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PubMed2003

Formation of a beta1 integrin signaling complex in Schwann cells is independent of rho.

Schwann cell adhesion to basal lamina is essential for peripheral nerve development. beta(1) integrin receptors for extracellular matrix cooperate with other receptors to transmit signals that coordinate cell cycle progression and initiation of differentiation, including myelin-specific gene expression. In Schwann cell/sensory neuron cocultures, beta(1) integrins complex with focal adhesion kinase (FAK), fyn kinase, paxillin, and schwannomin in response to basal lamina adhesion. To study the assembly of this signaling complex in Schwann cells (SCs), we induced beta(1) integrin clustering on suspended cells using an immobilized antibody and recovered a complex containing beta(1) integrin, FAK, paxillin, and schwannomin. In adherent subconfluent cells, the proteins colocalized to filopodia, ruffling membranes and focal contacts. We assessed the role of rhoGTPase in the process of integrin complex assembly by introducing C3 transferase (C3T), a rho inhibitor, into the cells. Although C3T caused dose-dependent morphological abnormalities, FAK, paxillin, and schwannomin were able to coimmunoprecipitate with beta(1) integrin. Additionally, colocalization of FAK, paxillin, and schwannomin with beta(1) integrin in filopodia and small focal contacts remained unchanged. We conclude that SCs do not require active rho to recruit signaling and structural proteins to beta(1) integrins clustered at the plasma membrane. Rho is required to establish large focal adhesions and to spread and stabilize plasma membrane extensions.

ADP Ribose Transferases↗
PubMed1998

[Type I collagen in xenogenic bone material regulates attachment and spreading of osteoblasts over the beta1 integrin subunit].

Xenogenic bone biomaterials have been proposed as an alternative to autografts or allografts in human bone restoring or in complement of prosthetic surgery. When appropriate treatments were applied, immunological, inflammatory, bacteriological or virological adverse responses can be prevented. However, these treatments may interact with type I collagen, the major component of the organic bone matrix. Type I collagen can bind osteoblasts via specific cell surface receptors, the integrins. In this work, two different xenogenic biomaterials were studied. Both biomaterials have a bovine bone origin. They displayed similar architectural organization with connected plates and rods and similar surface topography and roughness. They differed by the presence or not of collagen type I. The first one was characterized by preservation of the type I collagen matrix associated with spindle-shaped hydroxypatite crystals and the second was solely composed by heat-modified apatite crystals. Osteoblast-like cells (Saos-2) were cultured on both biomaterials and examined in scanning and transmission electron microscopy after 7 and 14 days. Both biomaterials were cytocompatible as demonstrated by good ultrastructural cell preservation. (1) At the surface of the collagen containing biomaterial, cells were elongated in shape and oriented according to the trabecular architecture and to the superficial collagen network. After 14 days of culture, cells were confluent and the biomaterial surface was hidden by the cell sheet. The beta 1 integrin subunit was detected by immunogold in transmission electron microscopy in close relationship with the superficial collagen fibres of the biomaterial and with the outer cell surface. When cultures were carried out in presence of anti beta 1 integrin subunit, cells were packed and piled up with lack of specific orientation. (2) At the surface of the deproteinized biomaterial, cells were globular without specific disposition and often partially attached to the surface. After 14 days of culture, large areas of the biomaterial surface remained uncovered. Anti beta 1 subunits conjugated with gold particles were detected around the cells but with no specific association with the deproteinized biomaterial. These results strongly suggest that presence of type I collagen fibres in the matrix of a bone biomaterial is of major interest to determine cell attachment, spreading and orientation via interaction between type I collagen and beta 1 integrin subunit of osteoblasts.

Animals↗
PubMed2004

Platelet-derived growth factor-BB modulates membrane mobility of beta1 integrins.

Platelet-derived growth factor (PDGF)-BB elicits a migratory response including reorganization of the actin cytoskeleton in different cell types. Here we have investigated the effects of PDGF-BB stimulation on beta(1) integrin containing focal adhesions in human diploid fibroblasts adhered to collagen type I. Stimulation with PDGF-BB dissociated focal adhesions and relocated beta(1) integrins from focal adhesions to the periphery of the cells. These changes were rapid and transient in character. Relocation of beta(1) integrins was prevented by inhibitors of phosphoinositide-3-kinase and protein kinase C. PDGF-BB stimulated fibroblasts exhibited an increased diffusion coefficient of cell surface beta(1) integrins as determined by fluorescence recovery of photobleaching. The cell surface expression of beta(1) integrins was not changed after stimulation with PDGF-BB. Our data suggest that PDGF-BB increases the dynamic properties of cell-surface beta(1) integrins, which most likely are important for the migratory response elicited by PDGF-BB.

