AU4500696A - Novel receptor tyrosine kinases - Google Patents

Description

NOVEL RECEPTOR TYROSINE KINASES

FIELDOFTHEINVENTION

The present invention relates to a novel muscle protein with tyrosine kinase activity, DN encoding the protein and its uses.

BACKGROUND AND PRIOR ART

The formation of 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. During differentiation a distinctive set of muscle specific proteins are expressed. There is also a loss of expression of particular receptor tyrosine kinases (RTKs). However, despite progress in the understanding of how environmental signals inhibit myoblast differentiation, the signalling pathways responsible for myogenesis are less well understood. Little is known of the signalling molecules involved in the maintenance and survival of post-mitotic myotubes.

Thus there remains a need to identify and understand signalling molecules which regulate the post-mitotic state. The role of such molecules is of potential significance in a wide variety of human diseases such as degenerative diseases (eg. muscular dystrophy) an cancers, especially sarcomas.

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.

Thus, 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. Desirably, 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. Preferably said antigenic determinant is specific for Nsk2 or Nskl. Also included in the invention are 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.

Also included in the invention are 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. Desirably, the vector is an expression vector which contains promoter compatible with a host cell operably linked to said nucleotide sequence. Desirably, the nucleotide sequence is a sequence of Figures 1 , 3b, 4a, 5a, 6a or 9 or a fragment or homologue thereof.

In another aspect, 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.

Brief Description of the Figures

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. In this variant, 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 10 shows:

A: Representation of the structure of the four distinct Nsk2 RTK isoforms identified fro analysis of mouse skeletal myotube cDNA clones.

B: representation of the structure of the two distinct truncated Nsk2 isoforms referred t as 2Ig and 4Ig hereafter identified from analysis of mouse skeletal myotube cDNA clone C: Whole cell lysates prepared from mouse skeletal myotubes were immunoprecipitate either with pre-immiune sera (PI) or immune sera (I) from a rabbit immunised with PPD-coupled synthetic peptide representing an extracellular sequence of Nsk2 RT

(aa341-352 of fig 2). Immunoprecipitates were subjected to an in vitro kinase assay in th presence of γ³²P ATP prior to SDS-PAGE and autoradiography essentially as described i

Reith et al., (1991; EMBO J. 9, 2451-2459).

D: Whole cell lysates of mouse skeletal myotubes were prepared, subjected to SDS-PAG and transferred to nitrocellulose essentially as described previously (Reith et al. , 1991 ibid). Filters were probed with either pre-immune sera (PI) or immune sera (I) from rabbit immunised with a PPD-coupled synthetic peptide representing amino acid sequence in the novel carboxy terminus of the 4Ig Nsk2 isoform (aa457-467 of fig. 5b). As further control, a third filter (IC) was probed with immune sera that had been pre-incubate with the free-peptide used for immunisation. Antibody binding was detected wit ^,25I-conjugated protein A and autoradiography.

E: Whole cell lysates of mouse skeletal myotubes were prepared, subjected to SDS-PAG and transferred to nitrocellulose essentially as described previously (Reith et al. , 1991, ibid). Filters were probed with either pre-immune sera (PI) or immune sera (I) from a rabbit immunised with a PPD-coupled synthetic peptide representing amino acid sequences in the novel carboxy terminus of the 2Ig Nsk2 isoform (aa 224-235, fig.6b). As a further control, 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 ^,25I-coηjugated 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.

DETAILED DESCRD TION OF PREFERRED EMBODIMENTS

The nucleotide sequences of the invention are preferably DNA sequences although they may also be RNA. In addition, 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. Currently the technology exists produce sufficient quantities of modified oligonucleotides for therapeutic use (e. Biosystems Reporter 1991). 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. For t purposes of the present invention, it is to be understood that the nucleotides describ herein may be modified by any method available in the art in order to enhance their in vi activity or lifespan.

Such modifications are of particular interest in the development of antisense nucleoti fragments. Such antisense 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 For fragments larger than about 50 nucleotides, 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. For smaller oligonucleotide fragments hybridisation conditions can be determine by reference to Meinkoth and Wahl, (Anal. Biochem. _ 1; 267-284 (1984)) incorporate by reference. SSC is defined as 0.15 M sodium chloride and 0.15 M sodium citrate at p 7.5.

