+

WO1996011664A2 - Nskz 2 recepteur tyrosine-kinase musculaire - Google Patents

Nskz 2 recepteur tyrosine-kinase musculaire Download PDF

Info

Publication number
WO1996011664A2
WO1996011664A2 PCT/US1995/013490 US9513490W WO9611664A2 WO 1996011664 A2 WO1996011664 A2 WO 1996011664A2 US 9513490 W US9513490 W US 9513490W WO 9611664 A2 WO9611664 A2 WO 9611664A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
nsk2
amino acids
sequence
tyrosine kinase
Prior art date
Application number
PCT/US1995/013490
Other languages
English (en)
Other versions
WO1996011664A3 (fr
Inventor
Alastair Reith
Original Assignee
Ludwig Institute For Cancer Research
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
Priority claimed from GB9420389A external-priority patent/GB9420389D0/en
Priority claimed from GBGB9510574.8A external-priority patent/GB9510574D0/en
Application filed by Ludwig Institute For Cancer Research filed Critical Ludwig Institute For Cancer Research
Priority to JP51346396A priority Critical patent/JP2002517166A/ja
Priority to EP95943567A priority patent/EP0800531A4/fr
Priority to AU45006/96A priority patent/AU4500696A/en
Publication of WO1996011664A2 publication Critical patent/WO1996011664A2/fr
Publication of WO1996011664A3 publication Critical patent/WO1996011664A3/fr

