WO1997014966A1

WO1997014966A1 - Method of activating a novel ligand regulatory pathway

Info

Publication number: WO1997014966A1
Application number: PCT/CA1996/000679
Authority: WO
Inventors: Anthony Pawson; Mark Henkemeyer
Original assignee: Mount Sinai Hospital Corporation
Priority date: 1995-10-13
Filing date: 1996-10-10
Publication date: 1997-04-24
Also published as: JPH11515105A; MX9802883A; EP1019728A1

Abstract

A novel ligand regulatory pathway is disclosed and methods of activating the novel pathway in a cell expressing a ligand for an Eph subfamily receptor tyrosine kinase protein. Methods are provided for identifying substances capable of activating the ligand regulatory pathway. Therapeutic methods for affecting neuronal development and regeneration and pharmaceutical compositions using the substances and Eph subfamily receptor tyrosine kinase proteins are also described.

Description

Title: Method of Activating a Novel Ligand Regulatory Pathway

FIELD OF THE INVENTION

The invention relates to a novel ligand regulatory pathway, to methods for identifying substances capable of activating the novel pathway, methods for assaying for agonists or antagonists of the novel pathway, and to methods and pharmaceutical compositions for affecting neuronal development and regeneration. BACKGROUND OF THE INVENTION

Embryonic development of multicellular organisms is a highly ordered process that requires coordination of individual cells. Every cell must decipher the numerous signals it receives and then properly execute commands in order to achieve the correct position and differentiated state in the animal. The exquisite controls over cell growth, determination, migration and adhesion are mediated by molecules located on the plasma membrane surface.

A class of membrane associated molecules known to regulate cellular interactions are receptor tyrosine kinase proteins. The evolutionary conservation of genes encoding receptor tyrosine kinase proteins and their targets has emphasized the importance of these proteins in intracellular communication, and has also provided model systems for genetic analysis of tyrosine kinase signalling pathways.

A growing number of closely related transmembrane receptor tyrosine kinase proteins containing cell adhesion-like domains on their extracelluar surface have been identified. Collectively, this group of proteins defines the Eph subfamily, which is made up of at least thirteen related but unique gene sequences in higher vertebrates (Hirai et al., Science 238:1717-1720, 1987; Letwin et al., Oncogene 3:621-627, 1988; Lindberg et al., Mol. Cell. Biol. 10:6316-6324, 1990; Lhotak et al, Mol. Cell. Biol. 11:2496-2502, 1991; Chan and Watt, Oncogene 6:1057-1061, 1991; Lai and Lemke, Neuron 6:691-704, 1991; Pasquale, Cell Regulation 2:523-534, 1991; Sajjadi et al, New Biologist 3:769-778, 1991; Wicks et al, PNAS 89:1611-1615, 1992; Gilardi-Hebenstreit et al., Oncogene 7:2499-2506, 1992; Bohme et al., Oncogene 8:2857-2862, 1993; Sajjadi and Pasquale, Oncogene 8:1801-1813, 1993). The presence of cell adhesion-like domains in this family of tyrosine kinases suggests that these proteins function in cell-cell interactions. The other major families of proteins implicated in cell adhesion include the cadherins, selectins, integrins, and those of the immunoglobulin superfamily (reviewed by Hynes, R.O. and Landers, A.D., Cell 68, 303-322, 1992). The extracelluar regions of cell adhesion molecules frequently contain peptide repeats, such as FN III motifs, epidermal growth factor (EGF) repeats, or Ig loops that may direct protein-protein interactions at the cell surface. A number of cell adhesion molecules in both vertebrates (Dodd, J. and Jessell, T.M., Science, 242, 692-699, 1988; Jessell, T.M., Neuron, 1, 3-13, 1988; Furley et al., Cell 61, 157- 170, 1990; Burns et al, Neuron, 7, 209-220, 1991) and invertebrates (Bastiani et al., Cell 48:745-755, 1987; Elkins et al, Cell 60:565-575, 1990; Grenningloh et al, Cold Spring Harb. Symp. Quant. Biol 55, 327-340, 1991; Nose et al, Cell 70:553-567, 1992) have been implicated in axonal growth cone guidance and pathway /target recognition. Other aspects of neuronal morphogenesis involving cell-cell interactions may also require the activities of cell adhesion molecules (Edelman and Thiery, In The Cell in Contact: Adhesions and Junctions as Morphogenetic Determinants, Wiley, New York, 1985; Hatta et al., Dev. Biol 120:215-227, 1987; Takeichi, Development 102:639-655, 1988; Takeichi, Annu. Rev. Biochem. 59:237-252 1990; Takeichi, Science 251:1451-1455, 1991; Edelman, Biochemistry 27:3533-3543, 1988; Grumet, Curr. Opin. NeurobioL 1:370-376, 1991; Hynes and Lander, Cell 68:303-322, 1992). For example, ectopic N-cadherin expression during gastrulation stage Xenopus embryos has been shown to interfere with segregation of the neural tube from the ectoderm (Derrick et al., Neuron 4:493-506, 1990; Fujimori et al., Development 110:97-104, 1990). Although many different types of cell adhesion molecules have been identified, Uttle is known about how these adhesive interactions are regulated and how they function in cell signalling pathways during normal development. A critical stage in the development of the nervous system is the projection of axons to their targets. Navigational decisions are made at the growth cones of the migrating axons. As axons grow their growth cones extend and retract filopodia and lamellipodia processes which are implicated in the navigational decisions and pathfinding abilities of migrating axons. Like peripheral nervous system axons, the growth cones of neurons associated with the central nervous system follow stereotyped pathways and apparently can selectively chose from a number of possible routes (reviewed by Goodman and Shatz, Cell 72:77-98, 1993). Early pathways in the vertebrate embryonic brain are thought to be arranged as a set of longitudinal tracts connected by commissures. However, the molecular mechanisms that underlay growth cone navigation axon pathfinding and commissure formation in development are poorly understood (Hynes, R.O. and Lander, A.D., 1992, Cell 68:303).

It is a fundamental principle of nervous system wiring that the projections of neurons from one region of the nervous system to another are organized topographically. During embryonic development a multitude of incoming axons must find and connect with a corresponding set of target cells to form a continuous topographic map. It has been suggested that formation and refinement of the topographic map of neurons may be directed in part by positional labels displayed on the surface of developing and migrating neurons. However, to date such positional labels have not been identified (Tessier-Lavigne, 1995, Cell 82:345-348). Recently, ligands for receptor tyrosine kinases of the Eph subfamily have been implicated as positional labels in the retinotectal system (Drescher et al., 1995 Cell 82:359-370). The developmental function of tyrosine kinases during axonogenesis has been studied in Drosophila. A function in axonal pathfinding is evident for the Drosophila abl tyrosine kinase when abl mutations are combined with mutations in other genes including the neural cell adhesion molecule, fasciclin I (fas I, Elkins et ah, Cell 60:565-575, 1990) or disabled (dab, Gertler et al., Cell 58:103-113, 1989). These studies have shown that the abl tyrosine kinase is specifically localized to the axonal compartment of the embryonic Central Nervous System (CNS) (Gertler et al, Cell 58:103-113, 1989). Moreover, genetic analysis has indicated that subcellular localization to axons is essential for abl function during development (Henkemeyer et al., Cell 63:949-960, 1990) and that mutations in second-site modifier genes including fas I and dab can reveal a role for abl in axonogenesis (Elkins et al, Cell 60:565-575, 1990; Gertler et al., Cell 58:103-113 1989). The requirement for tyrosine phosphorylation in axonal outgrowth and adhesion in Drosophila is strengthened by the identification in CNS axons of three transmembrane tyrosine phosphatases containing FN in motifs (Tian et al, Cell 67:675-685, 1991; Yang et al, Cell 67:661-673, 1991). SUMMARY OF THE INVENTION

The present inventors have identified and characterized a novel ligand regulatory pathway that plays a crucial role in cell-cell interactions and axonogenesis in the development and regeneration of the nervous system. The present inventors have determined that Eph subfamily receptor tyrosine kinases activate a ligand regulatory pathway in cells expressing ligands for the Eph subfamily receptor tyrosine kinases. Activation of the Ugand regulatory pathway results in downstream activation of a series of regulatory pathways in the cells that control gene expression, cell division, cytoskeletal architecture, cell metabohsm. cell migration and ceU-cell interactions. The ligand regulatory pathway may be activated by an Eph subfamily receptor tyrosine kinase lacking in an active catalytic kinase domain.

In particular, the inventors have demonstrated that expression of an Eph subfamily receptor tyrosine kinase is essential for formation of a commissure in the brain and that this essential function is independent of an intact catalytic kinase domain. The direct demonstration of a vital function in neuronal development for an Eph subfamily receptor tyrosine kinase is unprecedented, as is the showing of a function for a receptor tyrosine kinase which is mediated by the extracellular domain, independently of the catalytic kinase domain of the receptor. The inventors have demonstrated for the first time that a protein having the extracellular, transmembrane and juxtamembrane domains of an Eph subfamily receptor tyrosine kinase can provide a signal to a cell expressing a Ugand for the receptor tyrosine kinases and thereby activate a ligand regulatory pathway in the cell expressing the Ugand.

