, using known techniques such as those using anti-phosphotyrosine antibodies and labeUed phosphorous. For example, immunoblots of the complexes may be analyzed by autoradiography ( 32 P-labeUed samples) or may be blocked and probed with antiphosphotyrosine antibodies as described in Koch, CA. et al., 1989 (Mol. CeU. Biol. 9, 4131-4140).
  • Substances which bind to and activate the ligand may also be assayed by assaying for a biological affect on the ceU, for example inhibition or stimulation of cell proliferation, differentiation and migration.
  • Substances which bind to and activate the ligand will include Eph subfamily receptor tyrosine kinase proteins and portions of the proteins.
  • the method wiU permit identification of the minimum amino acid sequence of the protein which is required for Ugand binding and activation.
  • the invention further relates to a method for assaying a medium for an agonist or antagonist of a ligand regulatory pathway in a cell which comprises providing a cell expressing a Ugand for an Eph subfamUy receptor tyrosine kinase on the ceU surface, reacting the ceU with an Eph subfamUy receptor tyrosine kinase protein or part of a protein and a suspected agonist or antagonist under conditions which permit the formation of ligand- receptor tyrosine kinase protein complexes on the cell surface, and assaying for ligand-receptor tyrosine kinase protein complexes, for free receptor tyrosine kinase protein, for non-complexed proteins, for activation of the receptor tyrosine kinase protein, or for activation of the ligand.
  • Substances which activate the Ugand regulatory pathway such as Eph subfamily receptor tyrosine kinase proteins or parts thereof, and agonists or antagonists of the Ugand regulatory pathway may be used for affecting neuronal development or regeneration in a mammal.
  • the substances, agonists and antagonists may be used to stimulate or inhibit neuronal development, regeneration and axonal migration associated with neurodegenerative conditions and conditions involving trauma and injury to the nervous system, for example Alzheimer's disease, Parkinson's disease, Huntington's disease, demylinating diseases, such as multiple sclerosis, amyotrophic lateral sclerosis, bacterial and viral infections of the nervous system, deficiency diseases, such as Wernicke's disease and nutritional polyneuropathy, progressive supranuclear palsy, Shy Drager's syndrome, multistem degeneration and olivo ponto cerebellar atrophy, peripheral nerve damage, trauma and ischemia resulting from stroke.
  • demylinating diseases such as multiple sclerosis, amyotrophic lateral sclerosis, bacterial and viral infections of the nervous system
  • deficiency diseases such as Wernicke's disease and nutritional polyneuropathy, progressive supranuclear palsy, Shy Drager's syndrome, multistem degeneration and olivo ponto cere
  • the abiUty of substances, agonists, and antagonists identified using the methods of the invention to affect neuronal development or regeneration and to stimulate nerve regeneration may be confirmed in an animal model having an injured peripheral nervous system.
  • mammals having an injured peripheral nervous system include animals having damaged axons, such as axotomized facial neurons (Sendtner et al. Nature, 345, 440-441, 1990), neurodegenerative conditions (for example, the MPTP model as described in Langston J.W. et al, Symposium of Current Concepts and Controversies in Parkinson's Disease, Montebello, Quebec, Canada, 1983 and Tatton W.G. et al., Can. J. Neurol. Sci. 1992, 19), and traumatic and non-traumatic peripheral nerve damage (for example, animal stroke models such as the one described in MacMUlan et al. Brain Research 151:353-368 (1978)).
  • the present invention thus provides a method for affecting neuronal development or regeneration in a mammal comprising administering to a mammal an effective amount of a purified and isolated Eph subfamUy receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein, or a substance identified using the methods of the invention.
  • the invention also contemplates a method for stimulating or inhibiting axonogenesis in a mammal comprising administering to a mammal an effective amount of a purified and isolated Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein, or a substance identified using the methods of the invention.