Links

Classifications

    • 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
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel muscle protein with tyrosine kinase activity, DN encoding the protein and its uses.
  • vertebrate skeletal muscles involves many processes fundamental to ou understanding of developmental biology. This includes cell-cell signalling, differentiatio and morphogenesis. Individual skeletal muscles have distinctive muscle fibre composition that vary in energy metabolism, contraction rate and fatigue resistance. The variation i thought to reflect different populations of precursor myoblasts. During development o muscles waves of overlapping myoblast types can be observed.
  • Myoblasts undergo terminal differentiation to form myotubes by a process involvin cellular fusion.
  • Myoblasts can be cultivated in vitro in the presence of growth factors suc as fibroblast growth factor (FGF). Withdrawal of such factors or the absence of serum forces cells out of the cell cycle, resulting in terminal differentiation.
  • FGF fibroblast growth factor
  • During differentiation a distinctive set of muscle specific proteins are expressed.
  • RTKs receptor tyrosine kinases
  • Wilks et al (Proc. Natl. Acad. Sci. (1989) __ ⁇ ; 1603-1607) describe a general method t clone protein tyrosine kinase genes using degenerate oligonucleotide primers. Using modification of this approach, a gene which contains a tyrosine kinase domain has bee identified.
  • the gene which was initially called Msn-2, has homology with a recepto tyrosine kinase cloned from the electric organ of the electric ray, Torpedo calif ornic (Jennings et al, Proc. Natl. Acad. Sci. (1993) ___; 2895-2899).
  • the gene is now referred to herein as Nsk2.
  • the present invention provides a novel muscle RTK. It has also been surprisingly found that the Nsk2 gene is differentially spliced to produce at least eleven distinct gene products. In a further aspect of the invention, there is provided a related RTK which is called Nskl. The partial nucleotide sequence and translation product thereof is shown in Figure 9.
  • the present invention provides an isolated nucleic acid molecule, the complement of which is capable of selectively hybridizing to one of the nucleotide sequences of Figure 1 , 3b, 4a, 5a, 6a or 9 and fragments thereof capable of selectively hybridising to said sequences or their complement.
  • the nucleotide sequences are those of Figures 1, 4a, 5a, 6a or 9 or fragments thereof capable of selectively hybridizing to said sequences.
  • the invention also provides nucleic acid molecules in substantially isolated form, which are mammalian homologues of the Figure la, 3b, 4a, 5a, 6a or 9 sequences, including the human homologues, and fragments thereof capable of selectively hybridising to said homologues.
  • the invention further provides isolated nucleic acid molecules coding for the full codin sequence of Nskl, optionally with its 5' and/or 3' untranslated regions, as well a fragments of these nucleic acid molecules. These nucleic acid molecules may be obtaine by routine methodologies in the art, starting with probes based upon the sequence disclosed in Figure 9.
  • Such methods include making a cDNA library from cells whic express Nskl, and probing said library with a nucleic acid comprising all or part of th sequence of Figure 9.
  • the library may be made with a primer derived from part of the Figure 9 sequence to enrich the library for suitable clones. Details of methods for making cDNA libraries and the like may be found in standard reference books, e.g. Sambrook et al, 1987.
  • the invention further provides an isolated protein having one of the sequences set out in Figures 2, 3, 4b, 4c, 5b, 6b or 9.
  • the protein described in figure 2 (SEQ ID NO:2), may be presented as an additional variant, wherein the 20 amino acids of figure 3b are inserted between residues 209 and 210 of figure 2.
  • the invention also provides fragments of said proteins which encode at least one antigenic determinant.
  • said antigenic determinant is specific for Nsk2 or Nskl.
  • proteins which are mammalian homologues, preferably human homologues, of the proteins of Figures 2, 3, 4b, 4c, 5b, 6b or 9 and fragments thereof encoding an antigenic determinant specific for the Nsk2 or Nsk 1 homologue.
  • the invention also provides polypeptides and fragments thereof encoded by the entire Nskl gene.
  • the sequences of such polypeptides may be determined by translating the coding sequence of the gene, which may be determined as indicated above.
  • the invention also provides an antibody or fragment thereof capable of binding the kinase or fragments thereof, of the invention.
  • the antibody may be polyclonal or monoclonal.
  • nucleotide sequences encoding polypeptides (includin those of Figures 2, a modification of figure 2 to include the 60 nucleotides of figure 3b, 3a, 4b, 4c, 5b, 6b or 9) and fragments thereof of the invention, and vectors containing sai nucleotide sequences.
  • the vector is an expression vector which contains promoter compatible with a host cell operably linked to said nucleotide sequence.
  • the nucleotide sequence is a sequence of Figures 1 , 3b, 4a, 5a, 6a or 9 or a fragment or homologue thereof.
  • the invention provides a method of preparing a polypeptide or fragment thereof according to the invention which comprises culturing a host cell carrying a vector according to the invention under conditions suitable for expression of the polypeptide or fragment thereof, and recovering said polypeptide or fragment thereof from the culture.
  • Figure 1 shows the complete coding nucleotide and predicted amino acid sequence of the Nsk2 receptor tyrosine kinase (SEQ ID NO: l).
  • Figure 2 shows the full length Nsk2 receptor tyrosine kinase together with its various domains (SEQ ID NO:2).
  • Figure 3a shows the alternately spliced Nsk2 receptor tyrosine kinase isoform bearing a replacement within the extracellular domain (SEQ ID NO: 3).
  • Figure 3b presents a further, alternately spliced variant of the molecules of the inventio The splicing is within the extracellular domain.
  • a sequence of nucleotides provides a novel string of 20 amino acids is spliced in between nucleotides 6 and 674 of the sequence of SEQ ID NO: l. Overlining indicates sites of potenti glycosylation (SEQ ID NO: 4).
  • Figure 4a shows the nucleotide sequence and predicted amino acid sequence of a alternately spliced carboxy terminal domain of the Nsk2 receptor tyrosine kinase (SEQ I NO:5).
  • Figure 4b sets out the amino acid sequence of the full length Nsk2 RTK isoform bearin the alternately spliced carboxy terminus of Figure 4a (SEQ ID NO: 6).
  • Figure 4c sets out the amino acid sequence of the Nsk2 RTK isoform bearing both th replacement in the extracellular domain of Figure 3 and the alternately spliced carbox terminus of Figure 4a (SEQ ID NO:7).
  • Figure 5a shows the partial nucleotide and predicted amino acid sequence of an alternatel spliced truncated Nsk2 cDNA encoding a putative soluble extracellular domain (SEQ I NO:8).
  • Figure 5b sets out the amino acid sequence of the truncated Nsk2 isoform of Figure 5a and indicates the various domains and novel C-terminus (SEQ ID NO:9).
  • Figure 6a shows the partial nucleotide and predicted amino acid sequence of a further alternately spliced truncated Nsk2 cDNA (SEQ ID NO: 10).
  • Figure 6b shows the amino acid sequence of the trun-cated Nsk2 isoform of Figure 6a a indicates the various domains and novel C-terminus (SEQ ID NO: 11).
  • Figure 7 provides a summary of the differential splicing which occurs to produce isofor of Nsk2.
  • Figure 8 provides a comparison of the amino acid sequences of Nsk2 and the Torpedo RT of Jennings et al (ibid) (SEQ ID NO: 12).
  • Figure 9 shows the partial nucleotide and amino acid sequence of Nskl (SEQ ID NO: 13
