RESUMEN
Syndecan-4 is a cell surface heparan sulfate proteoglycan which, in cooperation with integrins, transduces signals for the assembly of focal adhesions and actin stress fibers in cells plated on fibronectin. The regulation of these cellular events is proposed to occur, in part, through the interaction of the cytoplasmic domains of these transmembrane receptors with intracellular proteins. To identify potential intracellular proteins that interact with the cytoplasmic domain of syndecan-4, we carried out a yeast two-hybrid screen in which the cytoplasmic domain of syndecan-4 was used as bait. As a result of this screen, we have identified a novel cellular protein that interacts with the cytoplasmic domain of syndecan-4 but not with those of the other three syndecan family members. The interaction involves both the membrane proximal and variable central regions of the cytoplasmic domain. We have named this cDNA and encoded protein syndesmos. Syndesmos is ubiquitously expressed and can be myristylated. Consistent with its myristylation and syndecan-4 association, syndesmos colocalizes with syndecan-4 in the ventral plasma membranes of cells plated on fibronectin. When overexpressed in NIH 3T3 cells, syndesmos enhances cell spreading, actin stress fiber and focal contact formation in a serum-independent manner.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Adhesión Celular/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteoglicanos/metabolismo , Células 3T3 , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Embrión de Pollo , Clonación Molecular , Citoesqueleto/metabolismo , ADN Complementario/genética , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/química , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteoglicanos/química , Sindecano-4 , Distribución Tisular , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The formation of focal adhesions and actin stress fibers on fibronectin is dependent on signaling through (&bgr;)1 integrins and the heparan sulfate proteoglycan syndecan-4, and we have analyzed the requirement of the glycosaminoglycan chains of syndecan-4 during these events. Chinese hamster ovary cells with mutations in key enzymes of the glycanation process do not synthesize glycosaminoglycan chains and are unable to assemble actin stress fibers and focal contacts when cultured on fibronectin. Transfection of the mutant cells with a cDNA that encodes the core protein of chicken syndecan-4 leads to the production of unglycanated core protein. The overexpression of syndecan-4 core protein in these mutant cells increases cell spreading and is sufficient for these cells to assemble actin stress fibers and focal adhesions similar to wild-type cells seeded on fibronectin and vitronectin matrices. Syndecan-4 core protein colocalizes to focal contacts in mutant cells that have been transfected with the syndecan-4 core protein cDNA. These data indicate an essential role for the core protein of syndecan-4 in the generation of signals leading to actin stress fiber and focal contact assembly.
Asunto(s)
Actinas/fisiología , Adhesión Celular/fisiología , Glicoproteínas de Membrana/fisiología , Proteoglicanos/fisiología , Animales , Células CHO , Pollos , Cricetinae , Glicosaminoglicanos/fisiología , Glicosilación , Glicoproteínas de Membrana/genética , Proteoglicanos/genética , Proteínas Recombinantes/metabolismo , Estrés Mecánico , Sindecano-4 , Transfección , Vinculina/fisiologíaRESUMEN
Cell surface heparan sulfate proteoglycans have been implicated as co-receptors facilitating cell adhesion and growth factor binding. Recent studies on the role of a family of transmembrane heparan sulfate proteoglycans, syndecans, in cell adhesion has identified one member, syndecan-4, to be present within focal contacts. The current study investigates the mechanisms regulating the association of syndecan-4 with focal contacts based upon its immunolocalization with vinculin in quiescent, serum-stimulated, and 12-0-tetradecanoylphorbol 13-acetate (TPA)-induced cultures. In quiescent cells, syndecan-4 did not localize to focal contacts. However, activation of protein kinase C by TPA or serum induces the active recruitment of syndecan-4 into focal contacts. This induction preferentially localizes syndecan-4 to focal contacts behind the leading lamella, the subnuclear region, and along the trailing edge of migratory cells. Focal contacts in either freshly adhered cells or in the leading lamellae of migrating cells did not stain for syndecan-4. In addition to the observed subcellular distribution and recruitment, syndecan-4 was observed to co-localize with endogenously synthesized fibronectin fibrils within focal contacts as well as with fibrils present in the matrix. These findings suggest that protein kinase C activation results in syndecan-4 recruitment to focal contacts and its association with sites of matrix deposition.
