The mouse Fgfrl1 gene coding for a novel FGF receptor-like protein

The mouse Fgfrl1 gene codes for a novel cell surface protein that is closely related to the family of the FGF receptors. It contains three extracellular Ig C2 loops and an acidic box, which share 29-33% sequence identity (48-50% similarity) with FGF receptors 1-4. The intracellular portion of the novel protein, however, lacks a tyrosine kinase domain required for signal transduction by transphosphorylation. The gene for Fgfrl1 comprises six exons and is located on mouse chromosome 5 in close proximity to the Idua gene for L-iduronidase.     

Characterization of FGFRL1, a novel fibroblast growth factor (FGF) receptor preferentially expressed in skeletal tissues

Clones for a novel transmembrane receptor termed FGFRL1 were isolated from a subtracted, cartilage-specific cDNA library prepared from chicken sterna. Homologous sequences were identified in other vertebrates, including man, mouse, rat and fish, but not in invertebrates such as Caenorhabditis elegans and Drosophila. FGFRL1 was expressed preferentially in skeletal tissues as demonstrated by Northern blotting and in situ hybridization. Small amounts of the FGFRL1 mRNA were also detected in other tissues such as skeletal muscle and heart. The novel protein contained three extracellular Ig-like domains that were related to the members of the fibroblast growth factor (FGF) receptor family. However, it lacked the intracellular protein tyrosine kinase domain required for signal transduction by transphosphorylation. When expressed in cultured cells as a fusion protein with green fluorescent protein, FGFRL1 was specifically localized to the plasma membrane where it might interact with FGF ligands. Recombinant FGFRL1 protein was produced in a baculovirus system with intact disulfide bonds. Similar to FGF receptors, the expressed protein interacted specifically with heparin and with FGF2. When overexpressed in MG-63 osteosarcoma cells, the novel receptor had a negative effect on cell proliferation. Taken together our data are consistent with the view that FGFRL1 acts as a decoy receptor for FGF ligands.     

Cloning multiple copies of a DNA segment

A method for self-ligation of DNA segments, which is based on the rotational non-equivalence of ends produced by AvaI cleavage, has been developed. Using this method and an initiator molecule to encourage the formation of long polymers, we have obtained a plasmid containing 34 repeats of a 123-bp rat DNA segment. All of the repeats are in the same orientation, and the plasmid is quite stable. It should be possible to polymerize any DNA segment by this method. Potential uses of the procedure include production of large amounts of small, homogeneous DNAs for physical studies such as X-ray crystallography, and increasing the expression of cloned genes in bacteria.     

Canopy1, a novel regulator of FGF signaling around the midbrain-hindbrain boundary in zebrafish

FGF signaling from the midbrain-hindbrain boundary (MHB, isthmus) plays a major role both in maintenance of the MHB and induction of the tectum and cerebellum. Since different levels of FGF signaling in the MHB result in a qualitative difference in inducing activity, FGF signaling in the MHB should be tightly regulated positively and negatively at multiple steps to ensure correct levels of FGF signaling. Factors that negatively regulate FGF signal around the MHB are reported. However, factors that ensure strong FGF signal in the MHB are largely unknown. Here we report the identification of Canopy1 (Cnpy1), a novel MHB-specific, Saposin-related protein that belongs to an evolutionarily conserved protein family. The cnpy1 gene was expressed specifically in the MHB of zebrafish embryos. Exogenous FGF8 induced expression of cnpy1 in the tectal primordial. Knockdown of cnpy1 resulted in MHB defects and impaired FGF signaling in a cell-autonomous manner. Cnpy1 is localized in the endoplasmic reticulum and interacts with FGFR1. This study highlights a positive-feedback loop between the FGFR pathway and Cnpy1 that may ensure the strength of FGF signaling in the MHB, leading to correct development of the tectum and cerebellum.     

ERK2 is required for FGF1-induced JNK1 phosphorylation in Xenopus oocyte expressing FGF receptor 1

A possible connection between the ERK2 and JNK1 MAP kinases transduction cascades was investigated in Xenopus oocytes expressing FGFR1 stimulated by FGF1. Injection of various inhibitors for the Shc/Grb2/Ras/Mos/MEK/ERK2 cascade blocked FGF1-induced germinal vesicle breakdown (GVBD), as well as ERK2 and JNK1 phosphorylation. JNK1 was found to be activated downstream of ERK2, since injection of an active ERK2 triggered JNK1 phosphorylation and inhibition of ERK2 either by a MEK inhibitor or the MKP3 phosphatase blocked JNK1 phosphorylation. These results demonstrated that in FGFR1 signalling JNK1 phosphorylation depends on ERK2.     