Accordingly, the present invention provides a method of activating a ligand regulatory pathway in a ceU, comprising reacting an Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein, with a cell expressing a Ugand for an Eph subfamily receptor tyrosine kinase on the surface of the cell thereby activating the ligand regulatory pathway in the cell. In an embodiment, the protein or part of the protein is lacking in catalytic kinase activity. In a further embodiment, the part of the protein comprises an extraceUular, transmembrane and juxtamembrane domain, or only an extracellular domain of an Eph subfamUy receptor tyrosine kinase, preferably Nuk.

The invention also provides a method for identifying a substance which is capable of binding to a Ugand for an Eph subfamily receptor tyrosine kinase and activating a ligand regulatory pathway in a cell, comprising reacting a cell expressing a ligand for an Eph subfamily receptor tyrosine kinase on the surface of the ceU, with at least one test substance, under conditions which permit the formation of substance-Ugand complexes, and assaying for substance-Ugand complexes, for free substance, for non-complexed Ugands, or for activation of the ligand.

Activation of the ligand may be assayed by measuring phosphorylation of the Ugand, or binding of SH2 domains to the ligand, or by assaying for a biological affect on the ceU, such as inhibition or stimulation of proliferation, differentiation or migration.

In an embodiment of the method, the substance is an Eph subfamily receptor tyrosine kinase protein, which is not the native receptor tyrosine kinase protein for the Ugand, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein. In a further embodiment the part of the protein comprises an extracellular, transmembrane and juxtamembrane domain. In a stiU further embodiment the part of the protein comprises an extraceUular domain. Another aspect of the invention provides a method for assaying a medium for an agonist or antagonist of a Ugand regulatory pathway in a cell which comprises providing a cell expressing a Ugand for an Eph subfamily receptor tyrosine kinase on the surface of the cell, reacting the ceU with an Eph subfamily receptor tyrosine kinase protein or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein, and a suspected agonist or antagonist, under conditions which permit the formation of ligand-receptor tyrosine kinase protein complexes on the cell surface, and assaying for ligand-receptor tyrosine kinase protein complexes, for free receptor tyrosine kinase protein, for non-complexed proteins, for activation of the receptor tyrosine kinase protein, or for activation of the Ugand. In an embodiment, activation of the ligand is assayed by measuring phosphorylation of the Ugand or binding of SH2 domains to the ligand or by assaying for a biological affect on the cell, such as inhibition or stimulation of proliferation, differentiation or migration.

The invention stiU further provides a method for affecting neuronal development or regeneration in a mammal comprising administering to a mammal an effective amount of a purified and isolated Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein. In an embodiment, the protein or part of the protein is lacking in a catalytic kinase domain. In another embodiment, the part of the protein comprises an extracellular, juxtamembrane or transmembrane domain. In a further embodiment, the part of the protein comprises at least one of an extracellular, juxtamembrane and transmembrane domain, preferably an extracellular domain. In yet another aspect, the invention provides a method for stimulating or inhibiting axonogenesis in a mammal comprising administering to a mammal an effective amount of a purified and isolated Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein. In an embodiment, the part of the protein comprises an extraceUular domain of an Eph subfamily receptor tyrosine kinase. In a further embodiment, the protein or part of the protein is lacking in a catalytic kinase domain.

The invention also relates to a pharmaceutical composition which comprises a purified and isolated Eph subfamily receptor tyrosine kinase protein or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein for affecting neuronal development or regeneration and a pharmaceutically acceptable carrier, diluent or excipient. The part of the protein may comprise an extraceUular domain of an Eph subfamily receptor tyrosine kinase, and the protein or part of the protein may be lacking in a catalytic kinase domain. DESCRIPTION OF THE DRAWINGS The invention wUl be better understood with reference to the drawings in which:

Figure 1 shows the amino acid sequences of members of the Eph subfamily of receptor tyrosine kinases, dots indicate spaces introduced in order to optimize alignment, conserved cysteine residues are marked with asterisks and, arrows indicate the boundaries of the catalytic kinase domain; Figure 2 shows the nucleotide sequence encoding the Nuk tyrosine kinase protein as shown in SEQ ID NO: 1;

Figure 3 shows the amino acid sequence of Nuk tyrosine kinase protein as shown in SEQ ID NO:2 and a schematic diagram of the regions of the Nuk receptor tyrosine kinase protein; Figure 4 shows a recombinant DNA molecule having a Nuk⁷ null mutation obtained by deletion of exon 2, corresponding to codons 29 to 50 as shown in SEQ ID NO: 1;

Figure 5 shows a recombinant DNA molecule encoding the Nuk² mutation in the ATP binding region of the kinase domain of Nuk protein, and a lac Z reporter gene;

Figure 6A is a photomicrograph showing a transverse section taken through the brain of heterozygous Nuk^J/+ mice across the anterior of the frontal lobes;

Figure 6B is a photomicrograph showing a transverse section taken through the brain of homozygous Nuk^J Wuk¹ mice across the anterior of the frontal lobes; Figure 6C is a photomicrograph showing a transverse section taken through the brain of homozygous Nuk⁷ /Nuk⁷ mice across the anterior of the frontal lobes;

Figure 6D is a photomicrograph showing a transverse section taken through the brain of homozygous Nuk² /Nuk² mice across the anterior of the frontal lobes (ac=anterior commissure, mt=medial tract);

Figure 7 A is a photomicrograph of a horizontal section taken through the brain of a Nuk⁷/+ mouse across the anterior of the frontal lobes, showing the medial tract of the anterior commissure;

Figure 7B is a photomicrograph of a horizontal section taken through the brain of a homozygous Nuk⁷ /Nuk⁷ mouse across the anterior of the frontal lobes, showing the absence of the medial tract of the anterior commissure;

Figure 8 shows horizontal sections taken through the brains of Nuk⁷/Nuk⁷ (bottom) and Nuk⁷/+ (top) mice injected in one frontal lobe with a fluorescent dye, fast blue;

Figure 9 is a diagram illustrating the fast blue tracing of the temporal lobe; Figure 10 is a diagram illustrating the axon pathways affected in Nuk/Sek4 double homozygotes;

Figure 11 shows an alignment of the amino acid sequences of ligands of the Eph subfamily of receptor tyrosine kinase proteins, amino acids identical in at least five out of nine proteins are shown in inverse type, the cysteine residues common to aU nine proteins are marked by asterisks;

Figure 12 is a diagram showing membrane anchored Ugands for Eph subfamily receptor tyrosine kinase proteins; and

Figure 13 is a diagram showing a potential signalling role for Lerks. DETAILED DESCRIPTION OF THE INVENTION As hereinbefore mentioned, the present inventors have identified and characterized a novel ligand regulatory pathway that plays a crucial role in cell-cell interactions and axonogenesis in the development and regeneration of the nervous system. The present inventors have determined that Eph subfamily receptor tyrosine kinases activate a Ugand regulatory pathway in ceUs expressing ligands for the Eph subfamily receptor tyrosine kinases.

Expression of an Eph subfamUy receptor tyrosine kinase, Nuk, was found to be essential for formation of at least one commissure in the brain, the medial tract of the anterior commissure. In nuU mice, lacking in Nuk expression the medial tract was found not to form. In Nuk²/Nuk² mice, expressing a fusion protein comprising the Nuk protein extracellular domain and β-galactosidase, the medial tract of the anterior commissure formed and was of a normal appearance. Therefore, the extraceUular domain of Nuk protein is required for formation of the medial tract of the anterior commissure. Nuk protein did not appear to be expressed in the medial tract of the anterior commissure, but expression was detected ventrally underlying the commissure. Ligands of Nuk protein are thought to be expressed in the medial tract of the commissure. Nuk protein also appears to play an important role in the formation of the habenular interpeduncle tract in the brain. Complete formation of the habenular interpeduncle tract was shown to require expression of at least two members of the Eph subfamily of receptor tyrosine kinase proteins and appeared to require expression of Nuk protein having a catalytic kinase domain. Both Nuk⁷/Nuk⁷ and Nuk² /Nuk² homozygotes exhibit a mild phenotype in the habenular interpeduncle tract, however, this phenotype is more severe in either Nuk⁷/Nuk⁷:Sek4/Sek4 and Nuk²/Nuk2:Sek4/Sek4 double homozygotes. The invention relates to a method of activating a Ugand regulatory pathway in a ceU, comprising reacting an Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein with a cell expressing a ligand for an Eph subfamUy receptor tyrosine kinase on the surface of the ceU thereby activating the Ugand regulatory pathway in the ceU. The term "Ugand regulatory pathway" used herein refers to the interactions of an

Eph subfamily receptor tyrosine kinase protein with a cell surface ligand for an Eph subfamUy receptor tyrosine kinase protein, to form a Ugand receptor tyrosine kinase protein eomplex thereby activating a series of downstream regulatory pathways in the Ugand expressing cell that affect the cell, for example by controlling gene expression, ceU division, cytoskeletal architecture, cell metabolism, migration, cell-cell interactions and spatial positioning. Examples of such downstream regulatory pathways are the GAP/Ras pathway, the pathway that regulates the breakdown of the polyphosphoinositides through phosphoUpase C (PLC) and the Src/ tyrosine kinase and Ras pathways.

"Eph subfamily receptor tyrosine kinase proteins" refers to proteins of the Eph subfamily which are characterised as encoding a structurally related cysteine rich extracellular domain containing a single immunoglobulin (Ig)-like loop near the N-terminus and two fibronectin in (FN UI) repeats adjacent to the plasma membrane. The structure of the extracellular region is thought to determine Ugand binding specificity. The intracellular regions contain the juxtamembrane and the catalytic kinase domain. Receptor mediated signal transduction is initiated in the receptor expressing cell by ligand binding to the extracellular domain, which facilitates dimerization of the receptor and autophosphorylation.

