RESUMO
The orphan receptor tyrosine kinase Tie-1 is expressed in endothelial cells and is essential for vascular development. Nothing is known about the signaling pathways utilized by this receptor. In this study we have used chimeric receptors composed of the TrkA ectodomain fused to the transmembrane and intracellular domains of Tie-1, or the related receptor Tie-2, to examine Tie-1 signaling capacity. In contrast to TrkA/Tie-2, the Tie-1 chimera was unable to phosphorylate cellular proteins or undergo autophosphorylation. Consistent with this Tie-1 exhibited negligible kinase activity. Co-immunoprecipitation analysis revealed Tie-1 was present in endothelial cells bound to Tie-2. Full-length Tie-1 and truncated receptor, formed by regulated endoproteolytic cleavage, were found to complex with Tie-2. Association was mediated by the intracellular domains of the receptors and did not require Tie-1 to be membrane-localized. Tie-1 bound to Tie-2 was not tyrosine-phosphorylated under basal conditions or following Tie-2 stimulation. This study provides the first evidence for the existence of a pre-formed complex of Tie-1 and Tie-2 in endothelial cells. The data suggest Tie-1 does not signal via ligand-induced kinase activation involving homo-oligomerization. The physical association between Tie-1 and Tie-2 is consistent with Tie-1 having a role in modulating Tie-2 signaling.
Assuntos
Proteínas de Transporte de Cátions , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas de Saccharomyces cerevisiae , Membrana Celular/metabolismo , Endotélio/metabolismo , Endotélio Vascular/citologia , Humanos , Ligantes , Fosforilação , Fosfotransferases/metabolismo , Fosfotirosina/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Receptor de TIE-1 , Receptor TIE-2 , Receptor trkA/química , Receptor trkA/metabolismo , Receptores de TIE , Transdução de Sinais , Transfecção , Veias Umbilicais/citologiaAssuntos
Anticorpos Monoclonais/genética , Anticorpos Antineoplásicos/genética , Escherichia coli/genética , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Fragmentos de Peptídeos/biossíntese , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Anticorpos Monoclonais/biossíntese , Anticorpos Antineoplásicos/biossíntese , Sequência de Bases , Clonagem Molecular , DNA/genética , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Aberrations in nuclear proto-oncogene organisation and/or gene expression have been implicated in cell transformation mediated by the v-abl gene. For example, it has been suggested that amplification of the c-myc proto-oncogene is a co-operative event in v-abl induced fibroblast transformation. We have investigated amplification of the c-myc, p53 and c-fos nuclear proto-oncogenes in several Abelson murine leukaemia virus (A-MuLV) transformed fibroblast lines. None of these proto-oncogenes were detectably rearranged or amplified in v-abl transformed Swiss 3T3 lines. In contrast, NIH3T3 fibroblasts transformed by the v-abl gene consistently showed a 4 to 16-fold amplification of the c-myc gene. These data show that c-myc gene amplification is not an obligatory event associated with A-MuLV transformation, but may be restricted to cell lines derived from NIH3T3. c-myc gene amplification also did not correlate with a reduced latency period for tumour induction in nude mice. In addition, c-myc amplification was not selected during tumourigenesis, indicating that this event is not required for A-MuLV transformed Swiss 3T3 cells to display a full tumourigenic phenotype.
Assuntos
Vírus da Leucemia Murina de Abelson/genética , Fibroblastos/microbiologia , Amplificação de Genes , Vírus da Leucemia Murina/genética , Proto-Oncogenes , Animais , Southern Blotting , Linhagem Celular Transformada , Fibroblastos/ultraestrutura , RNA Mensageiro/biossínteseRESUMO
Factor I is a serine proteinase of complement which together with one of several specific cofactors cleaves activation products of the third and fourth components of complement (C3b and C4b) and modulates the activity of C3 convertase. A heterodimer glycoprotein (Mr = 88,000), factor I is synthesized as a single-chain precursor, prepro-I, which undergoes intracellular proteolytic processing. The human hepatoma line HepG2, however, secretes predominantly the single-chain precursor pro-I. In order to determine the molecular basis for this apparent processing defect, factor I cDNA clones were isolated from a HepG2 mRNA-derived library. Sequencing of the largest insert, HI1971, revealed that it contains 14 base pairs of 5' untranslated region, the complete coding sequence for the 583-residue prepro-I (NH2-signal peptide-heavy chain-linking peptide-light chain-COOH), two polyadenylation signals within the 200-base pair 3' untranslated region, and a portion of poly(A) tail. Analysis of the derived protein structure 1) reveals a mosaic multidomain structure of the heavy chain; 2) demonstrates structural similarity between intracellular conversion of pro-I and activation of other serine proteinase zymogens; and 3) indicates that the light chain of factor I resembles most closely the active subunit of tissue plasminogen activator among all serine proteinases and factor D among complement proteinases. Furthermore, this protein sequence was compared to the sequences of factor I cDNA clones isolated from normal human liver libraries and found to be identical. By exclusion, this defines as cellular the basis for the inefficient processing of pro-I by the HepG2 line. Chromosomal localization by the somatic cell hybrid method maps the factor I gene to chromosome 4.