  • Figure 11 shows results obtained when levels of expression of the 23kDa 2Ig variant, and the 52kDa 4Ig variant in myoblasts, were measured.
  • Figure 12 shows the results obtained when full length, murine Nsk cDNA was expressed in an in vitro system.
  • Figure 13 is a summary of 11 possible isomeric variants of the Nsk 2 molecule described herein.
  • the nucleotide sequences of the invention are preferably DNA sequences although they may also be RNA.
  • fragments of said nucleotide sequences which are small enough to be produced synthetically may contain modifications which are suitable to (1) achieve resistance to degradation by DNases, (2) enhance the potency of the molecule and (3) to enhance uptake of the nucleotides by cells.
  • DNases a DNA sequence that is produced synthetically.
  • a number of different types of modification to nucleotides a known in the .art. These include methylphosphonate and phosphorothioate backbon addition of acridine or polylysine ch.ains at the 3' and/or 5' ends of the molecule.
  • the nucleotides describ herein may be modified by any method available in the art in order to enhance their in vi activity or lifespan.
  • antisense nucleoti fragments can be used to inhibit the transcription or translati of the Nsk2 or Nskl gene in vitro or in vivo. This use has applications for example studying myogenesis under conditions where the expression of the Nsk2 or Nskl ge product is selectively inhibited by introducing an effective amount of an antisen nucleotide fragment into a cell which expresses Nsk2.
  • Nucleotide fragments of the present invention will typically be from 10 to 2000 bases i length, eg. from 10 to 1,000, eg. 15-500, for example 16, 17, 18, 20, 25, 50, 100 or 20 nucleotides in length.
  • the nucleotide sequence of the invention may be single stranded or double stranded.
  • Preferred fragments of the invention include those encoding the following amino aci regions of the sequence of SEQ ID NO:2: aal-21, aa22-496 (including aa49-98, aal42-190 aa233-282 and aa401-450), aa497-517, aa518-871, aa518-576, aa577-858, aa674-693 an aa859-871.
  • a nucleotide sequence or fragment thereof is capable of selectively hybridising to the Nsk or Nskl sequence or its complements where, under high stringency conditions, t sequence or fragment thereof does not hybridise to genes normally found in associatio with Nsk2 or Nskl.
  • Stringent conditions will vary according to the size of the fragmen
  • high stringent conditions will typically b about 60°C at 0.2 X SSC, preferably 60°C at 0.1 x SSC, more preferably 65°C at 0.1 SSC.
  • SSC is defined as 0.15 M sodium chloride and 0.15 M sodium citrate at p 7.5.
  • nucleotide sequence or fragment thereof capable of selectively hybridising t the sequence of Figures 1, 3b, 4a, 5a, 6a or 9 will be at least 80 or 90% and mor preferably at least 95% homologous to the sequence of Seq. ID No. 1 over a region of a least 20, preferably at least 30, for instance 40, 60 or 100 or more contiguous nucleotides.
  • Homologues of the sequences according to the invention may be obtained by using th nucleotide sequences of Figure 1, 3b, 4a, 5a, 6a or 9 or fragments thereof as probes fo a mammalian genomic DNA or cDNA libraries prepared for example from differentiated muscle cells, myoblasts or embryonic cells.
  • the cDNA library may be prepared in an expression vector such as ⁇ gtl 1 and screened with antisera containing antibodies against Nsk2 or Nskl.
  • the fragments of the nucleotide sequence may be used as PCR primers directed against corresponding regions of the homologues and the homologues obtained by PCR.
  • the preparation of such libraries and suitable probing conditions can be determined by those of skill in the art by reference to standard textbooks, eg.
  • a polypeptide of the invention in substantially isolated form will generally comprise a polypeptide of a particular sequence in a preparation in which more than 90%, eg. 95%, 98% or 99% of the polypeptides in the preparation are those of the specified sequence.
  • a nucleotide sequence in substantially isolated form will generally comprise the sequence in a preparation in which more than 90% , eg. 95%, 98% or 99% of the nucleotides in the preparation are those of the specified sequence.
  • fragments of a polypeptide according to the invention will be at least 10, preferably at least 15, for example 20, 25, 30, 40, 50 or 60 amino acids in length. Such fragments will encode an antigenic determinant capable of stimulating the production of an antibody when introduced, optionally fixed to a suitable carrier, into a mammal.
  • the mammal will be of a different species from that from which the polypeptide sequence was derived.
  • the antigenic determinant is preferably specific for Nsk2 or Nskl. This can be determined by a sequence comparison of Nsk2 or Nskl with other RTKs including Torpedo RTK.
  • the specificity of the polypeptide can be tested by determining the specificity of antibodies against the polypeptide as described below.
  • any polypeptide with an antigenic determinant capable of provoking the production of an antibody specific for Nsk2 or Nskl i.e. an antibody with an affinity for Nsk2 or Nskl significantly higher than for other RTKs will be a polypeptide of the invention.
  • Suitable regions which contain Nsk2 specific antigenic determinants include aa674-693 and aa859-871 of Figure 2, as well as regions of the extracellular domain.
  • the alternate carboxy terminal domain of the receptor tyrosine kinase of Figures 4b and 4c, the carboxy termini of the soluble and truncated Nsk2 isoforms of Figs. 5b and 6b and the differentially spliced sequence DYKKENITT (SEQ ID NO: 14) are also regions of interest to which Nsk2 specific antibodies can be made.
  • a monoclonal antibody according to the invention may be prepared by convention hybridoma technology using the proteins or peptide fragments thereof, as an immunogen
  • Polyclonal antibodies may also be prepared by conventional means which compris inoculating a host animal, for example a rat or a rabbit, with a peptide of the invention an recovering immune serum.
  • Proteins or peptides of the invention may be presented a bacterial or baculoviral or mammalian (e.g. CHO derived) fusion proteins to use a immunogens.
  • monoclonal antibodies according to the invention which retain their antige binding activity, such a F(abM, F(ab 2 )' or Fv fragments form a further aspect of th invention.
  • monoclonal antibodies according to the invention may be analyze (eg. by DNA sequence analysis of the genes expressing such antibodies) and humanize antibody with complementarity determining regions of an antibody according to th invention may be made, for example in accordance with the methods disclosed in EP-A- 0239400 (Winter), or its U.S. equivalent, incorporated by reference.
  • Antibodies or fragments thereof will desirably be capable of binding an Nsk2 polypeptide or fragment thereof with an affinity significantly higher than their affinity to other RTKs.
  • the affinity for other RTKs will be at least 10 fold less than the affinity for Nsk2.
  • the affinity of an antibody may be determined by techniques available in the art.
  • antibodies or fragments thereof capable of binding an Nskl polypeptide or fragment thereof will have an affinity at least 10 fold higher for the Nskl polypeptide than for other RTKs.
  • Further antibodies of the invention may be made based on polypeptide sequences common to Nsk2 and Nskl . Such antibodies will be capable of identifying both targets.
  • Expression vectors containing nucleic acid molecules according to the invention may a contain an origin of replication compatible with a host cell in which the vector is desig to replicate.