Asunto(s)
Uniones Intercelulares/fisiología , Glicoproteínas de Membrana/metabolismo , Proteína Quinasa C/metabolismo , Proteoglicanos/metabolismo , Animales , Adhesión Celular , División Celular , Células Cultivadas , Embrión de Pollo , Activación Enzimática , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Fibronectinas/análisis , Fibronectinas/metabolismo , Uniones Intercelulares/ultraestructura , Cinética , Glicoproteínas de Membrana/análisis , Proteoglicanos/análisis , Piel/citología , Fenómenos Fisiológicos de la Piel , Sindecano-4 , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Vinculina/análisisRESUMEN
We have cloned and determined the genomic organization of the core protein of the chicken transmembrane proteoglycan, syndecan-4. Identification of the initial cDNA was accomplished using polyclonal antibodies directed against the cytoplasmic domain of murine syndecan-1 core protein. The cDNA for chicken syndecan-4 encodes a putative core protein of 197 amino acids which consists of a 19-amino acid signal peptide, a 125-amino acid ectodomain, a 25-amino acid transmembrane domain, and a 28-amino acid cytoplasmic domain. The predicted molecular mass of the mature core protein is 19,639 daltons. The ectodomain of chicken syndecan-4 core protein contains three potential sites for glycosaminoglycan attachment, two sites for N-glycosylation, and lacks a dibasic protease cleavage site proximal to the membrane-spanning region found in other syndecan family members. Comparison of the complete amino acid sequence with human syndecan-4 (amphlican (David, G., van der Schueren, B., Marynen, P., Cassiman, J. J., and van den Berghe, H. (1992) J. Cell Biol. 118, 961-969)) and rat syndecan-4 (ryudocan (Kojima, T., Shworak, N. W., and Rosenberg, R. D. (1992) J. Biol. Chem. 267, 4870-4877)) indicates an overall identity of 58 and 56%, respectively, with a 91 and 92% identity in the highly conserved transmembrane and cytoplasmic domains. The core protein of chicken syndecan-4 synthesized by chicken cells is modified with heparan sulfate side chains yielding a proteoglycan with a molecular mass of > 200 kDa in LMH cells (immortalized male leghorn LM strain hepatocytes) and primary skin fibroblasts. Syndecan-4 isolated from chondrocyte cultures runs as a diffuse band between 100 and 200 kDa. Northern analysis of chicken syndecan-4 indicates three messages with distinct sizes of 0.9, 1.3, and 2.9 kb and a wide mRNA tissue distribution. The chicken syndecan-4 gene is divided into 5 exons encoding distinct regions which contain the signal peptide, the glycosaminoglycan attachment sites, a small spacer of unknown function, the glycosylation sites and the transmembrane and cytoplasmic domains.
Asunto(s)
Glicoproteínas de Membrana/genética , Proteoglicanos/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Embrión de Pollo , Pollos , Clonación Molecular , ADN Complementario , Exones , Humanos , Intrones , Masculino , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Sindecano-4RESUMEN
Methods are described for the extraction and preparation of total nuclear proteins for high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The conditions for protein extraction and preparation limit both protease and phosphatase activity, allowing application of this technique to the reliable analysis of changes in nuclear protein composition and nuclear protein phosphorylation as well as other forms of post-translational modifications. Unlike other procedures for 2-D PAGE analysis of nuclear proteins the technique allows solubilization and extraction of all nuclear proteins along with removal of nucleic acids which interfere with isoelectric focusing and autoradiography of 32Pi-labeled proteins. It avoids lengthy dialysis in which precipitation of nuclear proteins often occurs and does not require precipitation and resolubilization of nuclear proteins to obtain sufficient protein concentrations for 2-D PAGE analysis; often impractical steps in which complete resolubilization of all proteins is not possible. It produces high resolution 2-D PAGE analysis in which identification of even low abundance proteins can be made, based on isoelectric point and molecular weight, allowing comparison with other studies.
Asunto(s)
Núcleo Celular/análisis , Proteínas Nucleares/aislamiento & purificación , Fosfoproteínas/aislamiento & purificación , Fraccionamiento Celular/métodos , Línea Celular , Células Clonales , Electroforesis en Gel Bidimensional , Humanos , Soluciones Isotónicas , Ácidos Nucleicos/aislamiento & purificación , Plata , Coloración y EtiquetadoRESUMEN
To test the recently developed method of exposing cells to volatile compounds, phytohemagglutinin-stimulated human peripheral lymphocyte cultures were exposed to gaseous methyl bromide, ethylene oxide, and propylene oxide, as well as diesel exhaust. The cultures were placed in sterile dialysis tubing and inserted into enclosed flasks containing additional culture medium. The test compounds (in gaseous state) were diluted with air and bubbled through the flasks for various lengths of time. The cells were then washed and incubated for a total of 75 h. The harvest was performed according to established procedures, and second-division cells were scored for induction of sister chromatid exchanges (SCEs). The SCE frequency was more than doubled in the cultures treated with ethylene oxide and propylene oxide; methyl bromide also induced SCEs. Cultures treated with diesel exhaust showed an increase in the SCE frequency in cells from two of four donors tested. These results further substantiate the use of this method for detecting the induction of SCEs by airborne genotoxins.