Journal articles on the topic 'Integrin alpha 5'
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1
Simon, E. E., C. H. Liu, M. Das, S. Nigam, T. J. Broekelmann, and J. A. McDonald. "Characterization of integrins in cultured human renal cortical tubule epithelial cells." American Journal of Physiology-Renal Physiology 267, no. 4 (October 1, 1994): F612—F623. http://dx.doi.org/10.1152/ajprenal.1994.267.4.f612.
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We have characterized the integrins present on cultured tubule epithelial cells from human renal cortexes, enriched for proximal cells, using fluorescence microscopy, immunoprecipitation, and cell adhesion assays. By immunofluorescence, the alpha 3-integrin subunit stained most intensely and was present on all cells predominantly at cell-cell contacts. The alpha 6-subunit was present on all cells in a pattern consistent with extracellular matrix contacts. The alpha 5-subunit was present on most cells in a cell-matrix contact pattern; alpha V-subunit was weakly positive and occasionally seen in cell-matrix contacts. The alpha 2-subunit was present on clusters of distal tubule cells, predominantly at cell-cell contacts. Immunoprecipitation revealed the predominant integrin to be alpha 3 beta 1 with some alpha 2 beta 1, presumably contributed by distal cells. The alpha 5 beta 1-, alpha 6 beta 1-, alpha 6 beta 4-, and alpha V beta 3-integrins, as well as trace amounts of alpha 1 beta 1-integrins, were also present. The alpha 4 beta 1-integrin was not detected. Initial attachment to fibronectin was mediated by alpha V beta 3- and alpha 5 beta 1-integrins; initial attachment to laminin was mediated by the alpha 6 beta 1- and alpha 3 beta 1- integrins and, in some preparations, by an unidentified integrin; and initial attachment to collagen type IV was mediated by alpha V beta 3-integrin and an unidentified beta 1-integrin. After extensively immunodepleting membrane extracts with anti-alpha 1, -alpha 2, -alpha 3, -alpha 4, -alpha 5, -alpha 6, and -alpha V antibodies, an anti-beta 1 antibody still precipitated an integrin. Its electrophoretic mobility differs from the laminin-binding alpha 7 beta 1-integrin. Thus we have identified many of the integrins on cortical tubule cells and their role in mediating initial attachment to extracellular matrix. However, the cell adhesion assays and immunoprecipitations suggest the presence of an unidentified beta 1-integrin that may mediate renal tubule cell attachment to laminin and collagen.
2
Wu, C., A. J. Fields, B. A. Kapteijn, and J. A. McDonald. "The role of alpha 4 beta 1 integrin in cell motility and fibronectin matrix assembly." Journal of Cell Science 108, no. 2 (February 1, 1995): 821–29. http://dx.doi.org/10.1242/jcs.108.2.821.
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The alpha 4 beta 1 integrin has been suggested to play important roles in embryogenesis and pathogenesis of many diseases which involve both cell adhesion and cell migration. Previous studies using anti-alpha 4 beta 1 antibodies and fibronectin (Fn) fragments have suggested that alpha 4 beta 1 integrins may be involved in cell motility on Fn and vascular cell adhesion molecule-1 (VCAM-1). However, the cells used in these studies also express other Fn integrin receptors including alpha 5 beta 1 integrin, which is known to function in cell motility on Fn. To test whether alpha 4 beta 1 integrins mediate cell motility on Fn and VCAM-1 in the absence of alpha 5 beta 1 integrin, we expressed human alpha 4 integrin in a Chinese hamster ovary (CHO) cell line that is deficient in alpha 5 beta 1 integrin (CHO B2). The parental alpha 5 deficient CHO B2 cells were unable to adhere, spread or migrate on Fn, nor could they assemble a fibrillar Fn matrix. Expression of alpha 4 beta 1 integrin in the CHO B2 cells enabled the cells to adhere, spread and migrate on Fn and on VCAM-1 but not to assemble a fibrillar Fn matrix. The cellular processes mediated by the interaction of alpha 4 beta 1 with Fn or VCAM-1 were inhibited by the CS1 peptide derived from the major alpha 4 beta 1 binding site on Fn. These findings demonstrate that alpha 4 beta 1 integrins not only function as cell adhesion receptors but also as cell motility receptors for Fn and VCAM-1 independent of alpha 5 beta 1. Moreover, they reveal important functional differences between Fn binding integrins. The alpha 4-positive, alpha 5-negative CHO cells described in this report will be useful tools in studying the mechanism of molecular signalling during integrin mediated cellular processes.
3
Muschler, J. L., and A. F. Horwitz. "Down-regulation of the chicken alpha 5 beta 1 integrin fibronectin receptor during development." Development 113, no. 1 (September 1, 1991): 327–37. http://dx.doi.org/10.1242/dev.113.1.327.
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We have characterized the diversity of the chicken beta 1 integrin family and studied the expression of individual receptors during development. The diversity of the beta 1 integrin family was investigated by affinity purifying the beta 1 integrins from a variety of adult and embryonic tissues. These purifications reveal the relative levels of expression and also the differential expression of the alpha subunits in those tissues. Monoclonal antibodies were generated against the prominent ‘band 1’ of the embryonic chicken integrins and used to characterize the expression of this alpha subunit in embryonic and adult tissues. This alpha subunit is shown to be the chicken homologue of human alpha 5 fibronectin receptor. The chicken alpha 5 beta 1 integrin is the most prominent beta 1 integrin in the embryo and is expressed on the majority of cell types through the day 17 stage. The distribution of this receptor in the embryo closely parallels the distribution of its ligand, fibronectin. In adult tissues, expression of this receptor is greatly diminished relative to the expression of other alpha subunits. The cell type distribution is highly restricted: limited primarily to the vasculature and to connective tissue regions. These studies reveal a prominent role for the alpha 5 beta 1 integrin in embryonic cell types and a down-regulation of this receptor on many cell types during development.
4
Milner, R., and C. Ffrench-Constant. "A developmental analysis of oligodendroglial integrins in primary cells: changes in alpha v-associated beta subunits during differentiation." Development 120, no. 12 (December 1, 1994): 3497–506. http://dx.doi.org/10.1242/dev.120.12.3497.
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We have examined the expression of integrins on primary oligodendroglial cells during the differentiation of the proliferative oligodendrocyte precursor (O-2A progenitor) cell to the postmitotic oligodendrocyte. Cells of the oligodendrocyte lineage expressed a limited repertoire of integrins: alpha 6 beta 1 and alpha v integrins including alpha v beta 1, alpha v beta 3 and alpha v beta 5, as well as a potentially novel integrin alpha v beta 80 kDa. Integrin expression was developmentally regulated; during differentiation alpha v beta 1 was reduced and alpha v beta 5 upregulated. These results suggest that laminin and vitronectin are important extracellular matrix ligands for oligodendrocytes, and provide a rational explanation for previous observations that RGD peptides inhibit the expression of myelin-specific genes. They also suggest a simple model by which switching of integrin beta subunits might regulate differentiation. As chimeric beta 1 integrins with a beta 5 cytoplasmic domain support proliferation less well than normal beta 1 integrins (Pasqualini and Hemler (1994), J. Cell Biol. 125, 447–460) the switch from alpha v beta 1 to alpha v beta 5 might play a key instructive role in the cessation of proliferation and subsequent differentiation.
5
Adams, J. C., and F. M. Watt. "Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes." Journal of Cell Biology 115, no. 3 (November 1, 1991): 829–41. http://dx.doi.org/10.1083/jcb.115.3.829.
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We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.
6
Jin, D. K., A. J. Fish, E. A. Wayner, M. Mauer, S. Setty, E. Tsilibary, and Y. Kim. "Distribution of integrin subunits in human diabetic kidneys." Journal of the American Society of Nephrology 7, no. 12 (December 1996): 2636–45. http://dx.doi.org/10.1681/asn.v7122636.
Full textAbstract:
Integrins are cell-surface protein receptors that participate in cell adhesion to multiple extracellular matrix ligands, and consist of alpha and beta chain heterodimers. This study examined altered integrin distribution in diabetic nephropathy by investigating 12 human diabetic kidney biopsies, which were compared with normal human kidney. Diabetic nephropathy is characterized by mesangial expansion and progressive thickening of the glomerular basement membrane. Based on morphometric studies of mesangial expansion, diabetic nephropathy was determined to be moderate or severe. Three different patterns (P) of altered intensity of integrin staining were observed. In the mesangial integrin P, the intensity of integrin subunit staining of mesangial cells (alpha 1, alpha 2, alpha 3, beta 1, alpha V, alpha V beta 5) was increased in moderate diabetic nephropathy and further increased in severe diabetic nephropathy. In the epithelial integrin P, integrin subunits localized to epithelial cells (alpha V, beta 3, alpha V beta 3, alpha V beta 5) were increased to the same extent in moderate and severe diabetic nephropathy. In the endothelial integrin P, integrin subunits localized to endothelial cells (alpha 3, alpha 5, alpha 6, beta 1) were increased in moderate diabetic nephropathy but returned to normal kidney staining intensity in severe diabetic nephropathy. From these observations, it was concluded that there is significant alteration in the expression of integrin subunits in diabetic nephropathy that is related to the severity of diabetic mesangial expansion. Additionally, the spectrum of integrin subunit alteration appears to be unique to individual glomerular cell types. Given the role of integrins in cell-surface interactions with extracellular matrix components, abnormalities in the expression of these molecules may be important in the pathogenesis of diabetic nephropathy.
7
Whittaker, C. A., and D. W. DeSimone. "Integrin alpha subunit mRNAs are differentially expressed in early Xenopus embryos." Development 117, no. 4 (April 1, 1993): 1239–49. http://dx.doi.org/10.1242/dev.117.4.1239.
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Adhesion of cells to extracellular matrix proteins is mediated, in large part, by transmembrane receptors of the integrin family. The identification of specific integrins expressed in early embryos is an important first step to understanding the roles of these receptors in developmental processes. We have used polymerase chain reaction methods and degenerate oligodeoxynucleotide primers to identify and clone Xenopus integrin alpha subunits from neurula-stage (stage 17) cDNA. Partial cDNAs encoding integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and an alpha IIb-related subunit were cloned and used to investigate integrin mRNA expression in early embryos by RNase protection assay and whole-mount in situ hybridization methods. Considerable integrin diversity is apparent early in development with integrins alpha 2, alpha 3, alpha 4, alpha 5 and alpha 6 each expressed by the end of gastrulation. Both alpha 3 and alpha 5 are expressed as maternal mRNAs. Zygotic expression of alpha 2, alpha 3, alpha 4 and alpha 6 transcripts begins during gastrulation. Integrin alpha 5 is expressed at relatively high levels during cleavage, blastula and gastrula stages suggesting that it may represent the major integrin expressed in the early embryo. We demonstrated previously that integrin beta 1 protein synthesis remains constant following induction of stage 8 animal cap cells with activin (Smith, J. C., Symes, K., Hynes, R. O. and DeSimone, D. W. (1990) Development 108, 289–298.). Here we report that integrin alpha 3, alpha 4 and alpha 6 mRNA levels increase following induction with 10 U/ml activin-A whereas alpha 5, beta 1 and beta 3 mRNA levels remain unchanged. Whole-mount in situ hybridization reveals that alpha 3 mRNAs are expressed by cells of the involuting mesoderm in the dorsal lip region of early gastrulae. As gastrulation proceeds, alpha 3 expression is localized to a stripe of presumptive notochordal cells along the dorsal midline. In neurulae, alpha 3 mRNA is highly expressed in the notochord but becomes progressively more restricted to the caudalmost portion of this tissue as development proceeds from tailbud to tadpole stages. In addition, alpha 3 is expressed in the forebrain region of later stage embryos. These data suggest that integrin-mediated adhesion may be involved in the process of mesoderm involution at gastrulation and the organization of tissues during embryogenesis.