  • Suitable host cells include prokaryotic or eukaryotic cells, such as bacte (eg. E.col ⁇ ), yeast, insect and mammalian (eg. Chinese Hamster ovary cells).
  • the vector When the vector is an expression vector it will also contain a promoter compatible with host cell operably linked to said nucleotide sequence.
  • "Operably linked” refers to juxtaposition wherein a promoter and a nucleotide carrying sequence are in a relations permitting the coding sequence to be expressed under the control of the promoter. Th may be elements such as a 5' non-coding sequence between the promoter and codi sequence. The 5' non-coding sequence may be heterologous or homologous to t promoter and/or coding sequence. The expression will desirably also contain 3' no coding sequence operably linked to the coding sequence. Where the expression vector a eukaryotic expression vector it may contain a polyadenylation signal 3' to the codi sequence and 3' non-coding sequence.
  • the promoter may be any suitable promoter available in the art. This includes regulatab promoters. Examples of suitable promoters include the E.coli 0-lactamase promoter, yeast ADH (alcohol dehydrogenase) promoter, a mammalian metallothionein promoter a viral promoters such as the SV40T antigen promoters or retroviral LTR promoters.
  • Nucleic acid molecules according to the invention may be produced by synthetic recombinant means known in the art and illustrated in the Example below.
  • the murine Nsk2 nucleotide sequence may be obtained using PCR primers directed t regions of the Nsk2 gene.
  • the primers can be used to amplify and clone the Nsk genomic DNA from a genomic DNA library or Nsk2 cDNA from a cDNA library derive from cells in which the gene is expressed.
  • Mammalian homologues may be obtained usin analogous procedures. Similar and analogous procedures may be used to obtain murin and other mammalian homologues of Nskl starting with the information in Figure 9.
  • Sequences capable of selectively hybridising to the nucleotide sequences of Figures 1, 3b, 4a, 5a, 6a or 9 may be made by any suitable technique available in the art.
  • the sequence of Figures 1 , 3b, 4a, 5a, 6a or 9 may be cloned from a murine genomic DNA library or suitable cDNA library.
  • a region of such a sequence may be modified by site directed mutagenesis.
  • a primer corresponding to the region of the sequence to be modified can be made which contains desired changes.
  • the primer can be used in conjunction with one or more other primers to perform a PCR on the original Figure 1, 3b, 4a, 5a, 6a or 9 sequence. This provides a new sequence containing desired nucleotide changes which is capable of selectively hybridising to the original sequence.
  • Fragments of the Figure 1, 3b, 4a, 5a, 6a or 9 sequences may be made synthetically or recombinantly, for example by restriction digestion or PCR using primers corresponding to the 3' and 5' ends of the desired fragment.
  • Polypeptides and fragments thereof according to the invention may be produced recombinantly using expression vectors of the invention or synthetically.
  • Recombinant production includes the cultivation of host cells carrying an expression vector according to the invention.
  • the vector may be introduced to the cells by any physical or biological means appropriate for the cell, eg. transfection or transformation.
  • the cells can be cultured under conditions known per se, which are suitable for the growth of the cells and expression of the protein or polypeptide.
  • the polypeptide may be recovered from the culture. This includes recovering the polypeptide from the medium in which the culture is grown or from the cells in the culture. The latter process may involve breaking open the cells by chemical or physical means.
  • the recovery of the polypeptide in substantially isolated form may include suitable purification means known per se in the art such chromatography (including HPLC), size fractionation on a suitable gel or column a affinity purification using an antibody capable of selectably binding an epitope present the polypeptide being recovered.
  • suitable purification means known per se in the art such chromatography (including HPLC), size fractionation on a suitable gel or column a affinity purification using an antibody capable of selectably binding an epitope present the polypeptide being recovered.
  • Polypeptides and fragments thereof and antibodies and fragments thereof capable of bindi said polypeptide may be used to study the role of Nsk2 in the growth and differentiati of mammalian skeletal muscle myotubes. They may be used as agonists or antagonists Nsk2 or Nskl in order to either enhance the Nsk2 or Nskl kinase activity in a cell or blo its activity. This may be achieved by introducing a polypeptide or fragment thereof of t invention into the environment of a cell in vitro or in vivo. The cell may be undifferentiated myoblast which is normally capable of differentiation to form a myotu under suitable conditions such as serum withdrawal. The myoblast can be exposed to su conditions in the presence of a peptide of the invention and the effect on differentiation c be observed.
  • Recombinant Nsk2 or Nsk 1 polypeptides, peptides, antibodies or fragments thereof m also be used in strategies to identify other components of the Nsk2 or Nskl signallin pathway.
  • antisera specific for the novel carboxy terminus of the solubl extracellular domain isoform of Nsk2 could be used to fix a recombinant extracellul domain to a solid support to create an affinity column to purify binding proteins.
  • Simil approaches can be used for screening cDNA expression libraries. Similar affinity method for which Nsk2 or Nskl antibodies would be required could be used to purify substrate of activated Nsk2 or Nskl receptors.
  • a suitable concentration of an agonist or antagonist o the invention will be from about 0.1 nM to about 10 ⁇ M, eg. from about 10 nm to 1 ⁇ M
  • Nsk2 include the following amino acid fragments as numbered i Figure 2: aal-21, aa22-496 (including aa49-98, aal42-190, aa233-282 and aa401-450) aa497-517, aa518-871, aa518-576, aa577-858, aa674-693 and aa859-871, as well as th novel carboxy terminal regions of Nsk2 alternate isoforms as set out in Figures 4b, 4c, 5 and 6b and the novel extracellular domain region as set forth in figure 3b. Th homologous regions of Nskl are also preferred.
  • Polypeptides and fragments thereof, and antibodies and fragments thereof may be prepared as a pharmaceutical formulation.
  • a formulation includes said polypeptide, antibody or fragments together with one or more pharmaceutically acceptable carriers or diluents.
  • Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral or parenteral (e.g. intramuscular or intravenous) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostatis and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the polypeptide to blood components or one or more organs.
  • RTKs receptor-like protein tyrosine kinase
  • one aspect of the invention is the inhibition of muscle RTK signalling. This can be accomplished via, e.g., antibodies against muscle RTK epitopes involved in activation, antisense nucleic acid molecules, expression of dominant, negative RTK receptors, and the delivery of solubilized RTK receptors to a target site of interest.
  • the RTKs of the invention are expressed in terminally differentiated skeletal myotubes in vitro, as well as in adult skeletal muscles, in vivo.
  • the muscle RTKs such as Nsk2 and Nskl may mediate cellular survival pathways in normal skeletal muscles. Stimulation, maintenance, or regulation of such pathways in general are expected to ameliorate effects of prolonged denervation following nerve injury, thereby facilitating survival of muscle tissue during nerve regeneration.
  • the necessary regulation of these pathways may be accomplished by, e.g., using agonist antibodies against RTKs which effectively catalyze the kinases.