Generally, a 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 λgtl 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. Sambrook et al, (Cold Spring Harbor, 1987). 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. Similarly, 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.

Generally, 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. That is, 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.

Fragments of monoclonal antibodies according to the invention which retain their antige binding activity, such a F(abM, F(ab₂)' or Fv fragments form a further aspect of th invention. In addition, 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. Preferably, 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. Likewise, 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. Preferably such antibodies or fragments thereof will have a 10 fold higher affinity to Nsk2 and Nskl than to other RTKs. 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.colϊ), yeast, insect and mammalian (eg. Chinese Hamster ovary cells).

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. For exampl 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. For example, 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.

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. For example, 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.

In an in vitro culture of myoblasts a suitable concentration of an agonist or antagonist o the invention will be from about 0.1 nM to about 10 μM, eg. from about 10 nm to 1 μM

H Preferred fragments of 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. 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. 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.

For example, 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. Analyses of prototype members of the superfamily of receptor-like protein tyrosine kinase (RTKs) have revealed that these membrane spanning molecules play key regulatory role in intercellular signalling pathways necessary for normal mammalian development and ar also frequent targets for subversion in transformed cells. The conserved nature of catalyti motifs, coupled with the application of PCR cDNA cloning strategies, has greatl facilitated the identification of RTKs in recent years, and we have taken this route to clon a partial cDNA of Nskl from early mouse embryos. The full coding sequence, isoformi variants and chromosomal location of Nsk2, which cross hybridise with Nskl on screening mouse genomic libraries have been identified and are part of the invention.

It is very well known that maintenance and subversion of growth factor signalling pathways are a requirement for cellular transformation by a wide variety of oncogenes, possibly reflecting some means for preventing carrying out of a natural apoptotic pathway. Thus, inhibition of signalling pathways involving the muscle RTKs, such as Nsk2 and Nskl, would be expected to inhibit tumor development. Thus, 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. One can analogize to the signalling properties of known RTKs, to conclude that 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. Similarly, 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

Prior studies have shown that 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. Further, it will be clea from the preceding disclosure that while "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.

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 locatio 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.

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.

The structural organisation of the Nsk2 extracellular domain appear to be unique amongst published RTKs, indicating that Nsk2 represents a novel subclass within this protein superfamily. The best homology is seen with 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. However, whilst the putative ligand binding domains of both 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. In agreement with this, 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. The intracellular domains of 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. Amongst mammalian RTKs, 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. Northern blot analysis identified two distinct Nsk2 transcripts co-expressed in skeleta myogenic cell types and 12.5 day gestation total embryo RNA. The precise relationshi between these mRNA species is not fully understood at the present time, but it i interesting to note that similar findings have been reported for Torpedo RTK in the electri ray (Jennings et al, 1993). Cross hybridisation to a Nsk2 related RTK is considered unlikely since cDNA probes corresponding to extracellular, intracellular or 3' untranslated regions of the Nsk2 RTK isoforms identified both transcripts in myotubes by northern blot analysis. Moreover, screening myotube cDNA libraries with the same cDNA probes identified only Nsk2 clones. 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. During skeletal myogenesis in vivo, 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. 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. In addition, the preferential expression of 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. In th respects, 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.

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 A discrete distribution of Nsk2 transcripts was also seen in epithelial components of variety of developing organs, including those of the kidney, lung and gut. Inductive eve by means of appropriate epithelial- mesenchymal interactions have long been known to essential for normal development of organs, and it is becoming clear that RTK signalli pathways can mediate inductive interactions, often by paracrine mechanisms in which t receptor tyrosine kinase and its relevant growth factor ligand are expressed in epithelia a esenchyme, respectively. The differential distribution of Nsk2 transcripts in embryon organ epithelia not only suggests roles for Nsk2 signalling in the regulation of epitheli - development, but also raises the possibility that the mesenchymal components of su embryonic tissues may provide a source of physiologic ligand(s) for this novel RTK. 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.

Examples

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).

EXAMPLE 1.

Identification of Nsk2

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 λG13 and XG23 carried the Nsk2 gene.

Complete Nsk2 receptor tyrosine kinase isoform coding sequences

The complete coding sequence of Nsk2 was determined from overlapping λG13 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).

