[The postman always rings twice, so does the receptor].
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Cell Transformation, Neoplastic↗
Biomedical subjects
F Coulier
Publications and source records attributed to F Coulier.
Receptor specificity of the fibroblast growth factor family.
Fibroblast growth factors (FGFs) are essential molecules for mammalian development. The nine known FGF ligands and the four signaling FGF receptors (and their alternatively spliced variants) are expressed in specific spatial and temporal patterns. The activity of this signaling pathway is regulated by ligand binding specificity, heparan sulfate proteoglycans, and the differential signaling capacity of individual FGF receptors. To determine potentially relevant ligand-receptor pairs we have engineered mitogenically responsive cell lines expressing the major splice variants of all the known FGF receptors. We have assayed the mitogenic activity of the nine known FGF ligands on these cell lines. These studies demonstrate that FGF 1 is the only FGF that can activate all FGF receptor splice variants. Using FGF 1 as an internal standard we have determined the relative activity of all the other members of the FGF family. These data should serve as a biochemical foundation for determining developmental, physiological, and pathophysiological processes that involve FGF signaling pathways.
Alternative Splicing↗
FGF6 modulates the expression of fibroblast growth factor receptors and myogenic genes in muscle cells.
Fgf6 is the only known member of the FGF family whose expression is restricted to the muscle cell lineage during development, suggesting it may have a role in myogenesis. Muscle satellite cells but not C2 myoblast cells were found to express Fgf6. We have used purified recombinant FGF6 protein to explore the effect of this factor on C2 cells in culture. FGF6 stimulated the proliferation of C2 myoblasts and, in combination with heparin, induced their morphological transformation. FGF6, added at 5 ng/ml and in the presence of heparin, increased the expression of a subset of muscle cell differentiation markers. In contrast, at 25 ng/ml, it down-regulated the expression of myogenic markers and myogenic transcription factors examined and delayed differentiation into myotubes of C2 cells. It also up-regulated the expression of FgfR1 and had an opposite effect on FgfR4. These results suggest that intramuscular FGF6 concentrations could influence the proliferation and differentiation processes taking place during development.
Animals↗
The human FGF9 gene maps to chromosomal region 13q11-q12.
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Chromosome Mapping↗
Expression of FGF and FGF receptor genes in human breast cancer.
The family of FGF growth factors is involved in several biological processes and might play an important role in tumorigenesis. We have studied the respective expression of 8 of the 9 characterized FGF genes, and of the 4 known FGF receptor genes, in a panel of 10 tumor-cell lines and 103 breast-tumor samples, using RT-PCR and Northern-blot analyses. FGF1 and FGF2 were expressed in almost all samples, while expression of FGF5, FGF6, FGF7, and FGF9 was more restricted. FGFR1, FGFR2 and FGFR4 were expressed at high levels in respectively 22%, 4% and 32% of tumors. FGFR3 expression was not detected. The transcript encoding an FGFR1 isoform with 2 immunoglobulin-like domains was the most prevalent.
Base Sequence↗
A diphtheria toxin/fibroblast growth factor 6 mitotoxin selectively kills fibroblast growth factor receptor-expressing cell lines.
The fibroblast growth factors (FGFs) constitute a family of nine polypeptides implicated in a number of physiological and pathological processes. They bind to at least three types of cell surface molecules, including low and high affinity receptor families. The role of FGFs and their receptors in human tumorigenesis has been suspected but not formally proven. FGF6 is an oncogene encoding a precursor protein of 208 amino acids that has been shown to bind to FGF receptors. Its normal function has not been identified, but its restricted pattern of expression suggests a role in muscle development or function. We have constructed, produced, and purified a diphtheria toxin/FGF6 mitotoxin that selectively kills FGF receptor-expressing cells. Interestingly, at least two cell lines that normally respond to FGF6 have been found resistant to DT/FGF6, suggesting that FGF6 acts on these cells through a transduction pathway that does not involve FGF receptor.