  • the loc application of ligands such as Nsk2 ligands, can accomplish this.
  • Other means achieving the stated goals will be clear to the skilled artisan, and need not be repeated her
  • RTKs such as Nsk2 are expressed in the epithelia of lactatin mammary glands, and in the seminiferous tubule of neonatal testis.
  • the determination o abnormal levels of RTK expression, especially Nsk2 expression in these tissues, may b used to diagnose disorders.
  • Hybridization, amplification, and immunoassays are example of the type of assay which may be used here. These assays and how to carry them out wil be clear to the skilled artisan and need not be elaborated herein.
  • muscle is used to modify the RTKs of th invention, the genes in question are expressed in cell types other than muscle cells.
  • Fo example mammary gland epithelia, and neural cells express the genes of interest. Othe cells may also express these genes.
  • Nsk2 The murine form of Nsk2 has been mapped to the distal portion of murine chromosom 13, as is reported in Oncogene 11:281-290, incorporated by reference. This location includes loci which are syntenic with human chromosome 5q. As an example HMGCR maps to 5q 13 - ql4, while IL-9 maps to 5ql5-21. Mutations have been mapped within this loci, which is correlatable with a form of limb girdle muscular dystrophy known as LGMD1A. This map location, together with the expression pattern of Nsk2, suggests involvement in muscular dystrophy. Thus, measurement of the RTKs, such as Nsk2, may be predective or diagnostic for muscular dystrophy.
  • Nsk2 The full length isoforms of Nsk2 bear all the structural motifs characteristic of transmembrane receptor tyrosine kinases, suggesting likely functions in intercellular signalling. Moreover, the distribution of Nsk2 transcripts during mouse development implicate functions for this novel RTK in skeletal myogenesis, neural development, and mesenchymal-epithelial interactions during fetal organogenesis. Both chromosomal mapping and nucleotide sequence comparisons demonstrate that Nskl (chr. 4) and Nsk2 (chr. 13) are distinct genes.
  • Nsk2 extracellular domain
  • Torpedo RTK cloned from the electric organ of Torpedo californica (Jennings et al, 1993), PNAS 90 2895-2899) and this, together with the preferential expression of both in skeletal muscle, suggests a close evolutionary relationship between these proteins.
  • Nsk2 and Torpedo RTK contain four Ig-like loops organised in a similar manner
  • a kringle-like protein binding motif that defines Torpedo RTK (Jennings et aL, 1993) is not present in Nsk2.
  • Torpedo RTK extracellular domains is consistent with a model proposed for kringle motifs as modular units subject to exon shuffling.
  • the boundaries of the region of non-homology between Nsk2 and Torpedo RTK extracellular domains represent exon-intron junctions, and sites of differential splicing, in the Nsk2 transcription unit.
  • Torpedo RTK and Nsk2 also exhibit structural similarities, including highly conserved tyrosine kinase domains and small kinase insert and carboxy terminal regions of identical size. However, little primary sequence homology is shared between these motifs, reinforcing the conclusion that Nsk2 is not the true mammalian homolog of Torpedo RTK.
  • the occurrence of a small kinase insert and short carboxy terminal tail in the Nsk2 intracellular domain is similar to that seen in the trk family of neurotrophin receptors with which the Nsk2 intracellular domain has next closest amino acid sequence homology.
  • the cloned cDNA sequences for each of the four Nsk2 RTK isoforms expressed in skeletal myotubes account for approximately 3.3kb of mRNA and include polyadenylation signals, but it is unlikely that the complete 5' untranslated region is represented in these clones.
  • Utilisation of alternate transcriptional starts, or further differentially processed variants, may account for the two transcripts detected in Nsk2 expressing cells and tissues.
  • Nsk2 transcripts were detected by RNA in situ hybridisation as early as the appearance of dermamyotome and subsequently exhibited a striated distribution within skeletal myofibers in the fetus.
  • Nsk2 Northern blot analysis demonstrated that expression persisted in skeletal muscle from adult mice. Moreover, the increased steady-state levels of Nsk2 transcripts observed on terminal differentiation of committed skeletal myoblasts in vitro, implicate this novel RTK in the formation and/or function of skeletal myotubes. In this respect, the expression profile of Nsk2 contrasts with those defined previously for RTKs that facilitate myoblast proliferation and which are down regulated on myotube formation. Increased expression of insulin-like growth factors and their receptors during myoblast differentiation has led to the proposal that IGF-mediated signalling may help promote myoblast differentiation in an autocrine manner. The differential expression of Nsk2 may reflect a role for this novel RTK as a positive mediator of myoblast differentiation.
  • Nsk2 in terminally differentiated myotubes, together with the known properties of prototype RTKs as mediators of cell survival, raises the interesting possibility that the N RTK may transduce signals necessary for survival of the post-mitotic myotube.
  • further analysis of the biologic properties and intracellular components of N signalling pathways in myogenic cells are likely to be of considerable interest in relati to the molecular mechanisms by which intercellular signalling pathways promote myobl proliferation and inhibit muscle-specific transcription factor activity.
  • Nsk2 RT Although expression in skeletal myogenic lineages is a major feature of the Nsk2 RT several additional sites of Nsk2 expression during embryonic development were identifi by RNA in situ hybridisation.
  • the expression in the periosteal layer of ossifying bon suggests further roles for Nsk2 in musculo-skeletal development, whilst the detection transcripts in cells of dorsal root ganglia of the trunk, cranial ganglia and enteric gang of the gut, implicate this novel RTK in major branches of the peripheral nervous syste
  • Nsk2 signalling T availability of physiologically relevant in vitro culture systems in which to analyse an manipulate Nsk2 signalling should facilitate the identification of such ligands an elucidation of the biologic properties of Nsk2 signalling in the regulation of cellular growt and differentiation.
  • the following Examples illustrate the invention.
  • Nsk2 The cloning, probing, sequencing and other methods used in the identification and analysi of Nsk2 are based on those general techniques described by Sambrook et al (ibid).
  • Nsk2 was cloned via a modification of the method described by Wilks et al. , ibid. Using degenerate oligonucleotides corresponding to conserved domains of tyrosine kinase, a gen fragment was obtained, which was used to screen a mouse genomic library constructed in the bacteriophage lambda vector LambdaDash. Screening was carried out at 0.1xSSC,0.1% SDS at 60°C. Two genomic clones, designated ⁇ G13 and XG23 carried the Nsk2 gene.
  • Nsk2 The complete coding sequence of Nsk2 was determined from overlapping ⁇ G13 and XG23 genomic subclones, and cDNA clones derived from a conditionally immortalised mouse fetal myoblast cell line (Morgan et al, (1994) Dev. Biol. 162: 486-498).
  • aal-21 . . . signal peptide bearing a 3-tubulin mRNA autoregulation signal aa22-496: extracellular region containing: aa49-98 . .
  • immunoglobulin-like domain aal42-190 immunoglobulin-like domain aa233-282: immunoglobulin-like domain aa401-450: immunoglobulin-like domain aa222-224: potential N-linked glycosylation site aa462-464: potential N-linked glycosylation site aa497-517: transmembrane domain aa518-871: intracellular region containing: aa518-576 juxtamembrane domain aa577-858 tyrosine kinase domain aa674-693 kinase insert domain aa859-871 carboxy terminal domain
