The correct attachment of a subset of muscles in the Drosophila embryo requires the expression and function of the RYK subfamily receptor tyrosine kinase gene derailed (drl). A second RYK homolog, doughnut (dnt), has been isolated from Drosophila. The Dnt protein exhibits 60% amino acid identity to Drl, and is structurally as similar to the mammalian RYK (for related to tyrosine kinase) proteins as is Drl, indicating an ancient duplication event. dnt is expressed in dynamic patterns in the embryonic epidermis; it is found at high levels in epithelia adjacent to cells that are invaginating the interior of the embryo, including ventral furrow, cephalic furrow, fore- and hind-gut, optic lobe and tracheal pits. dnt is capable of a partial rescue of the muscle attachment defect of drl-/- embryos, indicating that it encodes a receptor with a related and significantly overlapping biochemical function (Oates, 1998).

Wnt proteins are intercellular signals that regulate various aspects of animal development. In C. elegans, mutations in lin-17, a Frizzled-class Wnt receptor, and in lin-18 affect cell fate patterning in the P7.p vulval lineage. lin-18 encodes a member of the Ryk/Derailed family of tyrosine kinase-related receptors, found to function as Wnt receptors. Members of this family have nonactive kinase domains. The LIN-18 kinase domain is dispensable for LIN-18 function, while the Wnt binding WIF domain is required. Wnt proteins LIN-44, MOM-2, and CWN-2 redundantly regulate P7.p patterning. Genetic interactions indicate that LIN-17 and LIN-18 function independently of each other in parallel pathways, and different ligands display different receptor specificities. Thus, two independent Wnt signaling pathways, one employing a Ryk receptor and the other a Frizzled receptor, function in parallel to regulate cell fate patterning in the C. elegans vulva (Inoue, 2004).

Since lin-44(null) enhances lin-18(null) but not lin-17(null), lin-44 must function in parallel to lin-18. Similarly, since mom-2(null) enhances lin-17(null), mom-2 must function in parallel to lin-17. Based on these results, it is proposed that LIN-44 preferentially functions as the ligand for LIN-17/Frizzled and MOM-2 preferentially functions as the ligand for LIN-18/Ryk. Since lin-44 and mom-2 single mutant phenotypes are weaker than those of lin-17 and lin-18, each receptor likely transduces additional signals (including LIN-44/LIN-18 and MOM-2/LIN-17 combinations as well as CWN-2). A weak enhancement of lin-18(e620) by mom-2(RNAi) supports this possibility. The results do not rule out the possibility that LIN-44 or MOM-2 signals through a third pathway. However, the complete reversal of the P7.p orientation observed in the lin-17; lin-18 double mutant suggests that the two receptors account for most of the P7.p orienting activity. LIN-17 and LIN-44 are also required for other fate specifications in C. elegans, suggesting that LIN-17 acts as a LIN-44 receptor in multiple tissues. Sequence analysis suggests that CWN-2 is the ortholog of Wnt5, the ligand for Derailed in Drosophila. Therefore, the involvement of CWN-2 is consistent with it functioning as a LIN-18 ligand, although it was not possible to resolve the receptor specificity for this ligand. The orthology relationship of MOM-2 is not clear. MOM-2/Wnt and MOM-5/Frizzled are required for endoderm induction. However, no evidence of MOM-5 involvement in P7.p orientation was found, and LIN-18 is not required for endoderm induction (Inoue, 2004).

The C. elegans vulva is comprised of highly similar anterior and posterior halves that are arranged in a mirror symmetric pattern. The cell lineages that form each half of the vulva are identical, except that they occur in opposite orientations with respect to the anterior/posterior axis. Most vulval cell divisions produce sister cells that have asymmetric levels of POP-1 and that the asymmetry has opposite orientations in the two halves of the vulva. lin-17 (Frizzled type Wnt receptor) and lin-18 (Ryk/Derailed family) regulate the pattern of POP-1 localization and cell type specification in the posterior half of the vulva. In the absence of lin-17 and lin-18, posterior lineages are reversed and resemble anterior lineages. These experiments suggest that Wnt signaling pathways reorient cell lineages in the posterior half of the vulva from a default orientation displayed in the anterior half of the vulva (Deshpande, 2005).