Assuntos
Cromossomos Humanos Par 4 , DNA/análise , Endopeptidases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Fator I do Complemento , Humanos , Mosaicismo , Hibridização de Ácido NucleicoRESUMO
Site-directed mutagenesis was used to change Lys-128 of the simian virus 40 large-T nuclear location signal to Met, Ile, Arg, Gln, Asn, Leu, or His. Except for the large-T antigen of the Arg mutation, which was present in cytoplasmic and nuclear compartments, the resultant proteins were unable to enter the nucleus. By contrast, mutations at other sites within the signal were generally less severe in their effect. In some cases (Lys-128 to Gln, Asn, and His), the apparently cytoplasmic variants were able to support limited plasmid DNA replication, suggesting that low levels of large-T antigen undetectable by immunofluorescence were present in the nucleus. Such mutants did not support viral DNA replication. We conclude that there is a strong requirement for a basic residue at position 128 in the large-T nuclear location signal, with Lys the preferred residue.
Assuntos
Antígenos Virais de Tumores/genética , Núcleo Celular/metabolismo , Lisina , Mutação , Proteínas Oncogênicas Virais/genética , Proteínas Quinases/genética , Vírus 40 dos Símios/genética , Sequência de Aminoácidos , Animais , Antígenos Transformantes de Poliomavirus , Sequência de Bases , Linhagem Celular , PlasmídeosRESUMO
Prealbumin, a 55,000 Mr protein, is a normal constituent of human serum. In patients with familial amyloid polyneuropathy (FAP), an autosomal dominant disease, variant prealbumin molecules are found in association with systemic amyloid deposits. One variant prealbumin has a methionine for valine substitution at amino acid 30 and has been implicated in the pathogenesis of type 1 FAP. A prealbumin-specific complementary DNA clone has been isolated from an adult human liver library and used in Southern blot hybridization experiments to identify a unique NsiI restriction endonuclease site in the variant allele carried by type 1 FAP patients with the methionine for valine substitution. The complementary DNA clone has been used to analyse a panel of human-mouse and human--hamster somatic cell hybrid DNAs and localize the prealbumin gene to chromosome 18.
Assuntos
Amiloidose/genética , DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II , Doenças do Sistema Nervoso/genética , Pré-Albumina/genética , Alelos , Sequência de Aminoácidos , Mapeamento Cromossômico , Cromossomos Humanos 16-18 , Clonagem Molecular , Enzimas de Restrição do DNA , Feminino , Variação Genética , Humanos , Masculino , LinhagemRESUMO
A derivative of pBR322 has been constructed that contains both a unique EcoRI restriction site right at the beginning of the signal codons of the beta-lactamase (bla) gene and a unique BstEII site just at the end of the bla signal codons. Although the signal peptide encoded by the new plasmid differs from the wild type (pBR322) by 2 amino acid residues (Ser 2 to Arg 2 and Ala 23 to Gly 23), the synthesis, transport, and processing of the beta-lactamase remain unchanged in Escherichia coli. Two deletion mutants, in which the bla signal codons have been almost completely excised, have also been constructed. Bacteria containing either of these plasmids produce, but do not secrete, an active beta-lactamase. Last, the bla signal codons have been precisely joined to the cDNA version of the triose phosphate isomerase (tpi) gene from chicken. Expression of this fusion gene in E. coli gives a hybrid protein that is neither secreted into the periplasm nor proteolytically processed. This result supports the view that there are characteristics of the mature protein that are necessary for the secretion across the inner membrane of E. coli.