  • Nsk2 receptor tyrosine kinase was also identified in which a deletion of 24 nucleotides (1415-1438) results in replacement of aa457-465 with a single alanine (A) residue (SEQ ID NO:3). Both Nsk2 isoforms are expressed in fetal myoblasts and derivative myotubes.
  • Nsk2 receptor tyrosine kinase isoform was identified in fetal myotube derived cDNA clones in which nucleic acid sequence identity was seen to nucleotide 2649 (aa 868), after which a novel 483 nucleotide stretch was identified (SEQ ID NO:4). This encoded a further 13 amino acids, a stop codon and 3' untranslated region. In conjunction with the extracellular domain alternate splicing ( Figures 2 and 3a), this novel carboxy terminus predicts two further isoforms of the Nsk2 receptor tyrosine kinase ( Figures 4b and 4c). A fifth Nsk2 isoform encodes a putative soluble extracellular domain
  • nucleotide sequence of some cDNA clones isolated from a fetal myotube cDNA librar deviated further from the sequence of the full length receptor isoform shown in Figure 1
  • Nucleic acid sequence identity was seen to nucleotide 1414 (aa456) after which a novel 25 nucleotide span was identified. This encoded a further 11 amino acids, a stop codon, 3' untranslated region, polyadenylation signal and poly A tail (SEQ ID NO: 8).
  • thes cDNA clones predict a fifth Nsk2 isoform (SEQ ID NO: 9) predicted to encode a solubl extracellular domain of Nsk2 bearing only a single (aa222-224) putative N-linked glycosylation site.
  • nucleotide 673 of the Figure 1 sequence results from splicing at nucleotide 673 of the Figure 1 sequence.
  • the first 673 nucleotides of the full length sequence are spliced onto a 532 nucleotide sequence encoding a 36 amino acid C-terminal tail followed by a 3' untranslated region and polyadenylation signal. This is shown in SEQ ID NOS: 10 and 11, respectively.
  • This putative soluble extracellular domain isoform can be distinguished from that in SEQ ID NO:9 that it is predicted to encode only the first two Ig-like loops of the full length receptor, whereas that in Fig. 5b encodes all four Ig-like loops that characterise the full Nsk2 extracellular domain and a single putative N-linked glycosylation site.
  • Nsk2 isoforms result from differential splicing of the Nsk2 transcription unit
  • Nsk2 genomic clones Sequence analysis of Nsk2 genomic clones has confirmed that five of the six Nsk2 isoform discussed herein result from differential splicing of the Nsk2 transcription unit as outline in Figure 7. A 5' exon common to at least five isoforms ends at nucleotide 1414. Isoform arise as follows:
  • the carboxy terminus arises as a result of no splicing occurring between nucleotides 2649 and 2650.
  • the genomic organisation of nucleotides 2692-3250 is not known.
  • Nsk2 nucleotide and predicted amino acid sequences with GenEMBL a Swissport databases reveals closest homology with a previously published receptor tyrosi kinase cloned from the electric organ of the electric ray, Torpedo califomica (Jennings al ibid).
  • Figure 8 shows an optimally aligned comparison of Nsk2 and Torpedo RT amino acid sequences. Amino acid sequence identity (64%) and homology (78%) are qui high for genes of such distantly related species. However, a number of features sugge that Nsk2 is not simply the mammalian homologue of Torpedo RTK (Fig 8), namely: . . . . i) Nsk2 lacks a kringle domain in the extracellular region that is characteristic feature of Torpedo RTK;
  • kinase insert domains are not conserved (15% amino acid sequenc identity); and . . . . ii) carboxy terminal domains show little conservation (30% amino aci sequence identity)
  • Nsk2 the specific absence of a kringle domain in Nsk2 is consistent with the notio that the homology between Nsk2 and Torpedo RTK reflects conservation of exon sequence subjected to shuffling during evolution (i.e there may be no direct mammalian homologu of the Torpedo RTK protein).
  • Nsk2 is preferentially expressed in skeletal muscle
  • Nsk2 transcripts of approximately 6.6kb an 3.6kb in 12.5 day total embryo and adult skeletal muscle but not heart, spleen, brain, testi or liver RNA samples. Both fetal myoblast and adult myoblast cell lines express high levels of Nsk2 mRNA, the abundance of which increases markedly on terminal differentiation to post-mitotic myotubes.
  • the 6.6kb mRNA species corresponds to th alternately spliced carboxy terminal receptor tyrosine kinase isoform ( Figure 4a).
  • RNA in situ hybridisation analysis of E10-5 to El 7.5 mouse embryos has revealed Nsk2 receptor tyrosine kinase transcripts in developing myotome and derivative musculature of the trunk and limbs. Additional sites of expression are seen in epithelia of lung and kidney and neural cell types including dorsal root ganglia of the peripheral nervous system.
  • Nsk2 The full length isoforms of Nsk2 bear all the structural motifs characteristic of transmembrane receptor tyrosine kinases, typical of molecules which function as part of an intercellular signalling pathway necessary for the proliferation, survival and/or differentiation of cells expressing these receptors.
  • Nsk2 signalling activity may be induced by binding of specific growth factor(s) to the extracellular domain of the transmembrane isoforms.
  • the putative soluble extracellular domain isoform may also bind physiologic ligand(s) of the Nsk2 receptor. Such ligands may be further defined through use of the amino acid sequences presented here. EXAMPLE 2.
  • Nsk2 maps to mouse chromosome 13.
  • Nsk2 locus To determine the chromosomal location of the Nsk2 locus in the mouse genome, linkag relationships between Nsk2 and other loci mapped in recombinant inbred (RI) mouse strains sought.
  • An EcoRV restriction length polymorphism (RFLP) detected with this probe between C57bl/6 and DBA2/J inbred mouse strains was then used to determine the strain distribution pattern (SDP) of the Nsk2 locus in the BxD series of RI mice. Comparison of this SDP with others mapped in the BxD series identified linkage between Nsk2 and known loci found at the distal end of chromosome 13.
  • the Nsk2 locus can be mapped to the inclusive interval between DBBirl and Tel 13.
  • Nsk2 is preferentially expressed in skeletal muscle.
  • Nsk2 receptor tyrosine kinase To initiate analysis of the physiologic functions of the Nsk2 receptor tyrosine kinase, we performed Northern blot analysis on total cellular RNA isolated from adult mouse tissues and embryos. Two Nsk2-specific transcripts, of approximately 6.6kb and 3.6kb, were readily identified in 12.5 day gestation total embryo RNA. Amongst adult mouse tissues, Nsk2 transcripts were detected in adult skeletal muscle but not heart, brain, testis, lung, small intestine, kidney, spinal cord, cerebellum or newborn thymus. To investigate the preferential expression of Nsk2 in skeletal muscle in greater detail, we next analysed established skeletal myoblast cell lines and derivative myotube cultures were analyzed.
  • Nsk2 expression in in vitro myotube cultures facilitated the isolation of cDNA clones encompassing the entire coding sequence of the Nsk2 RTK.
  • polymorphic variants of the full length Nsk2 RTK were identified in skeletal myotubes.
  • Some cDNA clones carried an in frame deletion of 24 nucleotides (1415-1438), resulting in replacement of amino acids 457-465 with a single alanine residue C-terminal to the fourth Ig-like loop of the extracellular domain.
  • one of two putative sites of N-linked glycosylation in the Nsk2 extracellular domain was deleted in this isoform.
