Ror2, encoding a receptor-like tyrosine kinase, is required for cartilage and growth plate development.

Receptor tyrosine kinases often have critical roles in particular cell lineages by initiating signalling cascades in those lineages. Examples include the neural-specific TRK receptors, the VEGF and angiopoietin endothelial-specific receptors, and the muscle-specific MUSK receptor. Many lineage-restricted receptor tyrosine kinases were initially identified as 'orphans' homologous to known receptors, and only subsequently used to identify their unknown growth factors. Some receptor-tyrosine-kinase-like orphans still lack identified ligands as well as biological roles. Here we characterize one such orphan, encoded by Ror2 (ref. 12). We report that disruption of mouse Ror2 leads to profound skeletal abnormalities, with essentially all endochondrally derived bones foreshortened or misshapen, albeit to differing degrees. Further, we find that Ror2 is selectively expressed in the chondrocytes of all developing cartilage anlagen, where it essential during initial growth and patterning, as well as subsequently in the proliferating chondrocytes of mature growth plates, where it is required for normal expansion. Thus, Ror2 encodes a receptor-like tyrosine kinase that is selectively expressed in, and particularly important for, the chondrocyte lineage.

Dominant mutations in ROR2, encoding an orphan receptor tyrosine kinase, cause brachydactyly type B.

Inherited limb malformations provide a valuable resource for the identification of genes involved in limb development. Brachydactyly type B (BDB), an autosomal dominant disorder, is the most severe of the brachydactylies and characterized by terminal deficiency of the fingers and toes. In the typical form of BDB, the thumbs and big toes are spared, sometimes with broadening or partial duplication. The BDB1 locus was previously mapped to chromosome 9q22 within an interval of 7.5 cM (refs 9,10). Here we describe mutations in ROR2, which encodes the orphan receptor tyrosine kinase ROR2 (ref. 11), in three unrelated families with BDB1. We identified distinct heterozygous mutations (2 nonsense, 1 frameshift) within a 7-amino-acid segment of the 943-amino-acid protein, all of which predict truncation of the intracellular portion of the protein immediately after the tyrosine kinase domain. The localized nature of these mutations suggests that they confer a specific gain of function. We obtained further evidence for this by demonstrating that two patients heterozygous for 9q22 deletions including ROR2 do not exhibit BDB. Expression of the mouse mouse orthologue, Ror2, early in limb development indicates that BDB arises as a primary defect of skeletal patterning.

The receptor for ciliary neurotrophic factor.

Although neurotrophic factors were originally isolated on the basis of their ability to support the survival of neurons, these molecules are now thought to influence many aspects of the development and maintenance of the nervous system. Identifying the receptors for these neurotrophic factors should aid in identifying the cells on which these factors act and in understanding their precise mechanisms of action. A "tagged-ligand panning" procedure was used to clone a receptor for ciliary neurotrophic factor (CNTF). This receptor is expressed exclusively within the nervous system and skeletal muscle. The CNTF receptor has a structure unrelated to the receptors utilized by the nerve growth factor family of neurotrophic molecules, but instead is most homologous to the receptor for a cytokine, interleukin-6. This similarity suggestes that the CNTF receptor, like the interleukin-6 receptor, requires a second, signal-transducing component. In contrast to all known receptors, the CNTF receptor is anchored to cell membranes by a glycosyl-phosphatidylinositol linkage.

Identification of ubiquitin ligases required for skeletal muscle atrophy.

Skeletal muscle adapts to decreases in activity and load by undergoing atrophy. To identify candidate molecular mediators of muscle atrophy, we performed transcript profiling. Although many genes were up-regulated in a single rat model of atrophy, only a small subset was universal in all atrophy models. Two of these genes encode ubiquitin ligases: Muscle RING Finger 1 (MuRF1), and a gene we designate Muscle Atrophy F-box (MAFbx), the latter being a member of the SCF family of E3 ubiquitin ligases. Overexpression of MAFbx in myotubes produced atrophy, whereas mice deficient in either MAFbx or MuRF1 were found to be resistant to atrophy. These proteins are potential drug targets for the treatment of muscle atrophy.

Alternative forms of rat TrkC with different functional capabilities.