The complete coding nucleotide and predicted amino acid sequence of the full length Nsk2 receptor-like tyrosine kinase are shown in Figure 1. The following features of the full length protein are apparent (figure 2): 1 490

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

An alternative isoform of the 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.

A further 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

The 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). Thus, 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.

A sixth isoform encoding a truncated Nsk2.

In addition, a further isoform was identified which 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

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:

a) Soluble extracellular domain (Fig. 5a, 5b): no splicing occurs and the poly A signa results in transcriptional termination and polyadenylation.

b) Full length receptor protein tyrosine kinase (SEQ ID NO:2): splicing occurs a 1414n to an exon or exons encoding nucleotides 1415-1438. A further splice then occurs to the next exon encoding nucleotides 1439-1637.

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.

c) Deleted receptor protein tyrosine kinase isoform (Figure 3): splicing occurs directly from the 1414n splice site to the exon encoding nucleotides 1439-1637.

d) The alternate carboxy terminal receptor tyrosine kinase isoform (Figure 4b) arises by splicing at 2649n to an additional exon or exons encoding nucleotide sequences unique to this isoform.

e) Combination of the differential splicing outlined in c and d can give rise the fourth receptor tyrosine kinase isoform (Figure 4c).

2 U Nsk2 is somewhat homologous with Torpedo RTK

Comparison of 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;

. . . . ii) kinase insert domains are not conserved (15% amino acid sequenc identity); and . . . . iii) carboxy terminal domains show little conservation (30% amino aci sequence identity)

Furthermore, 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

Northern blot analysis identified specific 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). A mRNA species of approximately 2kb in myotube RNA corresponds to the solubl extracellular domain Nsk2 isoform cDNA clones (Figure 5a), whilst a mRNA species o approximately 1.3kb corresponds to the truncated extracellular domain isoform cDNA (Figure 6a). 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.

Functions of Nsk2 signalling path ay(s)

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. The preferential expression in skeletal muscle and differential expression on terminal differentiation of myoblasts, as well as in neural cells, suggests that the Nsk2 signalling pathway functions in the formation or survival of mammalian skeletal myotubes, .and in the formation and survival of nerve cells. By analogy with prototype receptor tyrosine kinases, 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.

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. A 1950bp genomic DNA fragment, encompassing intracellular and 3' untranslated regions of the Nsk2 transcription unit, identified Nsk2 as a single copy gene by genomic Southern blot analysis. 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.

EXAMPLE 3

Nsk2 is preferentially expressed in skeletal muscle.

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. Skeletal myoblasts derived from neonatal H-2K^b-tsA58 transgenic mice proliferate when cultured under conditions permissive for function of the thermolabile, interferon-inducible SV40 T antigen transgene. When grown under non-immortalising conditions, these cell undergo terminal differentiation to form striated multinucleated myotubes. Both 6.6kb an 3.6kb Nsk2 transcripts were readily detectable in total cellular RNA prepared from suc conditionally immortalised skeletal myoblast cell lines. Moreover, an increase in th steady-state level of Nsk2 transcripts was seen when these cell lines were induced to form myotubes by culture in the absence of IFN 7 at the non-permissive temperature for SV4 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. In good agreement with this, a similar differential expression profile was observed on in vitro differentiation of the spontaneously immortalised myogenic cell line C2C12. Taken together, these data indicate that increased steady-state levels of Nsk2 mRNA are an authentic and specific feature of skeletal myoblast differentiation.

EXAMPLE 4. Multiple Nsk2 RTK isoforms are expressed in skeletal myotubes

The relatively high levels of Nsk2 expression in in vitro myotube cultures facilitated the isolation of cDNA clones encompassing the entire coding sequence of the Nsk2 RTK. In the course of this analysis, 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. As a consequence, one of two putative sites of N-linked glycosylation in the Nsk2 extracellular domain was deleted in this isoform. A second polymorphism (Nsk2ΔC) 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. Taken together, the presence of both Nsk2ΔN an Nsk2ΔC polymorphisms indicate that as many as four distinct isoforms of the Nsk receptor tyrosine kinase are expressed in mammalian skeletal myotubes.

