Altered processing of integrin receptors during keratinocyte activation

We used monoclonal antibodies against specific integrin subunits to examine the role of integrin receptors in keratinocyte activation. We found that before activation, beta 1 subunits in keratinocytes showed a diffuse distribution, whereas after activation, keratinocytes organized beta 1 receptors into marginal adhesion plaques. In immunoprecipitation experiments with antibodies against beta 1 integrin subunits, we found mostly immature subunits synthesized in keratinocytes freshly harvested from skin. Moreover, integrin receptor complexes immunoprecipitated from these cells by monoclonal antibodies against alpha 2, alpha 3, or alpha 5 subunits contained only immature beta 1 subunits. With keratinocytes cultured 4-7 days, anti-beta 1 antibodies immunoprecipitated mostly mature beta 1 subunits, and integrin complexes immunoprecipitated from cultured cells by anti-alpha subunit antibodies contained mostly mature beta 1 subunits. Antibodies directed against beta 1 subunits also inhibited keratinocyte migration. Based on these results, we suggest that up-regulation of migration by activated keratinocytes depends on changes in processing of pre-beta 1 subunits to mature beta 1 subunits. We also studied the distribution of integrin subunits in skin and on keratinocytes migrating out of skin explants. Whereas beta 1, alpha 2, and alpha 3 subunits were detected in keratinocytes in skin and migrating out of explants, alpha 5 subunits were observed only in migrating cells.     

Skeletal myoblasts utilize a novel beta 1-series integrin and not alpha 6 beta 1 for binding to the E8 and T8 fragments of laminin.

The E8 fragment of laminin stimulates myoblast attachment and locomotion. Myoblast attachment to laminin/E8 was blocked by anti-integrin antibodies against beta 1-chains but not by antibodies against alpha 6-chains. By contrast, other cell lines (e.g. B16, HT1080, P19, F9, Pys2, 3T3, and 3T6) were blocked both by anti-beta 1 and anti-alpha 6. All cells tested also bound to approximately 125-kDa C-terminal fragments of E8 (T8 and T8'). Immunoprecipitation of surface-iodinated myoblasts revealed beta 1-, alpha 3-, and alpha 5-integrin chains and a novel chain that co-precipitated with anti-beta 1 antibodies running at approximately 95 kDa (reduced). I125-alpha 6 beta 1 was immunoprecipitated from cells whose attachment to E8 was blocked by anti-alpha 6 antibodies. By contrast, little alpha 6 beta 1 could be immunoprecipitated from myoblasts. beta 1-Integrin and the novel alpha-chain (alpha'), Mr approximately 120,000/approximately 95.000 (nonreduced/reduced), from myoblast lysates were retained during affinity chromatography on Engelbreth-Holm-Swarm-laminin affinity columns. beta 1, alpha 1, and the novel alpha' were retained from Rugli cell lysates on Engelbreth-Holm-Swarm-laminin columns. alpha 3 was not bound. When E8 was used as affinity matrix, only beta 1 and alpha' were retained. The N-terminal sequence of Rugli alpha' was homologous to alpha-chains of beta 1-series integrins and was most similar to alpha 6 (9 identical residues out of 14). However, there were distinctive differences; in particular, 2 residues were deleted in comparison with alpha 6.     

Activation and inhibition of phosphorylase kinase by monospecific antibodies against preparatively isolated alpha, beta and gamma subunits

