NOTE: First cloning of RYK was performed in 1992, ¹ with a PCR-based screen procedure (homology screen with primers based on highly conserved motifs common to all members of the protein tyrosine kinase [PTK] family), aimed to discover novel receptor tyrosine kinases. The new PTK-related molecule, RYK, was identified from murine peritoneal macrophage cDNA. The RYK sequence was characterized by unusual variations on the ordinarily highly conserved elements of the PTK family, prompting a specialized role in signal transduction within the cell. The human homologue of mouse Ryk has been cloned from a human hepatoma cDNA library by cross-species hybridization based on probing the tumor library with the mouse Ryk cDNA. ² Derailed and LIN-18 are orthologs of RYK respectively in Drosophila and Caenorhabditis.  GENE_NAME: RYK (receptor-like tyrosine kinase or related to receptor tyrosine kinases). CHROMOSOMAL LOCALIZATION: the RYK localization was determined by FISH analysis on normal human metaphase spreads and was shown to map to 3q22.2. ³

Figure 1: RYK localization by FISH

RYK localization on normal human metaphase with RTK15-EcoRV linearized clone 15.5BX3 as a probe. Image adapted from ³

LOCAL ORDER: gene sequence starts at 134157134 and ends at 134250744 bp from pter of the chromosome 3 (according to hg38). Source: Ensembl (RYK-208) ENST00000620660.4

Figure 2 Chromosomal band localization of RYK

RYK gene maps to 3q22.2

STRAND: reverse (-)

SIZE: 93611bp (including UTRs)

CODING REGION (source UCSC): Position: chr3:134,158,153-134,250,654. Size: 92502 bp.

RYKDNA FULL EXON-INTRON SEQUENCE: see the following internet address http://www.ensemble.org/id/ENSG00000163785 .

GENOMIC TOPOGRAPHY AND EXON-INTRON DISTRIBUTION OF RYK GENE: see Figure 3 modified from NCBI.

Figure 3. RYK genomic topography and  Exon-Intron distribution

TRANSCRIPT LENGTH: 2942 bps (Source: Ensembl RYK-208 ENST00000620660.4)

CODING EXON COUNT: 15 (TOTAL EXON COUNT: 15)

TRANSLATION LENGTH: 610 residues (Source: Ensembl RYK-208 ENST00000620660.4)

TRANSCRIPT VARIANTS: alternative splicing results in multiple transcript variants encoding distinct isoforms: this gene has 9 transcripts (splice variants) and 2 main isoforms. Source ENSG00000163785

Probing the genome by in situ hybridization two RYK loci (Ryk-1 locus and Ryk-2 locus) were identified on 3q22 (RYK gene) and on 17q13.2 (RYKP1 [RYK pseudogene 1 ]) ( PMID: 8386829 ; PMID: 7613029).The 3’ untranslated region human RYK cDNA-derived probe detects only the 3q22 locus and gives evidence that this is the only locus that encodes a functional protein in humans. The possibility has been proposed (PMID: 10454588) that a hypothetical product of the RYK2 locus (pp75) represents the Ryk-2 locus receptor and could be the kinase-active partner of RYK gene via a heterodimerization mechanism. However until now the cDNA for Ryk-2 has not been isolated.

From 58 known human receptor tyrosine kinases (RTKs), 8 are classed as pseudokinases ⁴ and four protein of this group are involved in Wnt signalling: RYK, ROR1, ROR2, PTK7. RYK, as well as other pseudokinases, has substitutions in conserved motifs and lacks both kinase activity and ATP binding ability.

PROTEIN SYMBOL: P34925-RYK_HUMAN (UniProtKB/Swiss-Prot).

RECOMMENDED NAME: Tyrosine-protein kinase RYK (UniProtKB/Swiss-Prot).

SIZE: 607 amino acids

MOLECULAR MASS: 67815 Da

STRUCTURE: see  https://www.uniprot.org/uniprot/P34925/protvista

Figure 4: Comparison between several examples of human RTK pseudo kinases in relation to the different protein domains composing them, located in the extracellular region (above), membrane and intracellular region(below). Domains are listed in the legend: RYK has 2 main functional domains: 1-pseudokinase (red), 2- Wnt Inhibitory Factor (WIF) domain.