EXAMPLE 5

Nsk2 expression during mouse embryogenesis

To investigate in more detail the expression of Nsk2 during skeletal muscle developmen in vivo, and to identify additional likely sites of Nsk2 function during mouse development 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.

Consistent with northern blot analysis, a major site of Nsk2 expression in the developing mouse embryo was in skeletal myogenic cell types. While no expression was observed in newly formed somites in E8.5 embryos, Nsk2 specific hybridisation was clearly visible at later stages in the dermamyotome component of differentiated somites. Subsequently, expression was maintained in muscles of both axial and appendicular skeletons in the fetus. Moreover, higher magnification of these developing muscle tissues, in both sagital and transverse planes of section, revealed a striated pattern of hybridisation associated with myofibers. Nsk2 hybridisation was also observed in the developing axial and appendicular skeletons. Expression was seen to be restricted to sites of ossification in the periostea layer of developing bones, including rib primordia and humerus, but not in vertebrae i which ossification is not yet initiated at this stage of development. In contrast to th distribution of Nsk2 transcripts in the developing musculo-skeletal system, no expressio was detected in cardiac muscle at any stage analysed.

RNA in situ hybridisation analysis also revealed a discrete distribution of Nsk2 transcript in a number of other tissues in the developing mouse embryo. In the central nervou system, Nsk2 transcripts were evident in the mantle and ependymal layers of the spinal cord and the choroid plexus. In the developing peripheral nervous system, 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. For example, 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.

EXAMPLE 6 Identification of Nskl.

To identify Nskl , PCR cloning of cDNA was carried out. To this end, 8.5 day gestation C57M/6 X C57W/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. 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.

A gene of novel nucleotide sequence, termed Nskl (neural fold/somite kinase one (1)) wa obtained. The nucleotide and predicted amino acid sequence of the Nskl partial cDNA ar presented in Figure 9. In addition to the conserved catalytic domain motif sequences Vl and IX by which it was cloned, Nskl contains all residues that define PTK catalytic motifs VII and VIII (Fig. 9). Moreover, subdomain VIII of Nskl includes a WMPPE motif that is a characteristic feature of receptor-like tyrosine kinases. In good agreement with this, comparison of the Nskl nucleotide and predicted amino acid sequences with GenEMBL and Swissprot databases revealed best homology with Torpedo RTK (Jennings et al., 1993), a receptor tyrosine kinase cloned from the electric ray, Torpedo califomica (Figure 9B).

To determine the chromosomal location of Nskl in the mouse genome, linkage relationships were established in recombinant inbred (RI) mouse strains. Genomic Southern blot analysis using the Nskl cDNA probe under high stringency hybridisation conditions (50% formamide, 42°C) and high stringency washes (0. 1 xSSC, 65°C), defined Nskl as a single copy gene and facilitated identification of an Xba\ restriction fragment length polymorphisms (RFLP) at the Nskl locus between C57bl/6 and DBA2J mouse strains. This RFLP was then used to determine the strain distribution pattern (SDP) of Nskl in the BxD series of RI mice. Comparison of this SDP with that of others in the BxD series established linkage between Nskl and yb2 (2/24, r =0.024), Mupl (0/25, r=0) and b (5/25, r=0.072) loci. Thus, the Nskl locus maps with confidence in the inclusive interval between Lyb2 and b on mouse chromosome 4.

The conservation of PTK catalytic domain motifs, coupled with the application of PCR-mediated cDNA cloning techniques, has led to a rapid increase in the identification of cloned members of the protein tyrosine kinase superfamily over the last several years. The data presented here demonstrate the feasibility of applying this approach directly to crude lysates prepared from dissected tissue fragments, and is validated by the cloning of PTKs known to be expressed at this stage of embryogenesis. c-kit transcripts have been observed in developing neural tube, but not somites, at E8.5 and high levels of FGFR1 expression have been detected in presomitic mesoderm and the rostral half of newly formed somites at E8.0-8.5. Similarly, the fyn locus is known to be transcription.ally active in neuroepithelia of neural folds and notochord in E8.5 embryos. By these criteria, it is expected that 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.

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. However, as with other RTKs cloned from lower vertebrates, it is likely to be difficult to know for certain whether Nskl is the true mammalian homolog of Toψedo RTK. In thi respect, it is interesting to note that 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.