Becaplermin↗
PubMed2006

Coculture with endothelial cells enhances vascular smooth muscle cell adhesion and spreading via activation of beta1-integrin and phosphatidylinositol 3-kinase/Akt.

The interactions between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) play significant roles in the homeostasis of the blood vessel during vascular remodeling. Cell adhesion and spreading are an essential process for VSMC migration, survival and proliferation in the events of vascular physiology and pathophysiology. However, effects of ECs on adhesion and spreading of VSMCs have not been characterized yet. Here, the interaction of ECs and VSMCs on adhesion and spreading of VSMCs were investigated by using a coculture system. The results showed that VSMCs cocultured with ECs exhibited a significant increase in the number of adherent and spreading cells, and much more mRNA (twofold, P<0.01) and protein (threefold, P<0.05) expression of beta(1)-integrin comparing to the control, i.e., VSMCs cultured alone. Furthermore, the enhanced functional activity of beta(1)-integrin expression was confirmed by FACS. A beta(1)-integrin blocking antibody (P5D2) could inhibit the EC-induced VSMC adhesion and spreading. It was demonstrated that in correspondence with enhanced cell adhesion, ECs also prompted focal adhesion complex assembly and stress fiber formation of VSMCs. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway was more pronouncedly activated in response to VSMC attachment. Our results for the first time show that coculture with ECs enhances VSMC adhesion and spreading by up-regulating beta(1)-integrin expression and activating the PI3K/Akt pathway, suggesting that the interaction between ECs and VSMCs serves an important role in vascular homeostasis and remodeling.

Cell Adhesion↗
PubMed2005

Differential effects of echistatin and thrombin on collagen production and prolidase activity in human dermal fibroblasts and their possible implication in beta1-integrin-mediated signaling.

Prolidase [E.C. 3.4.13.9] is a cytosolic imidodipeptidase that plays an important role in collagen biosynthesis. The enzyme contributes to the recovery of proline from protein degradation products (mainly collagen) for collagen resynthesis. Prolidase activity and collagen biosynthesis are supposed to be regulated by beta(1)-integrins, which initiate a signaling pathway in which several kinases and intracellular proteins are involved, including focal adhesion kinase pp125(FAK) (FAK), Src, Shc, growth factor receptor bound protein 2 (Grb-2), son of sevenless protein (SOS), Ras, Raf and mitogen-activated protein kinases (MAPK), extracellular-signal regulated kinase 1 (ERK(1)) and kinase 2 (ERK(2)). We studied the effects of echistatin, a well-known disintegrin and thrombin, a serine protease capable of activation of platelet integrin alpha(2)beta(1) receptor on collagen production, prolidase activity, expression of prolidase, beta(1)-integrin receptor, FAK, SOS-protein and phosphorylated MAP-kinases (ERK(1) and ERK(2)) in confluent human dermal fibroblasts. It has been found that treatment of the cells with 100nM echistatin contributes to inhibition of collagen production, as well as prolidase activity and expression compared to control cells. These phenomena were accompanied by a decrease in the expression of FAK, SOS-protein and phosphorylated MAP-kinases, ERK(1) and ERK(2). An opposite phenomenon was observed in fibroblasts treated with 0.1IU thrombin. In this case, a significant increase in collagen production and prolidase activity, accompanied by a distinct raise in the expression of prolidase, FAK and phosphorylated MAP-kinases and a slight increase in expression of SOS compared to controls were found. The results suggest that regulation of prolidase activity and collagen biosynthesis in human dermal fibroblasts may involve beta(1)-integrin-dependent signaling.

Cells, Cultured↗
PubMed2005

PKB mediates c-erbB2-induced epithelial beta1 integrin conformational inactivation through Rho-independent F-actin rearrangements.