Over a dozen members of the Eph subfamily have been identified (van der Geer et al., 1994, Annu. Rev. Cell Biol.. 10:251-237). Examples of Eph family members include mouse Nuk and its homologs Hek5, Cek5 in chickens (Pasquale, Cell Regulation 2:523-534, 1991), Sek3 in mice, and Erk in humans; Eek (Chan and Watt, Oncogene 6:1057-1061 1991); rat Elk and its homologs including Cek6a in chickens and xEK (Lhotak et al., 1991, Mol. CeU. Biol. 11:2496-2502); human Hek2 and its homologs including Sek4 in mice and CeklO in chickens; and human Htk and its homologs including Mykl in mice. The Eph faπύly member, Sek has been shown to be segmentally expressed in specific rhombomeres of the mouse hindbrain (Nieto et al, Development 116:1137-1150, 1992). Other members of the family include Eck (Lindberg and Hunter, 1990, Mol. Cell Biol. 10:6316-6324); Ceks 4, 6, 7, 8, 9 and 10 (Pasquale, 1991, Cell Regulation, 2:523-534) and Saajadi and Pasquale, 1993, Oncogene, 8, 1807-1813); Ehk 1 and 2 (Maisonpierre et al., 1992,, Oncogene, 8:3277-3288); Myk 1 and 2 (Andres et al., 1994); and Heks 4, 5, 7 (GenBank Accession No. L36644), 8 (GenBank Accession No. L36645) and 11 (GenBank Accession No. L36642) (Fox, et al., 1995, Oncogene, 10, 897-905). The amino acid sequences of some known members of the Eph subfamUy of receptor tyrosine kinases are described in Fox et al., 1995 (Oncogne 10, 897-905) and shown in Figure 1, which is excerpted from Fox et al., 1995, supra. Amino acid sequences for other Eph subfamUy receptors can be found in GenBank (e.g. Accession Nos. L25890 (Nuk), X13411 (rat Elk), U07695 (human Htk) and the pubUcations referred to therein).

Preferably, Eph subfamily receptor tyrosine kinases, or parts thereof, which bind to transmembrane Ugands are used in the present invention. For example, preferred Eph subfamily receptor tyrosine kinases, or parts thereof, used in the present invention include mouse Nuk and its homologs Hek5, Cek5 in chickens, and Erk; rat Elk and its homologs including Cekόa in chickens and xEK; human Hek2 and its homologs including Sek4 in mice and CeklO in chickens; and human Htk and its homologs including Mykl in mice. All the hallmarks of a receptor tyrosine kinase of the Eph subfamily family are exemplified in Nuk protein, including 20 cysteine residues whose position is conserved in the extraceUular domain of Eph famUy members (bold type, Figure 3), an immunoglobulin-Uke domain near the amino terminus (Ig-like), and two fibronectin type III repeats (FN III; between Nuk amino acids residues 330-420 and 444-534). The Ig-like domain of Nuk contains specific residues (Cys⁷⁰, Trp⁸⁰, Cys¹¹⁵) known to be conserved in the Ig superfamily (Williams and Barclay, Ann. Rev. Immunol. 6:381-405, 1988).

The cartoon in Figure 3 shows the location of the various domains of Nuk protein. FoUowing a 26 amino acid hydrophobic signal peptide, the Nuk protein extraceUuar domain is composed of an Ig-like domain and two FN III repeats. The Nuk protein extracelluar domain also contains 20 cysteines whose position is conserved in the Eph faπύly (Lhotak et al., Mol. Cell. Biol. 11:2496-2502, 1991). A hydrophobic transmembrane domain divides the Nuk protein into approximately two halves, a 548 amino acid extraceUuar region and a 419 amino acid cytoplasmic region which contains a tyrosine kinase catalytic domain.

Nuk is most highly related to the full length amino acid sequence of human Hek5 and also to chicken Cek5 (96% identity; Pasquale, Cell Regulation 2:523-534, 1991) and to short PCR products of mRNA from rats (Tyro 5; Lai and Lemke, Neuron 6:691-704, 1991) and humans (Erk; Chan and Watt, Oncogene 6:1057-1061 1991). The close identity between Nuk and CekS suggest they represent the mammaUan and avian orthologs of the same progenitor gene. The absence of fuU length cDNAs for Tyro 5 and Erk precludes the determination of whether these sequences correspond to the same or a closely related but different gene.

It wiU be appreciated that the Eph subfamily receptor tyrosine kinase protein for use in activating a Ugand regulatory pathway, as described herein, may be an isoform or a part of the protein having at least 20 contiguous amino acids of the protein. An isoform contains the same number and kinds of amino acids as the protein, but the isoform has a different molecular structure. The isoforms contemplated for use in the methods of the invention are isoforms having the same functional properties as the Eph subfamily receptor tyrosine kinase proteins. In a preferred embodiment, the part of the protein having at least 20 contiguous amino acids comprises an Eph subfamUy tyrosine kinase protein, preferably Nuk, lacking a catalytic kinase domain. For example, the part of the protein containing at least one of the extraceUular domain, the transmembrane domain and the juxtamembrane domain or parts thereof, preferably, the extraceUular domain is used in the methods herein. The extracellular domain is characterised by a cysteine rich region, whose position is conserved in the extracellular domain of Eph family members an immunoglobulin-like domain near the amino terminus (Ig-Uke), and two fibronectin type IU repeats (FN III). ExtraceUular domains of Eph subfamily receptor tyrosine kinase proteins may be identified based on the above-noted features and based on a comparison of the amino acid sequences of the extraceUular domains of known Eph subfamily receptor tyrosine kinase proteins. The extracellular domain may be generaUy defined as the region extraceUular to the transmembrane domain, which is indicated in bold underline in Figure 1.

The protein may also be a protein having substantial sequence identity with the sequence of an Eph subfamily receptor tyrosine kinase protein. The term "sequence having substantial identity" means those amino acid sequences having sUght or inconsequential sequence variations from the sequence of an Eph subfamily receptor tyrosine kinase protein. The variations may be attributable to local mutations or structural modifications. Suitable proteins may have over 95%, preferably over 97%, most preferably over 99% identity with an Eph subfamUy receptor tyrosine kinase protein. An Eph subfamUy receptor tyrosine kinase or part thereof, may be selected for use in the present invention based on the nature of the Ugand which is targeted or selected. The selection of a particular Ugand and complementary Eph subfamUy receptor tyrosine kinase in the method of the invention will aUow for the identification of specific substances that affect a Ugand regulatory pathway. An Eph subfamily receptor tyrosine kinase or part thereof may be prepared from

Eph subfamily receptor tyrosine kinase proteins isolated from cells which are known to express the proteins. Alternatively the protein or part of the protein may be prepared using recombinant DNA methods known in the art. By way of example, nucleic acid molecules having a sequence which codes for an Eph subfamUy receptor tyrosine kinase protein, or a part of the protein may be prepared and incorporated in a known manner into an appropriate expression vector which ensures good expression of the protein or part thereof. Possible expression vectors include but are not Umited to cosmids, plasmids, or modified viruses, so long as the vector is compatible with the host ceU used.

Suitable transcription and translation elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropriate transcription and translation elements is dependent on the host ceU chosen, and may be readUy accompUshed by one of ordinary skill in the art. Examples of such elements include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host ceU chosen and the vector employed, other genetic elements, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. It wiU also be appreciated that the necessary transcriptional and translation elements may be supplied by the native receptor tyrosine kinase protein and/ or its flanking regions.

The recombinant molecules may also contain a reporter gene which facilitates the selection of host ceUs transformed or transfected with a recombinant molecule. Examples of reporter genes are genes encoding a protein such as β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. In a preferred embodiment, the reporter gene is lac Z . Transcription of the reporter gene is monitored by changes in the concentration of the reporter protein such as β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, eiectroporation etc. Methods for transforming transfecting, etc. host ceUs to express foreign DNA are weU known in the art (see, e.g., Itakura et al., U.S. Patent No. 4,704,362; Hinnen et al., PNAS USA 75:1929-1933, 1978; Murray et al., U.S. Patent No. 4,801,542; Upshall et al., U.S. Patent No. 4,935,349; Hagen et al., U.S. Patent No. 4,784,950; Axel et al., U.S. Patent No. 4,399,216; Goeddel et al., U.S. Patent No. 4,766,075; and Sambrook et al. Molecular Cloning A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, all of which are incorporated herein by reference and see the detailed discussion below).

Suitable host cells include a wide variety of prokaryotic and eukaryotic host ceUs, including bacterial, mammalian, yeast or other fungi, viral, plant, or insect ceUs.

The Eph subfamily receptor tyrosine kinase protein or parts thereof may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).