We have identified transcripts encoding several different forms of rat TrkC, a member of the Trk family of receptor tyrosine kinases that serves as a receptor for neurotrophin-3. Some forms of TrkC lack the intracytoplasmic kinase domain and thus resemble previously defined truncated variants of TrkB. Other forms of TrkC contain variable-sized amino acid insertions within the tyrosine kinase domain. Transcripts encoding all forms of TrkC can be detected throughout the nervous system, displaying substantial overlap as well as mutually exclusive distribution patterns with transcripts for TrkB. Strikingly, only transcripts encoding the truncated forms of TrkB and TrkC are found in astrocytes, peripheral nerve, and nonneural tissues. Finally, forms of TrkC containing insertions within the kinase domain retain their ability to autophosphorylate in response to neurotrophin-3, but cannot mediate proliferation in fibroblasts or neuronal differentiation in PC12 cells.

Receptor tyrosine kinase specific for the skeletal muscle lineage: expression in embryonic muscle, at the neuromuscular junction, and after injury.

While a number of growth factors have been described that are highly specific for particular cell lineages, neither a factor nor a receptor uniquely specific to the skeletal muscle lineage has previously been described. Here we identify a receptor tyrosine kinase (RTK) specific to skeletal muscle, which we term "MuSK" for muscle-specific kinase. MuSK is expressed at low levels in proliferating myoblasts and is induced upon differentiation and fusion. In the embryo, it is specifically expressed in early myotomes and developing muscle. MuSK is then dramatically down-regulated in mature muscle, where it remains prominent only at the neuromuscular junction; MuSK is thus the only known RTK that localizes to the neuromuscular junction. Strikingly, MuSK expression is dramatically induced throughout the adult myofiber after denervation, block of electrical activity, or physical immobilization. In humans, MuSK maps to chromosome 9q31.3-32, which overlaps with the region reported to contain the Fukuyama muscular dystrophy mutation. Identification of MuSK introduces a novel receptor-factor system that seems sure to play an important and selective role in many aspects of skeletal muscle development and function.

Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis.

We report that the many Eph-related receptor tyrosine kinases, and their numerous membrane-bound ligands, can each be grouped into only two major specificity subclasses. Receptors in a given subclass bind most members of a corresponding ligand subclass. The physiological relevance of these groupings is suggested by viewing the collective distributions of all members of a subclass. These composite distributions, in contrast with less informative patterns seen with individual members of the family, reveal that the developing embryo is subdivided into domains defined by reciprocal and apparently mutually exclusive expression of a receptor subclass and its corresponding ligands. Receptors seem to encounter their ligands only at the interface between these domains. This reciprocal compartmentalization implicates the Eph family in the formation of spatial boundaries that may help to organize the developing body plan.

c-K-ras mutations in human carcinomas occur preferentially in codon 12.

A study was carried out to determine the frequency and distribution of mutations in the c-K-ras gene in human carcinoma tissue. The study was done on a total of 51 lung, colon and breast carcinoma tumors using a panel of oligonucleotides coding for the wild type and all possible mutations in codons 12 and 61 of c-K-ras gene. Four of 16 colon carcinomas, two of 27 lung carcinomas and one of eight breast carcinomas were found to contain mutations in codon 12. No mutations were found at position 61. Of the six possible amino acid replacements in codon 12, all but one was represented in the seven mutations identified.

Identification of full-length and truncated forms of Ehk-3, a novel member of the Eph receptor tyrosine kinase family.

Factors that bind and activate receptor tyrosine kinases are known to play key roles during development and in the adult. The Eph-related receptors constitute the largest known family of receptor tyrosine kinases. Members of the Eph family exhibit intriguing patterns of expression in the embryo, implicating them in a variety of developmental processes, and their expression is often restricted to particular subpopulations of postmitotic neurons in the adult. We describe the identification and characterization of a novel member of the Eph receptor family, which we have termed Ehk-3 for Eph Homologous Kinase 3. Ehk-3 displays all the major structural features shared by other members of the Eph family, including a cysteine-rich region and tandem fibronectin type-III domains in its extracellular portion. Ehk-3 is expressed in two forms in a developmentally-regulated fashion: a conventional full-length version containing the intracellular tyrosine kinase domain, as well as a truncated form that lacks this domain. Both forms of Ehk-3 are quite restricted to the nervous system in the adult, but Ehk-3 is more widely expressed in the embryo, suggesting that Ehk-3 mediates different functions during development and in the adult.

EphA2 promotes infiltrative invasion of glioma stem cells in vivo through cross-talk with Akt and regulates stem cell properties.

