WO1998020114A1 - Novel receptor tyrosine kinases - Google Patents

Abstract

A novel family of receptor tyrosine kinases is disclosed. These molecules, the nucleic acid molecules encoding them, and the uses of these, are features of the disclosed invention.

Description

NOVEL RECEPTOR TYROSINE KINASES

RELATED APPLICATIONS

This application is a continuation in part of PCT/US95/13490, filed October 10, 1995, designating the United States, which is entitled to the priority of British Application GB 94 20 389.0, filed October 10, 1994, and British Application GB 95 10 574.8, filed May 24, 1995, under 35 U.S.C. § 365(b). All applications referred to herein are incorporated by reference.

FIELD OF THE INVENTION

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

BACKGROUND AND PRIOR ART The formation of 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. 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) and cancers, especially sarcomas.

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.

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 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. 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 kinases or fragments thereof, of the invention. The antibody may be polyclonal or monoclonal .

Also included in the invention are 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. Desirably, the vector is an expression vector which contains a 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 invention. The splicing is within the extracellular domain. In this variant, 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:

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

B: representation of the structure of the two distinct truncated Nsk2 isoforms referred to as 2Ig and 4Ig hereafter identified from analysis of mouse skeletal myotube cDNA clones.

C: Whole cell lysates prepared from mouse skeletal myotubes were immunoprecipi tated either with pre-immiune sera (PI) or immune sera (I) from a rabbit immunised with a PPD-coupled synthetic peptide representing an extracellular sequence of

Nsk2 RTK (aa341-352 of fig 2) . Immunoprecipitates were subjected to an in vitro kinase assay in the presence of 7³²P ATP prior to SDS -PAGE and autoradiography essentially as described in Reith et al . , (1991; EMBO J. 9, 2451-2459) .

D: Whole cell lysates of mouse skeletal myotubes were prepared, subjected to SDS -PAGE 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 4Ig Nsk2 isoform (aa457-467 of fig. 5b) . 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 ¹²⁵I- conjugated protein A and autoradiography.

E: Whole cell lysates of mouse skeletal myotubes were prepared, subjected to SDS-PAGE 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 ¹²⁵I-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 β- dystro-glycan or utrophin.

Figure 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.

Figure 17, panels A and B, depict the localization of Nsk2 isoforms in transverse sections of the mdx EDL muscle.

Figure 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.

Figure 20, panels A through D, show parallel experiments on the 4Ig isoform.

DETAILED DESCRIPTION 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 to produce sufficient quantities of modified oligonucleotides for therapeutic use (e.g., Biosystems Reporter 1991) . A number of different types of modification to nucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotides described herein may be modified by any method available in the art in order to enhance their in vivo activity or lifespan.

Such modifications are of particular interest in the development of antisense nucleotide fragments. Such antisense 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. For smaller oligonucleotide fragments hybridisation conditions can be determined by reference to Meinkoth and Wahl, (Anal. Biochem. 132; 267-284 (1984)) incorporated by reference. SSC is defined as 0.15 M sodium chloride and 0.15 M sodium citrate at pH 7.5.

Generally, a 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 λgtll 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 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 .

Fragments of monoclonal antibodies according to the invention which retain their antigen binding activity, such a F(ab'), F(ab₂)' or Fv fragments form a further aspect of the invention. In addition, 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

(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 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) .

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 . Examples of 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. For example, 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. 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 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. For example, 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.

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

Preferred fragments of Nsk2 include the following amino acid fragments as numbered in 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 the novel carboxy terminal regions of

Nsk2 alternate isoforms as set out in Figures 4b, 4c, 5b and 6b and the novel extracellular domain region as set forth in figure 3b. 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. 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 kinases (RTKs) have revealed that these membrane spanning molecules play key regulatory roles in intercellular signalling pathways necessary for normal mammalian development and are also frequent targets for subversion in transformed cells. The conserved nature of catalytic motifs, coupled with the application of PCR cDNA cloning strategies, has greatly facilitated the identification of RTKs in recent years, and we have taken this route to clone a partial cDNA of Nskl from early mouse embryos. The full coding sequence, isoformic 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 agonistic antibodies against RTKs which effectively catalyze the kinases. Similarly, the local application of ligands, such as Nsk2 ligands, can accomplish this. Other means of achieving the stated goals will be clear to the skilled artisan, and need not be repeated here. Prior studies have shown that 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 .

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.

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 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. 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 skeletal myogenic cell types and 12.5 day gestation total embryo RNA. The precise relationship between these mRNA species is not fully understood at the present time, but it is interesting to note that similar findings have been reported for Torpedo RTK in the electric 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 si tu 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 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. 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 Nsk2 RTK may transduce signals necessary for survival of the post-mitotic myotube. In these respects, 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.

Although expression in skeletal myogenic lineages is a major feature of the Nsk2 RTK, several additional sites of Nsk2 expression during embryonic development were identified by 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. Inductive events by means of appropriate epithelial- mesenchymal interactions have long been known to be essential for normal development of organs, and it is becoming clear that RTK signalling pathways can mediate inductive interactions, often by paracrine mechanisms in which the receptor tyrosine kinase and its relevant growth factor ligand are expressed in epithelia and mesenchyme, respectively. The differential distribution of Nsk2 transcripts in embryonic organ epithelia not only suggests roles for Nsk2 signalling in the regulation of epithelial - development, but also raises the possibility that the mesenchymal components of such embryonic tissues may provide a source of physiologic ligand (s) for this novel RTK. The availability of physiologically relevant in vi tro culture systems in which to analyse and manipulate Nsk2 signalling should facilitate the identification of such ligands and elucidation of the biologic properties of Nsk2 signalling in the regulation of cellular growth and differentiation. The following Examples illustrate the invention. Examples

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) .

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 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 λG13 and λG23 carried the Nsk2 gene.

Complete Nsk2 receptor tyrosine kinase isoform coding sequences

The complete coding sequence of Nsk2 was determined from overlapping λG13 and λG23 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) :

aal-21: signal peptide bearing a β-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 library 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 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) . Thus, these cDNA clones predict a fifth Nsk2 isoform (SEQ ID NO: 9) predicted to encode a soluble 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 isoforms discussed herein result from differential splicing of the Nsk2 transcription unit as outlined in Figure 7. A 5' exon common to at least five isoforms ends at nucleotide 1414. 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. b) Full length receptor protein tyrosine kinase (SEQ ID NO: 2) : splicing occurs at 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) .

Nsk2 is somewhat homologous with Torpedo RTK

Comparison of Nsk2 nucleotide and predicted amino acid sequences with GenEMBL and Swissport databases reveals closest homology with a previously published receptor tyrosine kinase cloned from the electric organ of the electric ray, Torpedo californica (Jennings et al ibid) . Figure 8 shows an optimally aligned comparison of Nsk2 and Torpedo RTK amino acid sequences. Amino acid sequence identity (64%) and homology (78%) are quite high for genes of such distantly related species. However, a number of features suggest 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 a characteristic feature of Torpedo

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

Furthermore, the specific absence of a kringle domain in Nsk2 is consistent with the notion that the homology between 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) . A mRNA species of approximately 2kb in myotube RNA corresponds to the soluble extracellular domain Nsk2 isoform cDNA clones (Figure 5a), whilst a mRNA species of approximately 1.3kb corresponds to the truncated extracellular domain isoform cDNA (Figure 6a) . 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.

Functions of Nsk2 signalling pathway (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, linkage 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 Dl3Birl 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 cells undergo terminal differentiation to form striated multinucleated myotubes. Both 6.6kb and 3.6kb Nsk2 transcripts were readily detectable in total cellular RNA prepared from such conditionally immortalised skeletal myoblast cell lines. Moreover, an increase in the steady-state level of Nsk2 transcripts was seen when these cell lines were induced to form myotubes by culture in the absence of IFN γ 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. In good agreement with this, a similar differential expression profile was observed on in vi tro 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 vi tro 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 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. Taken together, the presence of both Nsk2ΔN and Nsk2ΔC polymorphisms indicate that as many as four distinct isoforms of the Nsk2 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 development in vivo, and to identify additional likely sites of Nsk2 function during mouse development, 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.

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 periosteal layer of developing bones, including rib primordia and humerus, but not in vertebrae in which ossification is not yet initiated at this stage of development. In contrast to the distribution of Nsk2 transcripts in the developing musculo-skeletal system, no expression was detected in cardiac muscle at any stage analysed.

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. In the central nervous 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 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. 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 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 . 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.

A gene of novel nucleotide sequence, termed Nskl (neural fold/somite kinase one (1) ) was obtained. The nucleotide and predicted amino acid sequence of the Nskl partial cDNA are presented in Figure 9. In addition to the conserved catalytic domain motif sequences Vlb 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 californica (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 Xfoal 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 Lyb2 (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 Ly.b2 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-ki t 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 transcriptionally 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 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.

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 Torpedo RTK. In this respect, it is interesting to note that 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.

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 N0:17) 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 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. 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) .

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 PEVY⁷⁵¹AIMRG (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 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)).

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 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.

Example 10

Expression of full length murine Nsk2 was carried out .

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 ³⁵S 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 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 : 1 , 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:l, 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 : 1 , 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.

Example 13

Studies were then carried out to determine what materials complex to the Nsk2 molecule and its isoforms. To do this, antibodies as described in example 7c, supra, were used. The polyclonal antisera will be referred to as SK20 hereafter. It recognizes Nsk2 RTK, and the 4Ig isoform. These can be distinguished on the basis of their different molecular weights .

Skeletal myotube cultures of cell line C2C12 were cultured, following art recognized methods. Two different recombinant 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. Cells were then washed with Tris-HCl, at pH 7.4, 100 mM KC1 , 1.25 mM CaCl₂, 5 mM MgCl₂. The washed cells were then extracted, using an extraction buffer, i.e., 20 mM Tris-HCl, at pH 7.5, 150 mM NaCl, 1.25 mM CaCl₂; 1 mM MgCl₂; 1% Triton X-100, 0.5% sodium doexycholate, and the protease mixture of Denzer et al . , J. Cell Biol. 131: 1547-1560 (1995) . 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.

When the data were analyzed, it was found that the an immunoreactive Nsk2 protein was co-precipitated only when agrin C95 0,0 isoform was used. These results are shown in figure 14.

When these experiments were repeated, using antibodies against β-dystroglycan or utrophin in the absence of added recombinant agrin, it was found that Nsk-2 precipitated with these as well. Figure 15 shows this. As these two proteins are known to be components of the so-called "DGC" (i.e., dystrophin-glycoprotein complex) , molecular defects in which are implicated in neuromuscular diseases, these data suggest several applications to the area of neuromuscular diseases, such as muscular dystrophies, including Duchenne's Muscular

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.