  • the invention still further relates to a pharmaceutical composition which comprises a purified and isolated Eph subfamily receptor tyrosine kinase protein or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein, or a substance identified using the methods of the invention, for affecting neuronal development or regeneration and a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical compositions may be used to stimulate or inhibit neuronal development, regeneration and axonal migration associated with neurodegenerative conditions and conditions involving trauma and injury to the nervous system as described above.
  • compositions of the invention are administered to subjects in a biologicaUy compatible form suitable for pharmaceutical administration in vivo.
  • biologically compatible form suitable for administration in vivo is meant a form of the protein to be administered in which any toxic effects are outweighed by the therapeutic effects of the protein.
  • subject is intended to include mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • Administration of a therapeuticaUy active amount of the pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • a therapeutically active amount of an Eph subfamily receptor tyrosine kinase protein may vary according to factors such as the condition, age, sex, and weight of the individual.
  • Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daUy or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the active compound e.g., protein
  • the active compound may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration inhalation, transdermal appUcation or rectal administration.
  • the active compound may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
  • the pharmaceutical compositions of the invention can be for oral, local, inhalant or intracerebral administration.
  • the pharmaceutical compositions of the invention are administered directly to the peripheral or central nervous system, for example by administration intracerebraUy.
  • the pharmaceutical composition of the invention can be administered to a subject in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier such as microporous or solid beads or liposomes.
  • pharmaceutically acceptable carrier as used herein is intended to include dUuents such as saline and aqueous buffer solutions. Liposomes include water-in-oil-in- water emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol 7:27).
  • the active compound may also be administered parenterally or intraperitoneaUy.
  • Dispersions can also be prepared in glycerol, Uquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • Pharmaceutical compositions suitable for injectable use include sterUe aqueous solutions (where water soluble) or dispersions and sterUe powders for the extemporaneous preparation of sterUe injectable solutions or dispersions. In aU cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the pharmaceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the Uke), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, asorbic acid, thimerosal, and the Uke.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • SterUe injectable solutions can be prepared by inco ⁇ orating active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, foUowed by filtered steriUzation.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (e.g., antibody) plus any additional desired ingredient from a previously sterile-fUtered solution thereof.
  • the composition may be orally administered, for example, with an inert dUuent or an assimUable edible carrier.
  • pharmaceutically acceptable carrier includes any and aU solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • the pharmaceutical compositions may be administered locally to stimulate axonogenesis and pathfinding in areas of the body in need thereof, for example in areas of local nerve injury or in areas where normal nerve pathway development has not occurred. It is also contemplated that the pharmaceutical compositions may be placed in a specific orientation or aUgnment along a presumptive pathway to stimulate axon pathfinding along that line, for example the pharmaceutical compositions may be present on microcarriers laid down along the pathway. In an embodiment, the pharmaceutical compositions may be used to stimulate formation of connections between areas of the brain, such as the area between the two hemispheres or between the thalamus and ventral midbrain. In an embodiment, the compositions may be used to stimulate formation of the medial tract of the anterior commissure or the habenular interpeduncle.
