Biomedical subjects

G Tarone

Publications and source records attributed to G Tarone.

At least 37 records · Page 2Linked to original sources

PubMed1998

Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival.

Adhesion of human primary skin fibroblasts and ECV304 endothelial cells to immobilized matrix proteins, beta1 or alphav integrin antibodies stimulates tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. This tyrosine phosphorylation is transiently induced, reaching maximal levels 30 min after adhesion, and it occurs in the absence of receptor ligands. Similar results were observed with EGF receptor-transfected NIH-3T3 cells. Use of a kinase-negative EGF receptor mutant demonstrates that the integrin-stimulated tyrosine phosphorylation is due to activation of the receptor's intrinsic kinase activity. Integrin-mediated EGF receptor activation leads to Erk-1/MAP kinase induction, as shown by treatment with the specific inhibitor tyrphostin AG1478 and by expression of a dominant-negative EGF receptor mutant. EGF receptor and Erk-1/MAP kinase activation by integrins does not lead per se to cell proliferation, but is important for entry into S phase in response to EGF or serum. EGF receptor activation is also required for extracellular matrix-mediated cell survival. Adhesion-dependent MAP kinase activation and survival are regulated through EGF receptor activation in cells expressing this molecule above a threshold level (5x10(3) receptors per cell). These results demonstrate that integrin-dependent EGF receptor activation is a novel signaling mechanism involved in cell survival and proliferation in response to extracellular matrix.

3T3 Cells↗

PubMed1998

Beta1B integrin interferes with matrix assembly but not with confluent monolayer polarity, and alters some morphogenetic properties of FRT epithelial cells.

Beta1B is a beta1 integrin splice variant that differs from the ubiquitous beta1A in the terminal portion of the cytosolic tail. The expression of this variant in CHO cells results in reduced fibroblast adhesion and motility (Balzac, E et al., J. Cell Biol. 127, 557-565 (1994)). We have evaluated the phenotypic changes induced by the expression of beta1B in the FRT epithelial cell line. Stable transfectants of FRT cells expressing beta1B or beta1A human integrins were obtained. The transfected integrins associated with the endogenous alpha subunits and were delivered to the plasma membrane. Beta1B expressing cells attached less efficiently and spread less on fibronectin, laminin or type IV collagen coated dishes. A great reduction of fibronectin fibrils associated to the basal membrane of non-confluent beta1B transfected cells was observed. This was paralleled by the disappearance of microfilament bundles and loss of basally located focal adhesions. On the contrary, upon beta1A transfection, a higher amount of fibronectin fibrils, together with microfilament bundles and focal adhesions, was observed. Expression of beta1B did not significantly modify the ability to manifest the polarized phenotype when cells were grown to confluence on filters in two-chamber-systems. Beta1B-transfected cells showed reduced motile properties when embedded as aggregates in type I collagen gels. Moreover, formation of polarized cysts in suspension culture was impaired. The results show that beta1B, by interfering with focal adhesion organization, microfilament and fibronectin assembly, cell spreading and migration, affects some morphogenetic properties of FRT epithelial cells.

Actin Cytoskeleton↗

PubMed1998

beta1-integrin cytoplasmic subdomains involved in dominant negative function.

The beta1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms ("common" region) and a distal subdomain specific for each isoform ("variable" region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used beta1A and beta1B isoforms as well as four mutants lacking the entire cytoplasmic domain (beta1TR), the variable region (beta1COM), or the common region (beta1 deltaCOM-B and beta1 deltaCOM-A). By expressing these constructs in Chinese hamster ovary and beta1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227-238, 1996), we show that beta1B, beta1COM, beta1 deltaCOM-B, and beta1 deltaCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, beta1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that beta1B interferes in a dominant negative manner with beta1A and beta3/beta5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the beta1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the beta1B isoform.

Actinin↗

PubMed1998

Myogenic conversion of NIH3T3 cells by exogenous MyoD family members: dissociation of terminal differentiation from myotube formation.

