WO1998020114A1 - Nouvelles tyrosine kinases receptrices - Google Patents

Nouvelles tyrosine kinases receptrices Download PDF

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WO1998020114A1
WO1998020114A1 PCT/US1997/019646 US9719646W WO9820114A1 WO 1998020114 A1 WO1998020114 A1 WO 1998020114A1 US 9719646 W US9719646 W US 9719646W WO 9820114 A1 WO9820114 A1 WO 9820114A1
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PCT/US1997/019646
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Alastair Reith
Markus Ruegg
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Ludwig Institute For Cancer Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators

Definitions

  • the present invention relates to a novel muscle protein with tyrosine kinase activity, DNA encoding the protein and its uses.
  • vertebrate skeletal muscles involves many processes fundamental to our understanding of developmental biology. This includes cell-cell signalling, differentiation and morphogenesis. Individual skeletal muscles have distinctive muscle fibre compositions that vary in energy metabolism, contraction rate and fatigue resistance. The variation is thought to reflect different populations of precursor myoblasts . During development of muscles waves of overlapping myoblast types can be observed.
  • Myoblasts undergo terminal differentiation to form myotubes by a process involving cellular fusion.
  • Myoblasts can be cultivated in vi tro in the presence of growth factors such 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) 86; 1603-1607) describe a general method to clone protein tyrosine kinase genes using degenerate oligonucleotide primers. Using a modification of this approach, a gene which contains a tyrosine kinase domain has been identified.
  • the gene which was initially called Msn-2, has homology with a receptor tyrosine kinase cloned from the electric organ of the electric ray, Torpedo californica (Jennings et al , Proc . Natl. Acad. Sci. (1993) 90; 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 coding sequence of Nskl, optionally with its 5' and/or 3' untranslated regions, as well as fragments of these nucleic acid molecules.
  • These nucleic acid molecules may be obtained by routine methodologies in the art, starting with probes based upon the sequences disclosed in Figure 9. Such methods include making a cDNA library from cells which express Nskl, and probing said library with a nucleic acid comprising all or part of the 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 kinases or fragments thereof, of the invention. The antibody may be polyclonal or monoclonal .
  • nucleotide sequences encoding polypeptides (including 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 said nucleotide sequences.
  • the vector is an expression vector which contains a promoter compatible with a host cell operably linked to said nucleotide sequence.
  • 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 invention.
  • the splicing is within the extracellular domain.
  • a sequence of 60 nucleotides provides a novel string of 20 amino acids is spliced in between nucleotides 673 and 674 of the sequence of SEQ ID NO:l. Overlining indicates sites of potential glycosylation (SEQ ID NO: 4) .
  • Figure 4a shows the nucleotide sequence and predicted amino acid sequence of an alternately spliced carboxy terminal domain of the Nsk2 receptor tyrosine kinase (SEQ ID NO: 5) .
  • Figure 4b sets out the amino acid sequence of the full length Nsk2 RTK isoform bearing 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 the replacement in the extracellular domain of Figure 3 and the alternately spliced carboxy terminus of Figure 4a (SEQ ID NO: 7) .
  • Figure 5a shows the partial nucleotide and predicted amino acid sequence of an alternately spliced truncated Nsk2 cDNA encoding a putative soluble extracellular domain (SEQ ID 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 truncated Nsk2 isoform of Figure 6a and 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 isoforms of Nsk2.
  • Figure 8 provides a comparison of the amino acid sequences of Nsk2 and the Torpedo RTK 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 10 shows:
  • Nsk2 RTK (aa341-352 of fig 2) .
  • Immunoprecipitates were subjected to an in vitro kinase assay in the presence of 7 32 P ATP prior to SDS -PAGE and autoradiography essentially as described in Reith et al . , (1991; EMBO J. 9, 2451-2459) .
  • PPD-coupled synthetic peptide representing amino acid sequences in the novel carboxy terminus of the 4Ig Nsk2 isoform (aa457-467 of fig. 5b) .
  • a third filter (IC) was probed with immune sera that had been pre-incubated with the free-peptide used for immunisation. Antibody binding was detected with 125 I- conjugated protein A and autoradiography.
  • 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.