Example 14

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. First, 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. Following immunization, 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.

The animals used were 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 . , Oncogene 12: 979-988 (1996), incorporated by reference, was used to prepare tissue extracts. Then 100 ug of total protein were applied to lanes of 10% SDS/polyacrylamide gels (or, 3-12% gradient gels in denervation experiments) , and were then transferred to nitrocellulose for immunoblotting. These nitrocellulose filters were blocked for two hours, in 5% (w/v) skim milk powder in "TBST" (20 mM Tris-HCl, pH 7.5; 150 mM NaCl, 0.1% (v/v) , Tween 20) , and then incubated for 12 hours at 4°C, in 5% (w/v) skim milk/TBST, containing the relevant antiserum. The antisera recognizing Nsk2 full length receptors, and the 2Ig isoform, were diluted 1:200, and that against 4Ig, at 1:500. The blots were washed, twice, for five minutes each time, in TBST, with shaking at room temperature. Next, these were incubated, with gentle shaking, for 1% hours at room temperature, with peroxidase-coupled anti -rabbit IgG, which had been diluted 1:100 with TBST. The filters were then washed, four times, 10 minutes each time, in TBST at room temperature. An electrochemiluminescence assay was used for visualization.

The results are presented in figure 16 which presents a number of panels.

Panel A shows results in normal (-) and denervated (+) EDL muscle following muscle denervation, but not devascularization. In normal muscle, there was little Nsk2 full length receptor protein, while both the 2Ig and 4Ig forms were found. In contrast, 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.

In panel C, the 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.

When mdx mice were used in the same experiments (panel D) , similar patterns were observed during the renervation. As these mice have a dystrophin deficient genetic background, the inducible level of expression was seen to be in addition to the higher, steady state levels of Nsk2 isoforms in unoperated muscle . Example 15

A series of immunohistochemical staining experiments were then carried out. The methodology described by Ganju et al . , Oncogene 9: 1613-1624 (1994), incorporated by reference, was used, with some modifications. Briefly, skeletal muscles were dissected, and then immediately frozen in liquid nitrogen- cooled isopentane. Then, 10 μm sections were mounted on potassium chromium sulphate/gelatin coated slides, followed by fixing for 30 minutes in 4% paraformaldehyde/PBS, followed by methanol for 15 minutes at -20°C. Slides were then rinsed, twice, in PBS supplemented with 1% bovine calf serum, 0.02% sodium azide, incubated for 10 minutes in this mixture, and then blocked for 20 minutes at room temperature with PBS containing 5% bovine calf serum. Next, the sections were incubated for 12 hours at 4°C, using the supplemented PBS discussed above, and polyclonal antibodies against Nsk2 , at a concentration of 4%. Controls were run, carrying out the entire protocol but for the use of the antibodies.

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. Next 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. In 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, and panel D shows staining after 21 days. Figure 14 shows hematoxylin staining, figure 15 shows 2Ig isoform distribution, and figure 16, 4Ig 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.

The work shown in figure 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.

The foregoing data have diagnostic and therapeutic ramifications, which will be clear to the skilled artisan. Nsk2 and its isoforms are clearly implicated in the regeneration of skeletal muscle in vivo. Hence, one can determine presence, each of, or degree of muscular-skeletal pathology, for example, congenital muscular dystrophies, or other muscle wasting conditions, such as cachexia in cancer patients (Toomey et al . , Cancer 76: 2418-2426 (1995)), AIDS, and inflammatory disorders via assaying a sample for presence, lack, or amount of any or all of Nsk2 , its 4Ig isoform, or its 2Ig isoform. As was noted, supra , there are various ways to kink this to some control value so as to draw valid conclusions therefrom. Further, 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.

The terms and expression which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expression of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

(1) GENERAL INFORMATION:

(i) APPLICANTS: Reith, Alastair; Ruegg, Markus

(ii) TITLE OF INVENTION: Novel Receptor Tyrosine Kinases

(iii) NUMBER OF SEQUENCES: 24

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Felfe & Lynch

(B) STREET: 805 Third Avenue

(C) CITY: New York City

(D) STATE: New York

(E) COUNTRY: USA

(F) ZIP: 10022

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.5 inch, 1.44 kb storage

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: PC-DOS

(D) SOFTWARE: Wordperfect

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/US95/13490

(B) FILING DATE: 10-October- 1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: UK 9420389.0

(B) FILING DATE: 10-October-1994

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION_. NUMBER: UK 9510574.8

(B) FILING DATE: 24-May-1995

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Hanson, Norman D.

(B) REGISTRATION NUMBER: 30,946

(C) REFERENCE/DOCKET NUMBER: LUD 5332.1-PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (212) 688-9200

(B) TELEFAX: (212) 838-3884 (2) INFORMATION FOR SEQUENCE ID NO:l: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3257 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ix) FEATURE:

(A) NAME/KEY: Figure 1: Ns 2 RTK (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