Diffuse infiltrative invasion is a major cause for the dismal prognosis of glioblastoma multiforme (GBM), but the underlying mechanisms remain incompletely understood. Using human glioma stem cells (GSCs) that recapitulate the invasive propensity of primary GBM, we find that EphA2 critically regulates GBM invasion in vivo. EphA2 was expressed in all seven GSC lines examined, and overexpression of EphA2 enhanced intracranial invasion. The effects required Akt-mediated phosphorylation of EphA2 on serine 897. In vitro the Akt-EphA2 signaling axis is maintained in the absence of ephrin-A ligands and is disrupted upon ligand stimulation. To test whether ephrin-As in tumor microenvironment can regulate GSC invasion, the newly established Efna1;Efna3;Efna4 triple knockout mice (TKO) were used in an ex vivo brain slice invasion assay. We observed significantly increased GSC invasion through the brain slices of TKO mice relative to wild-type (WT) littermates. Mechanistically EphA2 knockdown suppressed stem cell properties of GSCs, causing diminished self-renewal, reduced stem marker expression and decreased tumorigenicity. In a subset of GSCs, the reduced stem cell properties were associated with lower Sox2 expression. Overexpression of EphA2 promoted stem cell properties in a kinase-independent manner and increased Sox2 expression. Disruption of Akt-EphA2 cross-talk attenuated stem cell marker expression and neurosphere formation while having minimal effects on tumorigenesis. Taken together, the results show that EphA2 endows invasiveness of GSCs in vivo in cooperation with Akt and regulates glioma stem cell properties.

Immunological identification and distribution of parathyroid hormone-like protein polypeptides in normal and malignant tissues.

Monoclonal and polyclonal antibodies recognizing human parathyroid hormone-like protein (PTHLP) have been produced using a series of recombinant and synthetic PTHLP peptides. These antibodies have been used to develop a two-site immunometric enzyme immunoassay which detects PTHLP[1-87] and PTHLP[1-141] but not PTH. The immunoassay detected PTHLP in extracts of squamous carcinomas and normal tissues at concentrations from 7-515 ng PTHLP[1-87]/mg protein. Immunoblotting of the extract which showed the highest immunoreactivity, a squamous carcinoma of the lung from a patient with hypercalcemia, revealed a major band having an apparent mol wt of 26,500 and several other higher mol wt bands. Similar polypeptides were observed by immunoblotting cell extracts from a cell line, SCaBER, which secretes immunoreactive PTHLP into its medium and also from tumors in nude mice derived from this cell line. Chaotropic agents did not alter the immunoblotting pattern, and antibodies to three different epitopes of PTHLP recognized these bands, indicating PTHLP expression in the extracts. Immunohistochemical staining of normal human tissue with these antibodies revealed several PTHLP-containing tissues and confirmed the results of the immunoassay, suggesting a paracrine role for PTHLP. Staining was observed in several neoplastic tissues including squamous cell carcinomas, lung carcinoma, bladder carcinoma, osteogenic sarcoma, and adenocarcinoma of the colon.

Synthesis of a gene encoding parathyroid hormone-like protein-(1-141): purification and biological characterization of the expressed protein.

PTH-like proteins (PTHLP), which are associated with humoral hypercalcemia of malignancy, have recently been purified. Isolation of their corresponding cDNAs has revealed that they are derived from a single gene. In this report a synthetic gene encoding PTHLP-(1-141), a 141-amino acid protein corresponding to the most abundant PTHLP cDNA detected in human tumors, was expressed in bacteria and purified to homogeneity. Recombinant (r) PTHLP-(1-141) migrates with an aberrantly high mol wt on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, presumably as a result of its unusually basic pI. rPTHLP-(1-141), like PTH, induced hypercalcemia in rats, caused release of 45Ca from fetal rat bones, and stimulated the synthesis of cAMP by rat osteosarcoma cells and canine renal membrane preparations. A comparison of the abilities of rPTHLP-(1-141) and bovine PTH-(1-34) to stimulate cAMP synthesis indicated rPTHLP-(1-141) to be 5-fold more potent in the osteosarcoma assay, while nearly 30-fold less active in the renal membrane adenylate cyclase assay. Although 100-fold less potent than bovine PTH-(1-34) in promoting bone resorption, rPTHLP-(1-141) was a potent calcemic factor in vivo, inducing a rise in serum calcium from 10.4 to 14.5 mg/dl when infused into rats at 1.3 micrograms/h. These results support previous assumptions that PTHLP is the humoral factor responsible for humoral hypercalcemia of malignancy. In addition, they suggest substantial differences between PTHLP and PTH in the regulation of calcium homeostasis.