Myogenic regulatory factors (MRF) of the MyoD family regulate the skeletal muscle differentiation program. Non-muscle cells transfected with exogenous MRF either are converted to the myogenic lineage or fail to express the muscle phenotype, depending on the cell type analysed. We report here that MRF-induced myogenic conversion of NIH3T3 cells results in an incomplete reprogramming of these cells. Transfected cells withdrew from the cell cycle and underwent biochemical differentiation but, surprisingly, terminally differentiated myocytes absolutely failed to fuse into multinucleated myotubes. Analysis of muscle regulatory and structural gene expression failed to provide an explanation for the fusion defectiveness. However, myogenic derivatives of NIH3T3 cells were shown to be unable to accumulate the transcripts encoding muscle-specific isoforms of the integrin subunit beta1D and the transcription factor MEF2D1b2, that depend on muscle-specific alternative splicing. Our results suggest that the fusion into myotubes is under a distinct genetic control that might depend, at least partially, on differential splicing.

3T3 Cells↗

PubMed1998

Differential onset of expression of alpha 7 and beta 1D integrins during mouse heart and skeletal muscle development.

beta 1D is a recently identified isoform of the beta 1 integrin subunit selectively expressed in skeletal and cardiac muscles. In the present study we determined the temporal expression of beta 1D and its association with alpha subunits during mouse development. By immunohistochemistry and western blot analysis we demonstrated that beta 1D begins to be expressed in skeletal muscles of 17 days embryo (stage E17). Its level progressively increases reaching maximal values few days after birth and remaining high in adult mice. At earlier stages of development (E11-E17) the beta 1A isoform is expressed in skeletal muscle cells. After E17 beta 1A is downregulated and disappears from muscle fibers few days after birth. In cardiac muscle the regulation of the beta 1D expression is different: beta 1D and beta 1A are coexpressed in the heart of E11 embryo. Subsequently expression of beta 1A declines, while beta 1D increases until it becomes the unique beta 1 isoform in cardiomyocytes few days after birth. Previous studies (Belkin et al J. Cell Biol. 132: 211-226, 1996) demonstrated that beta 1D in adult mouse cardiomyocytes is exclusively associated with alpha 7B. Western blot analysis shows that alpha 7B starts to be expressed in the heart only at stage E17, while beta 1D is expressed already at E11 embryo, indicating that alpha subunits other than alpha 7 should associate with beta 1D in early developmental stages. To investigate this aspect, beta 1 associated alpha subunits were identified by western blotting from cardiomyocytes integrin complexes immunoprecipitated with alpha subunit specific antibodies. We found that, during cardiomyocyte development, beta 1D associates with several alpha subunits namely with alpha 5, alpha 6A and alpha 7B. In conclusion these data show that the expression of the beta 1D muscle specific integrin during development occurs much earlier in heart than in skeletal muscle and it can dimerize with different alpha subunits.

Alternative Splicing↗

PubMed1997

Soluble integrin ligands and growth factors independently rescue neuroblastoma cells from apoptosis under nonadherent conditions.

We have investigated the role of extracellular matrix (ECM) and growth factors in the survival of nonadherent human neuroblastoma cells (line SK-N-BE). Cells cultured in serum-free medium under nonadherent conditions died with apoptotic-like features (chromatin condensation and nuclear fragmentation). SK-N-BE cells underwent neuronal differentiation in response to retinoic acid (RA). While RA itself did not induce apoptosis, differentiation increased the susceptibility of SK-N-BE cells to detachment-induced apoptosis. The appearance of the apoptotic-like phenotype required the maintenance in suspension of SK-N-BE cells for at least 16 h (12.43 +/- 1.40% of cells undergoing apoptosis) and the percentage increased up to 46.84 +/- 3.15% after 24 h. Suspension-induced apoptosis did not depend on increased intracellular Ca2+ levels nor on de novo protein synthesis and was not associated with extensive DNA degradation. Stimulation by soluble collagen I rescued suspended cells from apoptosis, even in the absence of cell adhesion and spreading. The survival promoting effect of ECM was mediated by the integrin receptors, since (1) the protective effect of soluble collagen I was blocked by anti-integrin antibodies to beta 1 and alpha 1 subunits and (2) the antibody-induced clustering of alpha 1, alpha 3, alpha v, beta 1, and beta 3 integrins rescued SK-N-BE cells cultured in suspension from apoptosis. As expected, adhesion on immobilized ECM proteins, collagen I, or laminin (0.1 to 10 micrograms/ml) also rescued SK-N-BE cells from apoptosis in a dose-dependent manner. The de novo protein synthesis was required to promote the survival effect of ECM, since cycloheximide completely abolished the protective effect of collagen I and protection from apoptosis by ECM or by anti-beta 1 antibody was associated with the increased expression of bcl-2. In addition to integrin stimulation, serum, insulin, and nerve growth factor inhibited suspension-induced apoptosis of SK-N-BE cells. The survival effect of serum and growth factors did not require the synthesis of new proteins, unlike the ECM effect. These data show that matrix proteins can promote cell survival in neuronal cells via integrin receptors. This effect does not require cell adhesion and the subsequent changes in cell shape as it can be mediated by soluble integrin ligands in suspended cells and involves a signaling pathway different from that triggered by growth factors.