Homogeneous alpha and beta subunits were isolated for the first time in preparative amounts in the presence of sodium dodecyl sulfate. Analysis by analytical polyacrylamide electrophoresis, sedimentation velocity, and immunoprecipitation with monospecific antibodies indicated homogeneity. The apparent molecular masses of the purified subunits as determined electrophoretically in the presence of dodecyl sulfate are: alpha = 140.2 +/- 2.1 kDa and beta = 123 +/- 1.8 kDa. Amino acid analyses show that per 100 mol amino acid the alpha-subunit has a higher serine content (Ser alpha/Ser beta = 1.32, Ser alpha/Ser gamma = 1.42) and a lower aspartic acid/asparagine (Asx) content (AsX alpha/Asx beta = 0.76, Asx alpha/Asx gamma = 0.90) than the beta and gamma subunits. Monospecific antibodies against the purified alpha, beta and gamma subunits were produced in sheep [J. Immunol. Methods (1984) 70, 193-209] and their action on the catalytic activity of non-activated phosphorylase kinase assayed. It can be shown that certain antibody fractions of anti-alpha, anti-beta and anti-gamma inhibit the Ca2+-dependent and Ca2+-independent activity at pH 6.8 as well as at pH 8.2. Other antibody fractions against the beta and gamma subunits however activate the Ca2+-dependent activity at pH 6.8 threefold to fourfold, although they inhibit the activity at pH 8.2. These antibodies lead to a ca. five fold increase in the pH 6.8/8.2 activity ratio. Activating anti-beta can even overcome the inhibitory action of anti-alpha at pH 6.8. A kinetic analysis shows that inhibition is the result of a mixed type mechanism whereas activation is due to a fivefold to tenfold increase in V for phosphorylase b. The results illustrate the importance of possibly large, concerted conformational changes of phosphorylase kinase. It appears that activation or inhibition can be triggered by the antibody binding to conformational determinants of a single subunit type leading to a structural alteration of the holoenzyme.     

A streptococcal collagen-like protein interacts with the alpha2beta1 integrin and induces intracellular signaling

The streptococcal collagen-like proteins Scl1 and Scl2 are prokaryotic members of a large protein family with domains containing the repeating amino acid sequence (Gly-Xaa-Yaa)(n) that form a collagen-like triple-helical structure. Here, we test the hypothesis that Scl variant might interact with mammalian collagen-binding integrins. We show that the recombinant Scl protein p176 promotes adhesion and spreading of human lung fibroblast cells through an alpha2beta1 integrin-mediated interaction as shown in cell adhesion inhibition assays using anti-alpha2beta1 and anti-beta1 integrins monoclonal antibodies. Accordingly, C2C12 cells stably expressing alpha2beta1 integrin as the only collagen-binding integrin show productive cell adhesion activities on p176 that can be blocked by an anti-alpha2beta1 integrin antibody. In addition, p176 promotes tyrosine phosphorylation of p125(FAK) of C2C12 cells expressing alpha2beta1 integrin, whereas parental cells do not. Furthermore, C2C12 adhesion of human lung fibroblast cells to p176 induces phosphorylation of p125FAK, p130CAS, and p68Paxillin proteins. In a domain swapping experiment, we show that integrin binds to the collagenous domain of the Scl protein. Moreover, the recombinant inserted domain of the alpha2 integrin interacts with p176 with a relatively high affinity (K(D) = 17 nm). Attempts to identify the integrin sites in p176 suggest that more than one site may be involved. These studies, for the first time, suggest that the collagen-like proteins of prokaryotes retained not only structural but also functional characteristics of their eukaryotic counterparts.     

Promiscuity of GABAA-receptor beta 3 subunits as demonstrated by their presence in alpha 1, alpha 2 and alpha 3 subunit-containing receptor subpopulations.

Polyclonal antibodies were raised in rabbits against the GABAA-receptor beta 3 subunit peptide sequence, KQSMPREGHGRHMDR-NH2 coupled to keyhole limpet haemocyanin. These anti-beta 3 379-393 antibodies immunoprecipitated in a dose-dependent manner specific benzodiazepine agonist binding sites from Na+ deoxycholate extracts of bovine cerebral cortex. In immunoblots, anti-beta 3 379-393 antibodies recognised two species with Mr 59,900 and Mr 57,200 in all preparations tested, which included crude detergent-solubilised, benzodiazepine affinity chromatography-purified receptor, anti-alpha 1 324-341 antibody, anti-Cys alpha 2 414-424 antibody and anti-Cys alpha 3 454-467 antibody immunoaffinity-purified GABAA-receptor subpopulations. These results provide evidence for the ubiquity and promiscuity of the GABAA-receptor beta 3 subunit.     

Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells.