  • Figure 14 shows the result of Western blotting, using agrin isoforms.
  • Figure 15 shows the precipitation of NsK-2, with ⁇ - dystro-glycan or utrophin.
  • FIG 16 panels A through D, depicts a series of Western blots carried out on samples taken from murine EDL muscle of normal mice, and mdx mice, which are deficient in dystrophin.
  • FIG. 17 panels A and B, depict the localization of Nsk2 isoforms in transverse sections of the mdx EDL muscle.
  • FIG 18 panels A through D, show hematoxylin/eosin staining of murine EDL muscle during muscle degeneration and regeneration .
  • Figure 19 panels A through D, show localization of the 2Ig isoform during muscle degeneration/regeneration.
  • 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 which is a DNA sequence which is a DNA sequence which is RNA sequences of RNA 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 e.g., DNase
  • a number of different types of modification to nucleotides are known in the art.
  • nucleotides described herein may be modified by any method available in the art in order to enhance their in vivo activity or lifespan.
  • antisense nucleotide fragments can be used to inhibit the transcription or translation of the Nsk2 or Nskl gene in vitro or in vivo . This use has applications for example in studying myogenesis under conditions where the expression of the Nsk2 or Nskl gene product is selectively inhibited by introducing an effective amount of an antisense nucleotide fragment into a cell which expresses Nsk2.
  • Nucleotide fragments of the present invention will typically be from 10 to 2000 bases in length, eg. from 10 to 1,000, eg. 15-500, for example 16, 17, 18, 20, 25, 50, 100 or 200 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 acid 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 and aa859-871.
  • a nucleotide sequence or fragment thereof is capable of selectively hybridising to the Nsk2 or Nskl sequence or its complements where, under high stringency conditions, the sequence or fragment thereof does not hybridise to genes normally found in association with Nsk2 or Nskl.
  • Stringent conditions will vary according to the size of the fragment. For fragments larger than about 50 nucleotides, high stringent conditions will typically be about 60°C at 0.2 X SSC, preferably 60°C at 0.1 x SSC, more preferably 65°C at 0.1 x SSC.
  • SSC is defined as 0.15 M sodium chloride and 0.15 M sodium citrate at pH 7.5.
  • nucleotide sequence or fragment thereof capable of selectively hybridising to the sequence of Figures 1, 3b, 4a, 5a, 6a or 9 will be at least 80 or 90% and more preferably at least 95% homologous to the sequence of Seq. ID No. 1 over a region of at 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 the nucleotide sequences of Figure 1, 3b, 4a, 5a, 6a or 9 or fragments thereof as probes for 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 ⁇ gtll 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 are also regions of interest to which Nsk2 specific antibodies can be made .
  • a monoclonal antibody according to the invention may be prepared by conventional hybridoma technology using the proteins or peptide fragments thereof, as an immunogen.
  • Polyclonal antibodies may also be prepared by conventional means which comprise inoculating a host animal, for example a rat or a rabbit, with a peptide of the invention and recovering immune serum.
  • Proteins or peptides of the invention may be presented as bacterial or baculoviral or mammalian (e.g. CHO derived) fusion proteins to use as immunogens .
  • monoclonal antibodies according to the invention which retain their antigen binding activity, such a F(ab'), F(ab 2 )' or Fv fragments form a further aspect of the invention.
  • monoclonal antibodies according to the invention may be analyzed (eg. by DNA sequence analysis of the genes expressing such antibodies) and humanized antibody with complementarity determining regions of an antibody according to the invention may be made, for example in accordance with the methods disclosed in EP-A-0239400
  • 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 also contain an origin of replication compatible with a host cell in which the vector is designed to replicate.
  • Suitable host cells include prokaryotic or eukaryotic cells, such as bacterial (eg. E. coli ) , 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 the host cell operably linked to said nucleotide sequence. "Operably linked” refers to a juxtaposition wherein a promoter and a nucleotide carrying sequence are in a relationship permitting the coding sequence to be expressed under the control of the promoter. There may be elements such as a 5 ' non-coding sequence between the promoter and coding sequence. The 5' non-coding sequence may be heterologous or homologous to the promoter and/or coding sequence. The expression will desirably also contain 3' non-coding sequence operably linked to the coding sequence. Where the expression vector is a eukaryotic expression vector it may contain a polyadenylation signal 3' to the coding sequence and 3' non-coding sequence.