Apoptosis↗

PubMed1997

Muscle beta1D integrin reinforces the cytoskeleton-matrix link: modulation of integrin adhesive function by alternative splicing.

Expression of muscle-specific beta1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, beta1 integrin-minus cells leads to their phenotypic conversion. beta1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility compared with their beta1A-expressing counterparts. The transfected beta1D is targeted to focal adhesions and efficiently displaces the endogenous beta1A and alphavbeta3 integrins from the sites of cell-matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in beta1D transfectants. Whereas a significant part of cellular beta1A integrin is extractable in digitonin, the majority of the transfected beta1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton. Increased interaction of beta1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin. In contrast, beta1A interacts more strongly with alpha-actinin, than beta1D. Inside-out driven activation of the beta1D ectodomain increases ligand binding and fibronectin matrix assembly by beta1D transfectants. Phenotypic effects of beta1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of beta1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton- matrix link in muscle cells. This reflects the major role for beta1D integrin in muscle, where extremely stable association is required for contraction.

Actins↗

PubMed1997

Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly. Involvement of protein kinase C, Rho GTPase, and tyrosine phosphorylation.

A panel of antibodies to the alphaIIbbeta3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the alphaIIbbeta3 fibrinogen receptor. While some alphaIIbbeta3 antibodies were not able to induce p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, all the antibodies equally support cell adhesion but not spreading and assembly of actin stress fibers. Absence of stress fibers was also obtained by plating on antibodies directed to the hamster beta1 integrin. In contrast, cells plated on matrix proteins spread organizing actin stress fibers. Treatment with phorbol esters phorbol 12-myristate 13-acetate (PMA) induced cells to spread on antibodies-coated dishes but not to organize actin in stress fibers. The combination of PMA and cytotoxic necrotizing factor 1 (CNF1), a specific Rho activator, induced cell spreading and organization of stress fibers. PMA or the combination of PMA and CNF1 also increases tyrosine phosphorylation of p125FAK in response to antibodies that were otherwise unable to trigger this response. These data show that: 1) matrix proteins and antibodies differ in their ability to induce integrin-dependent actin cytoskeleton organization (while matrix induced stress fibers formation, antibodies did not); 2) p125FAK tyrosine phosphorylation is insufficient per se to trigger actin stress fibers formation since antibodies that activate p125FAK tyrosine phosphorylation did not lead to actin stress fibers assembly; and 3) the inability of anti-integrin antibodies to trigger stress fibers organization is overcome by concomitant activation of the protein kinase C (PKC) and Rho pathways; PKC activation leads to cell spreading and Rho activation is required to organize actin stress fibers.

Actins↗

PubMed1997

Actin cytoskeleton polymerization in Dbl-transformed NIH3T3 fibroblasts is dependent on cell adhesion to specific extracellular matrix proteins.

The Dbl oncogene is the putative exchange factor for two small GTP-binding proteins, RhoA and CDC42 which are involved in the polymerization of actin to produce stress fibers and filopodia, respectively. We report here that Dbl oncogene-transformed NIH3T3 cells show actin stress fibers only when cells are plated on fibronectin. Plating of cells on collagen I and IV as well as on poly-D-lysine and gelatin induces polymerization of actin to form filopodia, lamellipodia and membrane ruffles but not stress fibers. The putative collagen receptors, alpha1/beta1 and alpha2/beta1 integrins are expressed at reduced level in Dbl-transformed cells compared to untransformed NIH3T3 fibroblasts. Nevertheless, adhesion to collagens is not altered. Inhibitory monoclonal antibody to mouse integrin beta1 subunit blocked adhesion of both Dbl-transformed and untransformed NIH3T3 cells, demonstrating that adhesion to collagen I and IV is mediated by the beta1 family of integrins. Dbl product rapidly induces the depolymerization of actin stress fibers, rounding up of the cells, and formation of filopodia and lamellipodia when microinjected in NIH3T3 cells plated on gelatin. Thus, Dbl may exert its effect on actin cytoskeleton organization in response to extracellular proteins by altering integrin-mediated signalling pathways.