  • compositions of the invention may comprise ceUs or viruses, preferably retroviral vectors, transformed with nucleic acid molecules encoding a purified and isolated Eph subfamUy receptor tyrosine kinase protein, or an isoform or a part of the protein, or a substance identified using the methods of the invention, such that they express the protein, isoform, or a part of the protein, preferably the extracellular domain, or substance in vivo.
  • Viral vectors suitable for use in the present invention are weU known in the art including recombinant vaccinia viral vectors (U.S. Patent Nos.
  • compositions containing cells or viruses may be directly introduced into a subject as described herein.
  • Nucleic acid molecules encoding a purified and isolated Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein, or a substance identified using the methods of the invention may also be introduced into a subject using physical techniques such as microinjection and eiectroporation or chemical methods such as coprecipitation and incorporation of nucleic acids into liposomes. They may also be delivered in the form of an aerosol or by lavage.
  • the foUowing non-limiting examples are iUustrative of the present invention: EXAMPLES The foUowing materials and methods were utilized in the investigations outlined in the examples:
  • Nuk 7 A loss of function mutation in Nuk, designated Nuk 7 was generated in embryonic stem cells, and germline transmission of the null allele was obtained as described in co ⁇ pending International AppUcation PCT CA95/00254 and co-pending appUcation serial No. 08/235,407.
  • nuU mutation was obtained by deletion of exon 2, corresponding to codons 29 to 50, as shown in Figure 4.
  • Nuk+/- embryonic stem ceU Unes ES
  • 8 ceU embryos in vitro 8 ceU embryos in vitro
  • the resulting blastocysts were transferred into recipient females.
  • animals chimeric for ES and embryonic stem ceUs were recovered by scoring for eye pigment and coat colour. Breeding of these "aggregation chimeras" confirmed that the germ line of at least one founder mouse is derived completely from the ES ceUs.
  • Adult mice homozygous for the mutation did not express Nuk protein.
  • a targeted mutation, designated Nuk 2 was generated in the Nuk gene as described in co-pending International AppUcation PCT CA95/00254 and co-pending application serial No. 08/235,407 and shown in Figure 5.
  • a pPNT-LOX-Nuk 2 gene trap vector was used to delete the GXGXXG ATP binding region of the kinase domain (amino acids 623-707,) to create a Nuk-lac Z fusion receptor in ES ceUs. Chimeric animals were prepared as described above, by aggregating the ES ceUs with 8 ceU CDI embryos.
  • Nuk 2 mutation Animals generated with the Nuk 2 mutation provided Nuk expressing cells staining for ⁇ -galactosidase activity, providing a convenient marker for Nuk-positive ceUs in both heterozygous and homozygous backgrounds.
  • the Nuk 2 mutation led to the expression of a Nuk-beta galactosidase fusion protein in mouse heterozygous embryos, detected by a blue/green colour .
  • Nuk protein The role of Nuk protein, the extracellular domain of Nuk protein and the catalytic kinase domain of Nuk protein were investigated as foUows.
  • Loss of function Nuk mutant mice designated Nuk 7 were prepared as described herein. These mice may also be referred to as null mice as they do not express Nuk protein.
  • Nuk-lac Z fusion chimeric receptor mutant mice designated Nuk 2 were prepared as described above. These mice express a fusion protein having the entire extraceUular domain of Nuk, but lacking in the Nuk catalytic kinase domain, which is replaced by ⁇ -galactosidase. All mice, exhibited apparently normal appearance and behaviour.
  • Nuk,-Lac Z expression was detected in the mid line of the corpus caUosum.
  • the habenular interpeduncle tract which connects the thalamus to the ventral midbrain was defective in Nuk 2 /Sek4 and Nuk 1 / Sek4.
  • Careful analysis of Nuk protein using anti-Nuk antibodies and lac Z staining of Nuk 2 /Nuk 2 embryos showed that, during development, Nuk expression appears in the ventral midbrain and progresses towards the thalamus and axon migration occurred in the opposite direction, i.e. from the thalamus toward the ventral mid brain. This axon migration was dependent on the expression of Nuk protein having a catalytic kinase domain.
  • ORGANISM Mus musculus
  • CAAGCAGCAC CATCGGCCGT GTCCATCATG CACCAGGTGA GCCGCACTGT GGACAGCATC 1380
  • GTCACTGTGC AGGGCCTCAA AGCCGGCGCC ATCTATGTCT TCCAGGTGCG GGCACGCACC 1560
  • GTCTTCCTCA TCGCTGTGGT CGTCATTGCC ATCGTATGTA ACAGACGGGG GTTTGAGCGT 1740
  • MOLECULE TYPE protein
  • ORIGINAL SOURCE (A) ORGANISM: Mus musculus
  • Lys Val Asp Thr lie Ala Ala Asp Glu Ser Phe Ser Gin Val Asp Leu 145 150 155 160