  • the promoter may be any suitable promoter available in the art . This includes regulatable promoters .
  • suitable promoters include the E. coli -lactamase promoter, a yeast ADH (alcohol dehydrogenase) promoter, a mammalian metallothionein promoter and viral promoters such as the SV40T antigen promoters or retroviral LTR promoters.
  • Nucleic acid molecules according to the invention may be produced by synthetic or recombinant means known in the art and illustrated in the Example below.
  • the murine Nsk2 nucleotide sequence may be obtained using PCR primers directed to regions of the Nsk2 gene.
  • the primers can be used to amplify and clone the Nsk2 genomic DNA from a genomic DNA library or Nsk2 cDNA from a cDNA library derived from cells in which the gene is expressed.
  • Mammalian homologues may be obtained using analogous procedures. Similar and analogous procedures may be used to obtain murine 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.
  • 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 as chromatography (including HPLC) , size fractionation on a suitable gel or column and affinity purification using an antibody capable of selectably binding an epitope present on the polypeptide being recovered.
  • Polypeptides and fragments thereof and antibodies and fragments thereof capable of binding said polypeptide may be used to study the role of Nsk2 in the growth and differentiation of mammalian skeletal muscle myotubes. They may be used as agonists or antagonists of Nsk2 or Nskl in order to either enhance the Nsk2 or Nskl kinase activity in a cell or block its activity. This may be achieved by introducing a polypeptide or fragment thereof of the invention into the environment of a cell in vi tro or in vivo .
  • the cell may be an undifferentiated myoblast which is normally capable of differentiation to form a myotube under suitable conditions such as serum withdrawal .
  • the myoblast can be exposed to such conditions in the presence of a peptide of the invention and the effect on differentiation can be observed.
  • Recombinant Nsk2 or Nsk 1 polypeptides, peptides, antibodies or fragments thereof may also be used in strategies to identify other components of the Nsk2 or Nskl signalling pathway.
  • antisera specific for the novel carboxy terminus of the soluble extracellular domain isoform of Nsk2 could be used to fix a recombinant extracellular domain to a solid support to create an affinity column to purify binding proteins.
  • Similar approaches can be used for screening cDNA expression libraries.
  • Similar affinity methods for which Nsk2 or Nskl antibodies would be required could be used to purify substrates of activated Nsk2 or Nskl receptors.
  • a suitable concentration of an agonist or antagonist of the invention will be from about 0.1 nM to about 10 ⁇ M, eg. from about 10 nm to 1 ⁇ M.
  • Nsk2 Preferred fragments of Nsk2 include the following amino acid fragments as numbered in Figure 2: aal-21, aa22-496
  • the homologous regions of Nskl are also preferred.
  • Polypeptides and fragments thereof, and antibodies and fragments thereof may be prepared as a pharmaceutical formulation. Such 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.
  • 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 kinases
  • 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.
  • RTKs such as Nsk2 are expressed in the epithelia of lactating mammary glands, and in the seminiferous tubule of neonatal testis. The determination of abnormal levels of RTK expression, especially Nsk2 expression in these tissues, may be used to diagnose disorders.
  • Hybridization, amplification, and immunoassays are examples of the type of assay which may be used here. These assays and how to carry them out will be clear to the skilled artisan and need not be elaborated herein. Further, it will be clear from the preceding disclosure that while “muscle” is used to modify the RTKs of the invention, the genes in question are expressed in cell types other than muscle cells. For example mammary gland epithelia, and neural cells express the genes of interest. Other cells may also express these genes .
  • Nsk2 The murine form of Nsk2 has been mapped to the distal portion of murine chromosome 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 calif ornica (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.
  • This clear distinction between Nsk2 and 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 si tu 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 vi tro, 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 Nsk2 RTK may transduce signals necessary for survival of the post-mitotic myotube.
  • further analysis of the biologic properties and intracellular components of Nsk2 signalling pathways in myogenic cells are likely to be of considerable interest in relation to the molecular mechanisms by which intercellular signalling pathways promote myoblast proliferation and inhibit muscle-specific transcription factor activity.