  • Nsk2 ⁇ C A second polymorphism (Nsk2 ⁇ C) was identified in the carboxy terminal domain coding region of some Nsk2 cDNA clones. Nucleotide sequence identity was observed to residue 2649 of the full length Nsk2 receptor, after which a novel 483 nucleotides were present. Consequently, the three C-terminal amin acid residues of the full length Nsk2 RTK were replaced with a novel 13 amino acids, stop codon, and a 417 nucleotide (n) novel 3' untranslated region bearing both polyadenylation signal and poly A tail.
  • Nsk2 ⁇ N an Nsk2 ⁇ C polymorphisms indicate that as many as four distinct isoforms of the Nsk receptor tyrosine kinase are expressed in mammalian skeletal myotubes.
  • RNA in situ hybridisation was carried out on embryos isolated between 8.5-17.5 day gestation. The data presented below were obtained with a probe encompassing the entir intracellular domain and some 3' untranslated sequence of the full length Nsk2 RT (1675n-2668n). A second probe corresponding to extracellular, transmembrane and juxtamembrane regions (1261n-1680n) gave identical hybridisation patterns. In all cases, the corresponding sense control probe showed no specific hybridisation.
  • Nsk2 hybridisation was also observed in the developing axial and appendicular skeletons.
  • RNA in situ hybridisation analysis also revealed a discrete distribution of Nsk2 transcript in a number of other tissues in the developing mouse embryo.
  • Nsk2 transcripts were evident in the mantle and ependymal layers of the spinal cord and the choroid plexus.
  • Nsk2 specific hybridisation was observed in cells of dors ⁇ root ganglia and facial ganglia from E12.5 and enteric ganglia of the gut.
  • a marked differential distribution of Nsk2 transcripts was also detected between epithelial and mesenchymal components of a number of developing organs during fetal embryogenesis.
  • expression was seen specifically in epithelia of the primitive glomeruli within the cortical layer of the kidney, tracheal epithelia, segmented bronchi and terminal bronchioles of the lung, secretory epithelia within the mucosal lining of the gut, pancreatic islets, thymic rudiment and the dermis.
  • PCR cloning of cDNA was carried out.
  • PBS phosphate buffered saline
  • tissue fragment of the mid-third of the embryo (neural fold and somites) transferred in 5 microlitres Tris saline to a 0.5ml Eppendorf tube, incubated on dry ice for 60 minutes, and thawed.
  • Fully degenerate oligonucleotides corresponding to the DVWSF amino acid motif of conserved PTK catalytic subdomain IX were then used directly to prime cDNA synthesis on the freeze-thaw lysate.
  • First strand cDNA product was subjected to PCR using th same set of degenerate oligonucleotides, together with another set against the HRDL amin acid motif of conserved PTK catalytic subdomain Vlb for 30 cycles of 93°C for 1. minutes, 45°C for 1 minute, 63°C for 4 minutes.
  • PCR products were electrophoresed DNA fragments of approximately 210 nucleotides excised, cloned into the plasmid pKS + and the nucleotide sequence of clones determined.
  • Nskl neural fold/somite kinase one (1)
  • the nucleotide and predicted amino acid sequence of the Nskl partial cDNA ar presented in Figure 9.
  • Nskl contains all residues that define PTK catalytic motifs VII and VIII (Fig. 9).
  • subdomain VIII of Nskl includes a WMPPE motif that is a characteristic feature of receptor-like tyrosine kinases.
  • the fyn locus is known to be transcription.ally active in neuroepithelia of neural folds and notochord in E8.5 embryos.
  • Nskl also functions in some aspect of intercellular signalling at this stage of mouse embryogenesis.
  • Nskl likely encodes a low abundance mRNA at this stage of development since expression can be detected by the PCR screen described here, but no transcript is readily apparent by northern blot analysis of RNA prepared from total E8.5 mouse embryos.
  • RNA in situ hybridisation studies using Nskl probes excluding the highly conserved tyrosine kinase domain motifs will facilitate definition of the sites of expression of this RTK during mouse embryogenesis.
  • Nskl and Torpedo RTK The high homology shared between catalytic domain sequences of Nskl and Torpedo RTK implies a close evolutionary relationship between these two putative receptors.
  • RTKs are defined on the comparative nature of extracellular domain motifs and carboxy terminal and/or kinase insert domain sequences and so further insights to the relationship between these proteins awaits definition of the full coding sequence of the Nskl transcription unit.
  • Nsk2 shows a similar high degree of amino aci conservation with both Torpedo RTK and Nskl in catalytic domains Vlb-IX but, by variou other criteria, clearly encodes a receptor tyrosine kinase of a subclass distinct fro Torpedo RTK. This suggests that Nskl and Nsk2 represent a novel subfamily o mammalian receptor tyrosine kinases, with likely roles in embryonic development.
  • these antisera identify a protein(s) of approximately 130kDa with intrinsic capacity to autophosphorylate on tyrosine residues in vitro, following immunoprecipitation from lysates of mouse skeletal myotubes grown in vitro (Fig IOC). This result is consistent with the predicted catalytic motifs of the Nsk2 RTK isoforms. The size discrepancy between the primary Nsk2 RTK sequences (approx.
  • the BOkDa autophosphorylating band may represent post-translational glycosylation events (as suggested from the predicted amino acid sequence) and/or covalent linkage with another polypeptide (possibly the 23kDa truncated Nsk2 extracellular domain isoform (see B above).
  • Nsk2 RTK has best homology with transmembrane receptor kinase (trk) receptors, amongst the family of mammalian RTKS. Moreover, two tyrosine containing peptide motifs known to be functionally important for trk signalling are conserved in Nsk2 RTKS: i) . . Trk . IENPQY 490 FSDA (SEQ ID NO: 18)
  • Nsk2 polypeptides include those which incorporate Y 556 and Y 834 .
  • peptides of from 5 to 10, 20, 30, 40 or 50 amino acids in size encompassing one or other of these residues are preferred.
  • Antibodies capable of binding to such peptides are also preferred.
  • This example studied expression patterns of the proteins of the invention in various cell types.
  • the 231cDa 2Ig variant and the 52kDa 4Ig variant were expressed in myoblasts, and th steady state levels of both were found to increase in the myotubes referred to supra. N expression was found in COS or CHO cells.
  • Nsk2 RTK isoform whic contains regions I, II and III of figure 11 was cloned into a commercially availabl (Promega) SP64 based expression vector, and the resulting expression vector was subjecte to in vitro translation, in a coupled transcription/translation wheat germ extract syste (Promega), following the manufacturer's instructions.
  • the transcription/translation wa carried out in the presence of 35 S methionine. Aliquots of products were then subjected t SDS-PAGE, and then to autoradiography. These are shown in figure 12.
  • Results showed that the cDNA encoded a protein of about lOOKDa, which is consisten with what would be predicted from the full sequence information. In the absence o plasmid DNA, no 35 S methronine labelled protein were observed.
  • the alternate regions can yield up to eight isoformic variants of the membrane spanning isoforms, two isoformic variants of the soluble, extracellular domain, and one truncated extracellular domains.
  • "I” represents the insert between nucleotides 673 and 674
  • "II” is the full length molecule, and so forth.
  • Figure 13 summarizes all of the variants.
  • nucleotide 678 in figure 2 may be T, rather than G as presented. Thus, in SEQ ID NO:l, this nucleotide is presented as K
  • nucleotide 680 in figure 2 is G, and it can also be C.
  • this nucleotide is presented as S (III) nucleotide 1193 in figure 2 is T, and it can also be A.
  • SEQ ID NO: l this nucleotide is presented as W
  • T in figure 2 may also be C.
  • this nucleotide is presented as Y