ACGTTGTCCA GAAGCAACCT TTCCTTCCTG AGCCTGGATA TAATCATGAG 50

AGAGCTTGTC AACATTCCAC TGTTACAGAT GCTCACCCTG GTTGCCTTCA 100

GCGGGACTGA GAAACTTCCN AAACCCCCTG TCATCGCCAC GCCTCTTGAA 150

ACTGTAGATG CCTTGGTTGA AGAAGTAGCG ACTTTCATGT GTGCCGTGGA 200

ATCCTACCCT CAGCCCGAGA TTTCTTGGAC CAGAAATAAA ATTCTCATTA 250

AGCTGTTTGA CACCCGCTAC AGCATCCGGG AGAATGGTCA GCTCCTCACC 300

ATTCTGAGCG TGGAAGACAG TGATGATGGC ATCTACTGCT GCATAGCCAA 350

CAATGGAGTG GGAGGAGCCG TGGAGAGTTG TGGTGCCCTG CAAGTGAAGA 400

TGAAACCTAA AATAACTCGT CCTCCCATTA ATGTAAAAAT AATAGAGGGA 450

TTGAAGGCAG TTCTGCCGTG CACTACGATG GGTAACCCCA AACCATCTGT 500

GTCCTGGATC AAGGGGGACA ATGCTCTCAG GGAAAATTCC AGAATCGCAG 550

CTCTTGAATC TGGGAGCTTA AGGATCCATA ATGTGCAAAA GGAAGATGCA 600

GGACAGTACC GCTGTGTGGC CAAAAACAGC CTGGGCACAG CTTACTCCAA 650

ACTGGTGAAG CTGGAAGTGG AGGTTTTGGG AAGAATCCTG CGTGCTCCTG 700

AATCCCACAA TGTCACCTTT GGTTCCTTTG TAACCCTACG CTGCACAGAA 750

ATAGGCATCC CTGTCCCCAC CATCAGCTGG ATTGAAAACG GAAATGCTGT 800

TTCTTCAGGT TCCATTCAAG AGAGTGTGAA AGACCGAGTG ATTGACTCAA 850

GACTCCAGCT CTTCATCACA AAGCCAGGAC TCTACACATG CATAGCTACC 900

AATAAGCACG GAGAAAAGTT CAGTACCGCA AAGGCTGCAG CCACTGTCAG 950

CATAGCAGAA TGGAGTAAGT CACAGAAAGA CAGCCAAGGT TACTGTGCCC 1000

AGTACAGAGG GGAGGGTGTT TTGATGCAAG GCCCTGGCGA AAAGATGCTC 1050

TTGGTTTTTC TTCCAACAAC CTCCCACCGG GACCCCGAGG ACGCCCAGGA 1100

GCTGCTGATC CACACTGCGT GGAATGAACT GAAGGCTGTG AGTCCACTGT 1150

GCCGGCCAGC TGCTGAGGCT CTGCTGTGTT ACCACCTCTT CCTAGAGTGC 1200

AGCCCTGGAG TGGTACCTAC TCCCATGCCC ATTTGCAGAG AGTACTGCCT 1250

GGCGGTAAAG GAGCTCTTCT GTGCAAAGGA ATGGCAGGCA ATGGAAGGAA 1300

AGGCCCACCG GGGCCTCTAC AGATCTGGGA TGCATCTCCT TCCGGTACCA 1350

GAGTGCAGAA AGCTTCCCAG CATGCACCGG GACCCCACAG CCTGCACAAG 1400

ACTGCCATAT TTAGATTATA AAAAGGAAAA CATAACAACA TTCCCGTCAA 1450

TAACGTCCTC CAGGCCGAGC GCGGACATTC CAAACCTGCC TGCCTCCACC 1500

TCTTCCTTTG CCGTCTCGCC TGCGTACTCC ATGACCGTCA TCATCTCCAT 1550

CGTGTCCAGC TTGGCCCTGT TTGCTCTTCT CACCATCGTT ACTCTCTATT 1600

GCTGCCGAAG GAGGAAAGAA TGGAAAAATA AGAAAAGAGA GTCGACCGCG 1650

GTGACCCTCA CCACGTTGCC TTCCGAGCTC CTGCTGGATA GGCTCCATCC 1700

CAACCCCATG TACCAGAGGA TGCCACTCCT TCTGAATCCT AAGTTGCTCA 1750

GCCTGGAGTA TCCGAGGAAT AACATTGAGT ATGTCCGAGA CATCGGAGAG 1800

GGGGCGTTTG GAAGAGTCTT CCAAGCAAGG GCCCCTGGCT TGCTGCCTTA 1850

TGAACCTTTC ACTATGGTGG CCGTGAAGAT GCTTAAGGAA GAGGCCTCTG 1900

CAGACATGCA AGCGGACTTT CAGAGGGAGG CGGCCCTCAT GGCAGAGTTT 1950

GACAACCCCA ACATtGTGAA ACTCTTAGGT GTGTGTGCCG TTGGGAAGCC 2000

GATGTGTCTG CTCTTTGAAT ATATGGCCTA TGGTGACCTC AATGAGTTCC 2050

TCCGAAGTAT GTCCCCGCAC ACTGTTTGCA GCCTCAGCCA CAGTGACCTG 2100

TCCACGAGGG CTCGGGTGTC TAGCCCTGGT CCTCCACCAC TGTCCTGTGC 2150

AGAACAGCTC TGCATTGCCA GGCAGGTGGC AGCTGGCATG GCCTACCTTT 2200

CAGAGCGCAA GTTTGTCCAC CGGGACTTAG CTACCAGGAA CTGCCTGGTT 2250

GGGGAGACCA TGGTGGTGAA AATTGCAGAC TTTGGCCTCT CCAGGAACAT 2300

CTATTCCGCA GACTACTACA AAGCTGATGG AAATGACGCC ATCCCTATCC 2350

GCTGGATGCC GCCCGAGTCT ATCTTCTACA ACCGCTACAC CACGGAGTCG 2400

GATGTATGGG CCTATGGTGT GGTCCTCTGG GAGATCTTCT CCTATGGGCT 2450

GCAGCCCTAC TATGGAATGG CCCACGAGGA GGTCATTTAC TATGTGAGAG 2500

ATGGCAACAT CCTCGCCTGC CCTGAGAACT GCCCCTTGGA ACTGTACAAC 2550

CTCATGCGCC TGTGTTGGAG CAAGCTGCCT GCTGATAGAC CCAGCTTCTG 2600

CAGTATCCAC AGGATCCTGC AGCGCATGTG CGAGAGAGCA GAGGGAACGG 2650

TGGGTGTCTA AAGTTGACCA TTCTCAAACA ACACCCAGGA GGCTCTTTTC 2700

AGACTGTGAG CTGGAGGAAC CCTACCGCAG AGGCCGTGTA AGACCAGATA 2750 GGAGGAGTTT AACTCAGACA TCACGTGCCA GTTGATTGTT TGCCAGGAGA 2800

AACAGATGGT GAATATCCCA GGGTTAAAGA GTCACATCAA AATAGGTTGG 2850

AGATACAGGC TAGGAAAGAG GACAGCAGGT AGCTCCTCTC CCTCACAGGG 2900

GACCGCTTCT AATATATATT GCATAATAAG AACATCCCGG TTACGTTCCT 2950

ACATAATCTC TCAGAGCGAG ACTGCAGTGC TTAGGTTGAA TCCAAAAACT 3000

GGATGGGCAA CTTCATTTTT AACAGAAGAC ATCCTGCCCA TTGCAAAAGC 3050

AATGTGTCTT TGTGTATATT TAGGTAAAGG ACTGAAAACT AAAGATAGGA 3100

ATCCCTTCTT CCACCAGTCA AGACACGTGG CAGGGTCTTG CTGTTGTTTA 3150

GTTCTTCCTT GCACAGAATA TGTAACGTTG TATTTGCATT CTGGAATTGA 3200

GTATCTATTT TACTGATAGA CTTTTGAAGA ATAAAAAGTG GAAAGCCTGC 3250

AAAAAAA 3257

(2) INFORMATION FOR SEQUENCE ID NO : 2 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 871 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein ( ix) FEATURE :

(A) NAME/KEY: Figure 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Met Arg Glu Leu Val Asn He Pro Leu Leu Gin Met Leu Thr Leu 5 10 15

Val Ala Phe Ser Gly Thr Glu Lys Leu Pro Lys Pro Pro Val He 20 25 30

Ala Thr Pro Leu Glu Thr Val Asp Ala Leu Val Glu Glu Val Ala 35 40 45

Thr Phe Met Cys Ala Val Glu Ser Tyr Pro Gin Pro Glu He Ser 50 55 60

Trp Thr Arg Asn Lys He Leu He Lys Leu Phe Asp Thr Arg Tyr 65 70 75

Ser He Arg Glu Asn Gly Gin Leu Leu Thr He Leu Ser Val Glu 80 85 90

Asp Ser Asp Asp Gly He Tyr Cys Cys He Ala Asn Asn Gly Val 95 100 105

Gly Gly Ala Val Glu Ser Cys Gly Ala Leu Gin Val Lys Met Lys 110 115 120

Pro Lys He Thr Arg Pro Pro He Asn Val Lys He He Glu Gly 125 130 135

Leu Lys Ala Val Leu Pro Cys Thr Thr Met Gly Asn Pro Lys Pro 140 145 150

Ser Val Ser Trp He Lys Gly Asp Asn Ala Leu Arg Glu Asn Ser 155 160 165

Arg He Ala Ala Leu Glu Ser Gly Ser Leu Arg He His Asn Val 170 175 180

Gin Lys Glu Asp Ala Gly Gin Tyr Arg Cys Val Ala Lys Asn Ser 185 190 195 Leu Gly Thr Ala Tyr Ser Lys Leu Val Lys Leu Glu Val Glu Val

200 205 210

Leu Gly Arg He Leu Arg Ala Pro Glu Ser His Asn Val Thr Phe

215 220 225

Gly Ser Phe Val Thr Leu Arg Cys Thr Glu He Gly He Pro Val

230 235 240

Pro Thr He Ser Trp He Glu Asn Gly Asn Ala Val Ser Ser Gly

245 250 255

Ser He Gin Glu Ser Val Lys Asp Arg Val He Asp Ser Arg Leu

260 265 270

Gin Leu Phe He Thr Lys Pro Gly Leu Tyr Thr Cys He Ala Thr

275 280 285

Asn Lys His Gly Glu Lys Phe Ser Thr Ala Lys Ala Ala Ala Thr

290 295 300

Val Ser He Ala Glu Trp Ser Lys Ser Gin Lys Asp Ser Gin Gly

305 310 315

Tyr Cys Ala Gin Tyr Arg Gly Glu Gly Val Leu Met Gin Gly Pro

320 325 330

Gly Glu Lys Met Leu Leu Val Phe Leu Pro Thr Thr Ser His Arg

335 340 345

Asp Pro Glu Asp Ala Gin Glu Leu Leu He His Thr Ala Trp Asn

350 355 360

Glu Leu Lys Ala Val Ser Pro Leu Cys Arg Pro Ala Ala Glu Ala

365 370 375

Leu Leu Cys Tyr His Leu Phe Leu Glu Cys Ser Pro Gly Val Val

380 385 390

Pro Thr Pro Met Pro He Cys Arg Glu Tyr Cys Leu Ala Val Lys

395 400 405

Glu Leu Phe Cys Ala Lys Glu Trp Gin Ala Met Glu Gly Lys Ala

410 415 420

His Arg Gly Leu Tyr Arg Ser Gly Met His Leu Leu Pro Val Pro

425 430 435

Glu Cys Arg Lys Leu Pro Ser Met His Arg Asp Pro Thr Ala Cys

440 445 450

Thr Arg Leu Pro Tyr Leu Asp Tyr Lys Lys Glu Asn He Thr Thr

455 460 465

Phe Pro Ser He Thr Ser Ser Arg Pro Ser Ala Asp He Pro Asn

470 475 480

Leu Pro Ala Ser Thr Ser Ser Phe Ala Val Ser Pro Ala Tyr Ser

485 490 495

Met Thr Val He He Ser He Val Ser Ser Leu Ala Leu Phe Ala

500 505 510

Leu Leu Thr He Val Thr Leu Tyr Cys Cys Arg Arg Arg Lys Glu

515 520 525 Trp Lys Asn Lys Lys Arg Glu Ser Thr Ala Val Thr Leu Thr Thr

530 535 540

Leu Pro Ser Glu Leu Leu Leu Asp Arg Leu His Pro Asn Pro Met

545 550 555

Tyr Gin Arg Met Pro Leu Leu Leu Asn Pro Lys Leu Leu Ser Leu

560 565 570

Glu Tyr Pro Arg Asn Asn He Glu Tyr Val Arg Asp He Gly Glu

575 580 585

Gly Ala Phe Gly Arg Val Phe Gin Ala Arg Ala Pro Gly Leu Leu

590 595 600

Pro Tyr Glu Pro Phe Thr Met Val Ala Val Lys Met Leu Lys Glu

605 610 615

Glu Ala Ser Ala Asp Met Gin Ala Asp Phe Gin Arg Glu Ala Ala

620 625 630

Leu Met Ala Glu Phe Asp Asn Pro Asn He Val Lys Leu Leu Gly

635 640 645

Val Cys Ala Val Gly Lys Pro Met Cys Leu Leu Phe Glu Tyr Met

650 655 660

Ala Tyr Gly Asp Leu Asn Glu Phe Leu Arg Ser Met Ser Pro His

665 670 675

Thr Val Cys Ser Leu Ser His Ser Asp Leu Ser Thr Arg Ala Arg

680 685 690

Val Ser Ser Pro Gly Pro Pro Pro Leu Ser Cys Ala Glu Gin Leu

695 700 705

Cys He Ala Arg Gin Val Ala Ala Gly Met Ala Tyr Leu Ser Glu

710 715 720

Arg Lys Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val

725 730 735

Gly Glu Thr Met Val Val Lys He Ala Asp Phe Gly Leu Ser Arg

740 745 750

Asn He Tyr Ser Ala Asp Tyr Tyr Lys Ala Asp Gly Asn Asp Ala

755 760 765

He Pro He Arg Trp Met Pro Pro Glu Ser He Phe Tyr Asn Arg

770 775 780

Tyr Thr Thr Glu Ser Asp Val Trp Ala Tyr Gly Val Val Leu Trp

785 790 795

Glu He Phe Ser Tyr Gly Leu Gin Pro Tyr Tyr Gly Met Ala His

800 805 810

Glu Glu Val He Tyr Tyr Val Arg Asp Gly Asn He Leu Ala Cys

815 820 825

Pro Glu Asn Cys Pro Leu Glu Leu Tyr Asn Leu Met Arg Leu Cys

830 835 840

Trp Ser Lys Leu Pro Ala Asp Arg Pro Ser Phe Cys Ser He His

845 850 855 Arg He Leu Gin Arg Met Cys Glu Arg Ala Glu Gly Thr Val Gly 860 865 870

Val

(2) INFORMATION FOR SEQUENCE ID NO : 3 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 863 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: Figure 3a: Alternatively spliced Nsk2 RTK