By using the polymerase chain reaction with degenerate oligonucleotides based on highly conserved motifs held in common between all members of the protein tyrosine kinase (PTK) family, a PTK-related sequence was isolated from murine peritoneal macrophage cDNA. Full-length clones have been isolated that encompass the entire coding region of the mRNA, and the predicted amino acid sequence indicates that the protein encoded has the structure of a growth factor receptor PTK (RTK). This molecule has been dubbed RYK (for related to tyrosine kinase). The RYK-encoded protein bears a transmembrane domain, with a relatively small (183 amino acid) extracellular domain, containing five potential N-linked glycosylation sites. The intracellular domain of RYK is unique among the broader family of RTKs and has several unusual sequence idiosyncrasies in some of the most highly conserved elements of the PTK domain. These sequence differences call into question the potential catalytic activity of the RYK protein (Hovens, 1992).

A cDNA encoding the human homolog of mouse RYK (related to receptor tyrosine kinases) has been cloned from an interleukin 1 (IL-1)-stimulated human hepatoma cDNA library by cross-species hybridization using the mouse RYK cDNA as a probe. The sequence of the 3067-bp cDNA clone encoding human RYK predicts a transmembrane protein with a cytoplasmic domain that contains the consensus sequences (subdomains I-XI) of the protein tyrosine kinase (PTK) family. The highly conserved motif -D-F-G- (subdomain VII) of the catalytic domain of other receptor-type tyrosine kinases is altered to -D-N-A- in human RYK. In addition, a number of other changes are found in the ATP binding site (subdomains I and II) and the motif [-I-H-R-D-L-A-A-R-N-] found in subdomain VI. Comparison of the human and mouse RYK sequences shows a 92% conservation at the nucleotide level and 97% at the amino acid level. There was no significant homology between the extracellular domain of RYK and the other families of receptor tyrosine kinases described to date. RYK therefore appears to define a new subclass of receptor-type tyrosine kinases whose structure has remained highly conserved across species (Stacker, 1993).

Degenerate primers designed from conserved tyrosine kinase domains were used to identify and clone a novel receptor-like molecule (designated Nbtk-1) from an NB41 mouse neuroblastoma cell line. Nbtk-1 is related to the met proto-oncogene family of tyrosine kinase receptors. Transcripts of approximately 2.1 and 2.6 kb have been found in mouse cell lines and one transcript of approximately 3 kb in human cell lines and in a wide range of primary human tumors, such as neuroblastomas, primitive neuroectodermal tumors (PNETs), Wilms' tumors, and melanomas and in the corresponding normal human tissues. These observations suggest that Nbtk-1 may have important roles in normal and tumor cell growth (Maminta, 1992).

The gene encoding the murine RYK growth factor receptor protein tyrosine kinase has been mapped by genetic linkage analysis with recombinant inbred strains of mouse. Two distinct Ryk loci (Ryk-1 and Ryk-2) have been identified. Ryk-1 maps to Chromosome (Chr) 9, whereas Ryk-2 maps to Chr 12. A similar arrangement of RYK-related loci has previously been determined in the human. Synteny has already been established between murine Chr 9 in the region of Ryk-1, and human chromosome 3q11-12, the location of the human RYK-1 gene. However, the Ryk-2/RYK-2 loci on murine Chr 12 and human Chr 17p13.3 define a new region of synteny (Gough, 1995).

By screening for expressed sequences with conserved tyrosine kinase catalytic domains, an attempt was made to isolate novel receptor tyrosine kinases that may play roles in hematopoietic development. Among the known tyrosine kinases identified in this screen, a gene was found with characteristics of a receptor tyrosine kinase but unusual motifs in the catalytic domain. This gene is identical to ryk as described independently by other investigators. Chromosomal fluorescence in situ hybridization localization of human ryk was clarified by using monochromosomal hybrids, a process that places the gene as a single locus in 3q22. Although Northern analysis reveals widespread expression in adult mouse tissues, ryk expression is not ubiquitous. Expression increases in bone marrow cells from mice treated with 5-fluorouracil. Northern analysis on cell lines indicates expression in CD3-, CD4-, CD8- T cells (at a low level), pre-T cells, thymic epithelial cells, and mature myeloid cells, but not myeloid precursors or B cell precursors. Expression analysis with the use of RT-PCR on mouse bone marrow cells separated on the basis of cell surface markers (B220, CD4, CD8, Gr-1, Mac-1) reveals that this receptor is expressed in differentiated cells (Lin+) but is not expressed in the precursor cells (Lin-). Flow cytometric analysis with a monospecific anti-Ryk antibody demonstrates that Ryk+ cells constitute 36.7% and Lin+/Ryk+ cells constitute 33.7% of low density bone marrow cells, whereas Ryk+ cells represent only 0.3% of the Lin- population. It is concluded that during hematopoietic development, ryk expression is regulated by lineage commitment and stage of maturation (Simoneaux, 1995).