The mechanisms and receptors involved in phagocytosis by nonhematopoietic cells are not well understood. The involvement of the alpha 3 beta 1 integrin in phagocytosis of the extracellular matrix by human breast cancer cells was studied. The possible role of this integrin was suggested since alpha 3 and beta 1 but not alpha 2 subunits are concentrated at membrane sites where local degradation of fluorescently labeled gelatin occurs. Strikingly, anti-alpha 3 integrin monoclonal antibodies (mAbs) stimulate the phagocytosis of fluorescently labeled gelatin films, gelatin beads, and Matrigel films in a quantitative phagocytosis assay. Stimulation of the gelatin uptake by the anti-alpha 3 mAb is dose responsive, saturable, and time dependent. Antibodies against other integrin subunits have a lower stimulatory effect (anti-beta 1) or no significant effect (anti-alpha 2, -alpha 5, -alpha 6, and -alpha v) on gelatin phagocytosis. The synthetic HGD-6 human laminin peptide that binds specifically the alpha 3 beta 1 integrin, but not the scrambled HSGD-6 control peptide, also markedly stimulates gelatin uptake in a dose-responsive way. Furthermore, the stimulatory effects of the HGD-6 peptide and the anti-alpha 3 mAb are additive, suggesting that they might promote phagocytosis in different ways. Other laminin (YIGSR, IKVAV) and fibronectin (GRGDS) peptides have no effect on gelatin phagocytosis. Immunofluorescence shows that the alpha 3 and the beta 1, but not the alpha 2 integrin subunit, concentrate into patches on the cell surface after treatment with their respective mAbs. And, both gelatin and the alpha 3 beta 1 but not the alpha 2 beta 1 integrin are cointernalized and routed to acidic vesicles such as lysosomes. In conclusion, we demonstrate that human breast cancer cells locally degrade and phagocytose the extracellular matrix and show for the first time that the alpha 3 beta 1 integrin participates in this phagocytosis. We hypothesize that the anti-alpha 3 antibodies and the laminin peptide HGD-6 activate the alpha 3 beta 1 integrin, which results in a downstream signaling cascade stimulating phagocytosis.     

HIV-1 infection of human T lymphocytes results in enhanced alpha 5 beta 1 integrin expression.

Altered T cell adherence after human immunodeficiency virus 1 (HIV-1) infection may contribute to viral pathogenesis in the acquired immune deficiency syndrome. To address this hypothesis, we assessed mechanisms of T cell adherence to extracellular matrix proteins in vitro. We found that after HIV-1 infection, both chronically infected H9 CD4+ T cells and acutely infected primary peripheral blood lymphocytes acquired the ability to adhere to the extracellular matrix glycoprotein fibronectin, to a lesser extent to type IV collagen and laminin, but not to type I collagen. H9 cells chronically infected with two of the three HIV-1 strains studied showed approximately a sevenfold increase in attachment to fibronectin, while the same cells infected with the human retrovirus HIV-2 did not. Adhesion was accompanied by changes in morphology, including marked spreading and increased filopodia. These alterations were not blocked by the protein kinase C inhibitor H-7, which did inhibit TPA-induced T cell attachment to fibronectin. Monoclonal antibodies against both the alpha 5 and the beta 1 subunits of the classical fibronectin receptor as well as an Arg-Gly-Asp (RGD) peptide inhibited attachment, whereas anti-alpha 4 monoclonal antibodies and the CS1 peptide did not. Binding to collagen IV was also inhibited by the anti-beta 1 monoclonal antibody, but not the other antibodies. Cells metabolically labeled with [35S]methionine and analyzed by immunoprecipitation with polyclonal anti-beta 1 integrin antibody showed a 2.5-fold increase in integrin synthesis in infected cells compared to uninfected controls. This increase in synthesis was associated with an increase in cell surface expression of both alpha 5 and beta 1 integrins by FACS (registered trademark of Becton Dickinson for a fluorescence-activated cell sorter) analysis. Enhanced expression of integrins such as alpha 5 beta 1 may cause T cell adherence to a variety of tissues, where released viral gene products may induce some of the tissue-specific manifestations of HIV-1 infection.     

Involvement of host cell tyrosine phosphorylation in the invasion of HEp-2 cells by Bartonella bacilliformis

We have provided evidence that exposure of human cells to protein kinase inhibitors results in decreased invasion of these cells by Bartonella bacilliformis in a dose-dependent manner. Preincubation of human laryngeal epithelial cells in the presence of genistein, a tyrosine protein kinase inhibitor, decreased the invasion of these cells by B. bacilliformis significantly. Further, exposure of normal human umbilical vein endothelial cells to staurosporine, a potent inhibitor of protein kinase C and some tyrosine protein kinases, resulted in a considerable reduction in the number of organisms internalized by these cells. Moreover, Bartonella infection of HEp-2 cells induced tyrosine phosphorylation of several Triton X-100 soluble proteins with approximate molecular masses of 243, 215 179, 172 (doublet), 160, 145 and 110 kDa that were absent or reduced in the presence of genistein in cells after 1 h of infection. Exposure of HEp-2 cell monolayers to anti-alpha 5 and anti-beta 1 chain integrin monoclonal antibodies resulted in a moderate decrease in the invasion of these cells, suggesting a possible role of alpha 5 beta 1 integrins in the uptake of Bartonella into nucleated cells.     

