Increasing interest in understanding the molecular basis of tissue modeling and patterning processes in vertebrate development has led to the identification of protein families which direct cell movement in embryogenesis (reviewed by Bonhoeffer & Sanes, 1995, Curr. Opin. Neumbiol. 5 1-5). Apart from members of the fibroblast growth factor (FGF) and transforming growth factor beta (TOF-β) families, which are involved in mesoderm induction and patterning (Green & Smith, 1991, Trends in Genetics 7 245-250), proteins of the netrin, semaphorin and collapsin families are thought to control axon guidance and neural pathfinding (Kennedy & Tessier-Lavigne, 1995, Current Opinion in Neurobiology 5 83-90; Müller et al., 1996, Current Opinion in Genetics and Development 6 469-474).
Such growth factors and their cell surface receptors, as well as many other types of receptor-ligand pairs, have characteristic mechanisms for transducing the ligand-receptor binding effect into intracellular changes.
One major receptor type is the receptor protein-tyrosine kinase (RTK) family, the members of which include intracellular tyrosine kinase domains which are activated in response to ligand simulation, resulting in autophosphorylation of certain receptor tyrosine residues. The phosphorylated tyrosines in turn bind to and activate signaling molecules, thereby activating an intracellular signaling cascade.
Over 14 distinct groups of RTKs are known, and of these the largest group is the “Eph” family, which until comparatively recently were “orphan” receptors for which no ligand had been identified. However, the Eph family ligands are now known to represent a family of glycosyl phosphatidylinositol (GPI)-linked or transmembrane molecules.
Among RTKs which are implicated in the regulation of developmental patterning events (Pawson and Bemstein, 1990, Trends Genet. 6 350-356), members of the Eph-family RTKs have been linked to neurogenesis (Müller et al., 1996, supra; Tessier-Lavigne, M., 1995 Cell 82 345-348: Pandey et al., 1995 5 986-989; Nieto, M. A. 1996, 17 1039-1048) initially due to their spatially-restricted expression patterns during the development of the vertebrate nervous system (reviewed by Friedman & O'Leary, 1996, Current Opinion in Neurobiology 6 127-133). The characterisation of the expression patterns together with functional studies of Eph receptors has, in several cases, confirmed significant roles for Eph signaling in axon guidance, in particular, during the development of the retinotectal projection map (Cheng & Flanagan, 1994, Cell 79 157-168; Chang et al., 1995, Cell 82 371-381; Drescher, U., 1995, Cell 82 359-370; Winslow, et al. 1995, Neuron 14 973-981; Tessier-Lavigne, 1995, supra; Brennan et al., 1997, Development 124 655-664).
The results of studies utilizing overexpression of some family members including HEK, EPH, ERK and ECK in tumour-derived cell lines, tumour specimens and transfected cells implicates these receptors in oncogenesis (Hiral et al., 1989, Science 238 1717-20; Boyd et al., 1992, J. Biol. Chem. 267(5) 3262-7: Maru et al., 1990; Andres et al., 1994, Oncogene 9 1461-7).
HEK was first identified on the cell surface of a pre-B acute lymphoblastic leukemia cell line, LK63, using the III-A4 monoclonal antibody (Boyd et at., 1992, supra). Immunofluorescence studies with III-A4 revealed expression of HEK in blood samples from patients with acute leukemia, but not in normal adult tissues or blood cells (Boyd et al., 1992, supra; Wicks et al., 1992, Proc. Natl. Acad. Sci. (USA) 89(5) 1611-5).
A cDNA encoding HEK has been obtained and the nucleotide sequence of the entire coding region deduced as reported in Wicks et al., 1992, Proc. Natl. Acad. Sci USA (which is herein incorporated by reference), and in WO93/00425 (which is herein incorporated by reference).
In embryos, the expression patterns of the murine and chicken HEK homologues MEK 4 and CEK 4, and their recently identified respective ligands ELF1 and RAGS, suggest a role in the development of the retinotectal projection map. A soluble HEK ligand from human placenta conditioned medium has been identified using a biosensor-based affinity detection approach (Lackmann et al., 1995). The HEK ligand was identified by sequence homology as a soluble form of AL-1 (Winslow at al., 1995, Neuron 14 973-981), a member of the family of ligands for EPH Related Kinases (LERKS: Bohme et at., 1996, J. Biol. Chem. 271 24727-24752; Cerreti et al., 1996, Genomics 35 376-379), which for consistency with other members will hereinafter be referred to as LERK 7. This family of transmembrane or membrane-associated proteins were isolated as potential ligands for EPH-like RTKs through their interactions with recombinant EPH receptor family exodomains (Winslow et al. 1994: Beckmann et al., 1994, Embo Journal. 13 3757-62; Shao et al., 1995, Journal of Biological Chemistry 270 3467-70; Brambilia et al., 1995, Embo Journal 14 3116-3126).
Extremely high interspecies sequence similarities of the known Eph family members suggests that these receptors have evolutionarily conserved functions, but little is known about the actual protein structures or about the structure/function relationships between Eph-like receptors and their ligands. Typically, and as is the case with HEK, Eph RTKs have an exodomain which includes an N-terminal cysteine-rich region, the outer portion of which has been described as immunoglobulin-like (Ig-like), and two fibronectin ill regions (Pandey et at., 1995, Journal of Biological Chemistry 270 19201-19204; Tuzi & Gullick, 1994, British Journal of Cancer 69 417-421; Henkemeyer, M., 1994, Oncogene 9 1001-1014). Extensive crossreactivity of Eph receptor/ligand interactions has been observed with divalent receptor (ligand) fusion proteins containing the Fc domain of human IgG 1 (Beckmann et al., 1994, supra; Davis et al., 1994, Science 266 816-819; Pandey et al., 1994, Journal of Biological Chemistry 269 30154-30157; Cerretti et al. 1995, Molecular Immunology 32 1197-1205; Pandey et oh, 1995, Current Biology 5 986-989; Brambilla St al., 1995, supra).
All of the known ligands exist as membrane-associated forms, and dependence of receptor activation on membrane bound or oligomerised ligands (Winslow et al., 1995, supra; Davis et al., 1994, supra) was reported for most members of the Eph-like receptor and ligand families. The apparent receptor/ligand promiscuity of various receptors and ligands monitored with receptor or ligand Fc fusion constructs suggested that Eph family RTKs could be separated into two redundant sub-classes, based on affinity for transmembrane of GPI-linked respectively. Together with their overlapping expression patterns, this led to the formulation of a model in which promiscuous interactions within subclasses mediates formation of spatial boundaries and patterning events during development (Gale et at., 1996, Neuron 17 9-19).
This reported redundancy is at odds with several studies which demonstrate specialised functions of the homologous RTKs MEK4/CEK4/RTK2 and their corresponding ligands ELF1/RAGS/zEphL4 during the development of the retinoteotal projection map in mouse, chicken and zebrafish (Cheng et at., 1995, Cell 82 371-381; Drescher et al., 1995, Cell 82 359-370, Nakamoto et al., 1996, Cell 86 755-766; Brennan et at., 1997, Development 124 655-664).