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Dalton, S. L., E. Scharf, R. Briesewitz, E. E. Marcantonio, and R. K. Assoian. "Cell adhesion to extracellular matrix regulates the life cycle of integrins." Molecular Biology of the Cell 6, no. 12 (December 1995): 1781–91. http://dx.doi.org/10.1091/mbc.6.12.1781.
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The expression of alpha 5 beta 1 integrin on the surface of fibroblasts requires adhesion to substratum. We have examined the basis for this adhesion-dependent surface expression by comparing the life cycle of integrins in parallel cultures of adherent and nonadherent cells. Results of biosynthetic labeling experiments in NRK fibroblasts showed that the synthesis and biosynthetic processing of the beta 1 integrin subunit proceed in the absence of cell attachment; however, when examining the behavior of preexisting cell surface integrins, we observed that the alpha beta 1 integrins are internalized and degraded when adhesion to substratum is blocked. A kinetic analysis of integrin internalization in cycloheximide-treated NRK cells showed that each of the fibroblast integrins we examined (in both the beta 1 and beta 3 families) are lost from the cell surface after detachment from substratum. Thus, the default integrin life cycle in fibroblasts involves continuous synthesis, processing, transport to the cell surface, and internalization/degradation. Interestingly, studies with NIH-3T3 cells expressing alpha 1 beta 1 integrin showed that the loss of cell-surface alpha 5 beta 1 integrin is blocked by adhesion of cells to dishes coated with type IV collagen (a ligand for alpha 1 beta 1 integrin) as well as fibronectin. Similarly, adhesion of these cells to dishes coated with type IV collagen stabilizes the surface expression of alpha 5 beta 1 as well as alpha 1 beta 1 integrin. We propose that the adhesion of fibroblasts to extracellular matrix protein alters the integrin life cycle and permits retention of these proteins at the cell surface where they can play important roles in transmitting adhesion-dependent signals.
9
Hynes, R. O., E. E. Marcantonio, M. A. Stepp, L. A. Urry, and G. H. Yee. "Integrin heterodimer and receptor complexity in avian and mammalian cells." Journal of Cell Biology 109, no. 1 (July 1, 1989): 409–20. http://dx.doi.org/10.1083/jcb.109.1.409.
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We report data showing that the integrin receptor complex in chickens contains several discrete heterodimers all sharing the beta 1-integrin subunit combined separately with different alpha-subunits. Using antisera to synthetic peptides based on cDNA sequences of chicken and human alpha-integrin subunits to analyze the integrin complement of avian and mammalian cells, we show that band 2 of the chicken integrin complex contains alpha-subunits related to both alpha 3- and alpha 5-subunits of human integrins. alpha 3 beta 1 and alpha 5 beta 1 have both previously been shown in human cells to be fibronectin receptors and alpha 3 beta 1 can also act as a receptor for laminin and collagen. We also provide evidence for the presence, in band 1 of the chicken integrin complex, of a third integrin alpha-subunit which is also alpha 5 related. This integrin subunit exists in a separate heterodimer complex with beta 1 and binds to fibronectin-affinity columns. These results provide explanations for published data showing that the avian integrin complex contains receptor activity for a variety of extracellular matrix proteins. We conclude that the chicken integrin complex comprises a set of beta 1-integrin heterodimers equivalent to the human VLA antigens and includes at least two fibronectin receptors. Finally, we show that chicken embryo fibroblasts also contain a beta 3-class integrin related to the RGD receptors defined in various human cells.
10
Pilewski, J. M., J. D. Latoche, S. M. Arcasoy, and S. M. Albelda. "Expression of integrin cell adhesion receptors during human airway epithelial repair in vivo." American Journal of Physiology-Lung Cellular and Molecular Physiology 273, no. 1 (July 1, 1997): L256—L263. http://dx.doi.org/10.1152/ajplung.1997.273.1.l256.
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Airway epithelium is subject to injury during inflammation and exposure to a variety of inhaled and infectious agents. Little is known about the expression of integrins during human airway epithelial regeneration and differentiation after injury. We therefore characterized integrin subunit expression after mechanical injury in an in vivo xenograft model of human bronchial epithelium. On the migrating cells at the edges of surface epithelial wounds, there was increased expression of the alpha v-, beta 5-, beta 6-, and alpha 5-integrin subunits. During the later phase of repair, the increased expression of alpha v-, beta 5-, and beta 6-subunits persisted, but the expression of the beta 8-subunits was restricted to basal cells. In addition, there was a redistribution of the alpha 2- and alpha 6-collagen/laminin-binding integrins to suprabasal epithelial layers. There was no expression of the beta 3- or alpha 4-integrin subunit on reparative epithelium. A similar upregulation of alpha v-, beta 5-, and beta 6-integrin receptor subunits was observed in areas of undifferentiated airway from cystic fibrosis patients. Injured epithelium was found to be significantly more susceptible to gene transfer with a recombinant adenovirus, suggesting that the increased integrin expression has implications for the acquisition of adenovirus infections and for lung-directed gene therapy.
11
Hara, M., M. Yaar, A. Tang, M. S. Eller, W. Reenstra, and B. A. Gilchrest. "Role of integrins in melanocyte attachment and dendricity." Journal of Cell Science 107, no. 10 (October 1, 1994): 2739–48. http://dx.doi.org/10.1242/jcs.107.10.2739.
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Integrins are a family of proteins known to mediate attachment of cells to extracellular matrix materials. The substratum specificity and cation dependence of specific integrin heterodimers have been extensively characterized, and to a lesser degree specialized roles in cell attachment versus dendricity have been defined in some cell types. In the past decade, melanocyte attachment rate and morphology have been found to have strong substratum dependence, suggesting a major role for integrins in these processes. In order to investigate this aspect of pigment cell biology, human newborn melanocytes were subjected to flow cytometry analysis and plated on a variety of substrata under conditions known to promote or block the binding of specific integrin pairs. Melanocyte attachment to laminin and type IV collagen was promoted by Mg2+ and Mn2+ but not by Ca2+, in the range of concentrations examined. However, dendrite outgrowth from melanocytes already attached on laminin or type IV collagen was promoted by Ca2+ to a far greater degree than by Mg2+, and Mn2+ had no effect on dendrite outgrowth. Flow cytometry analysis revealed that melanocytes expressed beta 1, alpha 2, alpha 3, alpha 5, alpha 6 and alpha v integrin subunits as well as the alpha v beta 3 heterodimer. The influence of substratum on the profile of integrin expression was minimal, but alpha 6 and beta 1 integrins were observed by confocal microscopy to be expressed over the entire cell surface, while alpha 2, alpha 5 and alpha v beta 3 integrins localized along dendritic processes or at their tips. In accordance with the implications of these distribution patterns, anti-beta 1 and anti-alpha 6 integrin monoclonal antibodies blocked melanocyte attachment to laminin, while anti-alpha 2, anti-alpha 5 and anti-alpha v beta 3 inhibited dendrite outgrowth but did not block substratum attachment on either laminin or type IV collagen. On the basis of these data and the known characteristics of integrin molecules, we conclude that melanocyte attachment to laminin is mediated primarily by alpha 6 beta 1 integrin in a Ca(2+)-independent, Mg(2+)- and/or Mn(2+)-dependent manner, while dendrite outgrowth on laminin and type IV collagen requires extracellular Ca2+ and is mediated by alpha v beta 3 as well as alpha 2 and alpha 5 integrins.
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Zutter, MM. "Immunolocalization of integrin receptors in normal lymphoid tissues." Blood 77, no. 10 (May 15, 1991): 2231–36. http://dx.doi.org/10.1182/blood.v77.10.2231.2231.
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Abstract The integrin superfamily of cell adhesion receptors consists of heterodimeric glycoproteins composed of unique alpha and beta subunits. These receptors mediate cell-substrate and cell-cell adhesive properties for a variety of cell types. This investigation has focused on the histologic distribution of the beta 1 subfamily of integrins within lymphoid tissues including tonsil, lymph node, spleen, thymus, and appendix. The dendritic cells of both follicular center and thymic origin express the alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6, as well as the beta 1 integrin subunits. Most lymphoid cells in normal tissues do not express the alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits, or the alpha v beta 3 integrin. The beta 1 subunit is expressed by all lymphocytes but with variable intensity. Increased levels of the alpha 5 and beta 1 subunits are observed in the follicular light zone, suggesting a role for these integrins in B-cell activation. Although the alpha 4 subunit is expressed by all lymphoid cells, an increased expression of alpha 4 and decreased expression of beta 1 by the mantle zone B-cell compartment is noted in comparison with the decreased expression of alpha 4 and increased expression of beta 1 by follicular center B-cells. These studies suggest that alpha 4 may be paired with a beta subunit other than beta 1 on the mantle zone lymphoid population. Thus, integrin expression by cells of lymphoid tissues varies with location and function and differs significantly from integrin expression observed on circulating and cultured peripheral blood lymphocytes.
13
Zutter, MM. "Immunolocalization of integrin receptors in normal lymphoid tissues." Blood 77, no. 10 (May 15, 1991): 2231–36. http://dx.doi.org/10.1182/blood.v77.10.2231.bloodjournal77102231.
Full textAbstract:
The integrin superfamily of cell adhesion receptors consists of heterodimeric glycoproteins composed of unique alpha and beta subunits. These receptors mediate cell-substrate and cell-cell adhesive properties for a variety of cell types. This investigation has focused on the histologic distribution of the beta 1 subfamily of integrins within lymphoid tissues including tonsil, lymph node, spleen, thymus, and appendix. The dendritic cells of both follicular center and thymic origin express the alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6, as well as the beta 1 integrin subunits. Most lymphoid cells in normal tissues do not express the alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits, or the alpha v beta 3 integrin. The beta 1 subunit is expressed by all lymphocytes but with variable intensity. Increased levels of the alpha 5 and beta 1 subunits are observed in the follicular light zone, suggesting a role for these integrins in B-cell activation. Although the alpha 4 subunit is expressed by all lymphoid cells, an increased expression of alpha 4 and decreased expression of beta 1 by the mantle zone B-cell compartment is noted in comparison with the decreased expression of alpha 4 and increased expression of beta 1 by follicular center B-cells. These studies suggest that alpha 4 may be paired with a beta subunit other than beta 1 on the mantle zone lymphoid population. Thus, integrin expression by cells of lymphoid tissues varies with location and function and differs significantly from integrin expression observed on circulating and cultured peripheral blood lymphocytes.