Protein tyrosine kinases play an important role in cellular metabolism as key components of signal transduction pathways. An unusual receptor tyrosine kinase, H-RYK, has been isolated from a complimentary DNA library of SKOV-3, an epithelial ovarian cancer cell line, using a polymerase chain reaction-mediated approach. The primary structure of the predicted amino acid sequence of the protein shows a novel NH2-terminal region. The catalytic region shows homology to other tyrosine kinases, the closest homology being with v-sea (39%). A significant alteration in the catalytic domain is that the highly conserved 'DFG' triplet in subdomain VII is altered to 'DNA'. The gene was mapped to chromosome 3q22. Northern analysis has determined that a single transcript of 3.0 kb is expressed in heart, brain, lung, placenta, liver, muscle, kidney, and pancreas, with maximal expression in skeletal muscle. In situ hybridization analysis on human tissues demonstrates localization of message in the epithelial and stromal compartment of tissues such as brain, lung, colon, kidney, and breast. There is minimal to absent expression of H-RYK on the surface epithelium of ovaries. In benign and borderline tumors of the ovary, there is expression in the stromal compartment. However, in malignant tumors there is increased expression predominantly confined to the epithelium. Polyclonal antisera raised against synthetic peptides recognize a 100-kD protein in ovarian cancer cells and other cell lines. In contrast to other receptor tyrosine kinases, the receptor does not phosphorylate in an in vitro kinase assay. The expression of this unusual receptor tyrosine kinase in epithelial ovarian cancer suggests that it may be involved in tumor progression; this is an area in need of further investigation (Wang, 1996).

Receptor tyrosine kinase RYK is a mammalian homolog of Drosophila Derailed, which is involved in learning and memory and in axon guidance. A rat ryk gene has been cloned and its expression pattern in the central nervous system characterized. Northern blot analysis of the whole brain reveals that the RYK mRNA is abundant during the period from 13 to 18 embryonic days (E13-18); it decreases by E20. In the postnatal brain, the RYK signal is higher in postnatal one week (P1W) cerebrum and in P2W cerebellum than in later stages. In situ hybridization reveals that RYK is expressed throughout the central nervous system, mainly in the ventricular zone on E11 and E13. On E18 and E20, the remarkable level of RYK mRNA is detected in the ventricular zone as well as in the cortical plate of the forebrain. These two regions overlap the immunoreactive areas of nestin and MAP2, a neural stem cell marker and a mature neural marker, respectively. Moreover, the double-labeling analysis shows that the same cells express both RYK and nestin in the ventricular zone. In the postnatal brain, RYK is predominantly expressed in neurons of various regions. These observations suggest that RYK plays a contributory role as a multifunctional molecule in the differentiation and maturation of neuronal cells in the central nervous system (Kamitori, 1999).

The Ryk receptor belongs to the atypical receptor tyrosine kinase family. It is a new member of the family of Wnt receptor proteins. However, the molecular mechanisms by which the Ryk receptor functions remain unknown. Mammalian Ryk, unlike the Drosophila Ryk homolog Derailed, functions as a coreceptor along with Frizzled for Wnt ligands. Ryk also binds to Dishevelled, through which it activates the canonical Wnt pathway, providing a link between Wnt and Dishevelled. Transgenic mice expressing Ryk siRNA exhibit defects in axon guidance, and Ryk is required for neurite outgrowth induced by Wnt-3a and in the activation of T cell factor (TCF) induced by Wnt-1. Thus, Ryk appears to play a crucial role in Wnt-mediated signaling (Lu, 2004).

Ryk siRNA mice have defects in axon guidance of craniofacial motor nerves, ophthalmic nerves, and other nerves, suggesting an essential role of Ryk in axon guidance. Although there is no obvious deficiency in dorsal root ganglion neurite outgrowth in Ryk siRNA transgenic mice, dorsal root ganglion explants isolated from Ryk siRNA mice exhibit defects in neurite outgrowth in response to Wnt-3a stimulation. The lack of deficiency in DRG neurite outgrowth in Ryk siRNA mice is probably because NGF and other growth factors are also involved in inducing neurite outgrowth in vivo. The fact that the Wnt-3a-induces neurite outgrowth of dorsal root ganglion explants is inhibited in Ryk siRNA mice provides strong evidence that there is a functional interaction between Wnt and Ryk in neurite outgrowth (Lu, 2004).