Signalling from the growth factor receptor subunit and proto-oncogene c-erbB2 has been shown to inhibit the adhesive function of the collagen receptor integrin alpha(2)beta(1) in human mammary epithelial cells. This anti-adhesive effect is mediated by the MAP ERK kinase 1/2 (MEK1/2) and protein kinase B (PKB) pathways. Here, we show that both pathways mediate suppression of matrix adhesion by causing the extracellular domain of the beta(1) integrin subunit to adopt an inactive conformation. The conformational switch was also dependent on rapid and extensive actin depolymerisation. While neither activation nor inhibition of the Rho GTPase affected this rearrangement, Rho was found to be activated by c-erbB2 and to be necessary for conformation-dependent integrin inactivation and, apparently by a different mechanism, a delayed re-formation of stress fibers which did not restore integrin function. Interestingly, the initial actin depolymerisation as well as its effects on integrin function was shown to be mediated by PKB. These results demonstrate how oncogenic growth factor signalling inhibits matrix adhesion by multiple pathways converging on integrin conformation and how Rho signalling can profoundly influence integrin activation in a cytoskeleton-independent manner.

Actins↗
PubMed2003

Tensile force-dependent neurite elicitation via anti-beta1 integrin antibody-coated magnetic beads.

Previous work using glass microneedles to apply calibrated, localized force to neurons showed that tensile force is a sufficient signal for neurite initiation and elongation. However, previous studies did not examine the kinetics or probability of neurite initiation as a function of force or the rate of force application. Here we report the use of a new technique-magnetic bead force application-to systematically investigate the role of force in these phenomena with better ease of use and control over force than glass microneedles. Force-induced neurite initiation from embryonic chick forebrain neurons appeared to be a first-order random process whose rate increased with increasing force, and required the presence of peripheral microtubules. In addition, the probability of initiation was more than twofold lower for neurons exposed to rapid initial force ramps (450 pN/s) than for neurons exposed to slower ramps (1.5 and 11 pN/s). We observed a low force threshold for elongation (15-100 pN), which was not previously detected in chick forebrain neurites elongated by glass microneedles. Finally, neurites subjected to constant force elongated at variable instantaneous rates, and switched abruptly between elongation and retraction, similar to spontaneous, growth-cone-mediated outgrowth and microtubule dynamic instability.

Animals↗
PubMed2003

Role of multiple beta1 integrins in cell adhesion to the disintegrin domains of ADAMs 2 and 3.

ADAM disintegrin domains can support integrin-mediated cell adhesion. However, the profile of which integrins are employed for adhesion to a given disintegrin domain remains unclear. For example, we suggested that the disintegrin domains of mouse sperm ADAMs 2 and 3 can interact with the alpha6beta1 integrin on mouse eggs. Others concluded that these disintegrin domains interact instead with the alpha9beta1 integrin. To address these differing results, we first studied adhesion of mouse F9 embryonal carcinoma cells and human G361 melanoma cells to the disintegrin domains of mouse ADAMs 2 and 3. Both cell lines express alpha6beta1 and alpha9beta1 integrins at their surfaces. Antibodies to the alpha6 integrin subunit inhibited adhesion of both cell lines. An antibody that recognizes human alpha9 integrin inhibited adhesion of G361 cells. VLO5, a snake disintegrin that antagonizes alpha4beta1 and alpha9beta1 integrins, potently inhibited adhesion of both cell lines. We next explored expression of the alpha9 integrin subunit in mouse eggs. In contrast to our ability to detect alpha6beta1, we were unable to convincingly detect alpha9beta1 integrin on the surface of mouse eggs. Moreover, treatment of mouse eggs with 250 nm VLO5, which is 250 fold over its approximately IC(50) for inhibition of somatic cell adhesion, had minimal effect on sperm-egg binding or fusion. We did detect alpha9 integrin protein on epithelial cells of the oviduct. Additional studies showed that antibodies to the alpha6 and alpha7 integrins additively inhibited adhesion of mouse trophoblast stem cells and that an antibody to the alpha4 integrin inhibited adhesion of MOLT-3 cells to these disintegrin domains: Our data suggest that multiple integrins (on the same cell) can participate in adhesion to a given ADAM disintegrin domain and that interactions between ADAMs and integrins may be important for sperm transit through the oviduct.

ADAM Proteins↗
PubMed2000

Cytoplasmic domain mutants of beta1 integrin, expressed in beta 1-knockout lymphoma cells, have distinct effects on adhesion, invasion and metastasis.