EXAMPLE 7 Antibodies against Nsk2.

A: Rabbit polyclonal antisera raised against the peptide CGNKEVPPDFGS (SEQ ID NO: 15) i.e., representing amino acid residues 457-467 of the novel C terminus of the putative soluble extracellular domain of Nsk2 identify a polypeptide of approximately 52kDa (fig 10D) by western blot analysis of mouse skeletal myotube lysates in good agreement with the predicted open reading frame.

B: Rabbit polyclonal antisera raised against the peptide CTLDGERYDVGS (SEQ ID NO: 16) i.e., representing amino acid residues 224-235 of the novel C terminus of the truncated putative soluble extracellular domain of Nsk2 identify a polypeptide of approximately 23kDa (Fig. 10E) by western blot analysis of mouse skeletal myotube lysates in good agreement with the predicted open reading frame.

C: Rabbit polyclonal antisera raised against the peptide CTTSHRDPEDAQE (SEQ ID NO: 17) i.e., representing amino acid residues 341-352 of the full length Nsk2 RTK, alternately spliced Nsk2 RTK isoform, alternately spliced C terminal RTK isoforms and putative soluble extracellular domain isoform identify polypeptides of approximately 52kDa (presumed soluble extracellular domain isoform), 105kDa (in good agreement with predicted primary sequence of full length Nsk2 RTKs by western blot analysis of mouse skeletal myotube lysates. Moreover, 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. 105kDa) and 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).

The N-terminal C of the peptides made in (A) and (C) above are added synthetic residues.

EXAMPLE 8

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 . IENPQY⁴⁹⁰FSDA (SEQ ID NO: 18)

. . Nsk2 HPNPMY⁵⁵⁶QRMP (SEQ ID NO: 19) within the juxtamembrane domains of both RTKs. Y⁴⁹⁰ 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)). i) . . Trk PEVY^75IAIMRG (SEQ ID NO:20)

. . Nsk2 . . . LELY⁸³⁴NLMRL (SEQ ID NO:21) between catalytic motif domains X and XI in both trk and Nsk2. Y⁸³⁴ of trk has bee demonstrated to be an autophosphorylation site and mediate association with p85 subuni of PI-3 kinase. This association does not appear to be essential for the biologic signallin properties of trk (EMBO J. 13, 1585-1590 (1994); Neuron 12, 691-705 (1994)).

The conservation of motifs utilised by trk receptors is clearly suggestive that the above regions of Nsk2 are involved in autophosphorylation and also potentially associate with relevant substrate molecules. Thus preferred fragments of Nsk2 polypeptides include those which incorporate Y⁵⁵⁶ and Y⁸³⁴. For example, 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.

EXAMPLE 9

This example studied expression patterns of the proteins of the invention in various cell types.

Whole cell lysates of murine CsC12 myoblasts were prepared, as were derivative myotubes, in accordance with Oncogene 11:281-290 (1995) incorporated by reference herein. This reference will provide details of cell culture. Similarly, lysates of COS and CHO cells were prepared. Protein content in the lysates was quantitated, and equivalent amounts of total protein were then subjected to SDS-PAGE, followed by Western blotting. Blotting was carried out with the affinity purified antisera which had been raised against carboxy terminal regions of the 2Ig and 4Ig isoforms, respectively. Detection was via the well known ECL system. 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. These results are set forth in Figure 11.

EXAMPLE 10

Expression of full length murine Nsk2 was carried out. The 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 ³⁵S 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 ³⁵S methronine labelled protein were observed.

EXAMPLE 11

The work described in example 1, supra was continued, using the same cDNA library described therein, and the same techniques.

A total of eleven splice variants or isoforms were found, and these are depicted in figure

13. Analysis of these leads to the conclusion that alternate splicing occurs at three distinct sites in the full length molecule. The first variant region is inserted in between nucleotides

673 and 674 in the full length molecule. The result is an in frame, alternate region of 20 amino acids, which bear two potential N-linked glycosylation sites, which are located between residues 209 and 210 in the full length molecule.

In a second variant, alternate splicing leads to the deletion of nucleotides 1415-1438 of the full sequence. As a result, amino acids 457-465 of the full length molecule are replaced by a single alanine residue, and a potential N-glycosylation site is lost.