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

Met Arg Glu Leu Val Asn He Pro Leu Leu Gin Met Leu Thr Leu

5 10 15

Val Ala Phe Ser Gly Thr Glu Lys Leu Pro Lys Pro Pro Val He

20 25 30

Ala Thr Pro Leu Glu Thr Val Asp Ala Leu Val Glu Glu Val Ala

35 40 45

Thr Phe Met Cys Ala Val Glu Ser Tyr Pro Gin Pro Glu He Ser

50 55 60

Trp Thr Arg Asn Lys He Leu He Lys Leu Phe Asp Thr Arg Tyr

65 70 75

Ser He Arg Glu Asn Gly Gin Leu Leu Thr He Leu Ser Val Glu

80 85 90

Asp Ser Asp Asp Gly He Tyr Cys Cys He Ala Asn Asn Gly Val

95 100 105

Gly Gly Ala Val Glu Ser Cys Gly Ala Leu Gin Val Lys Met Lys

110 115 120

Pro Lys He Thr Arg Pro Pro He Asn Val Lys He He Glu Gly

125 130 135

Leu Lys Ala Val Leu Pro Cys Thr Thr Met Gly Asn Pro Lys Pro

140 145 150

Ser Val Ser Trp He Lys Gly Asp Asn Ala Leu Arg Glu Asn Ser

155 160 165

Arg He Ala Ala Leu Glu Ser Gly Ser Leu Arg He His Asn Val

170 175 180

Gin Lys Glu Asp Ala Gly Gin Tyr Arg Cys Val Ala Lys Asn Ser

185 190 195

Leu Gly Thr Ala Tyr Ser Lys Leu Val Lys Leu Glu Val Glu Val

200 205 210

Xaa Xaa Arg He Leu Arg Ala Pro Glu Ser His Asn Val Thr Phe

215 220 225

Gly Ser Phe Val Thr Leu Arg Cys Thr Glu He Gly He Pro Val

230 235 240 Pro Thr He Ser Trp He Glu Asn Gly Asn Ala Val Ser Ser Gly

245 250 255

Ser He Gin Glu Ser Val Lys Asp Arg Val He Asp Ser Arg Leu

260 265 270

Gin Leu Phe He Thr Lys Pro Gly Leu Tyr Thr Cys He Ala Thr

275 280 285

Asn Lys His Gly Glu Lys Phe Ser Thr Ala Lys Ala Ala Ala Thr

290 295 300

Val Ser He Ala Glu Trp Ser Lys Ser Gin Lys Asp Ser Gin Gly

305 310 315

Tyr Cys Ala Gin Tyr Arg Gly Glu Gly Val Leu Met Gin Gly Pro

320 325 330

Gly Glu Lys Met Leu Leu Val Phe Leu Pro Thr Thr Ser His Arg

335 340 345

Asp Pro Glu Asp Ala Gin Glu Leu Leu He His Thr Ala Trp Asn

350 355 360

Glu Leu Lys Ala Val Ser Pro Leu Cys Arg Pro Ala Ala Glu Ala

365 370 375

Leu Leu Cys Tyr His Leu Phe Leu Glu Cys Ser Pro Gly Val Val

380 385 390

Pro Thr Pro Met Pro He Cys Arg Glu Tyr Cys Leu Ala Val Lys

395 400 405

Glu Leu Phe Cys Ala Lys Glu Trp Gin Ala Met Glu Gly Lys Ala

410 415 420

His Arg Gly Leu Tyr Arg Ser Gly Met His Leu Leu Pro Val Pro

425 430 435

Glu Cys Arg Lys Leu Pro Ser Met His Arg Asp Pro Thr Ala Cys

440 445 450

Thr Arg Leu Pro Tyr Leu Ala Phe Pro Ser He Thr Ser Ser Arg

455 460 465

Pro Ser Ala Asp He Pro Asn Leu Pro Ala Ser Thr Ser Ser Phe

470 475 480

Ala Val Ser Pro Ala Tyr Ser Met Thr Val He He Ser He Val

485 490 495

Ser Ser Leu Ala Leu Phe Ala Leu Leu Thr He Val Thr Leu Tyr

500 505 510

Cys Cys Arg Arg Arg Lys Glu Trp Lys Asn Lys Lys Arg Glu Ser

515 520 525

Thr Ala Val Thr Leu Thr Thr Leu Pro Ser Glu Leu Leu Leu Asp

530 535 540

Arg Leu His Pro Asn Pro Met Tyr Gin Arg Met Pro Leu Leu Leu

545 550 555

Asn Pro Lys Leu Leu Ser Leu Glu Tyr Pro Arg Asn Asn He Glu

560 565 570 Tyr Val Arg Asp He Gly Glu Gly Ala Phe Gly Arg Val Phe Gin

575 580 585

Ala Arg Ala Pro Gly Leu Leu Pro Tyr Glu Pro Phe Thr Met Val

590 595 600

Ala Val Lys Met Leu Lys Glu Glu Ala Ser Ala Asp Met Gin Ala

605 610 615

Asp Phe Gin Arg Glu Ala Ala Leu Met Ala Glu Phe Asp Asn Pro

620 625 630

Asn He Val Lys Leu Leu Gly Val Cys Ala Val Gly Lys Pro Met

635 640 645

Cys Leu Leu Phe Glu Tyr Met Ala Tyr Gly Asp Leu Asn Glu Phe

650 655 660

Leu Arg Ser Met Ser Pro His Thr Val Cys Ser Leu Ser His Ser

665 670 675

Asp Leu Ser Thr Arg Ala Arg Val Ser Ser Pro Gly Pro Pro Pro

680 685 690

Leu Ser Cys Ala Glu Gin Leu Cys He Ala Arg Gin Val Ala Ala

695 700 705

Gly Met Ala Tyr Leu Ser Glu Arg Lys Phe Val His Arg Asp Leu

710 715 720

Ala Thr Arg Asn Cys Leu Val Gly Glu Thr Met Val Val Lys He

725 730 735

Ala Asp Phe Gly Leu Ser Arg Asn He Tyr Ser Ala Asp Tyr Tyr

740 745 750

Lys Ala Asp Gly Asn Asp Ala He Pro He Arg Trp Met Pro Pro

755 760 765

Glu Ser He Phe Tyr Asn Arg Tyr Thr Thr Glu Ser Asp Val Trp

770 775 780

Ala Tyr Gly Val Val Leu Trp Glu He Phe Ser Tyr Gly Leu Gin

785 790 795

Pro Tyr Tyr Gly Met Ala His Glu Glu Val He Tyr Tyr Val Arg

800 805 810

Asp Gly Asn He Leu Ala Cys Pro Glu Asn Cys Pro Leu Glu Leu

815 820 825

Tyr Asn Leu Met Arg Leu Cys Trp Ser Lys Leu Pro Ala Asp Arg

830 835 840

Pro Ser Phe Cys Ser He His Arg He Leu Gin Arg Met Cys Glu

845 850 855

Arg Ala Glu Gly Thr Val Gly Val 860 (2) INFORMATION FOR SEQUENCE ID NO : 4 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 86 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

( ix) FEATURE :

(A) NAME/KEY: Figure 3b: Alternately spliced Nsk 2 RTK (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

CTG GAA GTG GAG GCT CTT AAT ATG ACA AAT GCC ACA GAG AGA GAA 45 Leu Glu Val Glu Ala Leu Asn Met Thr Asn Ala Thr Glu Arg Glu

5 10 15

GAC AGA GAA CCT GAG CAG GAC GCT AAA GTT TTT GCA AGA AT 86

Asp Arg Glu Pro Glu Gin Asp Ala Lys Val Phe Ala Arg 20 25

(2) INFORMATION FOR SEQUENCE ID NO : 5 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 507 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ix) FEATURE:

(A) NAME/KEY: Figure 4a (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

ATG TGC GAG AGA GCA GAG GGA ACG ACA GAT GGC AGA GCC CAT TTC 45 Met Cys Glu Arg Ala Glu Gly Thr Thr Asp Gly Arg Ala His Phe 5 10 15

TTT TGG CCT CAC CAA TAC TAAGTGCATT AGCATTGTGA CCACTGACCA 93

Phe Trp Pro His Gin Tyr 20

CATGGTCGCC TAGAATACTT CATGCAGCTT GCACTTACAT CATTTGAATA 143

CTGTTTATGT TAAATATCTC TCAGTGTTTG GGGGACAGCT ATGATTTGTG 193

AGATACTAAT ATTTTTCTGT TGAGTGTATC ACTTGCTCAT TGCTTCATGA 243

AAGCACTTTA TAAAGCATTA AATTTTTTCT TACATATTTG AGAAATTGCT 293

ATCATGTTAA AATATATACA CTAGATTTCT GTGTTGTTGT TATCAGTTCT 343

TATTCATTGG AAAATGATGC TGAATTTTTA CACATTCAAA TGACTATTTT 393

TCCCTAGTTG ATATGTTAAT AATGTACAAC TGTATAAATT GACTTCCTAA 443

TATTGAAACA ACCTTGTATT CTGAGAATAA ACTCTCTTTG GATTTCAATC 493

TTAAAAAAAA AAAA 507

(2) INFORMATION FOR SEQUENCE ID NO : 6 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 881 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: Figure 4b (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :

Met Arg Glu Leu Val Asn He Pro Leu Leu Gin Met Leu Thr Leu 5 10 15 Val Ala Phe Ser Gly Thr Glu Lys Leu Pro Lys Pro Pro Val He

20 25 30

Ala Thr Pro Leu Glu Thr Val Asp Ala Leu Val Glu Glu Val Ala

35 40 45

Thr Phe Met Cys Ala Val Glu Ser Tyr Pro Gin Pro Glu He Ser

50 55 60

Trp Thr Arg Asn Lys He Leu He Lys Leu Phe Asp Thr Arg Tyr

65 70 75

Ser He Arg Glu Asn Gly Gin Leu Leu Thr He Leu Ser Val Glu

80 85 90

Asp Ser Asp Asp Gly He Tyr Cys Cys He Ala Asn Asn Gly Val

95 100 105

Gly Gly Ala Val Glu Ser Cys Gly Ala Leu Gin Val Lys Met Lys

110 115 120

Pro Lys He Thr Arg Pro Pro He Asn Val Lys He He Glu Gly

125 130 135

Leu Lys Ala Val Leu Pro Cys Thr Thr Met Gly Asn Pro Lys Pro

140 145 150

Ser Val Ser Trp He Lys Gly Asp Asn Ala Leu Arg Glu Asn Ser

155 160 165

Arg He Ala Ala Leu Glu Ser Gly Ser Leu Arg He His Asn Val

170 175 180

Gin Lys Glu Asp Ala Gly Gin Tyr Arg Cys Val Ala Lys Asn Ser

185 190 195

Leu Gly Thr Ala Tyr Ser Lys Leu Val Lys Leu Glu Val Glu Val

200 205 210

Leu Gly Arg He Leu Arg Ala Pro Glu Ser His Asn Val Thr Phe

215 220 225

Gly Ser Phe Val Thr Leu Arg Cys Thr Glu He Gly He Pro Val

230 235 240

Pro Thr He Ser Trp He Glu Asn Gly Asn Ala Val Ser Ser Gly

245 250 255

Ser He Gin Glu Ser Val Lys Asp Arg Val He Asp Ser Arg Leu

260 265 270

Gin Leu Phe He Thr Lys Pro Gly Leu Tyr Thr Cys He Ala Thr

275 280 285

Asn Lys His Gly Glu Lys Phe Ser Thr Ala Lys Ala Ala Ala Thr

290 295 300

Val Ser He Ala Glu Trp Ser Lys Ser Gin Lys Asp Ser Gin Gly

305 310 315

Tyr Cys Ala Gin Tyr Arg Gly Glu Gly Val Leu Met Gin Gly Pro

320 325 330

Gly Glu Lys Met Leu Leu Val Phe Leu Pro Thr Thr Ser His Arg

335 340 345 Asp Pro Glu Asp Ala Gin Glu Leu Leu He His Thr Ala Trp Asn 350 355 360