Structural requirements for beta 1 integrin cytoplasmic domain functions in adhesion, migration and signaling have been studied mainly for fibroblasts in vitro. The relevance for beta 1-dependent in vivo migration of lymphoid cells has not been assessed. To study this, we transfected beta 1 mutants into beta 1-deficient double knockout (DKO) ESb lymphoma cells, and tested the capacity of the cells to metastasize to liver and spleen. This was compared to alpha 4 beta 1-dependent invasion into cell monolayers in vitro and Mn2+-induced adhesion to fibronectin. Deletion of the five C-terminal residues or mutation of both threonines T788 and T789 to alanines blocked invasion and metastasis and greatly reduced adhesion, in line with known in vitro effects. However, mutations of the NPXY motif tyrosines had unexpected consequences. A Y783F mutation had no effect at all, but a Y783,795F double mutation strongly reduced Mn2+-induced adhesion, whereas it had limited effects on invasion and metastasis. Furthermore, cells expressing a beta 1 beta 2 chimeric subunit, which contains phenylalanines in the NPXY/F motifs, adhered poorly but invasion and metastasis was fully restored to the same levels as for cells expressing wild-type beta 1. We conclude that part of the functions of the beta 1 cytoplasmic domain that are required for adhesion are not essential for beta 1-dependent invasion and metastasis.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Beta1 integrin and alpha-dystroglycan binding sites are localized to different laminin-G-domain-like (LG) modules within the laminin alpha5 chain G domain.

Laminins are a group of extracellular-matrix proteins important in development and disease. They are heterotrimers, and specific domains in the different chains have specialized functions. The G domain of the alpha5 chain has now been produced in transfected mammalian cells as single modules and two tandem arrays, alpha5LG1-3 and alpha5LG4-5 (LG is laminin G domain-like). Using these fragments we produced specific polyclonal antibodies functional in immunoblotting and immunofluorescence studies and in solid-phase assays. Both alpha5LG tandem arrays had physiologically relevant affinities for sulphated ligands such as heparin and sulphatides. Cells adhered to these fragments and acquired a spread morphology when plated on alpha5LG1-3. Binding of integrins alpha3beta1 and alpha6beta1 was localized to the alpha5LG1-3 modules, and alpha-dystroglycan binding was localized to the alpha5LG4-5 modules, thus locating these activities to different LG modules within the laminin alpha5 G domain. However, both these activities were of relatively low affinity, indicating that integrin-mediated cell adhesion to the laminin 10/11 alpha5G domain depends on contributions from the other chains of the heterotrimer and that high-affinity alpha-dystroglycan binding could be dependent on specific Ca(2+)-ion-co-ordinating amino acids absent from alpha5LG4-5.

Animals↗
PubMed2003

NADPH oxidase activation in fibronectin adherent human neutrophils: A potential role for beta1 integrin ligation.

BACKGROUND: Hydrogen peroxide production by human neutrophils is tightly coupled to integrin ligation. This provides a means for spatial localization of oxidant production. However, integrin specificity and consequent signaling mechanisms for this process remain undefined. In the present study we demonstrate that otherwise unstimulated neutrophils adherent to fibronectin, a beta(1) ligand, but not fibrinogen, a beta(2) ligand, produce hydrogen peroxide in a time-dependent fashion. We hypothesized that signaling proceeded through focal, adhesionlike structures. METHODS: Triton-X insoluble (actin cytoskeleton) fractions from suspension phase cells and cells adherent to the beta(1) integrin ligand fibronectin were assessed for the presence of the integrin-associated kinase Pyk2, the scaffolding protein paxillin, and the downstream Src family kinase Lyn. Lysates were subjected to immunoprecipitation with anti-phosphotyrosine and probed for Pyk2, paxillin, and Lyn. Associations between focal adhesion kinase (FAK) and paxillin were determined by immunoprecipitation with anti-FAK and probing with anti-paxillin antibody. Activation of NADPH oxidase was determined by demonstration of redistribution of p47(phox) in Triton-X insoluble fractions. RESULTS: NADPH oxidase activation, as judged by H(2)O(2) production, occurred with fibronectin-, but not in suspension or fibrinogen-adherent cells. Cells adherent to fibronectin for 20 minutes demonstrated marked increases in Pyk2, paxillin, and Lyn activation in comparison to fibronectin-adherent and suspension phase cells. CONCLUSIONS: This study demonstrates that fibronectin adherence is a significant initiating factor for NADPH oxidase assembly in human neutrophils. This appears to be mediated by beta(1) integrins. We demonstrate formation of focal adhesions containing Pyk2/FAK, paxillin, and Lyn, and translocation of p47(phox).