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Cell Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne une nouvelle famille de récepteurs tyrosine-kinase musculaire. Les molécules d'acide nucléique codant ces derniers, ainsi que leurs utilisations.
PCT/US1995/013490 1994-10-10 1995-10-10 Nskz 2 recepteur tyrosine-kinase musculaire WO1996011664A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP51346396A JP2002517166A (ja) 1994-10-10 1995-10-10 新規な受容体チロシンキナーゼ
EP95943567A EP0800531A4 (fr) 1994-10-10 1995-10-10 Nouveaux recepteurs a activite tyrosine kinases
AU45006/96A AU4500696A (en) 1994-10-10 1995-10-10 Novel receptor tyrosine kinases

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9420389A GB9420389D0 (en) 1994-10-10 1994-10-10 Novel receptor tyrosine kinase
GB9420389.0 1994-10-10
GBGB9510574.8A GB9510574D0 (en) 1995-05-24 1995-05-24 Novel receptor tyrosine kinases
GB9510574.8 1995-05-24

Publications (2)

Publication Number Publication Date
WO1996011664A2 true WO1996011664A2 (fr) 1996-04-25
WO1996011664A3 WO1996011664A3 (fr) 1996-07-04

Family

ID=26305770

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/013490 WO1996011664A2 (fr) 1994-10-10 1995-10-10 Nskz 2 recepteur tyrosine-kinase musculaire

Country Status (4)

Country Link
EP (1) EP0800531A4 (fr)
JP (1) JP2002517166A (fr)
AU (1) AU4500696A (fr)
WO (1) WO1996011664A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997015318A1 (fr) * 1995-10-27 1997-05-01 Mount Sinai Hospital Corporation Peptides inhibiteurs d'une proteine contenant un domaine de liaison de phosphotyrosine
US5998187A (en) * 1992-07-30 1999-12-07 Mount Sinai Hospital Corporation Receptor tyrosine kinase
US6028053A (en) * 1995-10-27 2000-02-22 Mount Sinai Hospital Corporation Peptide inhibitors of a phosphotyrosine-binding domain containing protein

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2963395A (en) * 1994-07-20 1996-02-16 Genetics Institute Inc. Mlk receptor tyrosine kinases
JPH10505489A (ja) * 1994-07-20 1998-06-02 リジェネロン ファーマシューティカルズ， インコーポレイテッド 除神経筋キナーゼ（ｄｍｋ）、チロシンキナーゼスーパーファミリーのレセプター

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
MAMMALIAN GENOMICS, Vol. 6, No. 9, issued September 1995, REITH, "Isolation and Chromosomal Location of Nsk1, a Novel Murine Putative Receptor Tyrosine Kinase", page 689. *
ONCOGENE, Vol. 11, No. 2, issued 26 April 1995, GANJU et al., "Cloning and Developmental Expression of Nsk2, a Novel Receptor Tyrosine Kinase Implicated in Skeletal Myogenesis", pages 281-290. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 86, issued March 1989, WILKS, "Two Putative Protein-Tyrosine Kinases Identified by Application of the Polymerase Chain Reaction", pages 1603-1607. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 90, issued April 1993, JENNINGS et al., "Muscle-specific trk Related Receptor with a Kringle Domain Defines a Distinct Class of Receptor Tyrosine Kinases", pages 2895-2899. *
See also references of EP0800531A2 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5998187A (en) * 1992-07-30 1999-12-07 Mount Sinai Hospital Corporation Receptor tyrosine kinase
WO1997015318A1 (fr) * 1995-10-27 1997-05-01 Mount Sinai Hospital Corporation Peptides inhibiteurs d'une proteine contenant un domaine de liaison de phosphotyrosine
US6028053A (en) * 1995-10-27 2000-02-22 Mount Sinai Hospital Corporation Peptide inhibitors of a phosphotyrosine-binding domain containing protein

Also Published As

Publication number Publication date
EP0800531A2 (fr) 1997-10-15
AU4500696A (en) 1996-05-06
WO1996011664A3 (fr) 1996-07-04
EP0800531A4 (fr) 1999-09-22
JP2002517166A (ja) 2002-06-11

Similar Documents

Publication Publication Date Title
JP3039802B2 (ja) 繊維芽細胞成長因子のための受容体
US6610510B1 (en) Morphogenic proteins
WO2003101388A2 (fr) Element 11 famille 7 de support de solute humain (hslc7a11)
PL182324B1 (pl) Sekwencja polinukleotydowa kodujaca bialko ludzkiej protokadheryny pc3, wektor zawierajacy te sekwencje, komórka gospodarza transfekowana wektorem, bialko i sposób wytwarzania ludzkiej protokadheryny pc3 i przeciwcialo przeciwko ludzkiej pc3 PL
Lane et al. Characterization of a highly conserved human homolog to the chicken neural cell surface protein Bravo/Nr-CAM that maps to chromosome band 7q31
WO1998007736A9 (fr) Gene don-1, polypeptides et utilisations de ceux-ci
JPH10511936A (ja) ヒトソマトスタチン様受容体
WO2000008152A1 (fr) Nouveaux recepteurs de cytokine orphelins
EP1056852A1 (fr) Vaccins anti-angiogeniques: substances et methodes afferentes
KR100676229B1 (ko) 신경영양성 인자 수용체
EP0800531A2 (fr) Nouveaux recepteurs a activite tyrosine kinases
CA2389662A1 (fr) Pentraxine i et recepteur de pentraxine, inhibiteurs de ces proteines et compositions pharmaceutiques contenant ces composes
US5721113A (en) NERF genes
AU753400C (en) Orphan receptors
WO1998020114A1 (fr) Nouvelles tyrosine kinases receptrices
CN102443056A (zh) 表皮生长因子受体的外显子缺失变异体
KR20010012826A (ko) 신경교 세포주-유래 향신경성 인자 수용체
AU764484B2 (en) Orphan cytokine receptor
JPH10262680A (ja) Ｒｈｏ標的タンパク質ヒトｍＤｉａおよびその遺伝子
CA2009918A1 (fr) Acide nucleique encodant les proteines de canal sodique
CN1982455A (zh) 抑制Wnt活性的多核苷酸及其编码多肽和用途
CN1952134A (zh) 促进人体转录因子NF－κB活化的多核苷酸及其编码多肽和用途
WO2002022676A1 (fr) Proteine garante de longevite, sa sequence de codage et ses utilisations
JPH10179166A (ja) 受容体型チロシンキナーゼＥｒｂＢのリガンドをコードする遺伝子及びその蛋白質
CN1952132A (zh) 影响elk1活性的多核苷酸及其编码多肽和用途

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AU CA FI JP NO NZ US

AL Designated countries for regional patents

Kind code of ref document: A2

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

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1995943567

Country of ref document: EP

122 Ep: pct application non-entry in european phase
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
WWP Wipo information: published in national office

Ref document number: 1995943567

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1995943567

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载