  • Nsk2 RTK Nsk2 RTK
  • RNA in si tu hybridisation The expression in the periosteal layer of ossifying bones suggests further roles for Nsk2 in musculo-skeletal development, whilst the detection of transcripts in cells of dorsal root ganglia of the trunk, cranial ganglia and enteric ganglia of the gut, implicate this novel RTK in major branches of the peripheral nervous system.
  • a discrete distribution of Nsk2 transcripts was also seen in epithelial components of a variety of developing organs, including those of the kidney, lung and gut.
  • Nsk2 The cloning, probing, sequencing and other methods used in the identification and analysis 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 gene 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 ⁇ G23 carried the Nsk2 gene.
  • Nsk2 The complete coding sequence of Nsk2 was determined from overlapping ⁇ G13 and ⁇ G23 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 ⁇ -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 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
  • Nucleic acid sequence identity was seen to nucleotide 1414 (aa456) after which a novel 258 nucleotide span was identified. This encoded a further 11 amino acids, a stop codon, 3' untranslated region, polyadenylation signal and polyA tail (SEQ ID NO: 8) .
  • Nsk2 isoform predicted to encode a soluble 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 isoforms discussed herein result from differential splicing of the Nsk2 transcription unit as outlined in Figure 7.
  • Isoforms arise as follows: a) Soluble extracellular domain (Fig. 5a, 5b) : no splicing occurs and the poly A signal results in transcriptional termination and polyadenylation.
  • Nsk2 is somewhat homologous with Torpedo RTK
  • 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 a characteristic feature of Torpedo
  • RTK kinase insert domains are not conserved (15% amino acid sequence identity) ; and iii) carboxy terminal domains show little conservation (30% amino acid sequence identity)
  • Nsk2 and Torpedo RTK reflects conservation of exon sequences subjected to shuffling during evolution (i.e there may be no direct mammalian homologue of the Torpedo RTK protein) .
  • Nsk2 is preferentially expressed in skeletal muscle
  • Northern blot analysis identified specific Nsk2 transcripts of approximately 6.6kb and 3.6kb in 12.5 day total embryo and adult skeletal muscle but not heart, spleen, brain, testis 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 the alternately spliced carboxy terminal receptor tyrosine kinase isoform ( Figure 4a) .
  • RNA in si tu hybridisation analysis of E10-5 to E17.5 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 signalling pathway 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.
  • ligands may be further defined through use of the amino acid sequences presented here.
  • Nsk2 maps to mouse chromosome 13.
  • linkage 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.
  • RFLP restriction length polymorphism
  • 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 transcripts were induced to form myotubes by culture in the absence of IFN ⁇ at the non-permissive temperature for SV40 T antigen.
  • Northern blot analysis of other conditionally immortalised cell lines derived from the same transgenic mouse strain indicated that the differential expression of Nsk2 was not a general response to loss of SV40 T antigen, or removal of interferon.
  • a similar differential expression profile was observed on in vi tro differentiation of the spontaneously immortalised myogenic cell line C2C12.
  • Nsk2 RTK isoforms are expressed in skeletal myotubes
  • Nsk2 expression in in vi tro 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 amino acid residues of the full length Nsk2 RTK were replaced with a novel 13 amino acids, a stop codon, and a 417 nucleotide (n) novel 3' untranslated region bearing both a polyadenylation signal and polyA tail.
  • Nsk2 ⁇ N and Nsk2 ⁇ C polymorphisms indicate that as many as four distinct isoforms of the Nsk2 receptor tyrosine kinase are expressed in mammalian skeletal myotubes.
  • RNA in si tu hybridisation was carried out on embryos isolated between 8.5-17.5 days gestation. The data presented below were obtained with a probe encompassing the entire intracellular domain and some 3' untranslated sequence of the full length Nsk2 RTK (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 si tu hybridisation analysis also revealed a discrete distribution of Nsk2 transcripts 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 dorsal 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.
  • 8.5 day gestation C57bl/6 X C57bl/6 mouse embryos were dissected in phosphate buffered saline (PBS) , a 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 .
  • PBS phosphate buffered saline
  • First strand cDNA product was subjected to PCR using the same set of degenerate oligonucleotides, together with another set against the HRDL amino acid motif of conserved PTK catalytic subdomain Vlb for 30 cycles of 93 °C for 1.5 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 are 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.