Guidance cues along the longitudinal axis of the CNS are poorly understood. Wnt proteins attract ascending somatosensory axons to project from the spinal cord to the brain. Wnt proteins repel corticospinal tract (CST) axons in the opposite direction. Several Wnt genes were found to be expressed in the mouse spinal cord gray matter, cupping the dorsal funiculus, in an anterior-to-posterior decreasing gradient along the cervical and thoracic cord. Wnts repel CST axons in collagen gel assays through a conserved high-affinity receptor, Ryk, which is expressed in CST axons. Neonatal spinal cord secretes diffusible repellent(s) in an anterior-posterior graded fashion, with anterior cord being stronger, and the repulsive activity is blocked by antibodies to Ryk (anti-Ryk). Intrathecal injection of anti-Ryk blocks the posterior growth of CST axons. Therefore, Wnt proteins may have a general role in anterior-posterior guidance of multiple classes of axons (Liu, 2005).

Mammalian RYK is a receptor related to tyrosine kinase without detectable catalytic activity. Rat RYK is dominantly expressed in neural progenitor cells and mature neurons in the developing central nervous system. Mouse RYK has been found to bind to EphB2/B3 receptors, which have diverse functions during development. RYK, EphB2, EphB3, ephrinB1, and ephrinB2 are expressed in embryonic brain. In vitro analysis using COS-7 cells revealed binding between rat RYK and EphB3, and the RYK deletion mutant without extracellular leucine-rich motifs lacks this binding ability. To investigate the function of RYK in vivo, embryonic cortical slice cultures were analyzed after electroporation of expression plasmids for RYK or its deletion mutants. The results show that overexpression of RYK suppresses cell migration from the ventricular zone to the pial surface, however, overexpression of the RYK deletion mutant without leucine-rich motifs has no effect on cell migration. These results suggest that RYK regulates cell migration during mammalian cortical development through the binding to Eph receptors (Kamitori, 2005).

Ryk is novel Wnt receptor in both Caenorhabditis elegans and Drosophila melanogaster. Ryk-Wnt interactions have been shown to guide corticospinal axons down the embryonic mouse spinal cord. In Ryk-deficient mice, cortical axons project aberrantly across the major forebrain commissure, the corpus callosum. Many mouse mutants have been described in which loss-of-function mutations result in the inability of callosal axons to cross the midline, thereby forming Probst bundles on the ipsilateral side. In contrast, loss of Ryk does not interfere with the ability of callosal axons to cross the midline but impedes their escape from the midline into the contralateral side. Therefore, Ryk^-/- mice display a novel callosal guidance phenotype. Wnt5a acts as a chemorepulsive ligand for Ryk, driving callosal axons toward the contralateral hemisphere after crossing the midline. In addition, whereas callosal axons do cross the midline in Ryk^-/- embryos, they are defasciculated on the ipsilateral side, indicating that Ryk also promotes fasciculation of axons before midline crossing. In summary, this study expands the emerging role for Wnts in axon guidance and identifies Ryk as a key guidance receptor in the establishment of the corpus callosum. This analysis of Ryk function further advances understanding of the molecular mechanisms underlying the formation of this important commissure (Keeble, 2006).

Computational modelling has suggested that at least two counteracting forces are required for establishing topographic maps. Ephrin-family proteins are required for both anterior-posterior and medial-lateral topographic mapping, but the opposing forces have not been well characterized. Wnt-family proteins are recently discovered axon guidance cues. Wnt3 is expressed in a medial-lateral decreasing gradient in chick optic tectum and mouse superior colliculus. Retinal ganglion cell (RGC) axons from different dorsal-ventral positions show graded and biphasic response to Wnt3 in a concentration-dependent manner. Wnt3 repulsion is mediated by Ryk, expressed in a ventral-to-dorsal decreasing gradient, whereas attraction of dorsal axons at lower Wnt3 concentrations is mediated by Frizzled(s). Overexpression of Wnt3 in the lateral tectum repels the termination zones of dorsal RGC axons in vivo. Expression of a dominant-negative Ryk in dorsal RGC axons causes a medial shift of the termination zones, promoting medially directed interstitial branches and eliminating laterally directed branches. Therefore, a classical morphogen, Wnt3, acting as an axon guidance molecule, plays a role in retinotectal mapping along the medial-lateral axis, counterbalancing the medial-directed EphrinB1-EphB activity (Schmitt, 2006).