14
Rich, S., N. Van Nood, and H. M. Lee. "Role of alpha 5 beta 1 integrin in TGF-beta 1-costimulated CD8+ T cell growth and apoptosis." Journal of Immunology 157, no. 7 (October 1, 1996): 2916–23. http://dx.doi.org/10.4049/jimmunol.157.7.2916.
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Abstract TGF-beta 1 regulates cell growth, differentiation, and adhesion and is a potent immunosuppressant, in part through its well-recognized growth-inhibitory effects. However, certain T cell subsets, particularly of naive phenotype, can instead be costimulated to proliferate by TGF-beta 1. We have previously demonstrated that naive murine CD8+ T cells, TCR activated by platebound anti-CD3 Ab or SEB superantigen, are growth stimulated by TGF-beta 1, acquire a memory phenotype, express elevated IL-10 and TGF-beta 1, and cause T cell growth inhibition as effector CD8+ T cells. TGF-beta 1 causes growth among certain nonlymphoid cells in part by inducing or mimicking integrin activation. The present studies thus addressed mediation of TGF-beta 1-dependent growth and survival of anti-CD3-triggered CD8+ T cells via beta 1 integrins. TGF-beta 1 reduced anti-CD3-activated alpha 4 beta 1 integrin expression and constitutive adhesion to fibronectin, while initial alpha 5 beta 1 expression was heightened and adhesive function sustained. Fibronectin-based RGD peptides that bind alpha 5 beta 1 integrins and alpha 5 or beta 1 integrin chain-specific Abs blocked TGF-beta 1-dependent proliferation, while connecting segment-1 peptide that binds alpha 4 beta 1 integrin and alpha 4 chain-specific Abs had no effect. Cross-linked alpha 5- but not alpha 4-specific Ab mimicked TGF-beta 1 function by costimulating CD8+ T cell growth. TGF-beta 1 also caused RGD peptide-sensitive CD8+ T cell aggregation. Additionally, TGF-beta 1-costimulated proliferation correlated with TGF-beta 1 protection of CD8+ T cells from anti-CD3-induced apoptosis. RGD peptides and alpha 5 integrin-specific Ab abolished TGF-beta 1 prevention of activation-induced apoptosis. Therefore, TGF-beta 1 costimulates CD8+ T cell growth via activation of the alpha 5 beta 1 integrin and/or its ligand and supports sustained growth at least in part by alpha 5 beta 1-mediated protection from activation-induced apoptosis.
15
Kikkawa, Y., N. Sanzen, H. Fujiwara, A. Sonnenberg, and K. Sekiguchi. "Integrin binding specificity of laminin-10/11: laminin-10/11 are recognized by alpha 3 beta 1, alpha 6 beta 1 and alpha 6 beta 4 integrins." Journal of Cell Science 113, no. 5 (March 1, 2000): 869–76. http://dx.doi.org/10.1242/jcs.113.5.869.
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Laminin-10/11, the laminin isoforms containing the alpha 5 chain, are major components of basement membranes of many fetal and adult tissues. Laminin-10/11 purified from the conditioned medium of human lung carcinoma cells were potent in mediating adhesion of the carcinoma cells in an integrin alpha 3 beta 1-dependent manner. To further define the type(s) of integrins involved in cell adhesion to laminin-10/11, we examined the effects of a panel of function-blocking anti-integrin antibodies on the adhesion of different cell types to laminin-10/11. Although anti-integrin beta 1 antibody inhibited the adhesion of all cell types tested, anti-alpha 3 antibody inhibited the adhesion of carcinoma and glioma cells but not fibroblastic cells. Adhesion of fibroblastic cells was inhibited, however, by a combination of anti-alpha 3 and anti-alpha 6 antibodies, suggesting that both alpha 3 beta 1 and alpha 6 beta 1 integrins function as laminin-10/11 receptors in these cells. To explore this possibility, we examined the adhesion of K562 leukemic cells transfected with integrin alpha 3 or alpha 6 subunit to laminin-10/11 or other laminin isoforms. Laminin-10/11 were potent adhesive ligands for both the alpha 3 beta 11 and alpha 6 beta 1 transfectants, whereas laminin-5 was the preferred ligand for the alpha 3 beta 1 transfectants. Upon stimulation with the activating anti-integrin beta 1 antibody, both transfectants became more adherent to the substratum regardless of the type of laminins coated, although their preference for laminin isoforms remained unaltered. K562 cells transfected with alpha 6 and beta 4 subunits were also capable of adhering to laminin-10/11, indicating that integrin alpha 6 beta 4 is another receptor for laminin-10/11. Even with lung carcinoma cells, the alpha 6-containing integrins partly contributed to adhesion to laminin-10/11 at higher coating concentrations, although non-integrin receptor(s) might also be involved under such conditions. These results indicated that laminin-10/11 are potent and versatile adhesive ligands in basement membranes capable of binding to both alpha 3 beta 1 and alpha 6 beta 1 integrins with high avidity and also to alpha 6 beta 4 integrin.
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Díaz-González, F., J. Forsyth, B. Steiner, and M. H. Ginsberg. "Trans-dominant inhibition of integrin function." Molecular Biology of the Cell 7, no. 12 (December 1996): 1939–51. http://dx.doi.org/10.1091/mbc.7.12.1939.
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Occupancy of integrin adhesion receptors can alter the functions of other integrins and cause partition of the ligand-occupied integrin into focal adhesions. Ligand binding also changes the conformation of integrin extracellular domains. To explore the relationship between ligand-induced conformational change and integrin signaling, we examined the effect of ligands specific for integrin alpha IIb beta 3 on the functions of target integrins alpha 5 beta 1 and alpha 2 beta 1. We report that binding of integrin-specific ligands to a suppressive integrin can inhibit the function of other target integrins (trans-dominant inhibition). Trans-dominant inhibition is due to a blockade of integrin signaling. Furthermore, this inhibition involves both a conformational change in the extracellular domain and the presence of the beta cytoplasmic tail in the suppressive integrin. Similarly, ligand-induced recruitment of alpha IIb beta 3 to focal adhesions also involves a conformational rearrangement of its extracellular domain. These findings imply that the ligand-induced conformational changes can propagate from an integrin's extracellular to its intracellular face. Trans-dominant inhibition by integrin ligands may coordinate integrin signaling and can lead to unexpected biological effects of integrin-specific inhibitors.
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Yang, J. T., and R. O. Hynes. "Fibronectin receptor functions in embryonic cells deficient in alpha 5 beta 1 integrin can be replaced by alpha V integrins." Molecular Biology of the Cell 7, no. 11 (November 1996): 1737–48. http://dx.doi.org/10.1091/mbc.7.11.1737.
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alpha 5 beta 1 integrin mediates cell adhesion to extracellular matrix by interacting with fibronectin (FN). Mouse lines carrying null mutations in genes encoding either the alpha 5 integrin subunit or FN have been generated previously. Both mutations are embryonic lethal with overlapping defects, but the defects of alpha 5-null embryos are less severe. Primary embryonic cells lacking alpha 5 beta 1 are able to adhere to FN, form focal contacts, migrate on FN, and assemble FN matrix. These results suggest the involvement of (an)other FN receptors(s). In this study, we examined functions of alpha 4 beta 1 and alpha V integrins in embryonic cells lacking alpha 5 beta 1. Our analysis of cells lacking both alpha 4 beta 1 and alpha 5 beta 1 showed that alpha 4 beta 1 is also not required for these FN-dependent functions. Using alpha V-specific blocking reagents, we showed that alpha V integrins are required for alpha 5-null cells, but not wild-type cells, to adhere and spread on FN. Our data also showed that, although the expression levels of alpha V integrins on the wild-type and alpha 5-null cells are similar, there is an increase in recruitment of alpha V integrins into focal contacts in alpha 5-null cells plated on FN, indicating that alpha V integrins can compensate functionally for the loss of alpha 5 beta 1 in focal contacts of alpha 5-null cells. Finally, our data suggested possible roles for alpha V integrins in replacing the role of alpha 5 beta 1 in FN matrix assembly in vitro and in FN-dependent embryonic functions in vivo.
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Yang, J. T., H. Rayburn, and R. O. Hynes. "Embryonic mesodermal defects in alpha 5 integrin-deficient mice." Development 119, no. 4 (December 1, 1993): 1093–105. http://dx.doi.org/10.1242/dev.119.4.1093.
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A loss of function mutation of the murine alpha 5 integrin gene generated by gene targeting in embryonic stem cells is a recessive embryonic lethal. The mutant embryos start to show observable defects by day 9 of gestation and die around day 10–11. The alpha 5-null embryos have pronounced defects in posterior trunk and yolk sac mesodermal structures, suggesting a role for alpha 5 beta 1 integrin in mesoderm formation, movement or function. However, the embryos progress significantly further than embryos null for fibronectin, for which alpha 5 beta 1 integrin is a receptor, suggesting the involvement of other fibronectin receptors. In vitro studies on cells derived from the alpha 5-null embryos confirm that the alpha 5 beta 1 integrin is not expressed on mutant cells and show that the mutant cells are able to assemble fibronectin matrix, form focal contacts, and migrate on fibronectin despite the complete absence of the alpha 5 beta 1 fibronectin receptor integrin. All these functions have previously been thought to involve or require alpha 5 beta 1. The results presented show that these cellular functions involving fibronectin can proceed using other receptors.
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Bhatia, R., JB McCarthy, and CM Verfaillie. "Interferon-alpha restores normal beta 1 integrin-mediated inhibition of hematopoietic progenitor proliferation by the marrow microenvironment in chronic myelogenous leukemia." Blood 87, no. 9 (May 1, 1996): 3883–91. http://dx.doi.org/10.1182/blood.v87.9.3883.bloodjournal8793883.
Full textAbstract:
Chronic myelogenous leukemia (CML) progenitors show decreased adhesion to stroma and fibronectin (FN) through beta 1 integrin receptors. We have previously shown that interferon-alpha (IFN-alpha) restores beta 1 integrin-mediated adhesion of CML progenitors to stroma. Because beta1 integrins transmit proliferation inhibitory signals from the microenvironment to normal hematopoietic progenitors, we hypothesized that decreased integrin-mediated adhesion of CML progenitors contributes to their continuous proliferation when in contact with stroma and that IFN-alpha treatment, by restoring integrin-mediated adhesion, also restores integrin-mediated microenvironmental inhibition of CML progenitor proliferation. We show here that, in contrast to normal colony-forming cells (CFC), the percentage of malignant CML CFC in S-phase was not significantly reduced following coculture with stromal layers. However, IFN-alpha treatment resulted in a significant reduction in the proliferation of CML CFC on coculture with stroma. This effect was not because of a direct antiproliferative effect of IFN- alpha on CML CFC because the proliferation of IFN-alpha treated CML CFC kept in suspension culture was not reduced. We examined the role of restored signaling through beta 1 integrin receptors in IFN-alpha induced inhibition of CML progenitors in two sets of experiment. In the first set of experiments, we demonstrated that proliferation of IFN- alpha-treated CML CFC, but not untreated CML CFC, was significantly reduced following coculture with 33/66-kD and 75-kD FN fragments, recognized by alpha 4 beta 1 and alpha 5 beta 1 integrins respectively. In a second set of experiments, we demonstrate that direct stimulation of integrin receptors by crosslinking with blocking antibodies to alpha 4, alpha 5, and beta 1 integrins and secondary goat antimouse antibodies resulted in significant reduction in proliferation of normal and IFN-alpha treated CML progenitors but not untreated CML CFC. These studies indicate that CML hematopoietic progenitors are unresponsive to beta 1-integrin mediated proliferation inhibition and that IFN-alpha not only restores beta 1 integrin-mediated adhesion but also beta1- mediated microenvironmental inhibition of CML progenitor proliferation. These observations may explain, at least in part, the therapeutic efficacy of IFN-alpha in CML.