  • Nskl locus maps with confidence in the inclusive interval between Ly.b2 and b on mouse chromosome 4.
  • 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 si tu 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 acid conservation with both Torpedo RTK and Nskl in catalytic domains Vlb- IX but, by various other criteria, clearly encodes a receptor tyrosine kinase of a subclass distinct from Torpedo RTK. This suggests that Nskl and Nsk2 represent a novel subfamily of mammalian receptor tyrosine kinases, with likely roles in embryonic development.
  • these antisera identify a protein (s) of approximately l30kDa with intrinsic capacity to autophosphorylate on tyrosine residues in vi tro, following immunoprecipitation from lysates of mouse skeletal myotubes grown in vi tro (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 105kDa and the 130kDa 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 HPNPMY 556 QRMP (SEQ ID NO: 19) within the juxtamembrane domains of both RTKs.
  • Y 490 of trk has been demonstrated to be an autophosphorylation site and mediate association with SHC. This association contributes to the biologic signalling properties of trk (EMBO J. 13, 1585-1590 (1994); Neuron 12, 691-705 (1994)).
  • Trk PEVY 751 AIMRG SEQ ID NO: 20
  • Nsk2 LELY 834 NLMRL SEQ ID NO: 21
  • Y 834 of trk has been demonstrated to be an autophosphorylation site and mediate association with p85 subunit of PI-3 kinase. This association does not appear to be essential for the biologic signalling properties of trk (EMBO J. 13, 1585-1590 (1994); Neuron 12, 691-705 (1994)).
  • 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 23kDa 2Ig variant and the 52kDa 4Ig variant were expressed in myoblasts, and the steady state levels of both were found to increase in the myotubes referred to supra . No expression was found in COS or CHO cells. These results are set forth in Figure 11.
  • the Nsk2 RTK isoform which contains regions I, II and III of figure 11 was cloned into a commercially available (Promega) SP64 based expression vector, and the resulting expression vector was subjected to in vitro translation, in a coupled transcription/translation wheat germ extract system (Promega) , following the manufacturer's instructions. The transcription/translation was carried out in the presence of 35 S methionine. Aliquots of products were then subjected to 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 consistent with what would be predicted from the full sequence information. In the absence of plasmid DNA, no 35 S methronine labelled protein were observed.
  • the next three variants involve alternate carboxy terminal regions. Those shown in figures 5 and 6 are believed to create soluble extracellular domain isoforms. When combined, 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.
  • 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
  • nucleotide 1193 in figure 2 is T, and it can also be A. Thus, in SEQ ID NO : 1 , this nucleotide is presented as W
  • C950,0 agrin fragments referred to hereafter as "C950,0" and C95 4, 8 "were used. See Gesemann, et al . , J. Cell Biol. 128: 625-636 (1995), incorporated by reference. These were added at 10 nM. Gesemann describes a 95 kD carboxy terminal fragment of agrin, referred to as "C95". Within this fragment, splice variants are known, including C95 4,8, which contains 4 and 8 more amino acids at the A and B domains, respectively than does the C950,0 isoform. The mixtures were incubated for one hour, at 37°C.
  • Cell debris was removed via centrifuging the extract for 60 minutes, at 100,000 xg (4°C) . Following this, cells were lysed, and additional agrin protein was added to 100 nM. The mixture was incubated at room temperature for one hour. Agrin specific antibodies were then added, so as to purify any agrin containing complexes therefrom. The antibody containing mixture was incubated for one hour, at room temperature. Then, 150 ul of protein-G Sepharose beads were added, followed by an additional two hours of incubation. The beads were then washed, three times, in extraction buffer. Any bound protein was released by adding 150 ul of sample buffer, followed by five minutes of boiling.
  • the proteins were then subjected to SDS-PAGE, using a 3-12% gradient gel, followed by transfer to nitrocellulose for four hours at 300 mA.
  • the immunoprecipitates were then subjected to Western blotting, using affinity purified Nsk2 specific antiserum, i.e., SK20, diluted 1/200 which was then visualized with an electrochemiluminescence assay. Controls were also set up, wherein no exogenous agrin was added, and one where a whole cell lysate of myotubes were also used.