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Public Health (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Zoology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention porte sur une nouvelle voie de régulation de ligand ainsi que sur des procédés d'activation de cette nouvelle voie dans une cellule exprimant un ligand pour une protéine tyrosine kinase réceptrice de la sous-famille Eph. Elle concerne également des procédés permettant d'identifier des substances capables d'activer la voie de régulation du ligand. Elle a, en outre, trait à des techniques thérapeutiques visant à influer sur le développement et la régénération des neurones, ainsi qu'à des compositions pharmaceutiques utilisant ces substances et des protéines tyrosine kinase réceptrices de la sous-famille Eph.
PCT/CA1996/000679 1995-10-13 1996-10-10 Procede d'activation d'une nouvelle voie de regulation de ligand WO1997014966A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA 2234090 CA2234090A1 (fr) 1995-10-13 1996-10-10 Procede d'activation d'une nouvelle voie de regulation de ligand
JP9515370A JPH11515105A (ja) 1995-10-13 1996-10-10 新規リガンド調節経路の活性化方法
EP96932414A EP1019728A1 (fr) 1995-10-13 1996-10-10 Procede d'activation d'une nouvelle voie de regulation de ligand

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US551895P 1995-10-13 1995-10-13
US60/005,518 1995-10-13

Publications (2)

Publication Number Publication Date
WO1997014966A1 true WO1997014966A1 (fr) 1997-04-24
WO1997014966B1 WO1997014966B1 (fr) 1997-06-05

Family

ID=21716280

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA1996/000679 WO1997014966A1 (fr) 1995-10-13 1996-10-10 Procede d'activation d'une nouvelle voie de regulation de ligand

Country Status (3)

Country Link
EP (1) EP1019728A1 (fr)
JP (1) JPH11515105A (fr)
WO (1) WO1997014966A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998001548A1 (fr) * 1996-07-05 1998-01-15 Mount Sinai Hospital Corporation Recepteurs oligomerises modulant des voies regulees par des ligands transmembranaires pour des recepteurs tyrosine-kinases type elk
WO2000024413A1 (fr) * 1998-10-27 2000-05-04 The Walter And Eliza Hall Institute Of Medical Research Procede de traitement
WO2000037500A1 (fr) * 1998-12-18 2000-06-29 Mount Sinai Hospital Structure tridimensionnelle d'un domaine de motif alpha sterile
WO2002000939A2 (fr) * 2000-06-28 2002-01-03 Diadexus, Inc. Procede de diagnostic, de surveillance, de determination du stade, d'imagerie et de traitement du cancer du colon
US6514497B1 (en) 1997-10-02 2003-02-04 Millennium Pharmaceuticals, Inc. Inhibition of LERK-2-mediated cell adhesion
AU2004201391B2 (en) * 1998-10-27 2007-08-23 The Council Of The Queensland Institute Of Medical Research A method of the treatment
WO2024066489A1 (fr) * 2022-09-30 2024-04-04 苏州雅深智慧科技有限公司 Procédé de configuration pour base de données de recherche et de développement de médicament, et système

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993000425A1 (fr) * 1991-06-21 1993-01-07 Amrad Corporation Limited Nouvelle tyrosine kinase type recepteur et son utilisation
WO1995014776A1 (fr) * 1993-11-23 1995-06-01 Genentech, Inc. TYROSINE KINASES PROTEIQUES APPELEES Rse
WO1995027060A2 (fr) * 1994-04-04 1995-10-12 Regeneron Pharma Ligands de la famille des eph a activite biologique
WO1995028484A1 (fr) * 1994-04-15 1995-10-26 Amgen Inc. Nouvelles proteines tyrosine kinases receptrices analogues a l'eph, du type hek5, hek7, hek8, hek11
WO1995030326A1 (fr) * 1994-04-29 1995-11-09 Mount Sinai Hospital Corporation Tyrosine-kinase receptrice neuronale
WO1996026958A2 (fr) * 1995-02-27 1996-09-06 President And Fellows Of Harvard College L'elf-2, ligand du recepteur eph

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993000425A1 (fr) * 1991-06-21 1993-01-07 Amrad Corporation Limited Nouvelle tyrosine kinase type recepteur et son utilisation
WO1995014776A1 (fr) * 1993-11-23 1995-06-01 Genentech, Inc. TYROSINE KINASES PROTEIQUES APPELEES Rse
WO1995027060A2 (fr) * 1994-04-04 1995-10-12 Regeneron Pharma Ligands de la famille des eph a activite biologique
WO1995028484A1 (fr) * 1994-04-15 1995-10-26 Amgen Inc. Nouvelles proteines tyrosine kinases receptrices analogues a l'eph, du type hek5, hek7, hek8, hek11
WO1995030326A1 (fr) * 1994-04-29 1995-11-09 Mount Sinai Hospital Corporation Tyrosine-kinase receptrice neuronale
WO1996026958A2 (fr) * 1995-02-27 1996-09-06 President And Fellows Of Harvard College L'elf-2, ligand du recepteur eph