3T3 Cells↗
The human and mouse fibroblast growth factor 6 (FGF6) genes and their products: possible implication in muscle development.
FGF6 is structurally very similar to the other members of the FGF gene family, and particularly to the FGF4 gene, which was instrumental in its isolation. Its longest open reading frame encodes a 208 amino acid residues long protein, both in man and in the mouse. It is expressed as a 4.8 kb transcript in skeletal muscle. In developing muscle, expression starts at the myotomal stage and culminates in differentiated fetal muscle masses. In culture, FGF6 protein is mitogenic and has a transforming capacity for fibroblasts. It represses the terminal differentiation of myoblasts. Action of FGF6 could be mediated by the FGFR4 receptor, which binds FGF6 and whose gene is also expressed in developing skeletal muscle.
Animals↗
Expression of the Fgf6 gene is restricted to developing skeletal muscle in the mouse embryo.
Fgf6, a member of the Fibroblast Growth Factor (FGF) family, is developmentally regulated and its expression is highly restricted in the adult. To gain further insight into the role of Fgf6, we studied its expression during embryogenesis using RNA in situ hybridization. Fgf6 expression is restricted to developing skeletal muscle. Fgf6 transcripts are first detected in the somites at 9.5 days post-conceptus, and expression continues in developing skeletal muscles up to at least 16.5 days post-conceptus. Fgfr4 is a putative receptor for FGF6. Its pattern of expression during myogenesis overlaps that of Fgf6, but both genes are not expressed in exactly the same population of cells. In addition, recombinant FGF6 protein is able to repress the terminal differentiation of myoblasts in culture, providing additional support to the concept that FGF6 plays an important role in myogenesis.
Animals↗
Fibroblast growth factor receptor-4 shows novel features in genomic structure, ligand binding and signal transduction.
Fibroblast growth factor (FGF) receptor (FGFR) gene family consists of at least four receptor tyrosine kinases that transduce signals important in a variety of developmental and physiological processes related to cell growth and differentiation. Here we have characterized the binding of different FGFs to FGFR-4. Our results establish an FGF binding profile for FGFR-4 with aFGF having the highest affinity, followed by K-FGF/hst-1 and bFGF. In addition, FGF-6 was found to bind to FGFR-4 in ligand competition experiments. Interestingly, the FGFR-4 gene was found to encode only the prototype receptor in a region where both FGFR-1 and FGFR-2 show alternative splicing leading to differences in their ligand binding specificities and to secreted forms of these receptors. Ligands binding to FGFR-4 induced receptor autophosphorylation and phosphorylation of a set of cellular polypeptides, which differed from those phosphorylated in FGFR-1-expressing cells. Specifically, the FGFR-1-expressing cells showed a considerably more extensive tyrosine phosphorylation of PLC-gamma than the FGFR-4-expressing cells. Structural and functional specificity within the FGFR family exemplified by FGFR-4 may help to explain how FGFs perform their diverse functions.
Alternative Splicing↗
Overexpression of human TRK proto-oncogene into mouse cells using an inducible vector system.
The TRK proto-oncogene encodes a tyrosine kinase receptor for an, as yet, unidentified ligand. In order to help the identification of this ligand, we have constructed an expression vector capable of overexpressing the TRK protein in an inducible fashion. We report here the characterization of the TRK proto-oncogene products obtained from this expression vector.
Animals↗
The FGF6 gene within the FGF multigene family.
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3T3 Cells↗
Production and functional characterization of human recombinant FGF-6 protein.