Conjugates of the protein, or parts thereof, with other molecules, such as proteins or polypeptides, may be prepared and used in the methods described herein. This may be accompUshed, for example, by the synthesis of N-terminal or C-terminal fusion proteins. Thus, fusion proteins may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of an Eph subfamily receptor tyrosine kinase protein or parts thereof, and the sequence of a selected protein or marker protein with a desired biological function. The resultant fusion proteins contain Eph subfamily receptor tyrosine kinase protein or a part thereof fused to the selected protein or marker protein as described herein. Examples of proteins which may be used to prepare fusion proteins include immunoglobulins and parts thereof such as the constant region of immunoglobulin γl, and lymphokines such as gamma interferon, tumor necrosis factor, IL-1, IL-2.IL-3, 11-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, GM-CSF, CSF-1 and G-CSF. Sequences which encode the above-described proteins may generaUy be obtained from a variety of sources, including for example, depositories which contain plasmids encoding sequences including the American Type Culture Collection (ATCC, RockvUle Maryland), and the British Biotechnology Limited (Cowley, Oxford England). Examples of such plasmids include BBG 12 (containing the GM-CSF gene coding for the mature protein of 127 amino acids), BBG 6 (which contains sequences encoding gamma interferon), ATCC No. 39656 (which contains sequences encoding TNF), ATCC No. 20663 (which contains sequences encoding alpha interferon,) ATCC Nos. 31902 and 39517 (which contains sequences encoding beta interferon), ATCC No. 67024 (which contains a sequence which encodes Interleukin-lfi), ATCC Nos. 39405, 39452, 39516, 39626 and 39673 (which contains sequences encoding Interleukin-2), ATCC Nos. 59399, 59398, and 67326 (which contain sequences encoding Interleukin-3), ATCC Nos. 57592 (which contains sequences encoding Interleukin-4). ATCC Nos. 59394 and 59395 (which contain sequences encoding Interleukin-5), and ATCC No. 67153 (which contains sequences encoding Interleukin-6.

The Eph subfamUy receptor tyrosine kinase protein, isoforms or parts thereof, used in the method of the invention may be insolubilized. For example, the receptor protein or part thereof, preferably the extracellular domain, may be bound to a suitable carrier. Examples of suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, sUk, etc. The carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc. The insolubiUzed receptor tyrosine kinase protein may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling. The receptor tyrosine kinase protein or parts thereof may also be expressed on the surface of a ceU using the methods described herein.

Ligands for Eph subfamUy receptor tyrosine kinases may be identified based on homology with known Ugands and based on their interaction with the extraceUular domain of Eph subfamUy receptor tyrosine kinases. At least seven Ugands for Eph subfamily receptor tyrosine kinases have been identified, aU of which are membrane anchored via either a GPI linkage or transmembrane domain (see Figure 12), including B61 (Holzmann et al., 1990, Mol. Cell Biol. 10: 5830-5838 and Bartley et al., 1994 Nature 368:558-560), also known as LERK-1 (Beckmann et al, 1994, EMBO /. 13:3757-3762 and Davis et al., 1994 Science 266, 816-819) , LERK-2 (Beckmann et al., 1994, supra, and Davis et al., 1994 supra, also known as Eplg-2 (Fletcher et al, 1994, Oncogne 9:3241-3247), Cek5 Ugand, the chicken homolog of Lerk-2 and Elk-L, (Shao et al., 1994, /. Biol Chem. 269:26606-26609), ELF-1 (Cheng and Flanagan, 1994, Cell, 79:157-168), EHK1-L (Davis et al., 1994, supra), also known as LERK-3 (Kozlosky et al., 1995 Oncogne 10:299-306) and LERK-4 (Kozlosky et al, 1994, supra) ELF-1, AL-1 /RAGS (GPI- anchored, Drescher, et al, 1995, Cell, 82:359-370), LERK-4, HTKL/ELF-2/Lerk5, LERK- 2/CEK5-L/ELK-L (Tessier-Lavigne, M., 1995, supra). Ligands of Eph subfamily receptor tyrosine kinases show significant homology with each other. An aUgnment of the amino acid sequences of ligands of Eph subfamily receptor tyrosine kinases are shown in Figure 11 (excerpted from Drescher, et al., 1995, supra. Ligands for the Eph subfamily receptor tyrosine kinases are known to show promiscuous interactions with different Eph subfamily receptors (BrambUla et al., 1995, EMBO J. 14:3116-3126).

In an embodiment of the invention, the Ugands are ligands which are membrane anchored via a transmembrane domain. Preferably, the selected ligands are Elk- L/LERK2/Efl-3/Cek5-L; hHtk-L/ELF-2/LERK5 (Tessier-Lavigne, M., 1995, CeU 82:345-348), and hElk-L3/EU-6. These ligands have highly conserved cytoplasmic reions with multiple potential sites for phosphorylation. The amino acid sequences for hElk-L3, hHtk-L and hElk-L, and the extraceUular domains of the ligands can be found in GenBank (e.g. Accession Nos. L38734 (Htk) and L37361 (Efl-3)).

In the methods of the invention to activate a Ugand regulatory pathway in a ceU, the Ugand should be expressed on the surface of the cell. Preferably, the ceU is one which expresses native ligand. However, it will be appreciated that the invention also contemplates chimeric ceUs expressing a recombinant Ugand.

The invention also provides a method for identifying a substance which is capable of bmding to a Ugand for an Eph subfamUy receptor tyrosine kinase and activating a ligand regulatory pathway in a cell, comprising reacting a cell expressing a Ugand for an Eph subfamUy receptor tyrosine kinase with at least one substance which potentially can bind with the Ugand, under conditions which permit the formation of substance-Ugand complexes, and assaying for substance-Ugand complexes, for free substance, for non-complexed Ugands, or for activation of the ligand.

Activation of the ligand may be assayed by measuring phosphorylation of the Ugand, binding of SH2 domains to the Ugand, and where the Ugand is expressed on a ceU surface, by assaying for a biological affect on the ceU, such as inhibition or stimulation of proUferation, differentiation or migration. SH2-domains of cytoplasmic signalling proteins are known to bind to phosphorylated receptor tyrosine kinase proteins. In particular, the SH2 domains of p21 ^ras GTPase-activating protein (GAP), Src, and phosphoinositide-specific phosphoUpase C (PLCγ) may bind an Eph subfamily receptor tyrosine kinase protein. SH2 domains of cytoplasmic signalling proteins may bind to phosphorylated Ugands to mediate the interactions of the phophorylated Ugand with signalling proteins of the downstream regulatory pathways in the ceU.

Upon binding of a Ugand having an intracellular domain (e.g. Lerks such as Lerk2 and Lerk5) to an Eph subfamily receptor, a signal transduction event in the Ugand expressing cell may be initiated. This could occur by activation of one or more cytoplasmic tyrosine kinases which would phosphorylate the intracellular domain of the Ugand, which would then lead to the binding of SH2 domain-containing proteins to the phosphorylated activated ligand. A diagram of a potential signalling role for Lerks is shown in Figure 13.

In an embodiment, of the method, the substance is an Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein. In a further embodiment the part of the protein comprises an extraceUular domain. In a preferred embodiment, the substances is an Eph subfamUy receptor tyrosine kinase which is not the native receptor tyrosine kinase for the Ugand.

Conditions which permit the formation of substance-Ugand complexes may be selected having regard to factors such as the nature and amounts of the substance and the ligand.

The substance-Ugand complex, free substance or non-complexed Ugand may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel fUtration, fractionation,absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof. To facilitate the assay of the components, antibody against the Ugand or the substance, or a labelled Ugand, or a labelled substance may be utilized. Antibodies, receptor protein or substance may be labelled with a detectable substance as described above.

The substance used in the method of the invention may be insolubilized. For example, the receptor protein or substance may be bound to a suitable carrier. Examples of suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl ceUulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc. The insolubUized substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling. The substance may also be expressed on the surface of a cell using the methods described herein. Where the substance is expressed on the surface of a cell the presence of a substance which can bind to and be activated by the receptor tyrosine kinase protein may be identified by assaying for activation of the substance or by assaying for a biological affect on the cell. The above mentioned methods of the invention may be used to identify substances which bind with Ugands of the Eph subfamily of receptor tyrosine kinase proteins, thereby activating a ligand regulatory pathway in a cell, particularly those involved in neuronal development, axonal migration, pathfinding and regeneration. Identification and isolation of such substances will permit studies of the role of the substance in the developmental regulation of axonogenesis and neural regeneration, and permit the development of substances which affect these roles, such as functional or non-functional analogues of the extraceUular domain of an Eph subfamily receptor tyrosine kinase. It will be appreciated that such substances wiU be useful as pharmaceuticals to modulate axonogenesis, nerve ceU interactions and regeneration to treat conditions such as neurodegenerative diseases and cases of nerve injury.

Substances which bind to and activate the Ugand may be identified by assaying for protein tyrosine kinase activity i.e. by assaying for phosphorylation of the tyrosine residues of the Ugand, using known techniques such as those using anti-phosphotyrosine antibodies and labeUed phosphorous. For example, immunoblots of the complexes may be analyzed by autoradiography (³²P-labeUed samples) or may be blocked and probed with antiphosphotyrosine antibodies as described in Koch, CA. et al., 1989 (Mol. CeU. Biol. 9, 4131-4140).

Substances which bind to and activate the ligand may also be assayed by assaying for a biological affect on the ceU, for example inhibition or stimulation of cell proliferation, differentiation and migration. Substances which bind to and activate the ligand will include Eph subfamily receptor tyrosine kinase proteins and portions of the proteins. The method wiU permit identification of the minimum amino acid sequence of the protein which is required for Ugand binding and activation.