Hyperinsulinemia in transgenic mice carrying multiple copies of the human insulin gene

We are investigating human insulin gene expression in transgenic mice. An 8.8 kilobase (kb) human genomic DNA fragment, including the insulin gene (1.4 kb) and 2 kb of 5' human flanking sequences, was introduced into mouse embryos by pronuclear microinjection. Two lines of transgenic mice have been established, both of which carry the intact human gene in multiple copies. Animals from both lines have significantly higher insulin levels than control mice, and the degree of hyperinsulinemia shows a positive correlation with human gene copy number in the two lines. Expression of the human gene is confirmed by the detection of human C-peptide in plasma. Tissue specificity of expression is maintained, with human insulin mRNA detectable only in the pancreas. The transgenics maintain normal fasting blood glucose in spite of their high insulin levels, but preliminary studies show them to be glucose intolerant when given a glucose load. These mice provide a model system for further studies on the regulation of insulin gene expression and on the effects of chronic hyperinsulinemia on glucose homeostasis.     

Characterization of a cloned Bacillus subtilis gene that inhibits sporulation in multiple copies.

We have isolated a 1.0-kilobase fragment of the Bacillus subtilis chromosome which, when present in high-copy-number plasmids, caused a sporulation-proficient strain to become phenotypically sporulation deficient. This is referred to as the sporulation inhibition (Sin) phenotype. This DNA fragment, in multicopy, also inhibited the production of extracellular protease activity, which normally appears at the beginning of stationary growth. The origin of the fragment was mapped between the dnaE and spo0A genes on the B. subtilis chromosome, and its complete DNA sequence has been determined. By analysis of various deletions and a spontaneous mutant the Sin function was localized to an open reading frame (ORF) predicted from the DNA sequence. Inactivation of this ORF in the chromosome did not affect the ability of cells to sporulate. However, the late-growth-associated production of proteases and alpha-amylase was elevated in these cells. The predicted amino acid sequence of the protein encoded by this ORF had a DNA-binding domain, typically present in several regulatory proteins. We propose that the sin ORF encodes a regulatory protein that is involved in the transition from vegetative growth to sporulation.     

REX1, a novel gene required for DNA repair

Nucleotide excision repair is a major pathway for repairing UV light-induced DNA damage in most organisms. Using insertional mutagenesis, we isolated a UV-sensitive mutant of Chlamydomonas reinhardtii that is blocked in the excision of cyclobutane pyrimidine dimers. The mutant is also sensitive to the alkylating agent, methyl methanesulphonate. We have cloned REX1, a novel gene that rescues the mutant. The gene is unusual in a eukaryotic organism in that it is predicted to encode two different proteins, a small protein (8.9 kDa) and a larger protein (31.8 kDa). Neither protein is homologous to known DNA repair proteins. Partial complementation is achieved with subclones of the gene encoding only the 8.9-kDa protein. The 8.9-kDa protein has homologues in many organisms including Saccharomyces cerevisiae, Arabidopsis, and humans. The 31.8-kDa protein appears to be less conserved. These findings may be of general importance for DNA repair in other organisms.     

Receptor for activated C-kinase 1, a novel binding partner of adiponectin receptor 1

Adiponectin is an adipose tissue derived hormone with anti-diabetic and insulin-sensitizing properties. Two adiponectin receptors, AdipoR1 and AdipoR2, have recently been identified, yet the signaling pathways triggered through adiponectin receptors remain to be elucidated. Using a yeast two-hybrid screen, we identified an adaptor protein, receptor for activated protein kinase C1 (RACK1), as an interacting partner of human AdipoR1. RACK1 was confirmed to interact with AdipoR1 by co-immunoprecipitation and co-localization analysis in mammalian cells. The interaction was enhanced by adiponectin stimulation. In addition, the knockdown of RACK1 by RNA interference inhibited adiponectin-stimulated glucose uptake in HepG2 cells. These results suggest that RACK1 may act as a key bridging factor in adiponectin signaling transduction through interacting with AdipoR1.     

DIgR1, a novel membrane receptor of the immunoglobulin gene superfamily, is preferentially expressed by antigen-presenting cells

A novel membrane receptor of immunoglobulin gene superfamily (IgSF) has been identified from mouse dendritic cells (DC) and designated as DC-derived Ig-like receptor 1 (DIgR1). It encodes a 228-amino-acid (aa) residue polypeptide with a 21-aa signal peptide, a 20-aa transmembrane region, a 189-aa extracellular region, and a 19 aa intracellular region. Its extracellular region contains a single V domain of Ig. So it is a novel type I transmembrane glycoprotein of IgSF. DIgR1 shows significant homologies to human CMRF-35 antigens and polymeric immunoglobulin receptors (pIgR). The mRNA expression of DIgR1 was highly abundant in mouse spleen. The preferential expression of DIgR1 mRNA is observed in the known antigen-presenting cells (APC) including DC, monocytes/macrophages, and B lymphocytes. A 40 kDa of protein in NIH/3T3 cells transfected with the DIgR1 cDNA was detected by Western blot analysis using anti-DIgR1 polyclonal antibodies. The expression of DIgR1 protein on DC is not regulated by LPS stimulation. Further study should be conducted to investigate what were biological functions of DIgR1 in the immunobiology of APC.     

Constitutive, long-term production of human interferons by hamster cells containing multiple copies of a cloned interferon gene.