Cell Adhesion↗
PubMed2002

A functionally relevant conformational epitope on the CD9 tetraspanin depends on the association with activated beta1 integrin.

Tetraspanins associate on the cell membrane with several transmembrane proteins, including members of the integrin superfamily. The tetraspanin CD9 has been implicated in cell motility, metastasis, and sperm-egg fusion. In this study we characterize the first CD9 conformation-dependent epitope (detected by monoclonal antibody (mAb) PAINS-13) whose expression depends on changes in the activation state of associated beta(1) integrins. MAb PAINS-13 precipitates CD9 under conditions that preserve the association of this tetraspanin with integrins, but not under conditions that disrupt these interactions. Induction of activation of beta(1) integrins by temperature, divalent cation Mn(2+), or mAb TS2/16 correlated with enhanced expression of the PAINS-13 epitope on a variety of cells. Through the use of different K562 myeloid leukemia transfectant cells expressing specific members of the beta(1) integrin subfamily we show that the expression of the PAINS-13 epitope depends on CD9 association with alpha(6)beta(1) integrin. The mAb PAINS-13 reactivity has been mapped to the CD9 region comprising residues 112-154 in the NH(2) half of the large extracellular loop. Also, we show that the CD9 conformation recognized by mAb PAINS-13 is functionally relevant in beta(1) integrin-mediated cellular processes including wound healing migration, tubular morphogenesis, cell adhesion and spreading and in signal transduction involving phosphatidylinositol 3-kinase activation.

Animals↗
PubMed2003

Low density lipoprotein receptor-related protein-1 promotes beta1 integrin maturation and transport to the cell surface.

Low density lipoprotein receptor-related protein-1 (LRP-1) mediates the endocytosis of multiple plasma membrane proteins and thereby models the composition of the cell surface. LRP-1 also functions as a catabolic receptor for fibronectin, limiting fibronectin accumulation in association with cells. The goal of the present study was to determine whether LRP-1 regulates cell surface levels of the beta(1) integrin subunit. We hypothesized that LRP-1 may down-regulate cell surface beta(1) by promoting its internalization; however, unexpectedly, LRP-1 expression was associated with a substantial increase in cell surface beta(1) integrin in two separate cell lines, murine embryonic fibroblasts (MEFs) and CHO cells. The total amount of beta(1) integrin was unchanged because LRP-1-deficient cells retained increased amounts of beta(1) in the endoplasmic reticulum (ER). Expression of human LRP-1 in LRP-1-deficient MEFs reversed the shift in subcellular beta(1) integrin distribution. Metabolic labeling experiments demonstrated that the precursor form of newly synthesized beta(1) integrin (p105) is converted into mature beta(1) (p125) more slowly in LRP-1-deficient cells. Although low levels of cell surface beta(1) integrin, in LRP-1-deficient MEFs, were associated with decreased adhesion to fibronectin, the subcellular distribution of beta(1) integrin was most profoundly dependent on LRP-1 only after the cell cultures became confluent. A mutagen-treated CHO cell line, in which LRP-1 is expressed but retained in the secretory pathway, also demonstrated nearly complete ER retention of beta(1) integrin. These studies support a model in which LRP-1 either directly or indirectly promotes maturation of beta(1) integrin precursor and thereby increases the level of beta(1) integrin at the cell surface.

Animals↗
PubMed2003

Enterophilin-1 interacts with focal adhesion kinase and decreases beta1 integrins in intestinal Caco-2 cells.

Intestinal cell growth and differentiation are tightly regulated by growth factors and extracellular matrix components along the crypt-villus axis. We previously described enterophilin-1 (Ent-1) as a new intestinal protein associated with growth arrest and enterocyte differentiation. Ent-1 interacted with sorting nexin 1 and decreased cell surface epidermal growth factor receptor. Because beta(1) integrins are mostly found in vivo in the proliferative crypt cells, we investigated the role of Ent-1 in the fate of beta(1) integrin subunits. In undifferentiated intestinal Caco-2 cells, overexpression of Ent-1 induces a marked decrease of alpha(5)beta(1) integrin pools, whereas alpha(2)beta(1) integrin is weakly affected. Conversely, overexpression of sorting nexin 1 has no effect on integrin levels despite its ability to interact with Ent-1. Interestingly, we identified focal adhesion kinase as a new Ent-1 partner using yeast two-hybrid screening and co-precipitation experiments. Furthermore by confocal microscopy, we observed that Ent-1 and beta(1) integrins partly co-localize on vesicular structures, suggesting a role for Ent-1 in integrin trafficking. Because focal adhesion kinase is able to bind both Ent-1 and beta(1) integrins, the kinase might act as a molecular bridge between the two proteins. Altogether, these results support a role of Ent-1 in regulating beta(1) integrin expression that could favor intestinal differentiation.