The invention further relates to a method for assaying a medium for an agonist or antagonist of a ligand regulatory pathway in a cell which comprises providing a cell expressing a Ugand for an Eph subfamUy receptor tyrosine kinase on the ceU surface, reacting the ceU with an Eph subfamUy receptor tyrosine kinase protein or part of a protein and a suspected agonist or antagonist under conditions which permit the formation of ligand- receptor tyrosine kinase protein complexes on the cell surface, and assaying for ligand-receptor tyrosine kinase protein complexes, for free receptor tyrosine kinase protein, for non-complexed proteins, for activation of the receptor tyrosine kinase protein, or for activation of the ligand. Substances which activate the Ugand regulatory pathway, such as Eph subfamily receptor tyrosine kinase proteins or parts thereof, and agonists or antagonists of the Ugand regulatory pathway may be used for affecting neuronal development or regeneration in a mammal. The substances, agonists and antagonists may be used to stimulate or inhibit neuronal development, regeneration and axonal migration associated with neurodegenerative conditions and conditions involving trauma and injury to the nervous system, for example Alzheimer's disease, Parkinson's disease, Huntington's disease, demylinating diseases, such as multiple sclerosis, amyotrophic lateral sclerosis, bacterial and viral infections of the nervous system, deficiency diseases, such as Wernicke's disease and nutritional polyneuropathy, progressive supranuclear palsy, Shy Drager's syndrome, multistem degeneration and olivo ponto cerebellar atrophy, peripheral nerve damage, trauma and ischemia resulting from stroke.

The abiUty of substances, agonists, and antagonists identified using the methods of the invention to affect neuronal development or regeneration and to stimulate nerve regeneration, may be confirmed in an animal model having an injured peripheral nervous system. Examples of mammals having an injured peripheral nervous system include animals having damaged axons, such as axotomized facial neurons (Sendtner et al. Nature, 345, 440-441, 1990), neurodegenerative conditions (for example, the MPTP model as described in Langston J.W. et al, Symposium of Current Concepts and Controversies in Parkinson's Disease, Montebello, Quebec, Canada, 1983 and Tatton W.G. et al., Can. J. Neurol. Sci. 1992, 19), and traumatic and non-traumatic peripheral nerve damage (for example, animal stroke models such as the one described in MacMUlan et al. Brain Research 151:353-368 (1978)).

The present invention thus provides a method for affecting neuronal development or regeneration in a mammal comprising administering to a mammal an effective amount of a purified and isolated Eph subfamUy receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein, or a substance identified using the methods of the invention. The invention also contemplates a method for stimulating or inhibiting axonogenesis in a mammal comprising administering to a mammal an effective amount of a purified and isolated Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein, or a substance identified using the methods of the invention.

The invention still further relates to a pharmaceutical composition which comprises a purified and isolated Eph subfamily receptor tyrosine kinase protein or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein, or a substance identified using the methods of the invention, for affecting neuronal development or regeneration and a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical compositions may be used to stimulate or inhibit neuronal development, regeneration and axonal migration associated with neurodegenerative conditions and conditions involving trauma and injury to the nervous system as described above.

The compositions of the invention are administered to subjects in a biologicaUy compatible form suitable for pharmaceutical administration in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the protein to be administered in which any toxic effects are outweighed by the therapeutic effects of the protein. The term subject is intended to include mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Administration of a therapeuticaUy active amount of the pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of an Eph subfamily receptor tyrosine kinase protein may vary according to factors such as the condition, age, sex, and weight of the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daUy or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active compound (e.g., protein) may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration inhalation, transdermal appUcation or rectal administration. Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. The pharmaceutical compositions of the invention can be for oral, local, inhalant or intracerebral administration. Preferably, the pharmaceutical compositions of the invention are administered directly to the peripheral or central nervous system, for example by administration intracerebraUy.

The pharmaceutical composition of the invention can be administered to a subject in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier such as microporous or solid beads or liposomes. The term "pharmaceutically acceptable carrier" as used herein is intended to include dUuents such as saline and aqueous buffer solutions. Liposomes include water-in-oil-in- water emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol 7:27). The active compound may also be administered parenterally or intraperitoneaUy. Dispersions can also be prepared in glycerol, Uquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical compositions suitable for injectable use include sterUe aqueous solutions (where water soluble) or dispersions and sterUe powders for the extemporaneous preparation of sterUe injectable solutions or dispersions. In aU cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The pharmaceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the Uke), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, asorbic acid, thimerosal, and the Uke. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

SterUe injectable solutions can be prepared by incoφorating active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, foUowed by filtered steriUzation. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (e.g., antibody) plus any additional desired ingredient from a previously sterile-fUtered solution thereof.

When the active compound is suitably protected, as described above, the composition may be orally administered, for example, with an inert dUuent or an assimUable edible carrier. As used herein "pharmaceutically acceptable carrier" includes any and aU solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

It is contemplated that the pharmaceutical compositions may be administered locally to stimulate axonogenesis and pathfinding in areas of the body in need thereof, for example in areas of local nerve injury or in areas where normal nerve pathway development has not occurred. It is also contemplated that the pharmaceutical compositions may be placed in a specific orientation or aUgnment along a presumptive pathway to stimulate axon pathfinding along that line, for example the pharmaceutical compositions may be present on microcarriers laid down along the pathway. In an embodiment, the pharmaceutical compositions may be used to stimulate formation of connections between areas of the brain, such as the area between the two hemispheres or between the thalamus and ventral midbrain. In an embodiment, the compositions may be used to stimulate formation of the medial tract of the anterior commissure or the habenular interpeduncle.

It is also contemplated that the pharmaceutical compositions of the invention may comprise ceUs or viruses, preferably retroviral vectors, transformed with nucleic acid molecules encoding a purified and isolated Eph subfamUy receptor tyrosine kinase protein, or an isoform or a part of the protein, or a substance identified using the methods of the invention, such that they express the protein, isoform, or a part of the protein, preferably the extracellular domain, or substance in vivo. Viral vectors suitable for use in the present invention are weU known in the art including recombinant vaccinia viral vectors (U.S. Patent Nos. 4,603,112 and 4,769,330), recombinant pox virus vectors (PCT Publication No. WO 89/01973), and preferably, retroviral vectors ("Recombinant Retroviruses with Amphotropic and Ecotropic Host Ranges," PCT PubUcation No. WO 90/02806; "Retroviral Packaging CeU Lines and Processes of Using Same," PCT PubUcation No. WO 89/07150; and "Antisense RNA for Treatment of Retroviral Disease States," PCT Publication No. WO 87/03451). The compositions containing cells or viruses may be directly introduced into a subject as described herein. Nucleic acid molecules encoding a purified and isolated Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein, or a substance identified using the methods of the invention, may also be introduced into a subject using physical techniques such as microinjection and eiectroporation or chemical methods such as coprecipitation and incorporation of nucleic acids into liposomes. They may also be delivered in the form of an aerosol or by lavage.

The foUowing non-limiting examples are iUustrative of the present invention: EXAMPLES The foUowing materials and methods were utilized in the investigations outlined in the examples:

Cloning of Nuk The coding region of Nuk was cloned using the partial λQl, Nuk cDNA insert to probe a λgtlO expression Ubrary constructed from a mouse erythroleukemia cell line by screening with anti-phosphotyrosine antibodies (Ben-David et al., EMBO 10:317-325, 1991). Cloning of Nuk was carried out as described in Henkemeyer et al., 1994 (Oncogene 9:1001-1014) and in co-pending International AppUcation PCT CA95/00254 and co- pending appUcation serial No. 08/235,407, which are incorporated herein by reference. The nucleotide sequence encoding Nuk is shown in Figure 2 (SEQ. ID. NO: 1) and the amino acid sequence of Nuk protein is shown in Figure 3 (SEQ. ID. NO: 2). Generation of Loss of Function Nuk mutant

A loss of function mutation in Nuk, designated Nuk⁷ was generated in embryonic stem cells, and germline transmission of the null allele was obtained as described in co¬ pending International AppUcation PCT CA95/00254 and co-pending appUcation serial No. 08/235,407.

Briefly, the nuU mutation was obtained by deletion of exon 2, corresponding to codons 29 to 50, as shown in Figure 4. To obtain germ line transmission of the mutation Nuk+/- embryonic stem ceU Unes (ES) were aggregated with 8 ceU embryos in vitro and the resulting blastocysts were transferred into recipient females. Upon birth, animals chimeric for ES and embryonic stem ceUs were recovered by scoring for eye pigment and coat colour. Breeding of these "aggregation chimeras" confirmed that the germ line of at least one founder mouse is derived completely from the ES ceUs. Adult mice homozygous for the mutation did not express Nuk protein.

Generation of a Nuk-lac Z fusion chimeric receptor mutant

A targeted mutation, designated Nuk² was generated in the Nuk gene as described in co-pending International AppUcation PCT CA95/00254 and co-pending application serial No. 08/235,407 and shown in Figure 5. A pPNT-LOX-Nuk² gene trap vector was used to delete the GXGXXG ATP binding region of the kinase domain (amino acids 623-707,) to create a Nuk-lac Z fusion receptor in ES ceUs. Chimeric animals were prepared as described above, by aggregating the ES ceUs with 8 ceU CDI embryos.

Animals generated with the Nuk² mutation provided Nuk expressing cells staining for β-galactosidase activity, providing a convenient marker for Nuk-positive ceUs in both heterozygous and homozygous backgrounds. The Nuk² mutation led to the expression of a Nuk-beta galactosidase fusion protein in mouse heterozygous embryos, detected by a blue/green colour .