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Malek-Hedayat, S., and LH Rome. "Expression of a beta 1-related integrin by oligodendroglia in primary culture: evidence for a functional role in myelination." Journal of Cell Biology 124, no. 6 (March 15, 1994): 1039–46. http://dx.doi.org/10.1083/jcb.124.6.1039.
Full textAbstract:
We have investigated the expression of integrins by rat oligodendroglia grown in primary culture and the functional role of these proteins in myelinogenesis. Immunochemical analysis, using antibodies to a number of alpha and beta integrin subunits, revealed that oligodendrocytes express only one detectable integrin receptor complex (alpha OL beta OL). This complex is immunoprecipitated by a polyclonal anti-human beta 1 integrin subunit antibody. In contrast, astrocytes, the other major glial cell type in brain, express multiple integrins including alpha 1 beta 1, alpha 3 beta 1, and alpha 5 beta 1 complexes that are immunologically and electrophoretically indistinguishable from integrins expressed by rat fibroblasts. The beta subunit of the oligodendrocyte integrin (beta OL) and rat fibroblast beta 1 have different electrophoretic mobilities in SDS-PAGE. However, the two beta subunits appear to be highly related based on immunological cross-reactivity and one-dimensional peptide mapping. After removal of N-linked carbohydrate chains, beta OL and beta 1 comigrated in SDS-PAGE and peptide maps of the two deglycosylated subunits were identical, suggesting differential glycosylation of beta 1 and beta OL accounts entirely for their size differences. The oligodendrocyte alpha subunit, alpha OL, was not immunoprecipitated by antibodies against well characterized alpha chains which are known to associate with beta 1 (alpha 3, alpha 4, and alpha 5). However, an antibody to alpha 8, a more recently identified integrin subunit, did precipitate two integrin subunits with electrophoretic mobilities in SDS-PAGE identical to alpha OL and beta OL. Functional studies indicated that disruption of oligodendrocyte adhesion to a glial-derived matrix by an RGD-containing synthetic peptide resulted in a substantial decrease in the level of mRNAs for several myelin components including myelin basic protein (MBP), proteolipid protein (PLP), and cyclic nucleotide phosphodiesterase (CNP). These results suggest that integrin-mediated adhesion of oligodendrocytes may trigger signal(s) that induce the expression of myelin genes and thus influence oligodendrocyte differentiation.
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Sánchez-Mateos, P., M. R. Campanero, M. A. Balboa, and F. Sánchez-Madrid. "Co-clustering of beta 1 integrins, cytoskeletal proteins, and tyrosine-phosphorylated substrates during integrin-mediated leukocyte aggregation." Journal of Immunology 151, no. 7 (October 1, 1993): 3817–28. http://dx.doi.org/10.4049/jimmunol.151.7.3817.
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Abstract The involvement of the VLA-4 integrin in an alternative leukocyte homotypic adhesion mechanism that is LFA-1/ICAM-1 independent, has been previously reported. We describe here the localization of beta 1 and alpha 4 integrin subunits at sites of cell-cell contact, on both beta 1- and alpha 4-induced aggregates of B lymphoblastoid Ramos cells. Moreover, the distribution of different VLA-alpha subunits was also examined on beta 1-induced cell aggregates of alpha 2- and alpha 4-transfected K-562 cells. Both alpha 2 and alpha 4 integrin subunits were mainly localized at sites of intercellular boundaries, suggesting a possible role for these integrins in leukocyte intercellular adhesion. The fibronectin receptor alpha 5 subunit was either diffuse throughout the plasma membrane, or displayed some accumulation at sites of cell-cell contact. Even though homotypic aggregation of U-937 cells was induced with the anti-alpha 5 P1D6 mAb, the alpha 5 subunit showed only partial redistribution to regions of cell-cell contact, compared with the complete redistribution of the alpha 4 subunit in the alpha 4-induced aggregates. The reorganization of the actin-cytoskeleton was observed at sites of intercellular boundaries in both the anti-beta 1- and anti-alpha 4-induced cell aggregates. Hence, F-actin and the cytoskeletal protein talin co-localized with beta 1 and alpha 4 integrin clusters at sites of cell-cell contact. Signal transduction during VLA-mediated homotypic cell adhesion has also been investigated. We found co-localization of beta 1 and alpha 4 subunits with tyrosine-phosphorylated proteins at cell-cell contact regions during cell aggregation. These data indicate that VLA integrin-mediated leukocyte aggregation results in clustering of beta 1-integrins at sites of cell-cell contact, together with co-localization of cytoskeletal proteins. These results also suggest that protein tyrosine phosphorylation is an important signal transduction mechanism when VLA integrins participate in intercellular contacts.
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Wickham, T. J., E. J. Filardo, D. A. Cheresh, and G. R. Nemerow. "Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization." Journal of Cell Biology 127, no. 1 (October 1, 1994): 257–64. http://dx.doi.org/10.1083/jcb.127.1.257.
Full textAbstract:
Human adenovirus type 2 (Ad2) enters host cells by receptor-mediated endocytosis, an event mediated by the virus penton base binding to cell surface integrins alpha v beta 3 and alpha v beta 5. While both alpha v integrins promote virus internalization, alpha v beta 5 is involved in the subsequent event of membrane permeabilization. Cells transfected with the beta 5 or beta 3 subunit, expressing either alpha v beta 5 and alpha v beta 3, respectively, were capable of supporting Ad2 infection to varying degrees. In this case, cells expressing alpha v beta 5 were significantly more susceptible to Ad2-induced membrane permeabilization, as well as to Ad2 infection, than cells expressing alpha v beta 3. Adenovirus-mediated gene delivery was also more efficient in cells expressing alpha v beta 5. These results suggest that the interaction of alpha v beta 5 with Ad2 penton base facilitates the subsequent step of virus penetration into the cell. These studies provide evidence for the involvement of a cellular receptor in virus-mediated membrane permeabilization and suggest a novel biological role for integrin alpha v beta 5 in the infectious pathway of a human adenovirus.
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Klein, S., F. G. Giancotti, M. Presta, S. M. Albelda, C. A. Buck, and D. B. Rifkin. "Basic fibroblast growth factor modulates integrin expression in microvascular endothelial cells." Molecular Biology of the Cell 4, no. 10 (October 1993): 973–82. http://dx.doi.org/10.1091/mbc.4.10.973.
Full textAbstract:
During angiogenesis capillary endothelial cells undergo a coordinated set of modifications in their interactions with extracellular matrix components. In this study we have investigated the effect of the prototypical angiogenic factor basic fibroblast growth factor (bFGF) on the expression and function of several integrins in microvascular endothelial cells. Immunoprecipitation experiments with antibodies to individual subunits indicated that microvascular cells express at their surface several integrins. These include the alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1 laminin/collagen receptors; the alpha 6 beta 1 laminin receptor; the alpha 5 beta 1 and alpha v beta 1 fibronectin receptors; the alpha 6 beta 4 basement membrane receptor; and the alpha v beta 3 and alpha v beta 5 vitronectin receptors. Treatment with bFGF caused a significant increase in the surface expression of the alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha 6 beta 4, and alpha v beta 5 integrins. In contrast, the level of expression of the alpha 1 beta 1 and alpha v beta 3 integrins was decreased in bFGF-treated cells. Immunoprecipitation of metabolically labeled cells indicated that bFGF increases the biosynthesis of the alpha 3, alpha 5, alpha 6, beta 4, and beta 5 subunits and decreases the production of the alpha v and beta 3 subunits. These results suggest that bFGF modulates integrin expression by altering the biosynthesis of individual alpha or beta subunits. In accordance with the upregulation of several integrins observed in bFGF-treated cells, these cells adhered better to fibronectin, laminin, vitronectin, and type I collagen than did untreated cells. The largest differences in beta 1 integrin expression occurred approximately 72 h after exposure to bFGF, at a time when the expression of the endothelial cell-to-cell adhesion molecule endoCAM was also significantly upregulated. In contrast, a shorter exposure to bFGF (24-48 h) was required for the maximal induction of plasminogen activator production in the same cells. Taken together, these results show that bFGF causes significant changes in the level of expression and function of several integrins in microvascular endothelial cells.
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Tawil, N., P. Wilson, and S. Carbonetto. "Integrins in point contacts mediate cell spreading: factors that regulate integrin accumulation in point contacts vs. focal contacts." Journal of Cell Biology 120, no. 1 (January 1, 1993): 261–71. http://dx.doi.org/10.1083/jcb.120.1.261.
Full textAbstract:
We have studied the function and distribution of the alpha 1 beta 1, alpha 5 beta 1 and alpha 6 beta 1 heterodimers on type-1 astrocytes with antibodies specific for integrin subunits (alpha 1, alpha 5, alpha 6, and beta 1). The alpha 1 beta 1 heterodimer mediates adhesion to laminin and collagen, the alpha 5 beta 1 to fibronectin in an RGD-dependent manner. The alpha 5 beta 1 integrin is found in focal contacts in long-term cultures of well-spread astrocytes colocalizing with vinculin and the termini of actin stress fibers. alpha 1 beta 1 heterodimers can occasionally be found as small aggregates within focal contacts but they do not accumulate there. Instead, alpha 1 beta 1 integrins are found in punctate deposits called point contacts which are distributed over the upper and the lower cell surfaces whether laminin, collagen, fibronectin or polylysine is used as a substratum. Unlike focal contacts, point contacts contain clathrin but rarely codistribute with actin or vinculin. Two observations indicate that these point contacts are functional. First, mAb 3A3, directed against the rat alpha 1 subunit, inhibits the attachment of astrocytes to laminin and collagen. Second, during the spreading of astrocytes, a band of point contacts forms around the cell perimeter at a time when no focal contacts are visible. While alpha 1 beta 1 integrins are found only in point contacts in astrocytes, the alpha 6 beta 1 integrin, another laminin receptor, is localized within focal contacts. Moreover, alpha 1 beta 1 heterodimers accumulate in focal contacts in fibroblasts. Thus, the alpha subunit contributes, independent of its ligand, to functional integrin heterodimer accumulation in focal contacts or in point contacts. This accumulation varies among different cell types with apparently identical heterodimers as well as with the motile state (spreading vs. flattened) of the same cells.