Glu Leu Lys Ala Val Ser Pro Leu Cys Arg Pro Ala Ala Glu Ala 365 370 375

Leu Leu Cys Tyr His Leu Phe Leu Glu Cys Ser Pro Gly Val Val 380 385 390

Pro Thr Pro Met Pro He Cys Arg Glu Tyr Cys Leu Ala Val Lys 395 400 405

Glu Leu Phe Cys Ala Lys Glu Trp Gin Ala Met Glu Gly Lys Ala 410 415 420

His Arg Gly Leu Tyr Arg Ser Gly Met His Leu Leu Pro Val Pro 425 430 435

Glu Cys Arg Lys Leu Pro Ser Met His Arg Asp Pro Thr Ala Cys 440 445 450

Thr Arg Leu Pro Tyr Leu Asp Tyr Lys Lys Glu Asn He Thr Thr 455 460 465

Phe Pro Ser He Thr Ser Ser Arg Pro Ser Ala Asp He Pro Asn 470 475 480

Leu Pro Ala Ser Thr Ser Ser Phe Ala Val Ser Pro Ala Tyr Ser 485 490 495

Met Thr Val He He Ser He Val Ser Ser Leu Ala Leu Phe Ala 500 505 510

Leu Leu Thr He Val Thr Leu Tyr Cys Cys Arg Arg Arg Lys Glu 515 520 525

Trp Lys Asn Lys Lys Arg Glu Ser Thr Ala Val Thr Leu Thr Thr 530 535 540

Leu Pro Ser Glu Leu Leu Leu Asp Arg Leu His Pro Asn Pro Met 545 550 555

Tyr Gin Arg Met Pro Leu Leu Leu Asn Pro Lys Leu Leu Ser Leu 560 565 570

Glu Tyr Pro Arg Asn Asn He Glu Tyr Val Arg Asp He Gly Glu 575 580 585

Gly Ala Phe Gly Arg Val Phe Gin Ala Arg Ala Pro Gly Leu Leu 590 595 600

Pro Tyr Glu Pro Phe Thr Met Val Ala Val Lys Met Leu Lys Glu 605 610 615

Glu Ala Ser Ala Asp Met Gin Ala Asp Phe Gin Arg Glu Ala Ala 620 625 630

Leu Met Ala Glu Phe Asp Asn Pro Asn He Val Lys Leu Leu Gly 635 640 645

Val Cys Ala Val Gly Lys Pro Met Cys Leu Leu Phe Glu Tyr Met 650 655 660

Ala Tyr Gly Asp Leu Asn Glu Phe Leu Arg Ser Met Ser Pro His 665 670 675 Thr Val Cys Ser Leu Ser His Ser Asp Leu Ser Thr Arg Ala Arg

680 685 690

Val Ser Ser Pro Gly Pro Pro Pro Leu Ser Cys Ala Glu Gin Leu

695 700 705

Cys He Ala Arg Gin Val Ala Ala Gly Met Ala Tyr Leu Ser Glu

710 715 720

Arg Lys Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val

725 730 735

Gly Glu Thr Met Val Val Lys He Ala Asp Phe Gly Leu Ser Arg

740 745 750

Asn He Tyr Ser Ala Asp Tyr Tyr Lys Ala Asp Gly Asn Asp Ala

755 760 765

He Pro He Arg Trp Met Pro Pro Glu Ser He Phe Tyr Asn Arg

770 775 780

Tyr Thr Thr Glu Ser Asp Val Trp Ala Tyr Gly Val Val Leu Trp

785 790 795

Glu He Phe Ser Tyr Gly Leu Gin Pro Tyr Tyr Gly Met Ala His

800 805 810

Glu Glu Val He Tyr Tyr Val Arg Asp Gly Asn He Leu Ala Cys

815 820 825

Pro Glu Asn Cys Pro Leu Glu Leu Tyr Asn Leu Met Arg Leu Cys

830 835 840

Trp Ser Lys Leu Pro Ala Asp Arg Pro Ser Phe Cys Ser He His

845 850 855

Arg He Leu Gin Arg Met Cys Glu Arg Ala Glu Gly Thr Thr Asp

860 865 870

Gly Arg Ala His Phe Phe Trp Pro His Gin Tyr 875 880

(2) INFORMATION FOR SEQUENCE ID NO : 7 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 873 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: Figure 4c (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :

Met Arg Glu Leu Val Asn He Pro Leu Leu Gin Met Leu Thr Leu 5 10 15

Val Ala Phe Ser Gly Thr Glu Lys Leu Pro Lys Pro Pro Val He 20 25 30

Ala Thr Pro Leu Glu Thr Val Asp Ala Leu Val Glu Glu Val Ala 35 40 45

Thr Phe Met Cys Ala Val Glu Ser Tyr Pro Gin Pro Glu He Ser Trp Thr Arg Asn Lys He Leu He Lys Leu Phe Asp Thr Arg Tyr 65 70 75

Ser He Arg Glu Asn Gly Gin Leu Leu Thr He Leu Ser Val Glu 80 85 90

Asp Ser Asp Asp Gly He Tyr Cys Cys He Ala Asn Asn Gly Val 95 100 105

Gly Gly Ala Val Glu Ser Cys Gly Ala Leu Gin Val Lys Met Lys 110 115 120

Pro Lys He Thr Arg Pro Pro He Asn Val Lys He He Glu Gly 125 130 135

Leu Lys Ala Val Leu Pro Cys Thr Thr Met Gly Asn Pro Lys Pro 140 145 150

Ser Val Ser Trp He Lys Gly Asp Asn Ala Leu Arg Glu Asn Ser 155 160 165

Arg He Ala Ala Leu Glu Ser Gly Ser Leu Arg He His Asn Val 170 175 180

Gin Lys Glu Asp Ala Gly Gin Tyr Arg Cys Val Ala Lys Asn Ser 185 190 195

Leu Gly Thr Ala Tyr Ser Lys Leu Val Lys Leu Glu Val Glu Val 200 205 210

Leu Gly Arg He Leu Arg Ala Pro Glu Ser His Asn Val Thr Phe 215 220 225

Gly Ser Phe Val Thr Leu Arg Cys Thr Glu He Gly He Pro Val 230 235 240

Pro Thr He Ser Trp He Glu Asn Gly Asn Ala Val Ser Ser Gly 245 250 255

Ser He Gin Glu Ser Val Lys Asp Arg Val He Asp Ser Arg Leu 260 265 270

Gin Leu Phe He Thr Lys Pro Gly Leu Tyr Thr Cys He Ala Thr 275 280 285

Asn Lys His Gly Glu Lys Phe Ser Thr Ala Lys Ala Ala Ala Thr 290 295 300

Val Ser He Ala Glu Trp Ser Lys Ser Gin Lys Asp Ser Gin Gly 305 310 315

Tyr Cys Ala Gin Tyr Arg Gly Glu Gly Val Leu Met Gin Gly Pro 320 325 330

Gly Glu Lys Met Leu Leu Val Phe Leu Pro Thr Thr Ser His Arg 335 340 345

Asp Pro Glu Asp Ala Gin Glu Leu Leu He His Thr Ala Trp Asn 350 355 360

Glu Leu Lys Ala Val Ser Pro Leu Cys Arg Pro Ala Ala Glu Ala 365 370 375

Leu Leu Cys Tyr His Leu Phe Leu Glu Cys Ser Pro Gly Val Val 380 385 390 Pro Thr Pro Met Pro He Cys Arg Glu Tyr Cys Leu Ala Val Lys