Contributions of the Mac-1 glycoprotein family to adherence-dependent granulocyte functions: structure-function assessments employing subunit-specific monoclonal antibodies

MAb directed at the alpha-subunits of Mac-1 (alpha M), LFA-1 (alpha L), p150,95 (alpha X), or their common beta-subunit were used to characterize the contributions of the Mac-1 glycoprotein family to granulocyte adherence reactions. Inhibitory effects of these MAb in incubation experiments with normal granulocytes indicated distinct adhesive contributions of each subunit. Significantly greater adherence, and inhibition of adherence by anti alpha M, alpha X, and beta MAb, was observed under chemotactic conditions designed to "up-regulate" the surface expression of the alpha M beta and alpha X beta complexes. Adherence to protein-coated glass and binding of albumin-coated latex beads were significantly inhibited by anti-beta greater than anti-alpha M (OKM-10, M1/70, LM2/1.6 and OKM-1) greater than anti-alpha X greater than anti-alpha L MAb, but no effects of anti-HLA, AB, or anti-CR-1 MAb were evident. A similar rank order of inhibition was observed in granulocyte aggregation assays in response to C5a, PMA, or f-Met-Leu-Phe. Significant inhibition of directed migration by anti-beta or anti-alpha M (OKM-1 or OKM-10) MAb was observed in subagarose but not Boyden chemotaxis assays; inhibition was dependent on a continuous cell exposure to anti-Mac-1 alpha or beta during the assay, suggesting that a continuum of new Mac-1 expression is required for directed translocation. Phagocytosis of Oil-Red-O paraffin or zymosan selectively opsonized with C3-derived ligands was significantly inhibited by anti-alpha M MAb (OKM-10 greater than LM2/1.6 greater than M1/70 greater than OKM-1) or by combinations of anti-alpha M + anti-CR-1 MAb, but only minimal inhibitory effects of anti-beta MAb and no effects of anti-alpha L or anti-alpha X MAb were seen. Similarly, complement-dependent phagocytosis-associated lactoferrin release, ingestion, and intracellular killing of Staphylococcus aureus 502A, and binding of iC3b-opsonized SRBC, were significantly inhibited by anti-alpha M (OKM-10, M1/70) or combinations of anti-alpha M + anti-CR-1 MAb, but not by anti-beta, alpha L, or alpha X MAb. Notably, none of the anti-Mac-1 MAb demonstrated inhibitory effects in assays of adherence-independent functions including shape change, specific f-Met-Leu-3H-Phe binding, O-2 generation, chemiluminescence evolution, or lactoferrin release in response to PMA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Affinity chromatographic isolation of the melanoma adhesion receptor for the IIICS region of fibronectin and its identification as the integrin alpha 4 beta 1

Eukaryotic cells adhere to at least two different regions of the fibronectin molecule: a central domain present in all fibronectin isoforms, and the type III connecting segment domain (IIICS), the expression of which is controlled by complex alternative splicing of precursor mRNA. Using affinity chromatography on a matrix containing a synthetic peptide ligand (CS1) representing the strongest active site within the IIICS, we have isolated the human melanoma cell receptor recognizing this region of fibronectin. The receptor is a complex of two polypeptides with subunit molecular masses of 145 and 125 kDa. This heterodimeric structure resembles that of receptors for other extracellular matrix proteins. Immunological analysis with specific antibodies identified these polypeptides as the integrin subunits alpha 4 and beta 1. In addition, antifunctional monoclonal antibodies directed against either alpha 4 or beta 1, but not against other integrin subunits, were potent inhibitors of CS1-mediated melanoma cell spreading. Furthermore, when the function of the central cell-binding domain was blocked, anti-alpha 4 and anti-beta 1 antibodies abolished spreading of A375-M cells on fibronectin, indicating that alpha 4 beta 1 is an authentic fibronectin receptor. Taken together, these results identify the human fibronectin IIICS receptor as the integrin heterodimer alpha 4 beta 1.     