  • Dystrophy Specifically, a diagnosis of a neuromuscular disease can be seen, since changes in the level of Nsk2 in assays such as those described supra are correlatable to a pathological state. Further, the development of agonists of the Nsk2/DGC interaction may be seen as a therapeutic approach to these disorders.
  • mice In vivo experiments were then carried out, using a mouse model for muscular dystrophy. This is referred to as "mdx" hereafter. This is a model wherein the mice are deficient in dystrophin.
  • mdx a mouse model for muscular dystrophy.
  • polyclonal antiserum against each of RTK, the 4Ig isoform, and the 2Ig isoform were prepared, as described supra .
  • SK20 described supra, recognizes NsK.
  • SK15 recognizes the 4Ig isoform, while SK17 recognizes 2Ig. See examples 7A and 7B supra .
  • These antisera were affinity purified against immunizing peptides on a resin column.
  • the antisera were washed with 20 volumes of phosphate buffered saline, and then eluted with 0. IM triethylamine (pH 11.5), followed by neutralization with IM Tris-HCl, at pH 8.0, followed by extensive dialysis against PBS.
  • mice C57bll0 mice, and C57bll0 mdx mice.
  • the mice were treated to denerve and to devascularize the extensor digitorum longus muscle ("EDL") . These steps were carried out following Pastoret, et al . , Muscle and Nerve 18: 1147-54 (1995), incorporated by reference. One muscle per mouse was used in this procedure. These manipulations result in a single coordinated round of muscle fiber degeneration, and regeneration.
  • the muscle from denerved animals was removed at 21 days post operation.
  • Denerved and devascularized EDLs, and untreated EDLs were removed at 3 , 7, 21 and 60 days following the operations described supra and subjected to Western blotting. Briefly, Chen, et al .
  • TST 20 mM Tris-HCl, pH 7.5; 150 mM NaCl, 0.1% (v/v) , Tween 20
  • the filters were then washed, four times, 10 minutes each time, in TBST at room temperature. An electrochemiluminescence assay was used for visualization.
  • Panel A shows results in normal (-) and denervated (+) EDL muscle following muscle denervation, but not devascularization.
  • normal muscle there was little Nsk2 full length receptor protein, while both the 2Ig and 4Ig forms were found.
  • 21 days after denervation with no renervation, there were marked increases in both the Nsk2 , RTK and 2Ig levels, but none in the 4Ig level.
  • Results with the wild type (+) or mdx dystrophin deficient mouse (“mdx" in panel B) showed marked increase in steady state levels of RTK and 2Ig, and little change in the 4Ig level.
  • mice were both denerved and devascularized. Normal mice were used, and they show a transient increase in steady state levels of Nsk2. RTK and 2Ig proteins, at day 7. The operated muscle is predominantly newly generated skeletal myofibers, and renervation occurs around day 21, albeit with immature fibers. At day 21, steady state levels of Nsk2 and 2Ig decreased, but were greater than in unoperated contralateral controls. At day 60, the protein levels returned to normal. The steady state level of 4Ig protein remained constant throughout the time frame.
  • the slides were then rinsed, and incubated, twice (10 minutes each time) , in supplemented PBS followed by 90 minutes of incubation at room temperature with 10 u/ml of FITC coupled anti-rabbit IgG antiserum.
  • the sections were washed with the supplemented PBS, and mounted in glycerol containing 2.5% of 1, 4-diazabicyclo-2 , 2 , 2-octane, which is a standard, anti- fading agent.
  • the sections were then analyzed by conventional fluorescence microscopy. Sections for hematoxylin/eosin staining followed the protocol of Pastoret et al . , Muscle and Nerve 18: 1147-54 (1995).
  • Figures 17, 18, 19 and 20 present the results of these experiments.
  • Figure 17, panels A and B show 4Ig and 2Ig immunolocalization on transverse sections of EDL muscle of an mdx mouse.
  • panel A (4Ig staining)
  • a peripheral stain is seen in muscle fibers of normal morphology.
  • Regenerating muscle fibers in contrast, show a more general stain.