The fibroblast growth factor (FGF) gene family to date comprises seven members and has been implicated in a wide range of physiological and biological processes, including angiogenesis, morphogenesis, and tumorigenesis. The FGFs are mitogens for a broad range of cells of various embryological origins and can act as differentiation factors. The FGFs can bind to tyrosine kinase and non-tyrosine kinase transmembrane receptors; the physiological basis for this is still unknown. In order to study more thoroughly the activities of FGF-6, we have constructed a bacterial expression vector by inserting FGF-6 complementary DNA sequences into the T7 RNA polymerase-based pET3a vector. The resulting construct is able to drive the expression of a high amount of FGF-6 protein in Escherichia coli, which can be solubilized and purified through heparin-Sepharose chromatography and high salt elution. The purified FGF-6 protein displays a strong mitogenic activity on BALB/c 3T3 cells and is able to morphologically transform these cells. By contrast, adult bovine aortic endothelial cells, which normally require the presence of FGF-2 for their growth, show only a limited mitogenic response that is highly dependent on heparin concentration.
3T3 Cells↗
Putative structure of the FGF6 gene product and role of the signal peptide.
The human FGF6 gene is an oncogene related by sequence similarities to the fibroblast growth factor (FGF) gene family, which encodes mitogenic peptides implicated in various physiological processes including angiogenesis, morphogenesis, tissue regeneration and survival and oncogenesis. Nucleotide sequence analysis of the FGF6 gene and of cDNA clones revealed an open reading frame able to code for a protein of 208 residues. The FGF6 protein shares 32-70% residues with the other members of the family within the C-terminal two-thirds of the molecule. In vitro, three in-frame ATG codons are able to initiate the translation of three peptides of 175, 198 and 208 residues. These three peptides differ at their amino termini with respect to the relative position of a hydrophobic leader peptide, which extends from residues 16 to 40, and is therefore absent from the shorter (175 amino acids) form. In-vitro analysis indicates that this signal peptide is able to drive the FGF6 protein through the endoplasmic reticulum, where it becomes glycosylated. The presence of this signal peptide sequence appears essential for the in vivo transforming capacity of the FGF6 gene.
Amino Acid Sequence↗
Human trk oncogenes activated by point mutation, in-frame deletion, and duplication of the tyrosine kinase domain.
Malignant activation of the human trk proto-oncogene, a member of the tyrosine protein kinase receptor family, has been implicated in the development of certain human cancers, including colon and thyroid papillary carcinomas. trk oncogenes have also been identified in cultured cells transfected with various DNAs. In this study, we report the characterization of three in vitro-generated trk oncogenes, trk2, trk4, and trk5 (R. Oskam, F. Coulier, M. Ernst, D. Martin-Zanca, and M. Barbacid, Proc. Natl. Acad. Sci. USA 85:2964-2968, 1988), in an effort to understand the spectrum of mutational events that can activate the human trk gene. Nucleotide sequence analysis of cDNA clones of trk2 and trk4 revealed that these oncogenes were generated by a head-to-tail arrangement of two trk tyrosine protein kinase domains connected by a purine-rich region. These oncogenes code for cytoplasmic molecules of 67,000 (p67trk2) and 69,000 (p69trk4) daltons. In contrast, the product of the trk5 oncogene, gp95trk5, is a cell surface glycoprotein of 95,000 daltons. This oncogene was generated by a 153-base-pair in-frame deletion within sequences coding for the extracellular domain of the trk receptor. This activating deletion encompasses a triplet coding for one of the nine cysteine residues that the trk receptor shares with the product of the highly related trkB tyrosine protein kinase gene. Introduction of a single point mutation (TGT----AGT) in this codon resulted in a novel trk oncogene whose product, gp140S345, differs from the nontransforming trk proto-oncogene receptor in a single amino acid residue, Ser-345 instead of Cys-345. These results illustrate that multiple molecular mechanisms, including point mutation, internal deletion, and kinase domain duplication, can result in the malignant activation of the human trk proto-oncogene.
Amino Acid Sequence↗
trkB, a novel tyrosine protein kinase receptor expressed during mouse neural development.