1- Wnt-inhibitory factor-1 like domain (WIF1): (related to protein location 63>196). WIF1 sequence codes for a protein extracellular domain in metazoan RYK receptor tyrosine kinases. WIF-1 binds to Wnts ligands and inhibits their activity.

2- Pseudokinase domain of RYK (related to protein location 326 > 606). RYK pseudokinase sequence codes for an extracellular region with two leucine-rich motifs, a transmembrane segment, and an intracellular inactive pseudokinase domain, which shows similarity to tyrosine kinases but lacks crucial residues for catalytic activity and ATP binding.

RYK belongs to the RTK family, wich includes transmembrane glycoproteins typically endowed with an N-terminal extracellular ligand-binding domain and a C-terminal intracellular tyrosine kinase domain. ¹

As shown in figure 4 RYK shares with all other members of the RTKs family a hydrophobic transmembrane domain (lenght: 28 aa., from 187 to 215) and an intracellular kinase domain with all typical expected features. ⁵

However, there are strong structural differences with respect to the family to which RYK protein belongs, represented by a very short extracellular domain (183 aa) and an unusual kinase domain. RYK extracellular domain lacks typical features of all other RTKs (e.g.immunoglobulin-like domains or cysteine-rich domains ) but is equipped with 2 LRMs (Leucine-rich motifs) that are part of a WIF-type Wnt binding domain. In turn the intracellular kinase domain while showing a clear tyrosine specificity is almost unique in the family showing a sequence substitution (aspartate-asparagine-alanine [D-F-G motif] instead of aspartate-phenylalanine-glycine [D-N-A motif] where the Phe and Gly residues in the DFG motif of the activation domain are essential for catalytic activity of RTKs) in a key position for the catalytic activity and for the ATP binding. ⁶

Figure 5 shows a structural comparison between RYK and its catalytically active counterpart IRK (insulin receptor kinase); RYK in its activation loop configuration closely resemble inactive IRK. Structural elements in the RTK pseudokinase domains are not significantly more rigid than in catalytically active counterparts such as IRK. It's possible for the pseudokinases to keep conformational switching-based regulatory mechanisms typical of active kinases. RYK and the other members of the group of Wnt-binding pseudokinase ROR1,ROR2, PTK7 being no more static than typical kinases, could show dynamic characteristics very similar to those of IRK. RYK pseudokinase domains display conformational dynamics similar to those of IRK, despite mimicking an ATP-bound state which could control scaffolding functions in signaling. The RTK pseudokinases are likely to resemble canonical protein kinases such as IRK in their capabilities to undergo transitions between active and (family-specific) inactive conformations. ⁴ Despite their apparent lack of catalytic activity these pseudokinases are essential signaling molecules.

Figure 5: Close-up of residues surrounding the ATP-binding site for active and inactive IRK-insulin receptor kinase (black) and for RYK. AMP-PNP (analog of ATP) is solid when seen in the relevant crystal structure and transparent when not. Note the D-N-A motif instead of the canonical D-F-G. Adapted from ⁴

RYK is expressed in many different tissues both during development and in adults, suggesting a widespread function.

Figure 6 RYK gene expression in various tissues http://biogps.org/#goto=genereport&id=6259

RYK is located on the cell membrane as a single-pass type 1 membrane protein (protein spanning the membrane once, with its N terminus on the extracellular side of the membrane and removal of its signal sequence)