EXAMPLE 1

The role of Nuk protein, the extracellular domain of Nuk protein and the catalytic kinase domain of Nuk protein were investigated as foUows. Loss of function Nuk mutant mice, designated Nuk⁷ were prepared as described herein. These mice may also be referred to as null mice as they do not express Nuk protein. Nuk-lac Z fusion chimeric receptor mutant mice, designated Nuk² were prepared as described above. These mice express a fusion protein having the entire extraceUular domain of Nuk, but lacking in the Nuk catalytic kinase domain, which is replaced by β-galactosidase. All mice, exhibited apparently normal appearance and behaviour.

To analyze the brains of Nuk mutant mice, specimens were dissected and fixed in 4% paraformaldehyde in PBS. The fixed specimens were either embedded in paraffin and sectioned on a microtome or cryprotected in 30% sucrose and sectioned using a cryostat to obtain serial sections.

Serial sections were taken of a number of brains of heterozygous control and both Nuk⁷ and Nuk ²homozygous embryos at E14.5 to E18.5 days of embryonic development and of newborn and adult mice at 1 to 1.5 years of age. 6 to 30 μm thick coronal or horizontal sections were prepared and viewed on a compound microscope under bright field or polarized Ught. Figures 6A, 6B, 6C and 6D show photomicrographs of horizontal sections taken across the anterior of the temporal lobes at the level of the anterior commissure and pars posterior medial tract, which connects the frontal lobes. In heterozygous Nuk⁷/+ mice the pars posterior medial tract, and the pars anterior tract of the anterior commissure are clearly visible (Figure 6A) and appear the same as in wild type mice. Serial sections show that the pars posterior medial tract forms a continuous tract between the two frontal lobes. The entire medial tract is not visible in Figure 6A due to the plane of the section.

The presence of a continuous pars posterior medial tract communicating between the frontal lobes, was confirmed by dye injection experiments, which are illustrated diagramatically in Figure 9. Briefly, a fluorescent dye (fast blue) was injected into one temporal lobe of anaesthetized adult mice, either heterozygous or homozygous for the Nuk¹ mutation, approximately one year old, through standard surgical techniques. Mice were revived and the fast blue was aUowed to travel through the axons of the temporal neurons that received dye for 2 days, after which the mice were sacrificied, perfused with fixative, and the brains were coUected and post-fixed. After cryoprotection in 30% sucrose, serial sections were prepared and the brain sections were viewed by fluoresence microscopy. Where the dye was found to have been transported across to the opposite frontal lobe, the presence of an intact medial tract was confirmed. In homozygous Nuk¹ /Nuk⁷ nuU mice the pars posterior medial tract was found to be absent as shown in Figures 6B, 6C and 7B. Absence of the medial tract was confirmed by the inabiUty of dye injected into one frontal lobe to cross to the opposite frontal lobe as shown in Figure 8 (bottom). Absolutely no label was detected in the opposite frontal lobe, even when large amounts of dye were injected to maximize labeUing. In Nuk⁷/+ mice, however, small amounts of dye were sufficient to produce visible labeUing in the opposite frontal lobe, as shown in Figure 8 (top). Labeling was detected in the medial tract of Nuk⁷/+ mice but not in Nuk⁷/Nuk⁷ mice. This directly shows that expression of Nuk protein is required for the formation of the medial tract.

In homozygous Nuk²/Nuk² mice the medial tract was found to be present, as shown in Figure 6D and was shown by dye injection to form a continuous connection between the frontal lobes, as in the wUd type and Nuk⁷/* heterozygotes. This surprisingly indicates that the extraceUular domain of Nuk, in the absence of the catalytic kinase domain, is sufficient for formation of the medial tract. This is beUeved to be the first showing of a functional role for the extracellular domain of a receptor tyrosine kinase which is independent of the catalytic kinase domain. A role for the transmembrane and juxtamembrane domains of Nuk protein cannot be ruled out as the chimeric Nuk-β-galactosidase fusion protein has these domains in addition to the extraceUular domain. In view of the importance of Nuk protein in the formation of the pars posterior medial tract, a detaUed study of the expression of Nuk in this region of the brain was made by examining serial sections from the brains of Nuk²/Nuk² homozygous mice, which express a fusion protein comprising the Nuk extracellular transmembrane and juxtamembrane domains and β-galactosidase, which can readUy be detected in sections based on a blue green coloration, as described herein. Sections were taken from the brains of Nuk²/Nuk² mice and newborn pups and from embryos at various stages of gestation.

Nuk was not found to be expressed in the pars posterior medial tract of embryonic or adult Nuk² /Nuk² mice. Nuk expression was absent dorsal to the medial tract but apparent in the cells ventral to and underlying the medial tract. Nuk was generaUy found to be widely expressed in the brain, with an apparent increase in level posteriorly. Peripheral axons were found to express high levels of Nuk. In particular, the retinal ganglia ceUs of the eyes exhibited intense blue/green staining. The olfactory receptor neurons, the trigeminal gangUa and associated sensory whisker roots were also found to express Nuk. The corpus caUosum, the thick stratum of transversely-directed nerve fibres which connects the two hemispheres of the brain, was also stained for Nuk expression.

Further information about the role of Nuk protein in axonal pathfinding was obtained from examining the brains of mice having double mutations in Nuk and in Sek4, another member of the Eph subfamily of receptor tyrosine kinases. Mice bearing a Sek4 null mutation were prepared (Klein and Orioloi, European Molecular Biology Laboratory, Heidelberg, Germany). The Sek4 nuU mice, similar to the Nuk null mice, exhibited no obvious morphological or behavioral defects. However, Nuk^υSek4 double homozygous mutants died at birth. Nuk²/Sek* mice survived up to 3 months, con-firming that Nuk protein plays a crucial role which is independent of its catalytic kinase domain. An examination of coronal sections of the brains of newborn Nuk⁷/Sek* mice showed that, in addition to the anterior commissure defect found in Nuk¹/- mice, the corpus caUosum and habenular interpeduncle tracts were severly affected and failed to develop properly. The axon pathways affected in the Nuk/Sek double homozygotes is Ulustrated in Figure 10. The fibres of the anterior commissure appeared to be misdirected and oriented to the ventral-most floor of the brain. In addition, the fibres of the corpus caUosum had not joined up across the midline, but had pUed up against the lateral ventricles. Nuk,-Lac Z expression, based on blue/green staining, was detected in the mid line of the corpus caUosum. The habenular interpeduncle tract which connects the thalamus to the ventral midbrain, was defective in Nuk²/Sek4 and Nuk^{1 /}Sek4. Careful analysis of Nuk protein using anti-Nuk antibodies and lac Z staining of Nuk²/Nuk² embryos showed that, during development, Nuk expression appears in the ventral midbrain and progresses towards the thalamus and axon migration occurred in the opposite direction, i.e. from the thalamus toward the ventral mid brain. This axon migration was dependent on the expression of Nuk protein having a catalytic kinase domain.

Having iUustrated and described the principles of the invention in a preferred embodiment, it should be appreciated by those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. We claim aU modifications corning within the scope of the foUowing claims.

All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. The foUowing sequence Ustings form part of the appUcation.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANTS:

(A) NAME: Mount Sinai Hospital Corporation

(B) STREET: 600 University Avenue, Suite 970

(C) CITY: Toronto

(D) STATE: Ontario

(E) COUNTRY: Canada

<F) POSTAL CODE: M5G 1X5

(G) TELEPHONE NO. : (416) 586-3235

(H) TELEFAX NO.: (416) 586-3110

(A) NAME: Anthony Pawson

(B) STREET: 34 Glenwood Avenue

(C) CITY: Toronto

(D) STATE: Ontario

(E) COUNTRY: Canada

(F) POSTAL CODE (ZIP) : M6P 3C6

(A) NAME: Mark Henkemeyer

(B) STREET: Center for Developmental Biology, University of Texas

Southwestern Medical Center, 600 Harry Hines Blvd.

(C) CITY: Dallas

(D) STATE: Texas

(E) COUNTRY: U.S.A.

(F) POSTAL CODE (ZIP): 75235-9133

^*(ii) TITLE OF INVENTION: Method of Activating a Novel Ligand

Regulatory Pathway

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Bereskin & Parr

(B) STREET: 40 King Street West, Box 401

(C) CITY: Toronto

(D) STATE: Ontario

(E) COUNTRY: Canada

(F) ZIP: M5H 3Y2

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Kurdydyk, Linda M.