  • the pattern in panel B (2Ig staining) shows that normal myofibers show a peripheral stain, while regenerating areas show a general, uniformly distributed stain.
  • Figures 18-20 panels A-D presents transverse sections of the EDL muscle of wild type, C57bll0 mice. In each case, panel A shows normal muscle fibers from an unoperated, contralateral control .
  • Panel B shows staining three days after operation to denervate and devascularize
  • panel C show staining after 7 days
  • panel D shows staining after 21 days.
  • Figure 14 shows hematoxylin staining
  • figure 15 shows 2Ig isoform distribution
  • the hematoxylin/eosin staining pattern illustrates differing morphology of muscle fibers during degeneration/regeneration.
  • Panel A shows that normal fibers are characterized by peripheral nuclei. Three days after operation, large areas of necrotic tissue are present, while newly formed myofibers are abundant and show prominent, centrally located nuclei by day 7. At 21 days post operation, newly formed myofibers are more developed but, as compared, e.g., to panel A, are still immature.
  • FIG 15 demonstrates that, in necrotized areas (three days post operation) , 2Ig staining is reduced relative to normal muscle fibers, while by day 7, the isoform is seen throughout new myofibers. By day 21, the distribution is more peripheral, keeping with what is seen in panel A, the non-operated control.
  • Figure 16 shows that the distribution of 4Ig over time essentially parallels that of 2Ig.
  • Nsk2 and its isoforms are clearly implicated in the regeneration of skeletal muscle in vivo.
  • these assays can be used to determine the potential efficacy of a therapeutic agent, as per a method of screening. Expressed another way, one administers a potential therapeutic agent via any standard form of drug administration, and then determines the level of any or all of Nsk2 , 4Ig and 2Ig , comparing the value of values thus obtained to controls generated prior to administration of the drug. This permits the artisan to determine efficacy of a drug and/or evolution of a pathology. Other aspects of the invention will be clear to the artisan, and need not be repeated here.
  • NAME/KEY Figure 1: Ns 2 RTK (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
  • TAACGTCCTC CAGGCCGAGC GCGGACATTC CAAACCTGCC TGCCTCCACC 1500
  • CAGACATGCA AGCGGACTTT CAGAGGGAGG CGGCCCTCAT GGCAGAGTTT 1950
  • CTATTCCGCA GACTACTACA AAGCTGATGG AAATGACGCC ATCCCTATCC 2350
  • MOLECULE TYPE protein
  • FEATURE :
  • NAME/KEY Figure 3b: Alternately spliced Nsk 2 RTK (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
  • ATCATGTTAA AATATATACA CTAGATTTCT GTGTTGTTGT TATCAGTTCT 343
  • NAME/KEY peptide immunogen
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 15:
  • NAME/KEY peptide immunogen
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 16:

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Abstract

La présente invention concerne une nouvelle famille de tyrosine kinases réceptrices. La présente invention concerne également ces molécules, les molécules d'acide nucléique codant lesdites molécules et leur utilisation.
PCT/US1997/019646 1996-11-07 1997-10-29 Nouvelles tyrosine kinases receptrices WO1998020114A1 (fr)

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US5656473A (en) * 1993-07-21 1997-08-12 Regeneron Pharmaceuticals, Inc. Human Dmk receptor

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Publication number Priority date Publication date Assignee Title
US5656473A (en) * 1993-07-21 1997-08-12 Regeneron Pharmaceuticals, Inc. Human Dmk receptor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HUMAN MOLECULAR GENETICS, 1995, Vol. 4, No. 6, WHITING et al., "Characterization of Myotonic Dystrophy Kinase (DMK) Protein in Human and Rodent Muscle and Central Nervous Tissue", pages 1063-1072. *
NEURON, September 1995, Vol. 15, VALENZUELA et al., "Receptor Tyrosine Kinase Specific for the Skeletal Muscle Lineage: Expression in Embryonic Muscle, at the Neuromuscular Junction and after Injury", pages 573-584. *
PROC. NATL. ACAD. SCI. U.S.A., April 1993, Vol. 90, JENNINGS et al., "Muscle-Specific Trk-Related Receptor with a Kringle Domain Defines a Distinct Class of Receptor Tyrosine Kinases", pages 2895-2899. *

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