The next three variants, described, e.g., in figures 4, 5 and 6, 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. In the figures, "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.

EXAMPLE 12

Additional work was carried out to isolate and to sequence muscle RTKs in accordance with the invention. In so doing, there were six alternatives found. Specifically:

(I) 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

(II) nucleotide 680 in figure 2 is G, and it can also be C. Thus, in SEQ ID NO: 1 , this nucleotide is presented as S (III) nucleotide 1193 in figure 2 is T, and it can also be A. Thus, in SEQ ID NO: l, this nucleotide is presented as W

(IV) at nucleotide 1359, A in figure 2 may also be C. Thus, in SEQ ID NO: l, this nucleotide is presented as M

(V) at nucleotide 1563, G in figure 2 may also be T. Thus, in SEQ ID NO: l, this nucleotide is presented as K

(VI) at nucleotide 1589, T in figure 2 may also be C. Thus, in SEQ ID NO: l, this nucleotide is presented as Y

These alternates for nucleotides result in changes in the amino acid sequence. Thus, with reference to amino acid positions:

(I) 211:Leu or Phe

(II) 212:Gly or Ala

(III) 383:Leu or Gin

(IV) 439:Arg or Ser (V) 506: Leu or Phe

(VI) 515:Val or Ala

In each case, an "Xaa" is used to present these alternates. For convenience, the figures are not presented twice, to give all variants, as the foregoing information will guide the skilled artisan to what is encompassed.

Claims (12)

1. Isolated nucleic acid molecule which encodes a muscle receptor tyrosine kinase protein, the complement of which hybridizes, under stringent conditions, to at least one of the nucleotide sequences set forth in:

SEQ ID NO: 1;

SEQ ID NO:6; SEQ ID NO: 8; SEQ ID NO: 10; and SEQ ID NO: 13.

2. The isolated nucleic acid molecule of claim 1, selected from the group consisting of:

(i) the nucleic acid molcule consisting of the nucleotide sequence of SEQ ID NO:l;

(ii) the nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:6;

(iii) the nucleic molecule consisting of the nucleotide sequence of SEQ ID NO: 8;

(iv) the nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 10; and (v) the nucleic acid molecule consisting of the nucleotide sequence of

SEQ ID NO: 13.

3. Expression vector comprising the isolated nucleic acid molecule of claim 1, operably linked to a promoter.

4. Tranformed or transfected cell, transformed or transfected with the isolated nucl acid molecule of claim 1.

5. Transformed or transfected cell, transformed or transfected with the express vector of claim 3.

6. Isolated muscle receptor tyrosine kinase which is encoded by an isolated nucl acid molecule, the complementary nucleotide sequence of which hybridizes, un stringent conditions, with at least one of the nucleotide sequences set forth in:

SEQ ID NO: 1,

SEQ ID NO:6, SEQ ID NO: 8,

SEQ ID NO: 10, and SEQ ID NO: 13.

7. The isolated muscle receptor tyrosine kinase of claim 6, consisting of the ami acid sequence of:

SEQ ID NO:2,

SEQ ID NO:3,

SEQ ID NO:4,

SEQ ID NO:5,

SEQ ID NO:6, SEQ ID NO: 7,

SEQ ID NO:8,

SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO:ll.

8. Isolated polypeptide consisting of:

amino acids 1-21 of SEQ ID NO:2, amino acids 22-496 of SEQ ID NO:2, amino acids 49-98 of SEQ ID NO:2, amino acids 142-190 of SEQ ID NO:2, amino acids 233-282 of SEQ ID NO:2, amino acids 401-450 of SEQ ID NO:2, amino acids 497-517 of SEQ ID NO:2, amino acids 518-576 of SEQ ID NO:2, amino acids 518-871 of SEQ ID NO:2, amino acids 577-858 of SEQ ID NO: 2, amino acids 674-693 of SEQ ID NO: 2, and amino acids 859-871 of SEQ ID NO:2.

9. Isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

10. Immunogenic composition comprising the isolated polypeptide of claim 9, and an immunogenic carrier.

11. Antibody which specifically binds to a muscle receptor tyrosine kinase.

12. The antibody of claim 11, wherein said antibody is a monoclonal antibody.