Hybrid plasmids containing the mouse dihydrofolate reductase (dhfr) and a human interferon (either IFN-alpha 5 or IFN-gamma) coding sequence under the control of viral promoters were transfected into dhfr- Chinese hamster ovary (CHO) cells. dhfr+ colonies produced IFN at 10-1000 units X ml-1 X day-1. Clones selected in methotrexate had a 20-50-fold increase in the IFN-alpha 5 and dhfr DNA and mRNA content and secreted IFN at 20,000-100,000 units X ml-1 X day-1. SDS-polyacrylamide gel electrophoresis of partially purified 35S-HuIFN-gamma from CHO cells showed a multiple of labeled bands with a mobility corresponding to 22,400 to 23,400 daltons which was absent in the supernatants of non-transformed CHO cells. The higher apparent molecular weight of human IFN-gamma from CHO cells as compared to that of human IFN-gamma from E. coli (about 18,800) suggests that the former was glycosylated.     

A novel role for a nodal-related protein; Xnr3 regulates convergent extension movements via the FGF receptor

Convergent extension behaviour is critical for the formation of the vertebrate body axis. In Xenopus, components of the Wnt signaling pathway have been shown to be required for convergent extension movements but the relationship between cell fate and morphogenesis is little understood. We show by loss of function analysis that Xnr3 activates Xbra expression through FGFR1. We show that eFGF activity is not essential in the pathway, and that dishevelled acts downstream of Xnr3 and not in a parallel pathway. We provide evidence for the involvement of the EGF-CFC protein FRL1, and suggest that the pro-domain of Xnr3 may be required for its activity. Since Xnr3 is a direct target of the maternal betacatenin/XTcf3 signaling pathway, it provides the link between the initial, maternally controlled, allocation of cell fate, and the morphogenetic movements of cells derived from the organizer.     

Cloning of human DECTIN-1, a novel C-type lectin-like receptor gene expressed on dendritic cells

We identified a new Ca2+-dependent lectin-like receptor gene, DECTIN-1 (HGMW-approved symbol CLECSF12), the human orthologue of mouse Dectin-1, coding for a putative type II transmembrane glycoprotein with an extracellular C-type lectin-like domain. The gene structure and two alternative spliced forms of DECTIN-1 are described. The DECTIN-1 gene was localized in the natural killer gene complex on human Chromosome 12p12.3-p13.2, between OLR1 and CD94 (position 21.8 cM on the genetic map). The DECTIN-1 gene is highly expressed at the mRNA level in dendritic cells and is not further up-regulated during the maturation of these cells with tumor necrosis factor-alpha. The DECTIN-1 gene therefore represents a novel human member of the C-type lectin-like receptor gene family preferentially expressed in dendritic cells.     

An FGF1:FGF2 chimeric growth factor exhibits universal FGF receptor specificity, enhanced stability and augmented activity useful for epithelial proliferation and radioprotection

Structural instability of wild-type fibroblast growth factor (FGF)-1 and its dependence on exogenous heparin for optimal activity diminishes its potential utility as a therapeutic agent. Here we evaluated FGFC, an FGF1:FGF2 chimeric protein, for its receptor affinity, absolute heparin-dependence, stability and potential clinical applicability. Using BaF3 transfectants overexpressing each FGF receptor (FGFR) subtype, we found that, like FGF1, FGFC activates all of the FGFR subtypes (i.e., FGFR1c, FGFR1b, FGFR2c, FGFR2b, FGFR3c, FGFR3b and FGFR4) in the presence of heparin. Moreover, FGFC activates FGFRs even in the absence of heparin. FGFC stimulated keratinocytes proliferation much more strongly than FGF2, as would be expected from its ability to activate FGFR2b. FGFC showed greater structural stability, biological activity and resistance to trypsinization, and less loss in solution than FGF1 or FGF2. When FGFC was intraperitoneally administered to BALB/c mice prior to whole body gamma-irradiation, survival of small intestine crypts was significantly enhanced, as compared to control mice. These results suggest that FGFC could be useful in a variety of clinical applications, including promotion of wound healing and protection against radiation-induced damage.     

Ermap, a gene coding for a novel erythroid specific adhesion/receptor membrane protein

Ermap (erythroid membrane-associated protein), a gene coding for a novel transmembrane protein produced exclusively in erythroid cells, is described. It is mapped to murine Chromosome 4, 57 cM distal to the centromere. The initial cDNA clone was isolated from a day 9 murine embryonic erythroid cell cDNA library. The predicted peptide sequence suggests that ERMAP is a transmembrane protein with two extracellular immunoglobulin folds, as well as a highly conserved B30.2 domain and several phosphorylation consensus sequences in the cytoplasmic region. ERMAP shares a high homology throughout the entire peptide with butyrophilin, a glycoprotein essential for milk lipid droplet formation and release. A GFP-ERMAP fusion protein was localized to the plasma membrane and cytoplasmic vesicles in transiently transfected 293T cells. Northern blot analysis and in-situ hybridization demonstrated that Ermap expression was restricted to fetal and adult erythroid tissues. ERMAP is likely a novel adhesion/receptor molecule specific for erythroid cells.