Political identification and perceptions of homelessness: attributed causality and attitudes on public policy.

The study investigated relationships between political orientation, causal perceptions of poverty, and attitudes toward government programs for the poor. The test sample of 200 women and 200 men were recruited from introductory psychology classes. In support of hypotheses based on previous research and Weiner's attribution-emotion-action theory, when compared with self-identified Democrats, self-identified Republicans (a) were significantly more inclined to attribute homelessness to internal vs external factors and (b) expressed significantly less favorable attitudes toward publically funded programs for the homeless. Sex differences were nonsignificant. Conceptual-empirical and methodological implications are discussed. Limitations on inferences from these data and directions for inquiry into the development of individual difference in political cognitions and public policy attitudes are considered.

Expression of BMP-7 and USAG-1 (a BMP antagonist) in kidney development and injury.

Once developed, end-stage renal disease cannot be reversed by any current therapy. Bone morphogenetic protein-7 (BMP-7), however, is a possible treatment for reversing end-stage renal disease. Previously, we showed that the BMP antagonist uterine sensitization-associated gene-1 (USAG-1, also known as ectodin and sclerostin domain-containing 1) negatively regulates the renoprotective action of BMP-7. Here, we show that the ratio between USAG-1 and BMP-7 expression increased dramatically in the later stage of kidney development, with USAG-1 expression overlapping BMP-7 only in differentiated distal tubules. Examination of USAG-1 expression in developing kidney indicated that a mosaic of proximal and distal tubule marker-positive cells reside side by side in the immature nephron. This suggests that each cell controls its own fate for becoming a proximal or distal tubule cell. In kidney injury models, the ratio of USAG-1 to BMP-7 expression decreased with kidney damage but increased after subsequent kidney regeneration. Our study suggests that USAG-1 expression in a kidney biopsy could be useful in predicting outcome.

Four human carcinoma cell lines with novel mutations in position 12 of c-K-ras oncogene.

We have used synthetic oligonucleotides to probe for mutations affecting amino acid 12 of the c-K-ras gene in human cell line DNA. Of seven carcinoma cell lines tested, four were found to contain a mutation at this position. In each the nucleotide G was replaced with an A resulting in a Gly to Asp substitution in three cases (cell lines A427, A1165 and A1663) and Gly to Ser in the fourth (A549). Neither of these substitutions have been previously reported in either human tumor or human tumor-derived cell line DNA's. These results indicate that association between mutations involving position 12 of the human c-K-ras oncogene and carcinomas may be stronger than previously recognized.

Eukaryotic ribosomal subunit anti-association activity of calf liver is contained in a single polypeptide chain protein of Mr = 25,500 (eukaryotic initiation factor 6).

A protein factor that prevents the reassociation of eukaryotic 40 S and 60 S ribosomal subunits when the Mg+ concentration is raised from 1 to 5 mM has been purified to apparent electrophoretic homogeneity from postribosomal supernatant of calf liver extracts. The purified ribosomal subunit anti-association factor is a relatively heat-sensitive protein consisting of a single polypeptide chain of apparent Mr = 25,500. Direct assay for ribosomal subunit anti-association activity indicates that the majority (greater than 90% of such an activity in calf liver extracts can be accounted for by the presence of this 25,500-dalton protein factor in the postribosomal supernatant. The ribosomal salt wash protein fractions are virtually devoid of any significant ribosomal subunit anti-association or 80 S ribosome dissociation activity. The purified anti-association factor maintains a pool of ribosomal subunits by binding to 60 S ribosomal subunits and preventing them from reassociating with 40 S ribosomal subunits, rather than by dissociating 80 S monosomes. The factor neither binds to, nor seems to interact directly with, 40 S subunits. The properties of this factor are thus similar to wheat germ ribosome dissociation factor (eukaryotic initiation factor 6) described by Russell and Spremulli ((1979) J. Biol. Chem. 254, 8796-8800.

Identification of new oligodendrocyte- and myelin-specific genes by a differential screening approach.