395 400 405

Glu Leu Phe Cys Ala Lys Glu Trp Gin Ala Met Glu Gly Lys Ala

410 415 420

His Arg Gly Leu Tyr Arg Ser Gly Met His Leu Leu Pro Val Pro

425 430 435

Glu Cys Arg Lys Leu Pro Ser Met His Arg Asp Pro Thr Ala Cys

440 445 450

Thr Arg Leu Pro Tyr Leu Ala Phe Pro Ser He Thr Ser Ser Arg

455 460 465

Pro Ser Ala Asp He Pro Asn Leu Pro Ala Ser Thr Ser Ser Phe

470 475 480

Ala Val Ser Pro Ala Tyr Ser Met Thr Val He He Ser He Val

485 490 495

Ser Ser Leu Ala Leu Phe Ala Leu Leu Thr He Val Thr Leu Tyr

500 505 510

Cys Cys Arg Arg Arg Lys Glu Trp Lys Asn Lys Lys Arg Glu Ser

515 520 525

Thr Ala Val Thr Leu Thr Thr Leu Pro Ser Glu Leu Leu Leu Asp

530 535 540

Arg Leu His Pro Asn Pro Met Tyr Gin Arg Met Pro Leu Leu Leu

545 550 555

Asn Pro Lys Leu Leu Ser Leu Glu Tyr Pro Arg Asn Asn He Glu

560 565 570

Tyr Val Arg Asp He Gly Glu Gly Ala Phe Gly Arg Val Phe Gin

575 580 585

Ala Arg Ala Pro Gly Leu Leu Pro Tyr Glu Pro Phe Thr Met Val

590 595 600

Ala Val Lys Met Leu Lys Glu Glu Ala Ser Ala Asp Met Gin Ala

605 610 615

Asp Phe Gin Arg Glu Ala Ala Leu Met Ala Glu Phe Asp Asn Pro

620 625 630

Asn He Val Lys Leu Leu Gly Val Cys Ala Val Gly Lys Pro Met

635 640 645

Cys Leu Leu Phe Glu Tyr Met Ala Tyr Gly Asp Leu Asn Glu Phe

650 655 660

Leu Arg Ser Met Ser Pro His Thr Val Cys Ser Leu Ser His Ser

665 670 675

Asp Leu Ser Thr Arg Ala Arg Val Ser Ser Pro Gly Pro Pro Pro

680 685 690

Leu Ser Cys Ala Glu Gin Leu Cys He Ala Arg Gin Val Ala Ala

695 700 705

Gly Met Ala Tyr Leu Ser Glu Arg Lys Phe Val His Arg Asp Leu

710 715 720 Ala Thr Arg Asn Cys Leu Val Gly Glu Thr Met Val Val Lys He

725 730 735

Ala Asp Phe Gly Leu Ser Arg Asn He Tyr Ser Ala Asp Tyr Tyr

740 745 750

Lys Ala Asp Gly Asn Asp Ala He Pro He Arg Trp Met Pro Pro

755 760 765

Glu Ser He Phe Tyr Asn Arg Tyr Thr Thr Glu Ser Asp Val Trp

770 775 780

Ala Tyr Gly Val Val Leu Trp Glu He Phe Ser Tyr Gly Leu Gin

785 790 795

Pro Tyr Tyr Gly Met Ala His Glu Glu Val He Tyr Tyr Val Arg

800 805 810

Asp Gly Asn He Leu Ala Cys Pro Glu Asn Cys Pro Leu Glu Leu

815 820 825

Tyr Asn Leu Met Arg Leu Cys Trp Ser Lys Leu Pro Ala Asp Arg

830 835 840

Pro Ser Phe Cys Ser He His Arg He Leu Gin Arg Met Cys Glu

845 850 855

Arg Ala Glu Gly Thr Thr Asp Gly Arg Ala His Phe Phe Trp Pro

860 865 870

His Gin Tyr

(2) INFORMATION FOR SEQUENCE ID NO : 8 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 472 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ix) FEATURE:

(A) NAME/KEY: Figure 5a (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

AGC CCT GGA GTG GTA CCT ACT CCC ATG CCC ATT TGC AGA GAG TAC 45 Ser Pro Gly Val Val Pro Thr Pro Met Pro He Cys Arg Glu Tyr 5 10 15

TGC CTG GCG GTA AAG GAG CTC TTC TGT GCA AAG GAA TGG CAG GCA 90 Cys Leu Ala Val Lys Glu Leu Phe Cys Ala Lys Glu Trp Gin Ala 20 25 30

ATG GAA GGA AAG GCC CAC CGG GGC CTC TAC AGA TCT GGG ATG CAT 135 Met Glu Gly Lys Ala His Arg Gly Leu Tyr Arg Ser Gly Met His 35 40 45

CTC CTT CCG GTA CCA GAG TGC AGA AAG CTT CCC AGC ATG CAC CGG 180 Leu Leu Pro Val Pro Glu Cys Arg Lys Leu Pro Ser Met His Arg 50 55 60

GAC CCC ACA GCC TGC ACA AGA CTG CCA TAT TTA GGT AAC AAA GAA 225 Asp Pro Thr Ala Cys Thr Arg Leu Pro Tyr Leu Gly Asn Lys Glu 65 70 75 GTT CCT CCA GAC TTT GGA AGT 246 Val Pro Pro Asp Phe Gly Ser 80

TAAGGAAATG TTTACTGTTT CCAGTCAAGG AACGTTTGTA CTTAAAGCAC 296

CTAACATTTT TAGGCTTTCT AGGCTGTTCT AATAATGTTT TTTGCTTTGC 346

CATTTCTACA CACATAATTG CAGAATCCTA ATGCTCTCCT AATTGCATAG 396

GCTTTCATAA AGACTCATTT CATTTGTTTT CAAATAAATC AAAGGGCACT 446

TTTAAAAAAA AAAAAAAAAA AAAAAA 472

(2) INFORMATION FOR SEQUENCE ID NO : 9 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 467 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: Figure 5b (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :

Met Arg Glu Leu Val Asn He Pro Leu Leu Gin Met Leu Thr Leu 5 10 15

Val Ala Phe Ser Gly Thr Glu Lys Leu Pro Lys Pro Pro Val He 20 25 30

Ala Thr Pro Leu Glu Thr Val Asp Ala Leu Val Glu Glu Val Ala 35 40 45

Thr Phe Met Cys Ala Val Glu Ser Tyr Pro Gin Pro Glu He Ser 50 55 60

Trp Thr Arg Asn Lys He Leu He Lys Leu Phe Asp Thr Arg Tyr 65 70 75

Ser He Arg Glu Asn Gly Gin Leu Leu Thr He Leu Ser Val Glu 80 85 90

Asp Ser Asp Asp Gly He Tyr Cys Cys He Ala Asn Asn Gly Val 95 100 105

Gly Gly Ala Val Glu Ser Cys Gly Ala Leu Gin Val Lys Met Lys 110 115 120

Pro Lys He Thr Arg Pro Pro He Asn Val Lys He He Glu Gly 125 130 135

Leu Lys Ala Val Leu Pro Cys Thr Thr Met Gly Asn Pro Lys Pro 140 145 150

Ser Val Ser Trp He Lys Gly Asp Asn Ala Leu Arg Glu Asn Ser 155 160 165

Arg He Ala Ala Leu Glu Ser Gly Ser Leu Arg He His Asn Val 170 175 180

Gin Lys Glu Asp Ala Gly Gin Tyr Arg Cys Val Ala Lys Asn Ser 185 190 195

Leu Gly Thr Ala Tyr Ser Lys Leu Val Lys Leu Glu Val Glu Val 200 205 210

Leu Gly Arg He Leu Arg Ala Pro Glu Ser His Asn Val Thr Phe 215 220 225 Gly Ser Phe Val Thr Leu Arg Cys Thr Glu He Gly He Pro Val 230 235 240

Pro Thr He Ser Trp He Glu Asn Gly Asn Ala Val Ser Ser Gly 245 250 255

Ser He Gin Glu Ser Val Lys Asp Arg Val He Asp Ser Arg Leu 260 265 270

Gin Leu Phe He Thr Lys Pro Gly Leu Tyr Thr Cys He Ala Thr 275 280 285

Asn Lys His Gly Glu Lys Phe Ser Thr Ala Lys Ala Ala Ala Thr 290 295 300

Val Ser He Ala Glu Trp Ser Lys Ser Gin Lys Asp Ser Gin Gly 305 310 315

Tyr Cys Ala Gin Tyr Arg Gly Glu Gly Val Leu Met Gin Gly Pro 320 325 330

Gly Glu Lys Met Leu Leu Val Phe Leu Pro Thr Thr Ser His Arg 335 340 345

Asp Pro Glu Asp Ala Gin Glu Leu Leu He His Thr Ala Trp Asn 350 355 360

Glu Leu Lys Ala Val Ser Pro Leu Cys Arg Pro Ala Ala Glu Ala 365 370 375

Leu Leu Cys Tyr His Leu Phe Leu Glu Cys Ser Pro Gly Val Val 380 385 390

Pro Thr Pro Met Pro He Cys Arg Glu Tyr Cys Leu Ala Val Lys 395 400 405

Glu Leu Phe Cys Ala Lys Glu Trp Gin Ala Met Glu Gly Lys Ala 410 415 420

His Arg Gly Leu Tyr Arg Ser Gly Met His Leu Leu Pro Val Pro 425 430 435

Glu Cys Arg Lys Leu Pro Ser Met His Arg Asp Pro Thr Ala Cys 440 445 450

Thr Arg Leu Pro Tyr Leu Gly Asn Lys Glu Val Pro Pro Asp Phe 455 460 465

Gly Ser

(2) INFORMATION FOR SEQUENCE ID NO: 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 560 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: Figure 6a (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

TCC AAA CTG GTG AAG CTG GAA GTG GAG GGT AAG AGG CAG CAC GCA 45 Ser Lys Leu Val Lys Leu Glu Val Glu Gly Lys Arg Gin His Ala 5 10 15 CCC CTC ACT GTC TGCTGG GGA GGC TGT ACT TTG GAT GGA GAA AGA 90 Pro Leu Thr Val Cys Trp Gly Gly Cys Thr Leu Asp Gly Glu Arg 20 25 30

TAT GAT GTA GGA AGT TGT GGT TCA CCA GCT CTG AGG GCA AGG CTT 135 Tyr Asp Val Gly Ser Cys Gly Ser Pro Ala Leu Arg Ala Arg Leu 35 40 45

TGAAAACAGA GAAGTCAAAA GACAGTTCCC TAGCATAAAC GTTCATATCC 185

TATCTATTGC CCCAACTGCT GCTACGTTTG ACCGTGACAC CATTTTAGTG 235

TGACTTTCCA GCTCAAACAG TTCTCTTATT TATAAGATTG GCTACAAATA 285

AGTTTATCTT TTAATGCATT TGGATTGACT TTAAAACATA GGGAGATAGA 335

AAAAAAATTA ATCCTTTCCA AATTATGGCT TCACAAAGGA TGCAAATAAT 385

AAGCCAATGA ATCAAACATA TAAACATGGT CCTAATTCTG TGAGTTAGGT 435

AGCAATTAAT GTTATGAGCA TAGTAACAGT TTATCCAAAT ATAATGTATT 485

TTTAATGAGG TCATTCAATA AATTGCTGGG GAAAAATCTT CATTGAGAAA 535

TCAGAATCCA AAAAAAAAAA AAAAA 560

(2) INFORMATION FOR SEQUENCE ID NO: 11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 245 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: Figure 6b (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met Arg Glu Leu Val Asn He Pro Leu Leu Gin Met Leu Thr Leu

5 10 15

Val Ala Phe Ser Gly Thr Glu Lys Leu Pro Lys Pro Pro Val He

20 25 30

Ala Thr Pro Leu Glu Thr Val Asp Ala Leu Val Glu Glu Val Ala

35 40 45

Thr Phe Met Cys Ala Val Glu Ser Tyr Pro Gin Pro Glu He Ser

50 55 60

Trp Thr Arg Asn Lys He Leu He Lys Leu Phe Asp Thr Arg Tyr

65 70 75

Ser He Arg Glu Asn Gly Gin Leu Leu Thr He Leu Ser Val Glu

80 85 90

Asp Ser Asp Asp Gly He Tyr Cys Cys He Ala Asn Asn Gly Val

95 100 105

Gly Gly Ala Val Glu Ser Cys Gly Ala Leu Gin Val Lys Met Lys

110 115 120

Pro Lys He Thr Arg Pro Pro He Asn Val Lys He He Glu Gly

125 130 135

Leu Lys Ala Val Leu Pro Cys Thr Thr Met Gly Asn Pro Lys Pro

140 145 150

Ser Val Ser Trp He Lys Gly Asp Asn Ala Leu Arg Glu Asn Ser

155 160 165

Arg He Ala Ala Leu Glu Ser Gly Ser Leu Arg He His Asn Val

170 175 180 Gin Lys Glu Asp Ala Gly Gin Tyr Arg Cys Val Ala Lys Asn Ser 185 190 195