We have isolated a novel member of the tyrosine protein kinase family of cell surface receptors. This gene, designated trkB, is highly related to the human trk proto-oncogene. At the amino acid level, their respective products share a 57% homology in their extracellular regions including 9 of the 11 cysteines present in the trk proto-oncogene. This homology increases to 88% within their respective tyrosine kinase catalytic domains. Both trk and trkB are equally distantly related to the other members of this gene family of receptors. A biologically active cDNA clone of trkB can direct the synthesis of gp145trkB, a glycoprotein of 145 kd of which only 93 kd correspond to its polypeptide backbone. In adult mice, trkB is preferentially expressed in brain tissue, although significant levels of trkB RNA have also been observed in lung, muscle and ovaries. In addition, trkB transcripts can be detected in mid and late gestation embryos. The trkB locus exhibits a complex pattern of transcription. At least seven RNA species ranging in size from approximately 9 kb to 2 kb have been identified in brain. However, only a subset of these transcripts appears to be expressed in the other tissues. In situ hybridization analysis of 14 and 18 day old mouse embryos indicates that trkB transcripts are localized in the central (CNS) and peripheral (PNS) nervous systems, including brain, spinal cord, spinal and cranial ganglia, paravertebral trunk of the sympathetic nervous system and various innervation pathways. These results suggest that trkB may code for a novel cell surface receptor involved in neurogenesis.
Amino Acid Sequence↗
Mechanism of activation of the human trk oncogene.
The human trk oncogene was generated by a genetic rearrangement that replaced the extracellular domain of the normal trk tyrosine kinase receptor by sequences coding for the 221 amino-terminal residues of a nonmuscle tropomyosin. Molecular dissection of a cDNA clone of the trk oncogene indicated that both the tropomyosin and tyrosine kinase domains were required for proper transforming activity. Replacement of nonmuscle tropomyosin sequences with those of other tropomyosin isoforms had no deleterious effect. However, when tropomyosin sequences were replaced with those of another cytoskeletal gene, such as beta-actin or beta-globin, transforming activity was completely abolished. These results illustrate the important role of tropomyosin sequences in endowing the trk kinase with transforming properties. Functionally unrelated subdomains of the tropomyosin molecule were equally efficient in activating the trk gene. Moreover, the transforming activity of the trk oncogene was not affected when its subcellular localization was drastically altered. Therefore, tropomyosin sequences are likely to contribute to the malignant activation of the trk oncogene not by facilitating its interaction with defined cytoskeletal structures as initially suspected, but by allowing its kinase domain to fold into a constitutively active configuration.
Amino Acid Sequence↗
Characterization of the HST-related FGF.6 gene, a new member of the fibroblast growth factor gene family.
By screening a mouse cosmid library with a human HST probe under reduced conditions of stringency, we isolated several positive clones. One of them was identified as a new member of the fibroblast growth factor gene family, and called FGF.6. The human FGF.6 gene was subsequently isolated and sequenced. The deduced amino-acid sequence exhibited 70% identity with the HST gene product over the C-terminal two-thirds of the putative protein. FGF.6 was mapped to chromosome 12 at band p13 by in situ hybridization. The cloned normal human gene was able to transform mouse NIH3T3 fibroblasts using both focus- and tumorigenicity-assays.
Amino Acid Sequence↗
Frequent generation of oncogenes by in vitro recombination of TRK protooncogene sequences.
Transfection of NIH 3T3 cells with cDNA clones containing either the entire coding sequences or the tyrosine protein kinase domain of the human TRK protooncogene results in the frequent generation of transforming genes. Activation of most of these TRK oncogenes involves acquisition of DNA sequences. These sequences, unlike those present in the original human TRK oncogene, are not derived from tropomyosin genes. The products of these in vitro-generated TRK oncogenes retain the parental tyrosine protein kinase activity and contain an intact carboxyl terminus. However, they exhibit distinct biochemical properties. Whereas some of them are nonglycosylated cytoplasmic molecules, others were found to be transmembrane glycoproteins. These results suggest that TRK oncogenes may induce malignant transformation by allowing their tyrosine kinase to interact with various substrates depending on the nature of their activating sequences. If so, the TRK kinase may serve as a pleiotropic marker to identify various cellular proteins whose unscheduled phosphorylation on tyrosine residues contributes to neoplastic transformation.
Animals↗