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Filardo, E. J., S. L. Deming, and D. A. Cheresh. "Regulation of cell migration by the integrin beta subunit ectodomain." Journal of Cell Science 109, no. 6 (June 1, 1996): 1615–22. http://dx.doi.org/10.1242/jcs.109.6.1615.
Full textAbstract:
CS-1 melanoma cells transfected with cDNAs encoding either the beta 3 or beta 5 integrin subunit protein express alpha v beta 3 or alpha v beta 5, respectively, enabling them to adhere to vitronectin yet only alpha v beta 3 promotes cell spreading and migration on this substrate. Following exposure to insulin or insulin-like growth factor, alpha v beta 5-expressing CS-1 cells gain the ability to migrate on vitronectin. To identify structural regions in beta 3 or beta 5 that account for these distinct biological properties, CS-1 cells were transfected with one of two chimeric beta subunit proteins, in which the ecto- and cytoplasmic domains of beta 3 and beta 5 were exchanged (termed alpha v beta 3/5 or alpha v beta 5/3). Surprisingly, alpha v beta 3/5 expressing cells spread and migrate on vitronectin while cells expressing alpha v beta 5/3 do not unless they are exposed to cytokine. These findings suggest that the distinct migratory properties mediated by integrins alpha v beta 3 and alpha v beta 5 and their response to cytokine activation is determined by a sequence(s) within the ectodomain of the integrin beta subunit.
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Wu, C., A. E. Chung, and J. A. McDonald. "A novel role for alpha 3 beta 1 integrins in extracellular matrix assembly." Journal of Cell Science 108, no. 6 (June 1, 1995): 2511–23. http://dx.doi.org/10.1242/jcs.108.6.2511.
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To study the biological role of alpha 3 beta 1 integrins in cell adhesion, migration, and in the deposition of extracellular matrix, we stably expressed the human alpha 3 integrin subunit in the alpha 4, alpha 5 integrin deficient CHO cell line B2. The expression of alpha 3 beta 1 integrins enhanced cell adhesion on entactin (also known as nidogen), but not on fibronectin. Using recombinant GST-fusion proteins that span the entire length of the entactin molecule, we located cell adhesive activity to the G2 domain of entactin. These results suggest that the alpha 3 beta 1 integrin functions as an adhesion receptor interacting with the G2 domain of entactin. On the other hand, the expression of alpha 3 beta 1 integrins did not confer the ability to migrate on entactin. Strikingly, the expression of alpha 3 beta 1 dramatically increased the deposition of entactin and fibronectin into the pericellular matrix. This was accompanied by increased binding activity of the 29 kDa amino-terminal domain of fibronectin. Thus, similar to alpha 5 beta 1 integrins, alpha 3 beta 1 integrins can play an important role in modulating the assembly of pericellular matrices. However, unlike fibronectin deposition supported by alpha 5 beta 1, alpha 3 beta 1 supported fibronectin deposition into pericellular matrix was not inhibited by antibodies binding to the RGD containing cell adhesion domain of fibronectin, demonstrating that the two processes are mechanistically distinct. The role of alpha 3 beta 1 in pericellular matrix assembly potentially implicates this receptor in the assembly and/or recognition of entactin-containing pericellular matrices, an observation consistent with its apparent role in the renal glomerulus of the mammalian kidney.
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Herard, A. L., D. Pierrot, J. Hinnrasky, H. Kaplan, D. Sheppard, E. Puchelle, and J. M. Zahm. "Fibronectin and its alpha 5 beta 1-integrin receptor are involved in the wound-repair process of airway epithelium." American Journal of Physiology-Lung Cellular and Molecular Physiology 271, no. 5 (November 1, 1996): L726—L733. http://dx.doi.org/10.1152/ajplung.1996.271.5.l726.
Full textAbstract:
The cell migration that occurs during wound repair is dependent on modifications of the cell-matrix interaction in which extracellular matrix proteins and their receptors, the integrins, are involved. To study the interactions between airway epithelial cells and the extracellular matrix during the process of wound repair, we developed an in vitro wound model of human epithelial cells. Surface epithelial cells were dissociated from human nasal polyps and cultured on a type I collagen matrix. At confluency, a wound was made by the addition of 2 microliters of NaOH (1 N) to the cell culture. After the cell culture was washed, the wound area was recorded every 12 h for 96 h by a videomicroscopic technique. We calculated the wound-repair index that represents the decrease in the wound area per hour. Using immunofluorescence techniques, we first examined the localization, during wound repair, of fibronectin and of the beta 1-, alpha v-, alpha 2-, alpha 3-, and alpha 5-integrin subunits. Secondly, we carried out a series of wound-repair blocking experiments with the use of anti-integrin or anti-fibronectin antibodies diluted in the culture medium. We observed that fibronectin and the alpha 5- integrin subunit were exclusively expressed by the migratory cells in the wounded area. No difference in the localization of the alpha v-, alpha 2-, and alpha 3-integrin subunits was observed between the nonrepairing and repairing cells. The blocking experiments showed a significant decrease in the wound-repair index in the presence of either the anti-beta 1, -alpha 3, alpha 5, or the anti-fibronectin antibodies. Furthermore, the addition of fibronectin to the culture medium induced a significant increase in the wound repair index. These results suggest that fibronectin and the corresponding alpha 5 beta 1-integrin play an important role in the process of airway epithelium wound repair.
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Wennerberg, K., L. Lohikangas, D. Gullberg, M. Pfaff, S. Johansson, and R. Fässler. "Beta 1 integrin-dependent and -independent polymerization of fibronectin." Journal of Cell Biology 132, no. 1 (January 1, 1996): 227–38. http://dx.doi.org/10.1083/jcb.132.1.227.
Full textAbstract:
The mouse cell line GD25, which lacks expression of the beta 1 family of integrin heterodimers due to disruption of the beta 1 integrin subunit gene, was used for expression of full-length cDNA coding for splice variant A of the mouse beta 1 integrin subunit. In a stably transformed clone (GD25-beta 1A), the expressed protein was found to form functional heterodimeric receptors together with the subunits alpha 3, alpha 5, and alpha 6. Both GD25 and GD25-beta 1A attached to fibronectin and formed focal contacts which contained alpha v beta 3, but no detectable alpha 5 beta 1A. The presence of GRGDS peptide allowed alpha 5 beta 1A to locate to focal contacts of GD25-beta 1A cultured on fibronectin, while the beta 1-null GD25 cells were unable to attach under these conditions. Affinity chromatography revealed that alpha 5 beta 1A and alpha v beta 3 could bind to a large cell-binding fragment of fibronectin. alpha 5 beta 1A strongly promoted polymerization of fibronectin into a fibrillar network on top of the cells. Whereas little alpha v beta 3 was colocalized with the fibronectin fibrils in GD25-beta 1A cells, this integrin was able to support fibronectin fibril polymerization in GD25 cells. However, the alpha v beta 3-induced polymerization was less efficient and occurred mainly in dense cultures of the GD25 cells. Thus, while both alpha 5 beta 1A and alpha v beta 3 are able to support adhesion to fibronectin, alpha v beta 3 dominates in the formation of focal contacts, and alpha 5 beta 1A has a prime function in fibronectin matrix assembly. This is the first report on fibronectin matrix assembly in the absence of beta 1 integrins.
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Blystone, S. D., F. P. Lindberg, S. E. LaFlamme, and E. J. Brown. "Integrin beta 3 cytoplasmic tail is necessary and sufficient for regulation of alpha 5 beta 1 phagocytosis by alpha v beta 3 and integrin-associated protein." Journal of Cell Biology 130, no. 3 (August 1, 1995): 745–54. http://dx.doi.org/10.1083/jcb.130.3.745.
Full textAbstract:
Using a K562 cell transfection model, we have previously described a novel relationship between the integrins alpha v beta 3 and alpha 5 beta 1. alpha v beta 3 ligation was able to inhibit alpha 5 beta 1-mediated phagocytosis without effect on alpha 5 beta 1-mediated adhesion. The alpha v beta 3-dependent inhibition apparently required a signal transduction cascade as it was reversed by inhibitors of serine/threonine kinases. Now, we have studied the mechanisms of signal transduction in this system and have found that the beta 3 cytoplasmic tail is both necessary and sufficient for initiation of the signal leading to inhibition of alpha 5 beta 1 phagocytosis. Ligation of integrin-associated protein (IAP), which has been implicated in alpha v beta 3 signal transduction, mimics the effects of alpha v beta 3 ligation only when the beta 3 integrin with an intact cytoplasmic tail is present. Although fibronectin-mediated phagocytosis requires the high affinity conformation of alpha 5 beta 1, ligation of alpha v beta 3/IAP does not prevent acquisition of this high affinity state. We conclude that alpha v beta 3/IAP ligation initates a signal transduction cascade, dependent upon the beta 3 cytoplasmic tail, which inhibits the phagocytic function of alpha 5 beta 1 at a step subsequent to modulation of integrin affinity.
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Shibayama, H., S. Tagawa, H. Hattori, R. Inoue, S. Katagiri, and T. Kitani. "Laminin and fibronectin promote the chemotaxis of human malignant plasma cell lines." Blood 86, no. 2 (July 15, 1995): 719–25. http://dx.doi.org/10.1182/blood.v86.2.719.bloodjournal862719.
Full textAbstract:
We examined chemotaxis of human plasma cells (PCs) in response to extracellular matrix proteins (ECMs) in the human PC cell lines FR4ds and OPM-1ds. The FR4ds cells expressed beta 1+, beta 3-, alpha 2-, alpha 3-, alpha 4+, alpha 5+, alpha 6+, and alpha v+ integrins, whereas the OPM-1ds cells expressed beta 1+, beta 3-, alpha 2-, alpha 3+, alpha 4+, alpha 5-, alpha 6+, and alpha v+. Fibronectin (FN) and laminin (LN) promoted the chemotaxis of the PCs. An inhibitory assay with anti- integrin monoclonal antibodies (MoAbs) showed that anti-alpha 4 MoAb partially inhibited the chemotaxis of FR4ds and completely inhibited the chemotaxis of OPM-1ds. Anti-alpha 5 MoAb alone had no effect on either of these two lines. Nevertheless, anti-alpha 5 MoAb completely inhibited chemotaxis when it was added with anti-alpha 4 in FR4ds, demonstrating a novel complementary role of VLA-5 toward VLA-4 in the chemotaxis induced by FN. LN facilitated chemotaxis both in OPM-1ds expressing alpha 3 and alpha 6 integrins and in FR4ds expressing alpha 6 integrin alone. Anti-alpha 6 MoAb completely inhibited FR4ds chemotaxis, whereas anti-alpha 3 and -alpha 6 MoAb had synergistic inhibitory effects on the chemotaxis of OPM-1ds. These results indicated that the distribution of PCs in human tissue are determined by at least two factors: the concentration of the ECM proteins FN and LN and the expression of integrins.