Leu Gly Thr Ala Tyr Ser Lys Leu Val Lys Leu Glu Val Glu Gly 200 205 210

Lys Arg Gin His Ala Pro Leu Thr Val Cys Trp Gly Gly Cys Thr 215 220 225

Leu Asp Gly Glu Arg Tyr Asp Val Gly Ser Cys Gly Ser Pro Ala 230 235 240

Leu Arg Ala Arg Leu 245

(2) INFORMATION FOR SEQUENCE ID NO: 12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 961 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: Torpedo RTK (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 12 :

Val Asp He Pro Leu Leu Met He Phe Leu Val Thr Thr Gly Gly 5 10 15

Ser Ala Asp Gly He Leu Pro Lys Ala Pro Gin He Thr Ser Pro 20 25 30

Leu Glu Thr Val Asp Ala Leu Val Glu Glu Glu Ala Ser Phe Met 35 40 45

Cys Ala Val Asp Ser Tyr Pro Ala Ala Glu He Thr Trp Thr Arg 50 55 60

Asn Asn He Pro He Arg Pro Phe Asp Thr Arg Tyr Ser Thr Lys 65 70 80

Glu Asn Gly Gin He Leu Thr He Leu Ser Val Glu Asp Thr Asp 85 90 95

Asn Gly Val Tyr Cys Cys Thr Ala Asn Asn Gly Met Gly Ser Ser 100 105 110

Ala Gin Ser Cys Gly Ala Leu Gin Val Lys Met Lys Pro Lys He 115 120 125

He Arg Pro Pro Thr Asp Val Arg Ala Leu Leu Gly Ser Lys Val 130 135 140

Val Leu Pro Cys Ser Thr Met Gly Asn Pro Lys Pro Ala He Ser 145 150 155

Trp Phe Lys Asp Glu Thr Ala Leu Lys Asn Asp Gin Pro Arg Thr 160 165 170

Ser Val Leu Glu Ser Gly Asn Leu Arg He Arg Asn Val Gin Leu 175 180 190

Glu Asp Ala Gly Lys Tyr Arg Cys Leu Ala Arg Asn Ser Leu Gly 195 200 205 Phe Glu Tyr Ser Arg Ser Ala Ala Leu Glu Val Gin Val Ser Ala 210 215 220

Arg He Val Lys Ala Pro Thr Ser Gin Asn Val Ser Tyr Gly Ser 225 230 235

Glu Val He Leu Gin Cys Lys Ala Thr Gly Phe Pro He Pro Thr 240 245 250

He Lys Trp Leu Glu Asn Gly Arg Ala Val Pro Lys Gly Ser He 255 260 265

Gin Asn Arg He Lys Gly Glu Val Met Glu Ser Arg Leu Arg Val 270 275 280

Tyr Val Thr Arg Pro Ser Leu Phe Thr Cys Leu Thr Thr Asn Lys 285 290 300

His Asn Glu Gly Ser Thr Thr Ala Lys Ala Thr Ala Thr Leu Asp 305 310 315

He Lys Glu Trp Arg Leu Tyr Lys Gly Asp Leu Gly Tyr Cys Ser 320 325 330

Thr Tyr Arg Gly Glu Val Cys Gin Gly Leu Leu Gly Asn Gly Gin 335 340 345

Leu Val Phe Phe Asn Ser Ser Phe Ala Asp Ala Glu Gly Thr Gin 350 355 360

Glu Met Met Ala Arg Ser Thr Trp Thr Glu Leu Asp Gly Val Ser 365 370 375

Leu Leu Cys Lys Pro Ala Ala Glu Ser Leu Leu Cys His Phe He 380 385 390

Phe Gin Asp Cys Asn Pro Leu Gly Leu Gly Pro Thr Pro Lys Leu 395 400 405

Val Cys Arg Glu His Cys Leu Ala Val Lys Glu Leu Tyr Cys Tyr 410 415 420

Lys Glu Trp He Thr Met Glu Asp Asn Ser Arg He Gly Val Tyr 425 430 435

Ser Ala Gly Leu Ser Leu Pro Asp Cys Gin Arg Leu Pro Ser He 440 445 450

His His Asp Pro Glu Ala Cys Thr Arg Val Ser Phe Leu Asp Met 455 460 465

Lys Lys Gly Leu Val Thr Arg Met Cys Tyr Asn Asn Asn Gly Arg 470 475 480

Phe Tyr Gin Gly Ser Val Asn Val Thr Ala Ser Gly He Ser Cys 490 495 500

Gin Arg Trp Ser Glu Gin Ala Pro His Phe His Arg Arg Leu Pro 505 510 515

Glu He Phe Pro Glu Leu Ala Asn Ser Asp Asn Phe Cys Arg Asn 520 525 530

Pro Gly Gly Glu Ser Glu Arg Pro Trp Cys Tyr Thr Met Asp Arg 535 540 545 Asp He Arg Trp Glu Phe Cys Asn Val Pro Gin Cys He Asn Val

550 555 560

Ser Ser He Ser Glu Met Lys Pro Lys Thr Glu Thr Ala Asn Thr

565 570 575

Pro Ser Thr Ser Ala Thr Tyr Ser Met Thr Val He He Ser He

580 585 590

He Ser Ser Leu Ala Ala Ser He Leu Leu He He He He Leu

595 600 605

Thr Cys His His His Gin Lys Gly Leu Gin Thr Arg Lys Ser Tyr

610 615 620

Arg Thr Thr Glu Thr Pro Thr Leu Ala Thr Leu Pro Ser Glu Leu

625 630 635

Leu Leu Asp Arg Leu His Pro Asn Pro Met Tyr Gin Arg Leu Pro

640 645 650

Leu Leu Leu Asn Ala Lys Leu Leu Ser Leu Glu Tyr Pro Arg Asn

655 660 665

Asn He Glu Tyr Val Arg Asp He Gly Glu Gly Ala Phe Gly Arg

670 675 680

Val Phe Gin Ala Arg Ala Pro His Leu Leu Pro Gin Glu Thr Ser

685 690 695

Thr Met Val Ala Val Lys Met Leu Lys Glu Glu Ala Ser Pro Asp

700 705 710

Met Gin Ala Asp Phe Arg Arg Glu Ala Ala Leu Met Ala Glu Phe

715 720 725

Asn His Pro Asn He Val Lys Leu Leu Gly Val Cys Ala Val Gly

730 735 740

Lys Pro Met Cys Leu Leu Phe Glu Tyr Met Ala His Gly Asp Leu

745 750 755

Asn Glu Tyr Leu Arg Lys Arg Ser Pro He Thr Ala Arg Thr Leu

760 765 770

Arg Pro Ala Asn Cys Val Gly Trp Ser Ser Gly Trp Gly Lys Gly

775 780 785

Leu Thr Ala Leu Ser Cys Ala Asp Gin Leu Asn He Ala Lys Gin

790 795 800

He Ser Ala Gly Met Thr Tyr Leu Ser Glu Arg Lys Phe Val His

805 810 815

Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Lys Leu Val

820 825 830

Val Lys He Ala Asp Phe Gly Leu Ser Arg Asn He Tyr Ser Ala

835 840 845

Asp Tyr Tyr Lys Ala Asn Glu Asn Asp Ala He Pro He Arg Trp

850 855 860

Met Pro Pro Glu Ser He Phe Phe Asn Arg Tyr Thr Thr Glu Ser

865 870 875 Asp Val Trp Ala Tyr Gly Val Val Leu Trp Glu He Phe Ser Ser

880 885 890

Gly Met Gin Pro Tyr Tyr Gly Met Ala His Glu Glu Val He Tyr

895 900 905

Tyr Val Arg Asp Gly Asn He Leu Ser Cys Pro Glu Asn Cys Pro

910 915 920

Pro Glu Leu Tyr Asn Leu Met Arg Leu Cys Trp Ser Asn Met Pro

925 930 935

Ser Asp Arg Pro Thr Phe Ala Ser He His Arg He Leu Glu Arg

940 945 950

Met His Gin Arg Met Ala Ala Ala Leu Pro Val 955 960

(2) INFORMATION FOR SEQUENCE ID NO: 13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 201 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ix) FEATURE:

(A) NAME/KEY: Figure 9a (Nsk 1) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

CAC CGC GAC CTG GCC ACC CGG AAC TGC CTC GTA GGA GAG GAG ATG 45 His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Glu Met 5 10 15

GTG GTT AAG ATC GCC GAC TTC GGT TTA TCC AGA AAC ATC TAC TCT 90 Val Val Lys He Ala Asp Phe Gly Leu Ser Arg Asn He Tyr Ser 20 25 30

GCT GAC TAC TAC AAG GCC AAT GAG AAC GAT GCT ATC CCC ATC CGC 135 Ala Asp Tyr Tyr Lys Ala Asn Glu Asn Asp Ala He Pro He Arg 35 40 45

TGG ATG CCC CCG GAG TCC ATC TTC TAC AAC CGT TAC ACC ACG GAG 180 Trp Met Pro Pro Glu Ser He Phe Tyr Asn Arg Tyr Thr Thr Glu 50 55 60

TCG GAC GTC TGG TCC TAT GGA 201

Ser Asp Val Trp Ser Tyr Gly

(2) INFORMATION FOR SEQUENCE ID NO: 14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: page 10 of published PCT (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Asp Tyr Lys Lys Glu Asn He Thr Thr 5

(2) INFORMATION FOR SEQUENCE ID NO: 15: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: peptide immunogen (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Cys Gly Asn Lys Glu Val Pro Pro Asp Phe Gly Ser 5 10

(2) INFORMATION FOR SEQUENCE ID NO: 16: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: peptide immunogen (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Cys Thr Leu Asp Gly Glu Arg Tyr Asp Val Gly Ser 5 10

(2) INFORMATION FOR SEQUENCE ID NO: 17: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: peptide immunogen (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Cys Thr Thr Ser His Arg Asp Pro Glu Asp Ala Gin Glu 5 10 (2) INFORMATION FOR SEQUENCE ID NO: 18: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: peptide motif (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

He Glu Asn Pro Gin Tyr Phe Ser Asp Ala 5 10

(2) INFORMATION FOR SEQUENCE ID NO: 19: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: peptide motif (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