3T3 Cells↗

PubMed1996

Focal adhesion and stress fiber formation is regulated by tyrosine phosphatase activity.

Tyrosine phosphorylation of cytoskeletal proteins plays an important role in the regulation of focal adhesions and stress fiber organization. In the present study we examined the role of tyrosine phosphatases in this process using p125FAK and paxillin as substrates. We show that tyrosine phosphatase activity in Swiss 3T3 cells was markedly increased when actin stress fibers were disassembled by cell detachment from the substratum, by serum starvation, or by cytochalasin D treatment. This activity was blocked by phenylarsine oxide, an inhibitor of a specific class of tyrosine phosphatases characterized by two vicinal thiol groups in the active site. Phenylarsine oxide treatment of serum-starved cells induced increased tyrosine phosphorylation of p125FAK and paxillin in a dose-dependent manner and induced assembly of focal adhesions and actin stress fibers, showing that inhibition of one or more phenylarsine oxide-sensitive tyrosine phosphatases is a sufficient stimulus for triggering focal adhesion and actin stress fiber formation in adherent cells.

3T3 Cells↗

PubMed1996

Beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells.

The cytoplasmic domains of integrins provide attachment of these extracellular matrix receptors to the cytoskeleton and play a critical role in integrin-mediated signal transduction. In this report we describe the identification, expression, localization, and initial functional characterization of a novel form of beta 1 integrin, termed beta 1D. This isoform contains a unique alternatively spliced cytoplasmic domain of 50 amino acids, with the last 24 amino acids encoded by an additional exon. Of these 24 amino acids, 11 are conserved when compared to the beta 1A isoform, but 13 are unique (Zhidkova, N. I., A. M. Belkin, and R. Mayne. 1995. Biochem. Biophys. Res. Commun. 214:279-285; van der Flier, A., I. Kuikman, C. Baudoin, R, van der Neuf, and A. Sonnenberg. 1995. FEBS Lett. 369:340-344). Using an anti-peptide antibody against the beta 1D integrin subunit, we demonstrated that the beta 1D isoform is synthesized only in skeletal and cardiac muscles, while very low amounts of beta 1A were detected by immunoblot in striated muscles. Whereas beta 1A could not be detected in adult skeletal muscle fibers and cardiomyocytes by immunofluorescence, beta 1D was localized to the sarcolemma of both cell types. In skeletal muscle, beta 1D was concentrated in costameres, myotendinous, and neuromuscular junctions. In cardiac muscle this beta 1 isoform was found in costamers and intercalated discs. beta 1D was associated with alpha 7A and alpha 7B in adult skeletal muscle. In cardiomyocytes of adult heart, alpha 7B was the major partner for the beta 1D isoform. beta 1D could not be detected in proliferating C2C12 myoblasts, but it appeared immediately after myoblast fusion and its amount continued to rise during myotube growth and maturation. In contrast, expression of the beta 1A isoform was downregulated during myodifferentiation in culture and it was completely displaced by beta 1D in mature differentiated myotubes. We also analyzed some functional properties of the beta 1D integrin subunit. Expression of human beta 1D in CHO cells led to its localization at focal adhesions. Clustering of this integrin isoform on the cell surface stimulated tyrosine phosphorylation of pp125FAK (focal adhesion kinase) and caused transient activation of mitogen-activated protein (MAP) kinases. These data indicate that beta 1D and beta 1A integrin isoforms are functionally similar with regard to integrin-mediated signaling.

Amino Acid Sequence↗

PubMed1996

Alpha 2 beta 1 integrin is required for the collagen and FGF-1 induced cell dispersion in a rat bladder carcinoma cell line.