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Tsuchida, J., S. Ueki, Y. Takada, Y. Saito, and J. Takagi. "The ‘ligand-induced conformational change’ of alpha 5 beta 1 integrin. Relocation of alpha 5 subunit to uncover the beta 1 stalk region." Journal of Cell Science 111, no. 12 (June 15, 1998): 1759–66. http://dx.doi.org/10.1242/jcs.111.12.1759.
Full textAbstract:
Integrin heterodimers undergo a conformational change upon the binding of ligand to their extracellular domains. An anti-beta1 integrin monoclonal antibody AG89 can detect such a conformational change since it recognizes a ligand-inducible epitope in the stalk-like region of beta1 subunits. The binding of a 125I-labeled AG89 Fab fragment to alpha5 beta1 integrins on K562 cells was assessed and analyzed by the Scatchard method. High affinity binding sites for AG89 are present on cells treated with ligand peptide. In addition, results revealed that cells treated with EDTA also express AG89 binding sites with the same affinity although the number of binding sites is 4-fold lower. AG89 immunoprecipitated alpha5 beta1 complexes from surface-labeled K562 cells treated with ligand peptide. By contrast, it immunoprecipitated only beta1 chains when the ligand peptide was absent, suggesting that high affinity binding sites on EDTA-treated cells are associated with non-functional beta1 monomer. Additional studies show that the epitope for AG89 is constitutively exposed on mutant beta1 that cannot complex with alpha5. These data suggest that the AG89 epitope is masked by the alpha5 subunit. Ligand binding and integrin activation may uncover the beta1 stalk region by triggering a conformational shift of alpha5 relative to beta1.
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Aplin, J. D., A. Sattar, and A. P. Mould. "Variant choriocarcinoma (BeWo) cells that differ in adhesion and migration on fibronectin display conserved patterns of integrin expression." Journal of Cell Science 103, no. 2 (October 1, 1992): 435–44. http://dx.doi.org/10.1242/jcs.103.2.435.
Full textAbstract:
Starting from the BeWo choriocarcinoma cell line, two stable variant cell lines (epi and lc) were isolated. Epi cells displayed an epithelioid colony morphology while lc were fibroblastoid. lc cells attached and spread on fibronectin-coated surfaces at significantly lower density of fibronectin than epi or the parent cell line. lc also migrated more efficiently to fibronectin in a trans-filter assay than either epi or parent cells. Integrin expression by the cell lines was investigated by flow cytometry and immunoprecipitation from surface-labelled cells with a panel of subunit-specific antibodies. Integrins alpha 2 beta 1, alpha 5 beta 1, alpha v beta 1 and alpha 6 beta 4 were detected in each case, and levels of expression were identical in the two variant lines. Anti-functional antibodies were used to probe the role of integrins in fibronectin- and vitronectin-mediated adhesion. Complete inhibition of adhesion to fibronectin was observed with anti-beta 1 antibody, and partial inhibition with anti-alpha 5, suggesting that integrin alpha 5 beta 1 is mainly responsible for the interaction. Adhesion to vitronectin was inhibitable using anti-alpha v and anti-beta 1 antibodies, suggesting that integrin alpha v beta 1 is active in these cells as a vitronectin receptor. There was a correlation between the altered morphology of the variant cells and alterations in the distribution of integrin alpha 6 beta 4 and laminin in monolayer cultures. The results support the idea that fibronectin may mediate the migratory behaviour of extravillous trophoblast in vivo. Switch to a more migratory phenotype may be mediated by the selective activation of integrins and altered interaction with basement membrane.
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Lampugnani, M. G., M. Resnati, E. Dejana, and P. C. Marchisio. "The role of integrins in the maintenance of endothelial monolayer integrity." Journal of Cell Biology 112, no. 3 (February 1, 1991): 479–90. http://dx.doi.org/10.1083/jcb.112.3.479.
Full textAbstract:
This paper shows that, in confluent human umbilical vein endothelial cell (EC) monolayers, the integrin heterodimers alpha 2 beta 1 and alpha 5 beta 1, but not other members of the beta 1 subfamily, are located at cell-cell contact borders and not at cellular free edges. Also the alpha v chain, but not its most common partner beta 3, that is widely expressed in EC cell-matrix junctions, is found at cell-cell borders. In EC monolayers, the putative ligands of alpha 2 beta 1 and alpha 5 beta 1 receptors, i.e., laminin, collagen type IV, and fibronectin, are also organized in strands corresponding to cell-cell borders. The location of the above integrin receptors is not an artifact of in vitro culture since it has been noted also in explanted islets of the native umbilical vein endothelium. The integrins alpha 2 beta 1 and alpha 5 beta 1 play a role in the maintenance of endothelial monolayer continuity in vitro. Indeed, specific antibodies to alpha 2 beta 1, alpha 5 beta 1, and the synthetic peptide GRGDSP alter its continuity without any initial cell detachment. Moreover, antibodies to alpha 5 beta 1 increase the permeation of macromolecules across confluent EC monolayers. In contrast beta 3 antibodies were ineffective. It is suggested that the relocation of integrins to cell-cell borders is a feature of cells programmed to form polarized monolayers since integrins have a different distribution in nonpolar confluent dermal fibroblasts. The conclusion is that some members of the integrin superfamily collaborate with other intercellular molecules to form lateral junctions and to control both the monolayer integrity and the permeability properties of the vascular endothelial lining. This also suggest that integrins are adhesion molecules provided with a unique biochemical adaptability to different biological functions.
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Testaz, S., M. Delannet, and J. Duband. "Adhesion and migration of avian neural crest cells on fibronectin require the cooperating activities of multiple integrins of the (beta)1 and (beta)3 families." Journal of Cell Science 112, no. 24 (December 15, 1999): 4715–28. http://dx.doi.org/10.1242/jcs.112.24.4715.
Full textAbstract:
Based on genetic, functional and histological studies, the extracellular matrix molecule fibronectin has been proposed to play a key role in the migration of neural crest cells in the vertebrate embryo. In the present study, we have analyzed in vitro the repertoire and function of integrin receptors involved in the adhesive and locomotory responses of avian truncal neural crest cells to fibronectin. Immunoprecipitation experiments showed that neural crest cells express multiple integrins, namely (alpha)3(beta)1, (alpha)4(beta)1, (alpha)5(beta)1, (alpha)8(beta)1, (alpha)v(beta)1, (alpha)v(beta)3 and a (beta)8 integrin, as potential fibronectin receptors, and flow cytometry analyses revealed no major heterogeneity among the cell population for expression of integrin subunits. In addition, the integrin repertoire expressed by neural crest cells was found not to change dramatically during migration. At the cellular level, only (alpha)v(beta)1 and (alpha)v(beta)3 were concentrated in focal adhesion sites in connection with the actin microfilaments, whereas the other integrins were predominantly diffuse over the cell surface. In inhibition assays with function-perturbing antibodies, it appeared that complete abolition of cell spreading and migration could be achieved only by blocking multiple integrins of the (beta)1 and (beta)3 families, suggesting possible functional compensations between different integrins. In addition, these studies provided evidence for functional partitioning of integrins in cell adhesion and migration. While spreading was essentially mediated by (alpha)v(beta)1 and (alpha)8(beta)1, migration involved primarily (alpha)4(beta)1, (alpha)v(beta)3 and (alpha)8(beta)1 and, more indirectly, (alpha)3(beta)1. (alpha)5(beta)1 and the (beta)8 integrin were not found to play any major role in either adhesion or migration. Finally, consistent with the results of inhibition experiments, recruitment of (alpha)4(beta)1 and (alpha)v(beta)3, individually or in combination using antibodies or recombinant VCAM-1 and PECAM-1 molecules as a substratum, was required for migration but was not sufficient to produce migration of the cell population as efficiently as with fibronectin. In conclusion, our study indicates that neural crest cells express a multiplicity of fibronectin-binding integrins and suggests that dispersion of the cell population requires cooperation between distinct integrins regulating different events of cell adhesion, locomotion and, possibly, proliferation and survival.
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Watt, F. M., M. D. Kubler, N. A. Hotchin, L. J. Nicholson, and J. C. Adams. "Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions." Journal of Cell Science 106, no. 1 (September 1, 1993): 175–82. http://dx.doi.org/10.1242/jcs.106.1.175.
Full textAbstract:
Suspension-induced terminal differentiation of human epidermal keratinocytes can be inhibited by fibronectin through binding to the alpha 5 beta 1 integrin. We have investigated the effect of fibronectin on expression of integrins and proteins of the actin cytoskeleton and have explored the nature of the differentiation stimulus by testing different combinations of anti-integrin monoclonal antibodies or extracellular matrix proteins in the suspension assay. Fibronectin prolonged cell surface expression of beta 1 integrins but did not overcome the inhibition of intracellular transport of integrins that occurs when keratinocytes are placed in suspension. Fibronectin did not prevent the suspension-induced decline in the level of mRNAs encoding the beta 1 integrin subunit, actin, filamin and alpha-actinin; furthermore, the inhibition of terminal differentiation did not depend on the state of assembly of microfilaments or microtubules. Terminal differentiation could be partially inhibited by an adhesion-blocking monoclonal antibody to the beta 1 integrin subunit or by a combination of adhesion blocking antibodies recognising the alpha subunits that associate with beta 1 (alpha 2, alpha 3 and alpha 5). Although laminin and type IV collagen do not inhibit terminal differentiation individually, they were inhibitory when added to cells in combination with a low concentration of fibronectin. We conclude that the proportion of keratinocyte beta 1 integrins occupied by ligand can regulate the initiation of terminal differentiation independently of the state of assembly of the actin cytoskeleton.
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Gao, B., T. M. Curtis, F. A. Blumenstock, F. L. Minnear, and T. M. Saba. "Increased recycling of (alpha)5(beta)1 integrins by lung endothelial cells in response to tumor necrosis factor." Journal of Cell Science 113, no. 2 (January 15, 2000): 247–57. http://dx.doi.org/10.1242/jcs.113.2.247.
Full textAbstract:
Tumor necrosis factor (alpha) (TNF-(alpha) can change the interaction of lung endothelial cell monolayers with their extracellular matrix in association with an increase in endothelial monolayer protein permeability. Using immunofluorescence microscopy and flow cytometry, we determined if exposure of calf pulmonary artery endothelial monolayers to TNF-(alpha) may influence cell-matrix interactions by altering the clustering as well as internalization of the (α)5(beta)1 integrins (or fibronectin receptors) on the surface of endothelial cells. Immunofluorescence microscopy revealed that TNF-(alpha) caused an increase in the intracellular staining of (alpha)5(alpha)1 integrins within structures similar to endocytic vesicles as well as an increase in antibody-induced clustering of the integrins at the cell periphery. Flow cytometric analysis of endothelial cells incubated at 37 degrees C after antibody-labeling of their surface (alpha)5(beta)1 integrins at 4 degrees C confirmed an increase in the rate of (alpha)5(beta)1 integrin internalization which was at least 3 times greater after TNF-(α) exposure, based on the half-life for antibody-labeled surface integrins to reach equilibrium with non-labeled integrins within the intracellular pool. Interestingly, the total cell surface expression of (alpha)5(beta)1 integrins was relatively constant after TNF-(alpha) exposure despite the enhanced rate of internalization, suggesting an accelerated recycling of the internalized (alpha)5(beta)1 integrins back to the cell surface. This response was confirmed by the measurement of labeled integrin recycling, which showed a significant (P<0.01) increase in the rate of recycling of the internalized integrins in TNF-treated endothelial cells. Enhanced internalization and subsequent recycling of (alpha)5(beta)1 integrins by endothelial monolayers exposed to TNF-(alpha) may facilitate the redistribution of cell-surface integrins in response to this inflammatory cytokine and may also modify cell-matrix interactions leading to reduced integrity and increased protein permeability of the lung endothelial monolayers.
