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H-Ryk is an atypical receptor tyrosine kinase that is expressed in a differentiation-specific manner in epithelial tissues. We have previously shown by in situ hybridization and immunohistochemistry that H-Ryk is overexpressed in malignant ovarian tumors. In addition, we have demonstrated that overexpression of H-Ryk is transforming in vitro and in vivo. To evaluate whether expression of H-Ryk is a prognostic factor in epithelial ovarian cancer, we carried out a retrospective study of 88 primary malignant ovarian tumors (28 serous tumors, 11 mucinous tumors, 29 endometrioid tumors, 13 clear cell tumors, 3 malignant mixed Mullerian tumors, 1 mixed epithelial tumor, 1 primary peritoneal tumor, 1 undifferentiated tumor, and 1 transitional carcinoma) diagnosed between 1990 and 1993 using immunohistochemistry. On univariate analysis, overall survival decreased significantly with age (P = 0.01); in patients with International Federation of Gynecology and Obstetrics (FIGO) stage II (P = 0.008), FIGO stage III (P < 0.001), and FIGO stage IV (P < 0.001) disease; and in patients with residual disease (residual disease < or = 2 cm, P = 0.007; residual disease > 2 cm, P < 0.001) after surgery. In addition, overexpression of the H-Ryk receptor in malignant epithelium (P = 0.04) and blood vessel (P = 0.01) was associated with a significantly decreased overall survival. H-Ryk blood vessel overexpression (P = 0.03), residual disease > 2 cm (P = 0.006), and residual disease < or = 2 cm (P = 0.01) conferred a significantly shorter progression-free survival. No correlation was found between H-Ryk overexpression and age, histological subtype, degree of differentiation, FIGO stage, or residual disease. Overall, after adjustment for all of the prognostic factors by multivariate analysis (Cox proportional hazards model), residual disease was the most powerful prognostic indicator for overall survival (P < 0.001) and progression-free survival (P = 0.01) in this patient subset. This implies that H-Ryk acts cooperatively with other biological factors in the pathogenesis of ovarian cancer.

H-Ryk is an atypical receptor tyrosine kinase which differs from other members of this family at a number of conserved residues in the activation and nucleotide binding domains. Using a chimeric receptor approach, we demonstrate that H-Ryk has impaired catalytic activity. Despite the receptor's inability to undergo autophosphorylation or phosphorylate substrates, we demonstrate that ligand stimulation of the chimeric receptor results in activation of the mitogen-activated protein kinase pathway. The ability to transduce signals is abolished by mutation of the invariant lysine (K334A) in subdomain II of H-Ryk. Further, by in vitro mutagenesis, we show that the amino acid substitutions in the activation domain of H-Ryk account for the loss of catalytic activity. In addition to the essential aspartate residue, either phenylalanine or glycine is required in the activation domain to maintain proper conformation of the catalytic domain and thus ensure receptor autophosphorylation. Homology modelling of the catalytic domain of H-Ryk provides a rationale for these findings. Thus, the signalling properties of H-Ryk are divergent from those of other classical receptor tyrosine kinases.

Abnormalities in the function of receptor tyrosine kinases (RTKs) have been demonstrated to be important in the pathogenesis of cancer. H-Ryk, a new member of the RTK family, is an unusual RTK in that it is catalytically inactive because of amino acid substitutions of conserved residues in the catalytic domain. We show by immunohistochemistry that it is expressed in the epithelium, stroma, and blood vessels of normal tissues. Evaluation of a panel of 33 primary ovarian tumors (2 benign, 8 borderline, and 23 malignant) was performed. H-Ryk was overexpressed in borderline and malignant ovarian tumors. In serous and clear cell subtypes, there was increased expression in the epithelium, stroma, and blood vessels. Consistent with this observation, overexpression of H-Ryk in the mouse fibroblast cell line NIH3T3 induces anchorage-independent growth and tumorigenicity in nude mice. This implies that overexpression of the receptor can be transforming and may therefore be significant in the pathogenesis of ovarian cancer.

The discoidin domain receptor (DDR) is a new class of receptor tyrosine kinase that is distinguished by a unique extracellular domain homologous to the lectin Discoidin I found Dictyostelium discoideum. A cosmid was isolated from a human chromosome 6 cosmid library containing the DDR gene. A complete genomic contig of the DDR gene was constructed from seven subclones of the cosmid. The cosmid fragments were analyzed by PCR, sequencing, and comparison of genomic/cDNA sequence. The DDR gene is composed of 17 exons, ranging in size from 96 to 1014 bp, distributed along approximately 12 kb of genomic DNA. The extracellular domain is encoded by 8 exons of which three code for the discoidin domain. The transmembrane domain is encoded by 1 exon, the juxtamembrane by 3 exons, and the catalytic domain by 5 exons. The generation of the two splice variants of DDR, EDDR1 and EDDR2 are explained by the genomic structure. Exon 11 (111 bp in the juxtamembrane domain) is present in DDR and absent in the splice variant EDDR1. An inverted repeat of 20 bp was identified at the 3' exon-intron junction of exon 11, which results in a lariat loop-like secondary structure. EDDR2 is generated because of a cryptic splice acceptor site that results in an extra 18 bp (6 amino acids) inserted 5' of exon 14 in the catalytic domain. A polymorphic (GT)17 repeat was identified in intron 5 with a heterozygosity of 0.71. The exon-intron structure of the DDR gene will be helpful in further understanding of its function and explains the possible structural basis for the two splice variants.

Background: Protein tyrosine kinases play an important role in cellular metabolism as key components of signal transduction pathways. They are involved in cellular growth, differentiation, and development. Receptor tyrosine kinases (EGF receptor and c-erbB2) have been shown to be important in the pathogenesis of cancer. In ovarian cancer, overexpression of c-erbB2, a type I receptor, has been correlated with an adverse effect on survival of patients., Material and Methods: 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., Results: 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. A single transcript of 3.0 kb is expressed in heart, brain, lung, placenta, liver, muscle, kidney, and pancreas by Northern analysis with maximal expression in skeletal muscle. In situ hybridization analysis on human tissues demonstrated localization of message in the epithelial and stromal compartment of tissues such as brain, lung, colon, kidney, and breast. There was minimal to absent expression of H-RYK on surface epithelium of ovaries. In benign (3) and borderline tumors of the ovary (5), there was expression in the stromal compartment. However, in malignant tumors (24) there was 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 did not phosphorylate in an in vitro kinase assay., Conclusions: The expression of this unusual receptor tyrosine kinase in epithelial ovarian cancer suggests that it may be involved in tumor progression, which needs further investigation.