His Pro Asn Pro Met Tyr Gin Arg Met Pro 5 10

(2) INFORMATION FOR SEQUENCE ID NO: 20: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: peptide motif (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

Pro Glu Val Tyr Ala He Met Arg Gly 5

(2) INFORMATION FOR SEQUENCE ID NO: 21: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: peptide motif (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

Leu Glu Leu Tyr Asn Leu Met Arg Leu 5 (2) INFORMATION FOR SEQUENCE ID NO: 22: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 867 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: Figure 8 (MSN-2) ) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

Val Asn He Pro Leu Leu Gin Met Leu Thr Leu Val Ala Phe Ser 5 10 15

Gly Thr Glu Lys Leu Pro Lys Pro Pro Val He Ala Thr Pro Leu 20 25 30

Glu Thr Val Asp Ala Leu Val Glu Glu Val Ala Thr Phe Met Cys 35 40 45

Ala Val Glu Ser Tyr Pro Gin Pro Glu He Ser Trp Thr Arg Asn 50 55 60

Lys He Leu He Lys Leu Phe Asp Thr Arg Tyr Ser He Arg Glu 65 70 75

Asn Gly Gin Leu Leu Thr He Leu Ser Val Glu Asp Ser Asp Asp 80 85 90

Gly He Tyr Cys Cys He Ala Asn Asn Gly Val Gly Gly Ala Val 95 100 105

Glu Ser Cys Gly Ala Leu Gin Val Lys Met Lys Pro Lys He Thr 110 115 120

Arg Pro Pro He Asn Val Lys He He Glu Gly Leu Lys Ala Val 125 130 135

Leu Pro Cys Thr Thr Met Gly Asn Pro Lys Pro Ser Val Ser Trp 140 145 150

He Lys Gly Asp Asn Ala Leu Arg Glu Asn Ser Arg He Ala Ala 155 160 165

Leu Glu Ser Gly Ser Leu Arg He His Asn Val Gin Lys Glu Asp 170 175 180

Ala Gly Gin Tyr Arg Cys Val Ala Lys Asn Ser Leu Gly Thr Ala 185 190 195

Tyr Ser Lys Leu Val Lys Leu Glu Val Glu Val Leu Gly Arg He 200 205 210

Leu Arg Ala Pro Glu Ser His Asn Val Thr Phe Gly Ser Phe Val 215 220 225

Thr Leu Arg Cys Thr Glu He Gly He Pro Val Pro Thr He Ser 230 235 240

Trp He Glu Asn Gly Asn Ala Val Ser Ser Gly Ser He Gin Glu 245 250 255

Ser Val Lys Asp Arg Val He Asp Ser Arg Leu Gin Leu Phe He 260 265 270 Thr Lys Pro Gly Leu Tyr Thr Cys He Ala Thr Asn Lys His Gly 275 280 285

Glu Lys Phe Ser Thr Ala Lys Ala Ala Ala Thr Val Ser He Ala 290 295 300

Glu Trp Ser Lys Ser Gin Lys Asp Ser Gin Gly Tyr Cys Ala Gin 305 310 315

Tyr Arg Gly Glu Gly Val Leu Met Gin Gly Pro Gly Glu Lys Met 320 325 330

Leu Leu Val Phe Leu Pro Thr Thr Ser His Arg Asp Pro Glu Asp 335 340 345

Ala Gin Glu Leu Leu He His Thr Ala Trp Asn Glu Leu Lys Ala 350 355 360

Val Ser Pro Leu Cys Arg Pro Ala Ala Glu Ala Leu Leu Cys Tyr 365 370 375

His Leu Phe Leu Glu Cys Ser Pro Gly Val Val Pro Thr Pro Met 380 385 390

Pro He Cys Arg Glu Tyr Cys Leu Ala Val Lys Glu Leu Phe Cys 395 400 405

Ala Lys Glu Trp Gin Ala Met Glu Gly Lys Ala His Arg Gly Leu 410 415 420

Tyr Arg Ser Gly Met His Leu Leu Pro Val Pro Glu Cys Arg Lys 425 430 435

Leu Pro Ser Met His Arg Asp Pro Thr Ala Cys Thr Arg Leu Pro 440 445 450

Tyr Leu Asp Tyr Lys Lys Glu Asn He Thr Thr Phe Pro Ser He 455 460 465

Thr Ser Ser Arg Pro Ser Ala Asp He Pro Asn Leu Pro Ala Ser 470 475 480

Thr Ser Ser Phe Ala Val Ser Pro Ala Tyr Ser Met Thr Val He 485 490 495

He Ser He Val Ser Ser Leu Ala Leu Phe Ala Leu Leu Thr He 500 505 510

Val Thr Leu Tyr Cys Cys Arg Arg Arg Lys Glu Trp Lys Asn Lys 515 520 525

Lys Arg Glu Ser Thr Ala Val Thr Leu Thr Thr Leu Pro Ser Glu 530 535 540

Leu Leu Leu Asp Arg Leu His Pro Asn Pro Met Tyr Gin Arg Met 545 550 555

Pro Leu Leu Leu Asn Pro Lys Leu Leu Ser Leu Glu Tyr Pro Arg 560 565 570

Asn Asn He Glu Tyr Val Arg Asp He Gly Glu Gly Ala Phe Gly 575 580 585

Arg Val Phe Gin Ala Arg Ala Pro Gly Leu Leu Pro Tyr Glu Pro 590 595 600 Phe Thr Met Val Ala Val Lys Met Leu Lys Glu Glu Ala Ser Ala 605 610 615

Asp Met Gin Ala Asp Phe Gin Arg Glu Ala Ala Leu Met Ala Glu 620 625 630

Phe Asp Asn Pro Asn He Val Lys Leu Leu Gly Val Cys Ala Val 635 640 645

Gly Lys Pro Met Cys Leu Leu Phe Glu Tyr Met Ala Tyr Gly Asp 650 655 660

Leu Asn Glu Phe Leu Arg Ser Met Ser Pro His Thr Val Cys Ser 665 670 675

Leu Ser His Ser Asp Leu Ser Thr Arg Ala Arg Val Ser Ser Pro 680 685 690

Gly Pro Pro Pro Leu Ser Cys Ala Glu Gin Leu Cys He Ala Arg 695 700 705

Gin Val Ala Ala Gly Met Ala Tyr Leu Ser Glu Arg Lys Phe Val 710 715 720

His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Thr Met 725 730 735

Val Val Lys He Ala Asp Phe Gly Leu Ser Arg Asn He Tyr Ser 740 745 750

Ala Asp Tyr Tyr Lys Ala Asp Gly Asn Asp Ala He Pro He Arg 755 760 765

Trp Met Pro Pro Glu Ser He Phe Tyr Asn Arg Tyr Thr Thr Glu 770 775 780

Ser Asp Val Trp Ala Tyr Gly Val Val Leu Trp Glu He Phe Ser 785 790 795

Tyr Gly Leu Gin Pro Tyr Tyr Gly Met Ala His Glu Glu Val He 800 805 810

Tyr Tyr Val Arg Asp Gly Asn He Leu Ala Cys Pro Glu Asn Cys 815 820 825

Pro Leu Glu Leu Tyr Asn Leu Met Arg Leu Cys Trp Ser Lys Leu 830 835 840

Pro Ala Asp Arg Pro Ser Phe Cys Ser He His Arg He Leu Gin 845 850 855

Arg Met Cys Glu Arg Ala Glu Gly Thr Val Gly Val 860 865 (2) INFORMATION FOR SEQUENCE ID NO: 23: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 57 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: Figure 9b (Nsk 1: amino acids 5-67 of SEQ ID NO: 13) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Ala Thr Arg Asn Cys Leu Val Gly Glu Glu Met Val Val Lys He 5 10 15

Ala Asp Phe Gly Leu Ser Arg Asn He Tyr Ser Ala Asp Tyr Tyr 20 25 30

Lys Ala Asn Glu Asn Asp Ala He Pro He Arg Trp Met Pro Pro 35 40 45

Glu Ser He Phe Tyr Asn Arg Tyr Thr Thr Glu Ser 50 55

(2) INFORMATION FOR SEQUENCE ID NO: 24: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 57 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (ix) FEATURE:

(A) NAME/KEY: Figure 9b (Amino acids 819-875 of Torpedo RTK) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

Ala Thr Arg Asn Cys Leu Val Gly Glu Lys Leu Val Val Lys He 5 10 15

Ala Asp Phe Gly Leu Ser Arg Asn He Tyr Ser Ala Asp Tyr Tyr 20 25 30

Lys Ala Asn Glu Asn Asp Ala He Pro He Arg Trp Met Pro Pro 35 40 45

Glu Ser He Phe Phe Asn Arg Tyr Thr Thr Glu Ser 50 55

Claims

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 N0:1,

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 nucleic acid molecule of claim 1.

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

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

SEQ ID NO:l,

SEQ ID NO: 6,

SEQ ID NO: 8,

SEQ ID NO: 10, and

SEQ ID NO: 13.

The isolated muscle receptor tyrosine kinase of claim 6, consisting of the amino 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: 11.

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.

13. A method for screening for a neuromuscular disease, comprising assaying a sample taken from a subject believed to have a neuromuscular disease, determining level of at least one receptor tyrosine kinase in said sample and comparing said level to a control level, wherein variation therefrom is indicative of possible presence of a neuromuscular disease.

14. The method of claim 13, wherein said neuromuscular disease is muscular dystrophy.

15. The method of claim 13, wherein said receptor tyrosine kinase is Nsk2 or a truncated form thereof.

16. The method of claim 15, wherein said truncated form is 2Ig or 4Ig.

17. The method of claim 13, comprising determining said receptor tyrosine kinase via an immunoassay.

18. A method for monitoring progression or regression of a neuromuscular disease, comprising assaying a sample taken from a patient with a neuromuscular disease for a receptor tyrosine kinase, and comparing a value of said receptor tyrosine kinase to a value for said tyrosine kinase taken at a previous point in time, wherein variation therebetween is indicative of progression or regression of said neuromuscular disease.

19. A method for determining efficacy of a therapeutic agent in treatment of a neuromuscular disorder, comprising assaying a sample from a subject with a neuromuscular disorder for a receptor tyrosine kinase prior to administering said therapeutic agent, administering said therapeutic agent, and assaying a second sample taken from said subject following administration of said therapeutic agent for said receptor tyrosine kinase, wherein a change in said level is indicative of efficacy of said therapeutic agent.