The ability of polypeptide ligands to bind cells and thereby elicit a phenotypic response such as cell growth, survival or differentiation in such cells is often mediated through transmembrane tyrosine kinases. The extracellular portion of each receptor tyrosine kinase (RTK) is generally the most distinctive portion of the molecule, as it provides the protein with its ligand-recognizing characteristic. Binding of a ligand to the extracellular domain results in signal transduction via an intracellular tyrosine kinase catalytic domain which transmits a biological signal to intracellular target proteins. The particular array of sequence motifs of this cytoplasmic, catalytic domain determines its access to potential kinase substrates (Mohammadi, et al., 1990, Mol. Cell. Biol., 11: 5068-5078; Fantl, et al., 1992, Cell, 69: 413-413).
All known growth factor RTKs appear to undergo dimerization following ligand binding (Schlessinger, J., 1988, Trend Biochem. Sci. 13: 443-447; Ullrich and Schlessinger, 1990, Cell, 61: 203-212; Schlessinger and Ullrich, 1992, Neuron 9: 383-391); molecular interactions between dimerizing cytoplasmic domains lead to activation of kinase function. In some instances, such as the growth factor platelet derived growth factor (PDGF), the ligand is a dimer that binds two receptor molecules (Hart, et al., 1988, Science, 240: 1529-1531; Heldin, 1989, J. Biol. Chem. 264: 8905-8912) while, for example, in the case of EGF, the ligand is a monomer (Weber, et al., 1984, J. Biol. Chem., 259: 14631-14636).
The tissue distribution of a particular tyrosine kinase receptor within higher organisms provides relevant data as to the biological function of the receptor. The tyrosine kinase receptors for some growth and differentiation factors, such as fibroblast growth factor (FGF) are widely expressed and therefore appear to play some general role in tissue growth and maintenance. Members of the Trk RTK family (Glass & Yancopoulos, 1993, Trends in Cell Biol, 3: 262-268) of receptors are more generally limited to cells of the nervous system, and the neurotrophins which bind these receptors promote the differentiation of diverse groups of neurons in the brain and periphery (Lindsay, R. M., 1993, in Neurotrophic Factors, S. E. Loughlin & J. H. Fallon, eds., pp. 257-284 (San Diego, Calif.: Academic Press). The localization of one such Trk family receptor, trkB, in tissue provided some insight into the potential biological role of this receptor, as well as the ligands that bind this receptor (referred to herein as cognates). Thus, for example, in adult mice, trkB was found to be preferentially expressed in brain tissue, although significant levels of trkB mRNAs were also observed in lung, muscle, and ovaries. Further, trkB transcripts were detected in mid and late gestation embryos. In situ hybridization analysis of 14 and 18 day old mouse embryos indicated that trkB transcripts were localized in the central and peripheral nervous systems, including brain, spinal cord, spinal and cranial ganglia, paravertebral trunk of the sympathetic nervous system and various innervation pathways, suggesting that the trkB gene product may be a receptor involved in neurogenesis and early neural development as well as play a role in the adult nervous system.
The cellular environment in which an RTK is expressed may influence the biological response exhibited upon binding of a ligand to the receptor. Thus, for example, when a neuronal cell expressing a Trk receptor is exposed to a neurotrophin which binds that receptor, neuronal survival and differentiation results. When the same receptor is expressed by a fibroblast, exposure to the neurotrophin results in proliferation of the fibroblast (Glass, et al., 1991, Cell 66: 405-413). Thus, it appears that the extracellular domain provides the determining factor as to the ligand specificity, and once signal transduction is initiated the cellular environment will determine the phenotypic outcome of that signal transduction.
A number of RTK families have been identified based on sequence homologies in their intracellular domain. For example, two members of the TIE (tyrosine kinase with immunoglobulin and EGF homology domains) family, known as TIE-1 and TIE-2, have 79% sequence homology in their intracellular region (Maisonpierre, et al., 1993, Oncogene 8: 1631-1637). Although these receptors share similar motifs in their extracellular domain, only 32% of the sequences are identical. This indicates potentially divergent biological roles which are reflected in the fact that while both genes are widely expressed in endothelial cells of embryonic and postnatal tissue, significant levels of tie-2 transcripts are also present in other embryonic cell populations that include lens epithelium, heart epicardium and regions of mesenchyme.
The receptor and signal transduction pathways utilized by NGF involve the product of the trk proto-oncogene. (Kaplan et al., 1991, Nature 350: 156-160; Klein et al., 1991, Cell 65: 189-197). Klein et al. (1989, EMBO J. 8: 3701-3709) reported the isolation of trkB, which encodes a second member of the tyrosine protein kinase family of receptors found to be highly related to the human trk proto-oncogene. TrkB binds and mediates the functional responses to BDNF, NT-4, and to a lesser extent, NT-3 (Squinto, et al., 1991, Cell 65: 885-903; Ip, et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89: 3060-3064; Klein, et al., 1992, Neuron, 8: 947-956). At the amino acid level, the products of trk and trkB were found to share 57 percent homology in their extracellular regions, including 9 of the 11 cysteines present in trk. This homology was found to increase to 88 percent within their respective tyrosine kinase catalytic domains. The Trk gene family has now been expanded to include the trkC locus, with NT-3 having been identified as the preferred ligand for trkC (Lamballe, et al., 1991, Cell 66: 967-979).
Two novel human genes, ror1 and ror2, encode proteins which have in their cytoplasmic portion a region homologous to proteins differ considerably from the Trk family in their extracellular portion (Masiakowski and Carroll, 1992, J. Biol. Chem. 267: 26181-26190).
Another receptor having a kinase domain that is related to the Trk family has been identified in the electric ray Torpedo californica and may play a role in motor neuron induced synapses on muscle fibers. Jennings, et al. Proc. Natl. Acad. Sci. USA 90: 2895-2899 (1993). This kinase was isolated from the electric organ, tissue which is homologous to muscle. Like the rors, the tyrosine kinase domain of this protein is related to the Trk family, while the extracellular domain is somewhat divergent from the Trks. The protein was found to be expressed at high levels in Torpedo skeletal muscle, and at much lower levels in adult Torpedo brain, spinal cord, heart, liver and testis.
Because RTKs appear to mediate a number of important functions during development, the identification and isolation of novel RTKs may be used as a means of identifying new ligands that may play a crucial role in development. Often such novel RTKs are identified and isolated by searching for additional members of known families of tyrosine kinase receptors using, for example, PCR-based screens involving known regions of homology among Trk family members. (See, for example, Maisonpierre, et al., 1993, Oncogene 8: 1631-1637). Isolation of such so called "orphan" tyrosine kinase receptors, for which no ligand is known, and subsequent determination of the tissues in which such receptors are expressed, provides insight into the regulation of the growth, proliferation and regeneration of cells in target tissues. Further, such receptors may be used to isolate their cognate ligand, which may then be used to regulate the survival, growth and regeneration of cells expressing the receptor.
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