(B) REGISTRATION NUMBER: 34,971

(C) REFERENCE/DOCKET NUMBER: 3153-196

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (416) 364-7311

(B) TELEFAX: (416) 361-1398

(C) TELEX: 06-23115

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3105 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Mus musculus

(D) DEVELOPMENTAL STAGE: Embryo

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: lambda gtlO cDNA library

(B) CLONE: Combined PnUKRACE A2 and K2 AND cDNA clones

(viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT: Distal end of chromosome 4

(B) MAP POSITION: near the ahd-1 mutation

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

ATGGGAGCCC GGGTCCCCGT TCTGCCCGGG CTGGATGGCT CATTCTGCTG GCTGCTGCTG 60

CTGCCGCTGC TAGCCGCCGT GGAAGAAACC CTGATGGACT CTACGACAGC AACGGCTGAG 120

CTGGGCTGGA TGGTACATCC CCCATCAGGG TGGGAAGAGG TGAGCGGCTA CGACGAGAAC 180

ATGAACACTA TCCGTACCTA CCAGGTGTGC AATGTCTTTG AGTCAAGCCA GAACAACTGG 240

CTGCGGACCA AATTCATCCG GCGCCGTGGC GCCCACCGTA TCCACGTGGA GATGAAGTTC 300

TCGGTGCGTG ACTGCAGCAG CATTCCCAGC GTGCCGGGCT CCTGCAAGGA GACCTTCAAC 360

CTCTAC ACT ATGAGGCTGA TTTTGACTTA GCCACCAAAA CCTTTCCCAA CTGGATGGAG 420

AATCCGTGGG TGAAGGTGGA CACCATCGCG GCCGATGAGA GCTTCTCTCA GGTGGACCTG 480

GGTGGCCGCG TCATGAAAAT CAACACTGAG GTGCGAAGCT TCGGTCCTGT GTCCCGCAAT 540

GGTTTCTACC TGGCCTTCCA GGACTACGGC GGCTGTATGT CCCTCATTGC TGTGCGCGTC 600

TTCTACCGGA AGTGCCCCCG AATCATCCAG AATGGTGCCA TCTTCCAGGA GACACTATCG 660

GGGGCTGAGA GCACTTCGCT GGTGGCAGCT CGGGGCAGCT GCATCGCCAA TGCTGAAGAA 720

GTGGACGTGC CCATCAAACT CTACTGTAAC GGGGACGGCG AATGGCTGGT GCCCATCGGT 780

CGCTGCATGT GCAAGGCGGG CTTCGAGGCT GTGGAGAACG GCACCGTCTG CCGAGGTTGT 840

CCATCAGGAA CCTTCAAGGC CAACCAAGGG GACGAAGCCT GCACCCACTG TCCCATCAAC 900

AGCCGCACCA CCTCTGAGGG TGCCACCAAC TGTGTATGCC GCAACGGCTA CTACAGGGCC 960

GACCTGGACC CCTTAGACAT GCCTTGCACA ACCATCCCCT CTGCGCCCCA GGCTGTGATC 1020

TCCAGCGTCA ACGAGACATC CCTCATGCTA GAGTGGACCC CACCCCGAGA CTCCGGGGGT 1080

CGCGAGGATC TTGTTTACAA CATCATCTGC AAGAGCTGTG GCTCCGGCCG GGGCGCATGC 1140

ACGCGCTGCG GGGACAACGT GCAGTACGCG CCCCGCCAGC TGGGCCTGAC TGAGCCGCGC 1200

ATCTACATCA GTGACCTGCT GGCACACACG CAGTACACCT TCGAGATCCA GGCCGTGAAT 1260

GGTGTGACCG ACCAGAGTCC CTTCTCACCT CAGTTCGCCT CTGTGAACAT CACCACCAAC 1320

CAAGCAGCAC CATCGGCCGT GTCCATCATG CACCAGGTGA GCCGCACTGT GGACAGCATC 1380

ACCCTGTCGT GGTCCCAGCC AGACCAGCCC AACGGTGTGA TCCTGGACTA CGAGCTGCAG 1440 TACTATGAGA AGGAGCTCAG TGAGTACAAC GCCACGGCCA TAAAAAGCCC CACCAACACA 1500

GTCACTGTGC AGGGCCTCAA AGCCGGCGCC ATCTATGTCT TCCAGGTGCG GGCACGCACC 1560

GTTGCAGGCT ATGGGCGCTA CAGTGGCAAG ATGTACTTCC AAACCATGAC AGAAGCCGAG 1620

TACCAGACCA GCATCAAGGA AAAGCTACCC CTCATCGTTG GCTCCTCCGC CGCCGGCTTA 1680

GTCTTCCTCA TCGCTGTGGT CGTCATTGCC ATCGTATGTA ACAGACGGGG GTTTGAGCGT 1740

GCCGACTCAG AGTACACGGA CAAGCTACAG CACTACACCA GCGGACACAT GACCCCAGGC 1800

ATGAAGATCT ATATAGATCC TTTCACCTAT GAAGATCCTA ATGAGGCAGT GCGGGAGTTT 1860

GCCAAGGAAA TTGACATCTC CTGTGTCAAG ATTGAGCAGG TGATTGGAGC AGGGGAATTT 1920

GGTGAGGTCT GCAGTGGCCA TTTGAAGCTG CCAGGCAAGA GAGAGATCTT TGTAGCCATC 1980

AAGACCCTCA AGTCAGGATA CACGGAGAAA CAGCGCCGGG ACTTCCTGAG TGAGGCATCC 20 0

ATCATGGGCC AGTTCGACCA CCCCAATGTC ATCCATCTGG AAGGGGTTGT CACCAAGAGC 2100

ACACCTGTCA TGATCATCAC TGAATTCATG GAGAATGGAT CTCTGGACTC CTTCCTCCGG 2160

CAAAATGATG GGCAGTTCAC AGTCATCCAA CTGGTGGGCA TGCTGAGGGG CATTGCAGCC 2220

GGCATGAAGT ACCTGGCGGA CATGAACTAC GTGCACCGTG ACCTTGCTGC TCGAAACATC 2280

CTCGTCAACA GTAACCTGGT GTGTAAGGTG TCTGACTTTG GGCTCTCACG CTTCCTGGAG 2340

GATGACACGT CTGACCCCAC CTATACCAGC GCTCTGGGTG GGAAGATCCC CATCCGTTGG 2400

ACGGCACCGG AAGCCATCCA GTACCGGAAA TTCACCTCGG CCAGTGATGT GTGGAGCTAT 2460

GGCATCGTCA TGTGGGAGGT GATGTCCTAC GGGGAACGAC CCTACTGGGA CATGACCAAT 2520

CAAGACGTAA TCAACGCCAT TGAACAGGAC TACAGACTAC CTCCGCCCAT GGACTGCCCT 2580

AGCGCCCTGC ACCAGCTCAT GCTGGACTGC TGGCAGAAGG ACCGCAACCA CCGGCCCAAG 2640

TTCGGCCAGA TTGTCAACAC GCTGGACAAG ATGATCCGAA ACCCCAACAG CCTCAAAGCC 2700

ATGGCACCCC TGTCCTCTGG CATCAACCTG CCACTGCTGG ACCGCACGAT ACCGGACTAC 2760

ACCAGCTTTA ACACAGTGGA TGAGTGGCTA GAGGCCATCA AGATGGGCCA GTACAAGGAG 2820

AGCTTTGCCA ACGCCGGCTT CACCTCTTTC GACGTTGTAT CTCAGATGAT GATGGAGGAC 2880

ATTCTCCGCG TTGGGGTCAC TCTAGCTGGC CACCAGAAAA AAATCCTGAA CAGTATCCAG 2940

GTGATGCGGG CCCAGATGAA CCAGATCCAG TCTGTAGAGG TTTGACATTC GCCTGCCTCG 3000

GTTCTCCTCT TCCTCCACGC CGCCCCTGAG CCCCTACGTC GGTCCCTGCT GCTCTGTCAC 3060

TGCAGGTCAG CACTGCCAGG AGGCCACAGA CAACAGGAAG ACCAA 3105 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 994 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Mus musculus

(D) DEVELOPMENTAL STAGE: Embryo

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: lamda gtlO cDNA library

(B) CLONE: Combined pNukRACE A2 and K2 and cDNA clones

(viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT: Distal end of chromosome 4

(B) MAP POSITION: near the ahd-1 mutation

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Gly Ala Arg Val Pro Val Leu Pro Gly Leu Asp Gly Ser Phe Cys 1 5 10 15

Trp Leu Leu Leu Leu Pro Leu Leu Ala Ala Val Glu Glu Thr Leu Met 20 25 30

Asp Ser Thr Thr Ala Thr Ala Glu Leu Gly Trp Met Val His Pro Pro 35 40 45

Ser Gly Trp Glu Glu Val Ser Gly Tyr Asp Glu Asn Met Asn Thr lie 50 55 60

Arg Thr Tyr Gin Val Cys Asn Val Phe Glu Ser Ser Gin Asn Asn Trp 65 70 75 80

Leu Arg Thr Lys Phe lie Arg Arg Arg Gly Ala His Arg lie His Val 85 90 95

Glu Met Lys Phe Ser Val Arg Asp Cys Ser Ser lie Pro Ser Val Pro 100 105 110

Gly Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu Ala Asp Phe 115 120 125

Asp Leu Ala Thr Lys Thr Phe Pro Asn Trp Met Glu Asn Pro Trp Val 130 135 140

Lys Val Asp Thr lie Ala Ala Asp Glu Ser Phe Ser Gin Val Asp Leu 145 150 155 160

Gly Gly Arg Val Met Lys He Asn Thr Glu Val Arg Ser Phe Gly Pro 165 170 175

Val Ser Arg Asn Gly Phe Tyr Leu Ala Phe Gin Asp Tyr Gly Gly Cys 180 185 190

Met Ser Leu He Ala Val Arg Val Phe Tyr Arg Lys Cys Pro Arg He 195 200 205

He Gin Asn Gly Ala He Phe Gin Glu Thr Leu Ser Gly Ala Glu Ser 210 215 220

Thr Ser Leu Val Ala Ala Arg Gly Ser Cys He Ala Asn Ala Glu Glu 225 230 235 240

Val Asp Val Pro He Lys Leu Tyr Cys Asn Gly Asp Gly Glu Trp Leu 245 250 255

Val Pro He Gly Arg Cys Met Cys Lys Ala Gly Phe Glu Ala Val Glu 260 265 270

Asn Gly Thr Val Cys Arg Gly Cys Pro Ser Gly Thr Phe Lys Ala Asn 275 280 285 Gin Gly Asp Glu Ala Cys Thr His Cys Pro He Asn Ser Arg Thr Thr 290 295 300