We have isolated several new genes that are specifically expressed by oligodendrocytes in the CNS. This was achieved by differential screening of a rat spinal cord cDNA library with probes derived from normal and from oligodendrocyte-free spinal cord mRNAs. Four of these genes are exclusively expressed by oligodendrocytes: Three of these are not related to known genes, whereas one encodes the myelin oligodendrocyte glycoprotein (MOG). Four other genes are expressed by oligodendrocytes as well as by Schwann cells. One gene codes for apolipoprotein D, which is thought to be involved in lipid metabolism. A second cDNA sequence codes for the recently identified galactosylceramide-synthesizing enzyme UDP-galactose:ceramide galactosyltransferase. The third gene encodes a small protein with four putative transmembrane domains that is related to a T-lymphocyte-specific membrane protein, MAL. The fourth gene encodes the rat homologue of the stearyl-CoA-desaturase 2 (SCD2) gene, which is specifically expressed in the nervous system and involved in the synthesis and regulation of long-chain unsaturated fatty acids essential for myelination. Finally, we found that a member of the beta-tubulin family is highly expressed in oligodendrocytes as well as neurons. The identification of several new proteins that may play a role in myelin synthesis and sheath formation will lead to new insight into this complex mechanism.

Characterization of a rat gene, rMAL, encoding a protein with four hydrophobic domains in central and peripheral myelin.

Wrapping and compaction of myelin sheaths around axons require specific membrane and membrane-associated proteins. Transmembrane proteins like proteolipid protein (PLP), the peripheral myelin protein 22 (PMP-22) and P0 as well as myelin basic protein (MBP) are crucial for this process. We have isolated a rat cDNA, initially denominated NS 3, that is mainly expressed in the myelinating cells of the nervous system, the oligodendrocytes and Schwann cells. The cDNA encodes a highly hydrophobic protein of 16.8 kDa with four putative transmembrane domains. The putative NS 3 protein lacks a N-terminal hydrophobic leader sequence and has no consensus sequence for N-linked glycosylation. In contrast to PLP and PMP-22, the first and third putative transmembrane domain of the NS 3 protein contain charged amino acids, a feature which resembles the structure of gap junction proteins. Sequence analysis showed that NS 3 is the rat homolog of a human gene called MAL that was cloned from, and is expressed in various T-cell lines. Therefore, we call this gene rMAL (rat MAL). In the nervous system, the expression of rMAL, mRNA begins after birth and is highest during myelination. In situ hybridization shows that rMAL mRNA is exclusively expressed in white and gray matter oligodendrocytes in the CNS and in myelinating Schwann cells in peripheral nerves. Immunohistochemistry using a peptide-specific antibody localized the rMAL protein in the myelinated areas of the CNS and PNS. Furthermore, we demonstrate by immunoblot analysis that rMAL is a component of myelin. Its structure and distribution suggest that the rMAL protein might play an important role in compact myelin. We propose that the name rMAL protein refers to rat Myelin And Lymphocyte protein.

Ribosomal subunit antiassociation activity in rabbit reticulocyte lysates. Evidence for a low molecular weight ribosomal subunit antiassociation protein factor (Mr = 25,000).

A ribosomal subunit antiassociation activity has been purified from both the postribosomal supernatant and ribosomal salt-wash protein fractions of rabbit reticulocyte lysates. A majority (greater than 90%) of the activity is associated with a low molecular weight protein of Mr of approximately 25,000. A small but significant level of antiassociation activity (less than 10%) was found to be associated with higher molecular weight protein fractions. The purified 25,000-dalton antiassociation factor interacts with 60 S ribosomal subunits to prevent them from reassociating with 40 S ribosomal subunits. The factor does not seem to interact directly with 40 S subunits nor does it dissociate 80 S monosomes. The properties of this factor are thus similar to the eukaryotic initiation factor 6 isolated from both wheat germ and calf liver extracts.

Genomic organization and chromosomal localization of the human and mouse genes encoding the alpha receptor component for ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) has recently been found to share receptor components with, and to be structurally related to, a family of broadly acting cytokines, including interleukin-6, leukemia inhibitory factor, and oncostatin M. However, the CNTF receptor complex also includes a CNTF-specific component known as CNTF receptor alpha (CNTFR alpha). Here we describe the molecular cloning of the human and mouse genes encoding CNTFR. We report that the human and mouse genes have an identical intron-exon structure that correlates well with the domain structure of CNTFR alpha. That is, the signal peptide and the immunoglobulin-like domain are each encoded by single exons, the cytokine receptor-like domain is distributed among 4 exons, and the C-terminal glycosyl phosphatidylinositol recognition domain is encoded by the final coding exon. The position of the introns within the cytokine receptor-like domain corresponds to those found in other members of the cytokine receptor superfamily. Confirming a recent study using radiation hybrids, we have also mapped the human CNTFR gene to chromosome band 9p13 and the mouse gene to a syntenic region of chromosome 4.