We have investigated the role of integrins in the epithelial to mesenchymal transition (EMT) induced by either collagen or fibroblast growth factor-1 (FGF-1) in the rat bladder carcinoma cell line NBT-II. The major collagen-binding receptor is the alpha 2 beta 1 integrin. An increase in expression of alpha 2 beta 1 integrin coincided with EMT induced by either collagen or FGF-1. When both inducers were present, a further increase in alpha 2 expression was observed which correlated with an enhancement in the speed of locomotion. Overexpression of human alpha 2 in NBT-II cells did not trigger EMT but rendered cells more sensitive to the dispersing effect of collagen and FGF-1. Anti-human alpha 2 blocking antibodies affected cell scattering and motility induced by either collagen or FGF-1. These data demonstrate that alpha 2 beta 1 integrin is the mediator of the cell scattering effect induced by collagen. They also indicate that a functional alpha 2 integrin is essential for the motile behavior of NBT-II cells during the FGF-1 induced EMT.

Animals↗

PubMed1995

p125FAK tyrosine phosphorylation and focal adhesion assembly: studies with phosphotyrosine phosphatase inhibitors.

p125FAK is a major tyrosine kinase phosphorylated in response to integrin-dependent adhesion. In this study we use vanadate and phenylarsine oxide (PAO), known inhibitors of phosphotyrosine phosphatases (PTPases), as a tool to artificially modulate p125FAK phosphorylation in human endothelial and in Chinese hamster ovary cells. Vanadate treatment strongly upregulates in a dose-dependent manner the level of tyrosine phosphorylation of several proteins in adherent cells. PAO induces a more restricted profile of tyrosine-phosphorylated proteins, increasing primarily a broad band of 120-140 kDa. Maximal stimulation of p125FAK tyrosine phosphorylation is reached at 10 microM PAO. In contrast, in vanadate-treated cells the p125FAK tyrosine phosphorylation shows a biphasic curve, being increased at high doses of vanadate (100 microM) and downregulated at low doses (25 microM). Immunofluorescence analysis of cells treated with PTPase inhibitors showed a direct correlation between the level of p125FAK tyrosine phosphorylation and the assembly of focal adhesions and actin stress fibers. Downregulation of p125FAK tyrosine phosphorylation is observed by treating cells with cytochalasin D (CD), a drug known to rapidly disrupt the actin cytoskeleton. When PTPase inhibitors are added in combination to CD, the level of tyrosine phosphorylation of p125FAK remains high and focal adhesions and actin stress fibers are preserved from the CD-mediated disruption. Based on these data we suggest that assembly of actin cytoskeleton plays an important role in inhibiting PTPases involved in p125FAK tyrosine phosphorylation.

Actins↗

PubMed1995

Ciliary neurotrophic factor-induced gene expression in human neuroblastoma cell lines.

We have analyzed the response of the human neuroblastoma cell lines SK-N-SH (clone SY5Y) and SK-N-BE to the ciliary neurotrophic factor CNTF. In both cell lines CNTF induced the expression of the mRNA for two transcription factors, c-fos and NGF1A. The induction was rapid and transient reaching a maximum between 30 and 60 min after exposure to CNTF and subsequently declining. The level of induction of both c-fos and NGF1A mRNAs was much higher in SK-N-BE neuroblastoma cells compared to the SY5Y. Both cells express comparable levels of the transcript for the CNTF receptor-alpha. This mRNA was down regulated after 5 days of CNTF stimulation in both cell lines. CNTF also induced increased levels of the transcript for the growth cone associated protein GAP43 in SK-N-BE, but not in SY5Y cells. Induction followed a slower kinetic compared to that observed for c-fos and NGF1A. In fact, the GAP43 mRNA levels increased during 2 days of exposure to CNTF. Morphological analysis of CNTF treated cells showed that SK-N-BE undergo significant differentiation in response to CNTF (increased number of cells with neurites and increased neurite length) while SY5Y did not show appreciable morphological differentiation. These data shows that CNTF may elicit different response in neuroblastoma cell lines.

Cell Division↗

PubMed1994

Internalization of the alpha 5 beta 1 integrin does not depend on "NPXY" signals.