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Pacifici, R., J. Roman, R. Kimble, R. Civitelli, C. M. Brownfield, and C. Bizzarri. "Ligand binding to monocyte alpha 5 beta 1 integrin activates the alpha 2 beta 1 receptor via the alpha 5 subunit cytoplasmic domain and protein kinase C." Journal of Immunology 153, no. 5 (September 1, 1994): 2222–33. http://dx.doi.org/10.4049/jimmunol.153.5.2222.
Full textAbstract:
Abstract Regulation of the functional status of integrin receptors plays a critical role in inflammation and tissue remodeling, as it affects cell adherence and cytokine secretion. We have previously shown that in monocytes the binding of collagen to the alpha 2 beta 1 integrin induces the release of IL-1, an event that is potentiated by binding of fibronectin (Fn) to the alpha 5 beta 1 integrin. In this study, we have investigated the mechanisms leading to this phenomenon. Fn binding to alpha 5 beta 1 induced intracellular signals which increased the alpha 2 beta 1-dependent adhesiveness of monocytes to collagen without modifications of alpha 2 beta 1 expression. By using Abs against the intracellular region of the alpha 5 subunit of the alpha 5 beta 1 receptor, and specific inhibitors of protein kinase C (PKC), we found that the potentiation effect of Fn on monocyte IL-1 production and their adherence to collagen was dependent on an intact alpha 5 subunit cytoplasmic domain, and required PKC activation. Although the alpha 2 beta 1 could be activated by several intracellular second messengers, including protein kinase A and intracellular calcium, the potentiating effect of Fn was mediated only by PKC. These data provide an example of a novel regulatory mechanism: potentiation of beta 1 integrin-mediated events as a result of ligand binding to another integrin of the same class. They also show that the intracellular region of alpha 5 beta 1 plays a critical role in transducing signals generated by ligand binding to alpha 5 beta 1.
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Vogel, B. E., S. J. Lee, A. Hildebrand, W. Craig, M. D. Pierschbacher, F. Wong-Staal, and E. Ruoslahti. "A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin." Journal of Cell Biology 121, no. 2 (April 15, 1993): 461–68. http://dx.doi.org/10.1083/jcb.121.2.461.
Full textAbstract:
Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD-containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.
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Mannion, B. A., F. Berditchevski, S. K. Kraeft, L. B. Chen, and M. E. Hemler. "Transmembrane-4 superfamily proteins CD81 (TAPA-1), CD82, CD63, and CD53 specifically associated with integrin alpha 4 beta 1 (CD49d/CD29)." Journal of Immunology 157, no. 5 (September 1, 1996): 2039–47. http://dx.doi.org/10.4049/jimmunol.157.5.2039.
Full textAbstract:
Abstract Anti-alpha 4 integrin mAb coprecipitated CD81 (TAPA-1), a 25-kDa cell surface protein, from various alpha 4 beta1 -positive hemopoietic cell lines, including Molt4, Jurkat, Ramos, and alpha 4-transfected K562 (KX4C4) cells. In reciprocal experiments, the integrin alpha 4 beta 1 (VLA4, CD49d/CD29) could be reprecipitated from CD81 immunoprecipitates. Anti-alpha 4 integrin mAb also coprecipitated CD81 from the alpha 4 beta 7-positive B cell line RPMI 8866. In contrast, no CD81 was identified in alpha 2 beta 1, alpha 5 beta 1, or alpha L beta 2 immunoprecipitates. Abs to other members of the transmembrane-4 superfamily, including CD53, CD63, and CD82, also coprecipitated alpha 4 beta 1. As shown by confocal microscopy, CD81 and CD82 colocalized with alpha 4 beta 1 in cell surface clusters. The cytoplasmic domain of the alpha 4 integrin was not necessary for alpha 4 beta 1/CD81 association, nor was the association influenced by divalent cations, EDTA, integrin-activating mAb, or alpha 4 subunit cleavage. Notably, two independent alpha 4 adhesion-deficient mutants (D346E and D408E) were deficient in their ability to associate with CD81. Thus, CD81 and other transmembrane-4 superfamily members may participate in functionally relevant interactions with alpha 4 beta 1 and other integrins.
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Grassi, M., G. Moens, P. Rousselle, J. P. Thiery, and J. Jouanneau. "The SFL activity secreted by metastatic carcinoma cells is related to laminin 5 and mediates cell scattering in an integrin-independent manner." Journal of Cell Science 112, no. 15 (August 1, 1999): 2511–20. http://dx.doi.org/10.1242/jcs.112.15.2511.
Full textAbstract:
We have previously reported that an in vivo-selected metastatic variant of NBT-II rat carcinoma cells, M-NBT-II, produces and secretes a factor with cell-scattering activity, SFL, that is potentially involved in tumor progression. This biological activity was purified and characterized as a laminin 5 (LN5) -related protein. This SFL/LN5 protein consists of the (alpha)3, (beta)3 and (gamma)2 chains of expected sizes. Laminin 5 is a multifunctional secreted glycoprotein thought to be involved in cell adhesion and migration, mainly via its interaction with (alpha)3(beta)1 and (alpha)6(beta)4 integrins. SFL/LN5, and purified human laminin 5, induced the scattering and motility of MDCK cells and the formation of actin stress fibers and focal contacts in A549 cells. These events were dependent on activation of the small GTP-binding protein Rho. (Alpha)v colocalized with vinculin in the focal contacts of activated cells whereas (alpha)3 and (alpha)6 integrins did not. Blocking antibodies directed against (alpha)3 and (alpha)6 integrins or the laminin 5 integrin-binding site did not abolish SFL/LN5 biological activity, which, in contrast, was completely inhibited by heparin. Thus, SFL/LN5 activity in epithelial cell scattering and cytoskeletal reorganization is probably independent of integrin receptors.
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Koivunen, E., B. Wang, and E. Ruoslahti. "Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library." Journal of Cell Biology 124, no. 3 (February 1, 1994): 373–80. http://dx.doi.org/10.1083/jcb.124.3.373.
Full textAbstract:
Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin.
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Salter, D. M., J. L. Godolphin, and M. S. Gourlay. "Chondrocyte heterogeneity: immunohistologically defined variation of integrin expression at different sites in human fetal knees." Journal of Histochemistry & Cytochemistry 43, no. 4 (April 1995): 447–57. http://dx.doi.org/10.1177/43.4.7897185.
Full textAbstract:
During development and at maturity different forms of cartilage vary in morphology and macromolecular content. This reflects heterogeneity of chondrocyte activity, in part involving differential interactions with the adjacent extracellular matrix via specialized cell surface receptors such as integrins. We undertook an immunohistological study on a series of human fetal knee joints to assess variation in the expression of integrins by chondrocytes and potential matrix ligands in articular, epiphyseal, growth plate, and meniscal cartilage. The results show that articular chondrocytes (beta 1+, beta 5 alpha V+, alpha 1+, alpha 2+/-, alpha 5+, weakly alpha 6+, alpha V+) differed from epiphyseal (beta 1+, beta 5 alpha V+, alpha 1+/-, alpha 2+/-, alpha 5+, alpha 6+, alpha V+) growth plate (beta 1+, beta 5 alpha V+, alpha 1-, alpha 2-, alpha 5+, alpha 6+, alpha V+), and meniscal cells (beta 1+, beta 5 alpha V+, alpha 1+, strongly alpha 2+, alpha 5+, alpha 6+, alpha V+ in expression of integrin subunits. There was no expression of beta 3, beta 4, beta 6, or alpha 3 by chondrocytes. These results differ from previous reports on the expression of integrins by adult articular cartilage, where alpha 2 and alpha 6 are not seen. Variation in distribution of matrix ligands was also seen. Fibronectin, laminin and Type VI collagen were expressed in all cartilages but there was restricted expression of tenascin, ED-A and ED-B fibronectin isoforms (articular cartilage and meniscus), and vitronectin (absent from growth plate cartilage). Regulated expression of integrins by chondrocytes, associated with changes in the pericellular matrix composition, is of potential importance in control of cartilage differentiation and function in health and disease.
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Martel, V., L. Vignoud, S. Dupe, P. Frachet, M. R. Block, and C. Albiges-Rizo. "Talin controls the exit of the integrin alpha 5 beta 1 from an early compartment of the secretory pathway." Journal of Cell Science 113, no. 11 (June 1, 2000): 1951–61. http://dx.doi.org/10.1242/jcs.113.11.1951.
Full textAbstract:
Talin is a major cytosolic protein that links the intracellular domains of beta1 and beta3 integrins to the cytoskeleton. It is required for focal adhesion assembly. However, its downregulation not only slows down cell spreading and organization of focal adhesions but also impairs the maturation of some beta1 integrins, including the fibronectin receptor alpha5beta1. To investigate this, we characterized the beta1 integrin synthesized in cells expressing talin anti-sense RNA (AT22 cells). We identified a large intracellular pool of beta1 integrins that is abnormally accumulated in an earlier compartment of the secretory pathway. In this report, we show that in talin-deficient AT22 cells, the aberrant glycosylation of integrin receptors is accompanied by a delay in the export of the integrin alpha5beta1. In normal cells, talin was found associated with beta1 integrins in an enriched membrane fraction containing Golgi and endoplasmic reticulum. Finally, microinjection of anti-talin antibodies resulted in accumulation of the integrins within the cells. These data strongly suggest that talin plays a specific role in the export of newly synthesized integrins. We propose that talin binding to the integrin may disclose a diphenylalanine export signal, which is present in the membrane-proximal GFFKR motif conserved in all integrin alpha chains.
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Damsky, C. H., C. Librach, K. H. Lim, M. L. Fitzgerald, M. T. McMaster, M. Janatpour, Y. Zhou, S. K. Logan, and S. J. Fisher. "Integrin switching regulates normal trophoblast invasion." Development 120, no. 12 (December 1, 1994): 3657–66. http://dx.doi.org/10.1242/dev.120.12.3657.