Adenosine triphosphatase and nucleotide binding activity of isolated beta-subunit preparations from Escherichia coli F1F0-ATP synthase

Adenosine triphosphatase activity and nucleotide binding affinity of isolated beta-subunit preparations from Escherichia coli F1F0-ATP synthase were studied. The aim was to find out whether isolated beta-subunit would provide an experimental model in which effects of mutations on catalysis per se, unencumbered by complications due to their effects on positive catalytic cooperativity, could be studied. Three types of purified, isolated beta-subunit preparations were studied. Type I-beta was from a strain lacking all F1F0 subunits except beta and epsilon. Type II-beta was from F1 carrying the alpha S375F mutation which blocks positive catalytic cooperativity. Type III-beta was from normal F1. Type I- and II-beta had very low ATPase activity (less than 10(-4) s-1) which was azide-insensitive, aurovertin-insensitive, and unaffected by anti-beta antibody. Type I-beta activity was EDTA-insensitive. We conclude that isolated beta-subunit from E. coli F1F0 has zero or at most very low intrinsic ATPase activity. Type III-beta had low ATPase activity (8.4 x 10(-5) s-1 to 1.1 x 10(-3) s-1 in seven different preparations). This activity was aurovertin-sensitive, but varied in azide sensitivity from 0 to 34% inhibited. The azide-sensitive component, like F1 and alpha 3 beta 3 gamma oligomer, was inhibited by anti-beta and anti-alpha antibodies. The azide-insensitive component was stimulated by anti-beta and unaffected by anti-alpha. We show here that (alpha beta)-oligomer has ATPase activity which is azide-insensitive, aurovertin-sensitive, stimulated by anti-beta, and unaffected by anti-alpha. The intrinsic ATPase activity of Type III-beta could be due to contaminating (alpha beta)-oligomer plus alpha 3 beta 3 gamma-oligomer. Isolated beta had very low affinity for nucleotide as compared to the first catalytic site on F1. Taken together with the very low ATPase activity of isolated beta (even if real), the work shows that isolated beta is not a good experimental model of F1 catalysis.     

Monoclonal antibodies to Escherichia coli F1-ATPase. Correlation of binding site location with interspecies cross-reactivity and effects on enzyme activity

Twenty-one hybridoma cell lines which secret antibodies to the subunits of the Escherichia coli F1-ATPase were produced. Included within the set are four antibodies which are specific for alpha, six for beta, three for gamma, four for delta and four for epsilon. The antibodies were divided into binding competition subgroups. Two such competition subgroups are represented for the alpha, beta, and epsilon subunits, one for delta and three for gamma. The ability to bind intact F1-ATPase was demonstrated for some of the antibodies to alpha and beta, and for all of those to delta, while the antibodies to gamma and epsilon gave unclear results. All of the antibodies to alpha and beta which bound ATPase were found to have effects on the ATPase activity of purified E. coli F1-ATPase. One of those to alpha inhibited activity by about 30%. Another anti-alpha was mildly stimulatory. The four antibodies to beta which bound ATPase inhibited activity by 90%. In contrast, membrane-bound ATPase was hardly affected by the antibodies to alpha, but was inhibited by 40-60% by the antibodies to beta. The other antibodies to alpha and beta bound only free subunits, or partially dissociated ATPase, suggesting that their epitopes are buried between subunits in ATPase. These antibodies had no effects on activity. The ability of the antibodies to recognize ATPase subunits present in crude extracts from mitochondria, chloroplasts, and a variety of bacteria was tested using nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels. One anti-beta specifically recognized proteins in the range of 50,000-60,000 daltons in each of the extracts, although the reaction with mitochondrial beta was weak. Some of the other antibodies had limited cross-reaction, but most were specific for the E. coli protein. In some species, those proteins which were recognized by the anti-beta ran with a higher apparent molecular weight than proteins which were recognized by an anti-alpha. All antibodies which exhibited cross-reactivity were found to recognize sites which were not exposed in intact ATPase, implying that the surfaces which lie between subunits are most highly conserved.     