Ryk is a widely expressed member of the general PTK family, with all of the features of a growth factor receptor, and more specifically is an atypical member of the family of growth factor receptor protein tyrosine kinases with structural differences from other members in the activation and nucleotide binding domains already mentioned above. It belongs to a subfamily whose members (the Wnt-binding ROR1,  ROR2, PTK7 and few others) do not appear to be regulated by phosphorylation in the activation segment. ¹ Ligand stimulation of the RYK receptor results in activation of the mitogen-activated protein kinase (MAPK) pathway. ⁶ In 1999, RYK’s function emerged from the fruitfly, Drosophila melanogaster, in which the RYK ortholog (Derailed) was found to be a chemorepulsive on the axon growth. ⁷ The Derailed ligand was identified in 2003 as Wnt5 ⁸ and in 2009 Wnt5a/Ryk interactions were recognized as necessary for chemorepulsive axon guidance in the developing mammalian brain and spinal cord ⁹. In vertebrates, more diverse roles are emerging for RYK in a range of neurodevelopmental processes; RYK is important for establishing planar polarity during neural tube formation. ¹⁰ It is as well essential in skeletal (e.g.osteoclast differentiation), ¹¹ craniofacial, and cardiac development. Undoubtedly, given these premises, RYK is involved in signal transduction despite its lack of kinase activity. Derailed, the Drosophila RYK ortholog, contains an extracellular Wnt-interacting WIF domain with ability to bind the fruitfly D-Wnt-5 protein. ⁸ This indicates Derailed is a D-Wnt-5 receptor. In 2005 proof has been given that in mammals too (mouse) RYK is a high-affinity receptor for Wnt5a. ¹² Wnt5a is the ligand and Ryk is the receptor binding it with its extracellular WIF domain. ¹³ Also the other pseudokinases RORs and PTK7 are receptors or co-receptors for WNTs but RYK is special as the only one having a WIF (for WNT-Inhibitory Factor) as the binding motif. RORs have an extracellular cysteine-rich domain (CRD) related to that seen in FZDs and PTK7 has a multiple immunoglobulin-like domain (see FIG. 7). The correlations between WNTs and their ligands are complex as suggested by the fact that RYK appears to form WNT-dependent complexes with FZDs. ¹⁴ The kinase domain lacks enzymatic activity but it is peculiar in the fact of being cleaved by a gamma-secretase with a product that migrates to nucleus. This could be an alternative way to regulate Wnt-linked signaling. Recent developments confirm that WNTs cross-link RTK pseudokinase co-receptors into signaling complexes with FZDs, within which they might allosterically regulate other key components. ¹⁵ The complexity of Wnt signaling was well known as well as the concept that Wnt family are key regulators of pivotal developmental processes (embryonic patterning, specification of cell fate, determination of tissue polarity, development of mammalian central nervous system). Three interconnected signaling pathways (canonical, noncanonical, and Wnt/calcium pathways) are known for Wnts ligand system and each of them is activated by a subset of receptors, configuring the Wnt signaling as highly regulated at the level of ligand–receptor interactions. RYK associates molecularly to FZD receptors and can modulate canonical (beta-catenin dependent) and non-canonical (beta-catenin independent) pathways. ¹⁶

Figure 7 Extracellular domains of WNTs-related pseudokinase

RYK with respect to the other Wnt-binding pseudokinases is characterized by the exclusive extracellular WIF domain.

A few years after the discovery of RYK extensive cross-species reactivity of a mouse RYK kinase subdomain probe demonstrated that RYK and related family members encode a class of widely conserved proteins. ¹⁷ 210 orthologues and 53 paralogues are known (ENSG00000163785). 354 organisms have orthologs with human gene RYK. The RYK gene is conserved in chimpanzee, dog, cow, mouse, rat, chicken, zebrafish, fruit fly, mosquito, Caenorhabditis elegans and frog.

Very little is known about mutations of pathogenetic significance. The only observation reported in the literature concerns a survey carried out on a large sample (354) of Vietnamese, Japanese, and Caucasian patients with cleft lip and/or palate (CL/P) gathered in a group of 1647 tests comprising patient relatives and normal adult controls. ¹⁸ Only 1 out of the patient samples tested harbored a RYK missense mutation, 1355G.A (Y452C). Moreover, the unaffected father of the patient carried the same mutation. The effect of this mutation could be a decrease in the gene function and this mutation in RYK may explain a small subset of CL/P.