Full textAbstract:
Cells invade extracellular matrices in a regulated manner at specific times and places during normal development. A dramatic example is trophoblast invasion of the uterine wall. Previous studies have shown that differentiation of trophoblasts to an invasive phenotype is accompanied by temporally and spatially regulated switching of their integrin repertoire. In the first trimester human placenta, alpha 6 integrins are restricted to cytotrophoblast (CTB) stem cells and downregulated in invasive CTBs, whereas alpha 5 beta 1 and alpha 1 beta 1 integrins are upregulated in differentiating and invasive CTBs. The goal of the present study was to determine whether these changes have functional consequences for CTB invasiveness. Using an in vitro invasion model, we determined first that aggregates of invading first trimester CTBs in vitro undergo the same pattern of integrin switching as was observed in situ, thereby validating the utility of the model. We then showed that antibody perturbation of interactions involving laminin or collagen type IV and their integrin alpha 1/beta 1 receptor inhibited invasion by CTBs, whereas perturbing interactions between fibronectin and the alpha 5/beta 1 fibronectin receptor accelerated invasion. Finally, we report that later gestation CTBs, which display greatly decreased invasive capacity, are also unable to upregulate alpha 1 beta 1 complexes, providing further evidence that this integrin is critical for CTB invasion. This gestational regulation is transcriptional. These data indicate that integrin switching observed during differentiation in situ has significant functional consequences for CTB invasion. The data suggest further that differentiating CTBs upregulate counterbalancing invasion-accelerating and invasion-restraining adhesion mechanisms. We propose that this contributes to regulating the depth of CTB invasion during normal implantation.
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Mojcik, CF, DR Salomon, AC Chang, and EM Shevach. "Differential expression of integrins on human thymocyte subpopulations." Blood 86, no. 11 (December 1, 1995): 4206–17. http://dx.doi.org/10.1182/blood.v86.11.4206.bloodjournal86114206.
Full textAbstract:
Integrins represent a candidate group of cell surface receptors that may control the homing and population of the thymus by T-cell precursors and the subsequent migration of developing thymocytes through the thymic architecture. We have used multiparameter flow cytometric methods to characterize the expression of several members of the integrin family (alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, alpha 6 beta 1, and alpha L beta 2) on thymocyte subpopulations and have correlated integrin expression with other well-defined thymocyte differentiation markers. alpha 4 beta 1 was expressed by all thymocytes, but expression was highest on CD4-CD8- double-negative (DN) cells, high on CD+CD8+ double-positive (DP) cells, and lowest on mature single-positive (SP) cells, alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1 were present on 13%, 63%, and 26% of thymocytes, respectively, with maximal levels of expression on DN and SP cells, and low levels of expression on DP cells. Simultaneous analysis of alpha 4 beta 1, alpha 5 beta 1, and CD3 expression suggested a pathway of T-cell differentiation in the thymus in which the majority of the DN cells were alpha 4 beta 1hi alpha 5 beta 1hi, the DP cells alpha 4 beta 1hi alpha 5 beta 1lo/-, and the most mature SP cells were alpha 4 beta 1int. The stage-specific expression of integrins strongly implies their functional involvement during T-cell maturation in the thymus.
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Ylanne, J., DA Cheresh, and I. Virtanen. "Localization of beta 1, beta 3, alpha 5, alpha v, and alpha IIb subunits of the integrin family in spreading human erythroleukemia cells." Blood 76, no. 3 (August 1, 1990): 570–77. http://dx.doi.org/10.1182/blood.v76.3.570.570.
Full textAbstract:
Abstract The localization of five integrin subunit proteins was studied in human erythroleukemia (HEL) cells spreading on various culture substrata in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) and the absence of serum. The cells readily adhered on fibronectin, but TPA was needed for adherence on vitronectin and for the spreading of the cells on both substrata. Indirect immunofluorescence microscopy showed that in the spread cells cultured on vitronectin or fibronectin for 2 hours, beta 1, beta 3, alpha 5, and alpha IIb integrin subunits were localized at focal adhesions as identified by talin-immunoreactivity. The alpha v integrin immunoreactivity was initially found at the focal adhesions when the cells were cultured on vitronectin, but was also found later in cells cultured on fibronectin. The alpha IIb integrin immunoreactivity disappeared from focal adhesions within 24 hours. The alpha 5 and beta 1 integrin immunoreactivities disappeared from the focal adhesions in cells cultured on vitronectin, but not in cells cultured on fibronectin. When the cells were plated on glass substratum in the presence of TPA, they spread much slower than on vitronectin or fibronectin, but some cells showed focal adhesions after only 8 hours in culture. In this case, the alpha v and beta 3 integrin subunits were found at focal adhesions. After TPA treatment, HEL cells deposited thrombospondin-immunoreactive material onto their culture substratum, but synthesis of fibronectin, vitronectin, fibrinogen, or von Willebrand factor was not detected. Thus, the results suggest that TPA would activate several integrin receptors in HEL cells and also stimulate the secretion of thrombospondin, which might be used as an adhesion ligand for the integrin vitronectin receptor alpha v/beta 3 complex.
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Ylanne, J., DA Cheresh, and I. Virtanen. "Localization of beta 1, beta 3, alpha 5, alpha v, and alpha IIb subunits of the integrin family in spreading human erythroleukemia cells." Blood 76, no. 3 (August 1, 1990): 570–77. http://dx.doi.org/10.1182/blood.v76.3.570.bloodjournal763570.
Full textAbstract:
The localization of five integrin subunit proteins was studied in human erythroleukemia (HEL) cells spreading on various culture substrata in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) and the absence of serum. The cells readily adhered on fibronectin, but TPA was needed for adherence on vitronectin and for the spreading of the cells on both substrata. Indirect immunofluorescence microscopy showed that in the spread cells cultured on vitronectin or fibronectin for 2 hours, beta 1, beta 3, alpha 5, and alpha IIb integrin subunits were localized at focal adhesions as identified by talin-immunoreactivity. The alpha v integrin immunoreactivity was initially found at the focal adhesions when the cells were cultured on vitronectin, but was also found later in cells cultured on fibronectin. The alpha IIb integrin immunoreactivity disappeared from focal adhesions within 24 hours. The alpha 5 and beta 1 integrin immunoreactivities disappeared from the focal adhesions in cells cultured on vitronectin, but not in cells cultured on fibronectin. When the cells were plated on glass substratum in the presence of TPA, they spread much slower than on vitronectin or fibronectin, but some cells showed focal adhesions after only 8 hours in culture. In this case, the alpha v and beta 3 integrin subunits were found at focal adhesions. After TPA treatment, HEL cells deposited thrombospondin-immunoreactive material onto their culture substratum, but synthesis of fibronectin, vitronectin, fibrinogen, or von Willebrand factor was not detected. Thus, the results suggest that TPA would activate several integrin receptors in HEL cells and also stimulate the secretion of thrombospondin, which might be used as an adhesion ligand for the integrin vitronectin receptor alpha v/beta 3 complex.
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Maguire, J. E., K. M. Danahey, L. C. Burkly, and G. A. van Seventer. "T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells." Journal of Experimental Medicine 182, no. 6 (December 1, 1995): 2079–90. http://dx.doi.org/10.1084/jem.182.6.2079.
Full textAbstract:
The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.
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Davey, G., M. Buzzai, and R. K. Assoian. "Reduced expression of (alpha)5(beta)1 integrin prevents spreading-dependent cell proliferation." Journal of Cell Science 112, no. 24 (December 15, 1999): 4663–72. http://dx.doi.org/10.1242/jcs.112.24.4663.
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Cell adhesion to substratum results in the initiation of integrin signaling and an integrin-dependent organization of the cytoskeleton (cell spreading). To address the potential relationships between these events and cell proliferation, we transfected NRK fibroblasts with an antisense cDNA encoding a 1.3 kb ATG-spanning portion of (alpha)5 integrin subunit and obtained stable clones in which the surface expression of (alpha)5(beta)1 integrin was selectively reduced. (alpha)5-antisense NRK cells are less spread than the control transfectants, have poorly defined stress fibers, and an increased amount of cortical actin. The antisense clones remained anchorage-dependent, but they proliferated very slowly. Serum dose-response curves showed that they have an impaired response to mitogens. Importantly, cell spreading and stress fiber formation could be completely restored by plating the antisense cells on collagen, but cell spreading failed to rescue proliferation. These data indicate that cell spreading can be uncoupled from cell proliferation and that cytoskeletal organization is important for NRK cell proliferation because it enforces the proliferative effect of (alpha)5(beta)1 integrin. Our results also indicate that reduced surface expression of (alpha)5(beta)1 integrin is not sufficient to confer the anchorage-independent phenotype to nontransformed cells.
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Bauer, JS, J. Varner, C. Schreiner, L. Kornberg, R. Nicholas, and RL Juliano. "Functional role of the cytoplasmic domain of the integrin alpha 5 subunit." Journal of Cell Biology 122, no. 1 (July 1, 1993): 209–21. http://dx.doi.org/10.1083/jcb.122.1.209.
Full textAbstract:
The purpose of this study was to explore the functional role of the cytoplasmic domain of the alpha subunit of the alpha 5/beta 1 integrin, a fibronectin receptor. Mutant CHO cells that express very low levels of endogenous hamster alpha 5 subunit (CHO clone B2) were transfected with an expression vector containing full-length or truncated human alpha 5 cDNAs to form chimeric human alpha 5/hamster beta 1 integrins. Three transfectants were examined: B2a27 expresses a full-length human alpha 5 subunit with 27 amino acids in the cytoplasmic domain; B2a10 expresses an alpha 5 with a 17-amino acid cytoplasmic truncation; B2a1 expresses an alpha 5 with a 26-amino acid truncation. Levels of alpha 5/beta 1 surface expression in B2a27 and B2a10 cells were similar to that in wild type CHO cells. The expression of alpha 5/beta 1 in B2a1 cells was less, amounting to 15-20% of WT levels, despite message levels that were three to five times greater than those of B2a27. The transfectants were used to examine the role of the alpha 5 cytoplasmic domain in cell adhesion, cell motility, cytoskeletal organization, and integrin-mediated tyrosine phosphorylation. The adhesion characteristics of B2a27 and B2a10 cells on fibronectin substrata were similar to each other and to wild type CHO cells. B2a1 cells displayed slight reductions in the strength and rate of adhesion to fibronectin. Cell motility in the presence of fibronectin was similar for B2a27, B2a10, and wild type CHO cells, while the B2a1 cells were substantially less motile. Comparable degrees of cell spreading and extensive organization of actin filaments were observed for B2a27, B2a10, and wild type CHO cells on fibronectin substrata. The B2a1 cells spread to a lesser degree, and some organization of actin was observed; the untransfected B2 cells remained round on fibronectin substrata and showed no actin reorganization. Since the reduced motility and cell spreading observed in the B2a1 cells might be due either to reduced surface expression of alpha 5/beta 1 or to the truncation in the alpha 5 cytoplasmic domain, we used flow cytometric cell sorting to select populations of B2a1 and B2a27 cells expressing similar levels of cell surface alpha 5. The deficits in spreading and motility were present in B2a1 cells expressing high levels of alpha 5. Thus the region of the alpha 5 cytoplasmic domain adjacent to the membrane seems to play an important role in cytoskeletal organization and cell motility. We also examined whether alpha subunit truncation would affect integrin-mediated tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)