Activation of protein kinase C by phorbol esters modulates alpha2beta1 integrin on MCF-7 breast cancer cells

Cellular adhesions to other cells and to the extracellular matrix play crucial roles in the malignant progression of cancer. In this study, we investigated the role of protein kinase C (PKC) in the regulation of cell-substratum adhesion by the breast adenocarcinoma cell line MCF-7. A PKC activator, 12-O-tetradecanoylphorbol-l, 3-acetate (TPA), stimulated cell adhesion to laminin and collagen I in a dose-dependent manner over a 1- to 4-h interval. This enhanced adhesion was mediated by alpha2beta1 integrin, since both anti-alpha2 and anti-beta1 blocking antibodies each completely abrogated the TPA-induced adhesion. FACS analysis determined that TPA treatment does not change the cell surface expression of alpha2beta1 integrin over a 4-h time interval. However, alpha2beta1 levels were increased after 24 h of TPA treatment. Thus, the enhanced avidity of alpha2beta1-dependent cellular adhesion preceded the induction of alpha2beta1 cell surface expression. Northern blot analysis revealed that mRNA levels of both alpha2 and beta1 subunits were increased after exposure to TPA for 4 h, indicating that the induction of alpha2beta1 mRNA preceded that of its cell surface expression. This further suggested that the TPA-induced avidity of alpha2beta1 was independent of increased expression of alpha2beta1. Pretreatment of cells with the PKC inhibitor calphostin C partially antagonized the TPA-induced increase in expression of alpha2beta1 integrin expression and of alpha2beta1-mediated cellular adhesion. To identify a possible mechanism by which TPA could be acting to promote the rapid induction of alpha2beta1 adhesion, we treated the cells with the Rho-GTPase inhibitor Clostridium botulinumexotoxin C3. C3 inhibited TPA-induced adhesion to laminin and collagen I in a dose-dependant manner, suggesting a likely role for Rho in TPA-induced adhesion. Together, these results suggest that PKC can modulate the alpha2beta1-dependent adhesion of MCF-7 cells by two distinct mechanisms: altering the gene expression of integrins alpha2 and beta1 and altering the avidity of the alpha2beta1 integrin by a Rho-dependant mechanism.     

Chick integrin alpha V subunit molecular analysis reveals high conservation of structural domains and association with multiple beta subunits in embryo fibroblasts.

We have cloned and characterized a chick homologue of the human vitronectin receptor alpha subunit (alpha v) whose primary sequence is 83% identical with its human counterpart but less than 40% identical with any other known integrin alpha subunit. Comparison of the chick and human sequences reveals several highly conserved regions, including the cytoplasmic domain. The putative ligand binding domain contains alpha v-specific residues that may contribute to ligand binding specificity. These are concentrated in three regions that are located before and between the first three Ca2+ binding domains. Polyclonal antibodies raised against two peptides deduced from the putative cytoplasmic and extracellular domains of the chick alpha v sequence recognize specifically integrin heterodimers in chick embryo fibroblasts. At least three putative beta subunits coimmunoprecipitate with the chick alpha v subunit. In addition to a protein with the same molecular weight as beta 3 (94K), protein bands of Mr 84K and 110K are also coprecipitated. By successive immunodepletions, we demonstrate that this latter Mr 110K subunit is beta 1, which appears to be one of the alpha v-associated subunits in chick embryo fibroblasts.     

Real-time monitoring approach: assessment of effects of antibodies on the adhesion of NCI-H460 cancer cells to the extracellular matrix

This paper presents an in situ impedance chip system, which allows studying the effects of drugs on the behaviors of living cell in real-time. A new label-free measurement approach is introduced, which enables to assess the extent of the adhesion of a cell population to the extracellular matrix (ECM)-coated microelectrode array, by adding together all the impedance changes from individually controlled microelectrodes. A high sensitivity was demonstrated and allowed us to monitor cell attachment with the resolution of a "few cells". The dose and pre-incubation time effects of antibodies against beta1-integrin and its subunit alpha2beta1-integrin on the adhesion behavior of NCI-H460 lung cancer cells to collagen type I was extensively studied. Results show that both anti-beta1-integrin and anti-alpha2beta1-integrin inhibit NCI-H460 cells attachment to collagen I. This indicates that beta1-integrin is present on the surface of NCI-H460 cancer cells, and that its subunit alpha2beta1 significantly affects NCI-H460 cells attachment on collagen I.     