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
TOYOSHIMA, HIDEO ET AL: "Differently spliced cDNAs of human leukocyte tyrosine kinase receptor tyrosine kinase predict receptor proteins with and without a tyrosine kinase domain and a soluble receptor protein", PROC. NATL. ACAD. SCI. U. S. A. (1993), 90(12), 5404-8 CODEN: PNASA6;ISSN: 0027-8424, 1993, XP002024806 *
VETTER, MONICA L. ET AL: ".beta. PDGF receptor mutants defective for mitogenesis promote neurite outgrowth in PC12 cells", CURR. BIOL. (1995), 5(2), 168-78 CODEN: CUBLE2;ISSN: 0960-9822, 1995, XP002024805 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998001548A1 (fr) * 1996-07-05 1998-01-15 Mount Sinai Hospital Corporation Recepteurs oligomerises modulant des voies regulees par des ligands transmembranaires pour des recepteurs tyrosine-kinases type elk
US6413730B1 (en) 1996-07-05 2002-07-02 Mount Sinai Hospital Corporation Method for identifying compounds that inhibit or enhance activation of a transmembrane ligand for a receptor tyrosine kinase
US6514497B1 (en) 1997-10-02 2003-02-04 Millennium Pharmaceuticals, Inc. Inhibition of LERK-2-mediated cell adhesion
WO2000024413A1 (fr) * 1998-10-27 2000-05-04 The Walter And Eliza Hall Institute Of Medical Research Procede de traitement
AU2004201391B2 (en) * 1998-10-27 2007-08-23 The Council Of The Queensland Institute Of Medical Research A method of the treatment
US7897570B2 (en) 1998-10-27 2011-03-01 The Walter And Eliza Hall Institute Of Medical Research Method of treatment
WO2000037500A1 (fr) * 1998-12-18 2000-06-29 Mount Sinai Hospital Structure tridimensionnelle d'un domaine de motif alpha sterile
WO2002000939A2 (fr) * 2000-06-28 2002-01-03 Diadexus, Inc. Procede de diagnostic, de surveillance, de determination du stade, d'imagerie et de traitement du cancer du colon
WO2002000939A3 (fr) * 2000-06-28 2003-12-11 Diadexus Inc Procede de diagnostic, de surveillance, de determination du stade, d'imagerie et de traitement du cancer du colon
WO2024066489A1 (fr) * 2022-09-30 2024-04-04 苏州雅深智慧科技有限公司 Procédé de configuration pour base de données de recherche et de développement de médicament, et système

Also Published As

Publication number Publication date
JPH11515105A (ja) 1999-12-21
EP1019728A1 (fr) 2000-07-19
MX9802883A (es) 1998-11-29

Similar Documents

Publication Publication Date Title
Biggs 3rd et al. Primary structure, expression, and signal-dependent tyrosine phosphorylation of a Drosophila homolog of extracellular signal-regulated kinase.
Leonard et al. Jaks and STATs: biological implications
Flanagan et al. Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the SId mutant
Meyer et al. Multiple essential functions of neuregulin in development
US6451997B1 (en) Kits for detecting chromosomal rearrangements
DE69736807T2 (de) Lipid kinase
KR100453499B1 (ko) 신경영양성 인자 ｎｎｔ-1
US6413730B1 (en) Method for identifying compounds that inhibit or enhance activation of a transmembrane ligand for a receptor tyrosine kinase
KR20180019521A (ko) 인간화된 sirpa-il15 녹인 마우스 및 이의 이용 방법
Benjamin The hr-t gene of polyoma virus
AU710856B2 (en) The irs family of genes
US6696548B2 (en) Antibodies for recognition of alk protein tyrosine/kinase receptor
WO1997014966A1 (fr) Procede d'activation d'une nouvelle voie de regulation de ligand
US7205113B2 (en) Methods of modulation of the immune system
US6107472A (en) Receptor-type tyrosine kinase-like molecules
US6218356B1 (en) Neural receptor tyrosine kinase
DE69733414T2 (de) Protein tyrosin-phosphatasen von hematopoietischen zellen
CA2234090A1 (fr) Procede d'activation d'une nouvelle voie de regulation de ligand
MXPA98002883A (en) Method to activate a regulatory route of noved ligands
US20040234540A1 (en) Diagnosis and treatment of ALK-7 related disorders
Garbers The regulation of spermatozoon function by the egg
CA2039540A1 (fr) Recepteur beta-adrenergique modifie
AU664752B2 (en) Receptor-type tyrosine kinase-like molecules
White Molecular mechanisms of signal transduction by tyrosine kinase receptors
Fantin Molecular, cellular, and physiological characterization of the insulin receptor substrate 4

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP MX

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2234090

Country of ref document: CA

Kind code of ref document: A

Ref document number: 2234090

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 1997 515370

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: PA/a/1998/002883

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 1996932414

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1996932414

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1996932414

Country of ref document: EP