Ser Glu Gly Ala Thr Asn Cys Val Cys Arg Asn Gly Tyr Tyr Arg Ala 305 310 315 320

Asp Leu Asp Pro Leu Asp Met Pro Cys Thr Thr He Pro Ser Ala Pro 325 330 335

Gin Ala Val He Ser Ser Val Asn Glu Thr Ser Leu Met Leu Glu Trp 340 345 350

Thr Pro Pro Arg Asp Ser Gly Gly Arg Glu Asp Leu Val Tyr Asn He 355 360 365

He Cys Lys Ser Cys Gly Ser Gly Arg Gly Ala Cys Thr Arg Cys Gly 370 375 380

Asp Asn Val Gin Tyr Ala Pro Arg Gin Leu Gly Leu Thr Glu Pro Arg 385 390 395 400

He Tyr He Ser Asp Leu Leu Ala His Thr Gin Tyr Thr Phe Glu He 405 410 415

Gin Ala Val Asn Gly Val Thr Asp Gin Ser Pro Phe Ser Pro Gin Phe 420 425 430

Ala Ser Val Asn He Thr Thr Asn Gin Ala Ala Pro Ser Ala Val Ser 435 440 445 lie Met His Gin Val Ser Arg Thr Val Asp Ser He Thr Leu Ser Trp 450 455 460

Ser Gin Pro Asp Gin Pro Asn Gly Val He Leu Asp Tyr Glu Leu Gin 465 470 475 480

Tyr Tyr Glu Lys Glu Leu Ser Glu Tyr Asn Ala Thr Ala He Lys Ser 485 490 495

Pro Thr Asn Thr Val Thr Val Gin Gly Leu Lys Ala Gly Ala He Tyr 500 505 510

Val Phe Gin Val Arg Ala Arg Thr Val Ala Gly Tyr Gly Arg Tyr Ser 515 520 525

Gly Lys Met Tyr Phe Gin Thr Met Thr Glu Ala Glu Tyr Gin Thr Ser 530 535 540

He Lys Glu Lys Leu Pro Leu He Val Gly Ser Ser Ala Ala Gly Leu 545 550 555 560

Val Phe Leu He Ala Val Val Val He Ala He Val Cys Asn Arg Arg 565 570 575

Gly Phe Glu Arg Ala Asp Ser Glu Tyr Thr Asp Lys Leu Gin His Tyr 580 585 590

Thr Ser Gly His Met Thr Pro Gly Met Lys He Tyr He Asp Pro Phe 595 600 605

Thr Tyr Glu Asp Pro Asn Glu Ala Val Arg Glu Phe Ala Lys Glu He 610 615 620

Asp He Ser Cys Val Lys He Glu Gin Val He Gly Ala Gly Glu Phe 625 630 635 640

Gly Glu Val Cys Ser Gly His Leu Lys Leu Pro Gly Lys Arg Glu He 645 650 655

Phe Val Ala He Lys Thr Leu Lys Ser Gly Tyr Thr Glu Lys Gin Arg 660 665 670

Arg Asp Phe Leu Ser Glu Ala Ser He Met Gly Gin Phe Asp His Pro

675 680 685

Asn Val He His Leu Glu Gly Val Val Thr Lys Ser Thr Pro Val Met

690 695 700

He He Thr Glu Phe Met Glu Asn Gly Ser Leu Asp Ser Phe Leu Arg 705 710 715 720

Gin Asn Asp Gly Gin Phe Thr Val He Gin Leu Val Gly Met Leu Arg 725 730 735

Gly He Ala Ala Gly Met Lys Tyr Leu Ala Asp Met Asn Tyr Val His 740 745 750

Arg Asp Leu Ala Ala Arg Asn He Leu Val Asn Ser Asn Leu Val Cys 755 760 765

Lys Val Ser Asp Phe Gly Leu Ser Arg Phe Leu Glu Asp Asp Thr Ser 770 775 780

Asp Pro Thr Tyr Thr Ser Ala Leu Gly Gly Lys He Pro He Arg Trp 785 790 795 800

Thr Ala Pro Glu Ala He Gin Tyr Arg Lys Phe Thr Ser Ala Ser Asp 805 810 815

Val Trp Ser Tyr Gly He Val Met Trp Glu Val Met Ser Tyr Gly Glu 820 825 830

Arg Pro Tyr Trp Asp Met Thr Asn Gin Asp Val He Asn Ala He Glu 835 840 845

Gin Asp Tyr Arg Leu Pro Pro Pro Met Asp Cys Pro Ser Ala Leu His 850 855 860

Gin Leu Met Leu Asp Cys Trp Gin Lys Asp Arg Asn His Arg Pro Lys 865 870 875 880

Phe Gly Gin He Val Asn Thr Leu Asp Lys Met He Arg Asn Pro Asn 885 890 895

Ser Leu Lys Ala Met Ala Pro Leu Ser Ser Gly He Asn Leu Pro Leu 900 905 910

Leu Asp Arg Thr He Pro Asp Tyr Thr Ser Phe Asn Thr Val Asp Glu 915 920 925

Trp Leu Glu Ala He Lys Met Gly Gin Tyr Lys Glu Ser Phe Ala Asn 930 935 940

Ala Gly Phe Thr Ser Phe Asp Val Val Ser Gin Met Met Met Glu Asp 945 950 955 960

He Leu Arg Val Gly Val Thr Leu Ala Gly His Gin Lys Lys He Leu 965 970 975

Asn Ser He Gin Val Met Arg Ala Gin Met Asn Gin He Gin Ser Val 980 985 990

Glu Val

Claims

WE CLAIM:

1. A method of activating a ligand regulatory pathway in a cell, comprising reacting an Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein with a cell expressing a ligand for an Eph subfamily receptor tyrosine kinase on the cell surface thereby activating the Ugand regulatory pathway in the ceU.

2. A method as claimed in claim 1 wherein the protein or part of the protein is lacking in catalytic kinase activity.

3. A method as claimed in claim 1 wherein the part of the protein comprises an extraceUular domain of an Eph subfamily receptor tyrosine kinase.

4. A method for identifying a substance which is capable of binding to a Ugand for an Eph subfamily receptor tyrosine kinase and activating a ligand regulatory pathway in a ceU, comprising reacting a cell expressing a Ugand for an Eph subfamily receptor tyrosine kinase with at least one substance which potentially can bind with the ligand, under conditions which permit the formation of substance-Ugand complexes, and assaying for substance-Ugand complexes, for free substance, for non-complexed Ugands, or for activation of the Ugand.

5. A method as claimed in claim 4 wherein activation of the ligand is assayed by measuring phosphorylation of the ligand or binding of SH2 domains to the ligand, or by assaying for a biological affect on the ceU.

6. A method as claimed in claim 4 wherein the substance is an Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein.

7. A method as claimed in claim 4 wherein the part of the protein comprises an extracellular domain.

8. A method as claimed in claim 4 wherein the biological affect on the cell is inhibition or stimulation of proliferation, differentiation or migration.

9. A method for assaying a medium for an agonist or antagonist of a Ugand regulatory pathway in a cell which comprises providing a ceU expressing a Ugand for an Eph subfamUy receptor tyrosine kinase on the cell surface, reacting the cell with an Eph subfamily receptor tyrosine kinase protein and a suspected agonist or antagonist under conditions which permit the formation of ligand-receptor tyrosine kinase protein complexes on the cell surface, and assaying for ligand-receptor tyrosine kinase protein complexes, for free receptor tyrosine kinase protein, for non-complexed proteins, for activation of the receptor tyrosine kinase protein, or for activation of the ligand.

10. A method as claimed in claim 9 wherein activation of the ligand is assayed by measuring phosphorylation of the Ugand or binding of SH2 domains to the Ugand or by assaying for a biological affect on the ceU.

11. A method as claimed in claim 9 wherem the biological affect on the cell is inhibition or stimulation of proUferation, differentiation or migration.

12. A method for affecting neuronal development or regeneration in a mammal comprising administering to a mammal an effective amount of a purified and isolated Eph subfamUy receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein.

13. A method as claimed in claim 12 wherein the part of the protein comprises an extraceUular domain of an Eph subfamily receptor tyrosine kinase.

14. A method as claimed in claim 12 wherein the protein or part of the protein is lacking in a catalytic kinase domain.

15. A method for stimulating or inhibiting axonogenesis in a mammal comprising administering to a mammal an effective amount of a purified and isolated Eph subfamily receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein.

16. A method as claimed in claim 15 wherein the part of the protein comprises about an extraceUular domain of an Eph subfamUy receptor tyrosine kinase.

17. A method as claimed in claim 15 wherein the protein or part of the protein is lacking in a catalytic kinase domain.

18. A pharmaceutical composition which comprises a purified and isolated Eph subfamily receptor tyrosine kinase protein or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein for affecting neuronal development or regeneration and a pharmaceutically acceptable carrier, diluent or excipient.

19. A pharmaceutical composition as claimed in claim 18 wherein the part of the protein comprises an extraceUular domain of an Eph subfamUy receptor tyrosine kinase.

20. A pharmaceutical composition as claimed in claim 18 wherein the protein or part of the protein is lacking in a catalytic kinase domain.