The alpha 5 beta 1 integrin is a constitutively internalized fibronectin receptor. It contains in the cytoplasmic tail of its beta 1 subunit two NPXY sequences which have been proposed to mediate internalization. Indeed a NPXY motif constitutes the internalization signal for the Low Density Lipoprotein (LDL) and insulin receptors. To learn more about the putative role of the two NPXY sequences in internalization of the alpha 5 beta 1 receptor, we have made and expressed mutants of the human beta 1 subunit in Chinese Hamster Ovary (CHO) cells, in which the two tyrosines of the NPXY motifs were replaced by serine residues. A cytoplasmic variant beta 1B which does not contain any NPXY sequence was also analyzed. Our results indicate that the NPXY mutants and the cytoplasmic variant are still internalized. Thus in the alpha 5 beta 1 receptor, the highly conserved NPXY sequences do not function as internalization motifs.

Amino Acid Sequence↗

PubMed1994

Alpha v integrin subunit is predominantly located in nervous tissue and skeletal muscle during mouse development.

Alpha v integrin subunit can dimerize with different beta subunits to form receptors for several matrix proteins. The function of these receptors in vivo is still largely unknown. We examined the localization of alpha v integrin during mouse development and showed that its distribution is dynamically regulated in the glia of the central nervous system and in skeletal muscle. Immunoreactivity in the neural tube was firstly localized at embryonic day 10.5 (E10.5) around cell bodies lining the lumen and along tiny fibres extending towards the outer margin. At E12.5 alpha v distribution follows the highly defined pattern of the radial glia: fascicles of immunoreactive fibres form parallel palisades, in particular along the hindbrain and the spinal cord. At E15.5, although with weaker intensity, alpha v was still detectable in radial glia fibres, and it codistributed with glial fibrillary acidic protein positive fascicles. After birth (P8) alpha v immunoreactivity in the brain and spinal cord decreased dramatically, but remained high in the radial glia of the cerebellum. In adult mice alpha v reactivity in the central nervous system disappeared. During myogenesis alpha v appears at E10.5 in myotomal cells and from E12.5 alpha v was evident in myoblasts and in myotubes. In the developing skeletal muscle of E15.5 embryos, immunoreactivity became more concentrated in the apical portion of the myotubes. In adult striated muscle the amount of alpha v subunit dramatically declined and immunostaining was no longer detectable. During development, alpha v was weakly evident in other sites including heart and endothelia of blood vessels, mesonephric tubula, smooth muscle of the digestive tract, and bronchia. Comparative analysis of the localization of alpha v, alpha 3, and alpha 5 integrin subunits indicated that alpha v has a unique and highly regulated distribution pattern. The distribution in the nervous system is consistent with a role of alpha v in neuron-glia interaction during the organization of the neuronal layers in the brain cortex and in the cerebellum. Moreover, alpha v is likely to be involved in the myotendinous junction during embryonic life, suggesting a dual functional role of this integrin in muscle and nervous tissue.

Amino Acid Sequence↗

PubMed1994

Role of tyrosine phosphorylation in matrix-induced neurite outgrowth in human neuroblastoma cells.

Adhesion of human neuroblastoma cells (SK-N-SH clone SY5Y) to laminin or collagen type IV promotes tyrosine phosphorylation of a group of proteins with molecular mass ranging from 100 to 130 kDa and of a protein of 180 kDa. The same pattern of tyrosine phosphorylation was observed when SY5Y cells were allowed to adhere to culture dishes coated with monoclonal antibodies directed to the integrin subunits expressed in the cells, alpha 1, alpha 3, and beta 1, indicating that these receptors are responsible for this signaling mechanism. Using specific antibodies we identified the focal adhesion kinase p125FAK as a component of the 100- to 130-kDa phosphoproteins. Treatment with genistein or herbimycin A, two specific tyrosine kinase inhibitors, greatly reduced the tyrosine phosphorylation of the 100- to 130- and the 180-kDa proteins in response to laminin or collagen IV. Concomitantly, neurite outgrowth on the matrix proteins was strongly inhibited. This effect was observed in two distinct neuroblastoma cell lines, SY5Y and SK-N-BE. Genistein and herbimycin A treatment did not affect cell viability nor cause retraction of preformed neurites. These data suggest that matrix-induced tyrosine phosphorylation events are involved in neurite extension.

Cell Adhesion↗

PubMed1994

Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility.

The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125-kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration.

Animals↗