Monoclonal antibodies to rabbit skeletal muscle phosphorylase kinase. Probes for studies of subunit function

Monoclonal antibodies to rabbit skeletal muscle phosphorylase kinase were produced by the conventional hybridoma cell technique. 90 out of 600 hybridomas were found to produce phosphorylase kinase binding antibodies from which only five secreted also phosphorylase kinase activity affecting antibodies. Three of them were cloned; two hybridomas resisted all cloning efforts. Employing immunoblot technique all monoclonal antibodies show cross-reactivity with the alpha, beta, and gamma subunits of phosphorylase kinase indicating that similar, if not identical, epitopes are present on these three subunits. No cross-reactivity with delta is observed. Monoclonal antibodies secreted by two clones which bind to the alpha subunit stimulate the Ca2+-independent A0 activity of phosphorylase kinase more than 30-fold, whereas all other monoclonal antibodies obtained are ineffective in this respect. Monoclonal antibodies binding to the beta subunit inhibit the Ca2+-dependent activities significantly. Antibody produced by one hybridoma binds to the alpha, beta, and gamma subunits with approximately the same affinity. Based on the dual function of calmodulin in phosphorylase kinase (Hessová, Z., Varsányi, M., and Heilmeyer, L.M.G., Jr. (1985) Eur. J. Biochem. 146, 107-115) we conclude that binding of anti-alpha monoclonal antibodies to a regulatory domain in the alpha subunit results in an uncoupling of the inhibitory function of the Ca2+-free delta from the holoenzyme which leads to a concomitant increase in A0 activity. Furthermore, binding of anti-beta monoclonal antibodies to the beta subunit prevents a signal transfer from the Ca2+-saturated delta to the catalytic site of the holoenzyme which inhibits the Ca2+-dependent activities.     

Identification of a novel recognition sequence for the integrin alpha 4 beta 1 in the COOH-terminal heparin-binding domain of fibronectin.

The type III connecting segment of fibronectin contains two cell binding sites, represented by the peptides CS1 and CS5, that are recognized by the integrin receptor alpha 4 beta 1. Using assays measuring the spreading of A375-SM human melanoma cells, we now report that the adhesion promoting activity of a 29 kDa protease fragment of fibronectin containing the COOH-terminal heparin-binding domain (HepII), but lacking CS1 and CS5, is completely sensitive to anti-alpha 4 and anti-beta 1 antibodies, suggesting that HepII contains a third alpha 4 beta 1-binding sequence. Examination of the primary structure of HepII revealed a sequence with homology to CS1. A 19mer peptide spanning this region (designated H1) was found to support cell spreading to the same level as the 29 kDa fragment. H1-dependent adhesion was completely sensitive to anti-alpha 4 and anti-beta 1 antibodies. When soluble peptides were tested for their ability to block cell spreading on the 29 kDa fragment, a 13mer peptide comprising the central core of H1 was found to be completely inhibitory. The active region of H1 was localized to the pentapeptide IDAPS, which is homologous to LDVPS from the active site of CS1. Taken together, these results identify a novel peptide sequence in the HepII region of fibronectin that supports alpha 4 beta 1-dependent cell adhesion.     

Tight association of DNA polymerase alpha with granular structures in the nuclear matrix of chick embryo cell: immunocytochemical detection with monoclonal antibody against DNA polymerase alpha

Immunofluorescent methods using a monoclonal antibody against chick DNA polymerase alpha and a rabbit antibody against chick DNA polymerase beta demonstrated that both DNA polymerases alpha and beta are present mainly in nuclei of cultured chick embryo cells. Fluorescence produced by anti-DNA polymerase alpha was more intense in the small granules than in other parts of the nucleus but, fluorescence produced by anti-DNA polymerase beta was distributed evenly in the nucleus. Cells first were treated with Nonidet P-40, followed by treatment with 50 micrograms/ml pancreatic DNase and 2 M NaCl in order to prepare the nuclear matrix. Fluorescence produced by anti-DNA polymerase alpha was still detectable in the granules after these treatments, but most of the fluorescence produced by anti-DNA polymerase beta disappeared. Our results indicate that a part of DNA polymerase alpha is tightly bound to a special structure present in the nuclear matrix which presumably is the DNA replication machinery.     

Integrin alpha6beta4-erbB2 complex inhibits haptotaxis by up-regulating E-cadherin cell-cell junctions in keratinocytes

Keratinocyte integrins alpha6beta4 and alpha3beta1 bind laminin-5, a component of basement membranes. We previously demonstrated that in keratinocytes, haptotactic migration on laminin-5 was stimulated by anti-beta1 integrin-activating antibody TS2/16, whereas antibodies to alpha6 and beta4, respectively, blocked TS2/16-induced, alpha3beta1-dependent migration. Moreover, alpha6beta4-associated haptotaxis inhibition was linked to a phosphatidylinositol 3-kinase (PI3K) pathway and required erbB2 activation. erbB2, the ligand-less member of the epidermal growth factor receptor family, was shown to form a complex with the hemidesmosomal integrin alpha6beta4. Here, we demonstrate that alpha6beta4 inhibitory effects on haptotaxis are abolished by an anti-E-cadherin antibody, which interferes with cell-cell adhesion. Furthermore, antibodies to alpha6 and beta4 stimulated adhesion to an E-cadherin-Fc recombinant protein. In addition, anti-alpha6/beta4 antibodies increased colony size in plated cells, stimulated cell-cell aggregation, and up-regulated E-cadherin localization to cell-cell contacts. These effects were abolished when erbB2 or PI3K were blocked. These results indicate that stimulation of alpha6beta4 increases E-cadherin-mediated cell-cell adhesion and that this mechanism depends on erbB2 activation. The molecule that links alpha6beta4 with E-cadherin may be the small GTPase cdc42, an effector of PI3K, because dominant-negative cdc42 abolished the inhibitory effect of anti-alpha6/beta4 antibodies and increased basal migration, whereas constitutively active cdc42 prevented the TS2/16-induced increase in haptotaxis. These findings suggest a model whereby alpha6beta4 can augment cell-cell adhesion and slow down haptotaxis over laminin-5 and point to the alpha6beta4-erbB2 heterodimer as an important signaling complex for the formation of cohesive keratinocyte layers.     

Multiple cell surface receptors for the short arms of laminin: alpha 1 beta 1 integrin and RGD-dependent proteins mediate cell attachment only to domains III in murine tumor laminin

Cell surface molecules that interact with the cross formed by the three short arms of murine tumor laminin were studied using thermal perturbation, antibody and peptide blocking, and affinity chromatography. Several potential receptors for the laminin short arms were revealed that differed from those mediating cell attachment to the E8 (long arm) fragment. Two cell lines, Rugli and L8 attached well to E1-X (short arm) fragments of laminin. This attachment was blocked by antibodies against alpha 1 integrin chains. Other cells were unable to attach strongly to E1-X, but attached to P1. This attachment was unaffected by anti-beta 1 integrin antibodies, but specifically blocked by the peptide GRGDS. By contrast, binding of Rugli cells was RGD independent and blocked by anti-beta 1 integrin antibodies. G7 and C2C12 myoblasts were very sensitive to GRGDS (ID50 approximately 2 micrograms.ml-1) for attachment to P1 which implied that a non-beta 1 series integrin, possibly alpha v beta 3, was involved. On heat denaturation of P1(3) attachment remained sensitive to RGDS and ID50 was unchanged. On heat denaturation of E1-X, attachment remained sensitive to RGDS but the ID50 increased to approximately 200 micrograms.ml-1. Cellular beta 1 integrins were retained on laminin affinity columns. A beta 1 integrin with an approximately 190 kD alpha-chain could be isolated from Rugli cells whose attachment could be blocked by anti-alpha 1 antibodies and not from cells blocked by RGDS peptides. Anti-alpha 1 antibodies blocked Rugli attachment to native laminin, but only when the E8 cell binding sites on laminin were also blocked. Thus, a receptor related to alpha 1 beta 1 integrin can function simultaneously with a receptor for E8. Anti-alpha 1 also blocked attachment to heated laminin, suggesting that the heat-stable attachment activity in laminin involved the E1-X binding site. Thus, at least two putative receptors mediate attachment to the short arms of laminin. One, related to alpha 1 beta 1 integrin, recognizes RGDS-independent sites in E1-X defined by P1 (within domains III, IIIa, IIIb), and one is an RGD-dependent molecule recognizing sites in P1, and is not a beta 1 integrin.