Caco-2 Cells↗
PubMed2004

Allosteric beta1 integrin antibodies that stabilize the low affinity state by preventing the swing-out of the hybrid domain.

The ligand binding function of integrins can be modulated by various monoclonal antibodies by both direct and indirect mechanisms. We have characterized an anti-beta(1) antibody, SG/19, that had been reported to inhibit the function of the beta(1) integrin on the cell surface. SG/19 recognized the wild type beta(1) subunit that exists in a conformational equilibrium between the high and low affinity states but bound poorly to a mutant beta(1) integrin that had been locked in a high affinity state. Epitope mapping of SG/19 revealed that Thr(82) in the beta(1) subunit, located at the outer face of the boundary between the I-like and hybrid domains, was the key binding determinant for this antibody. Direct visualization of the alpha (5)beta(1) headpiece fragment in complex with SG/19 Fab with electron microscopy confirmed the location of the binding surface and showed that the ligand binding site is not occluded by the bound Fab. Surface plasmon resonance showed that alpha (5)beta(1) integrin bound by SG/19 maintained a low affinity toward its physiological ligand fibronectin (Fn) whereas binding by function-blocking anti-alpha(5) antibodies resulted in a complete loss of fibronectin binding. Thus a class of the anti-beta antibodies represented by SG/19 attenuate the ligand binding function by restricting the conformational shift to the high affinity state involving the swing-out of the hybrid domain without directly interfering with ligand docking.

Allosteric Regulation↗
PubMed2004

Expression of the vacuolar H+-ATPase 16-kDa subunit results in the Triton X-100-insoluble aggregation of beta1 integrin and reduction of its cell surface expression.

Vacuolar H(+)-ATPase functions as a vacuolar proton pump and is responsible for acidification of intracellular compartments such as the endoplasmic reticulum, Golgi, lysosomes, and endosomes. Previous reports have demonstrated that a 16-kDa subunit (16K) of vacuolar H(+)-ATPase via one of its transmembrane domains, TMD4, strongly associates with beta(1) integrin, affecting beta(1) integrin N-linked glycosylation and inhibiting its function as a matrix adhesion receptor. Because of this dramatic inhibition of beta(1) integrin-mediated HEK-293 cell motility by 16K expression, we investigated the mechanism by which 16 kDa was having this effect. Using HT1080 cells whose alpha(5)beta(1) integrin-mediated adhesion to fibronectin has been extensively studied, the expression of 16 kDa also resulted in reduced cell spreading on fibronectin-coated substrates. A pulse-chase study of beta(1) integrin biosynthesis indicated that 16K expression down-regulated the level of the 110-kDa biosynthetic form of beta(1) integrin (premature form) and, consequently, the level of the 130-kDa form of beta(1) integrin (mature form). Further experiments showed that the normal levels of association between the premature beta(1) integrin form and calnexin were significantly decreased by the expression of either 16 kDa or TMD4. Expression of 16 kDa also resulted in a Triton X-100-insoluble aggregation of an unusual 87-kDa form of beta(1) integrin. Interestingly, both Western blotting and a pulse-chase experiment showed co-immunoprecipitation of calnexin and 16K. These results indicate that 16K expression inhibits beta(1) integrin surface expression and spreading on matrix by a novel mechanism that results in reduced levels of functional beta(1) integrin.

Biotinylation↗
PubMed2004

Identification of novel beta1 integrin binding sites in the type 1 and type 2 repeats of thrombospondin-1.

In addition to the three known beta(1) integrin recognition sites in the N-module of thrombospondin-1 (TSP1), we found that beta(1) integrins mediate cell adhesion to the type 1 and type 2 repeats. The type 1 repeats of TSP1 differ from typical integrin ligands in that recognition is pan-beta(1)-specific. Adhesion of cells that express one dominant beta(1) integrin on immobilized type 1 repeats is specifically inhibited by antagonists of that integrin, whereas adhesion of cells that express several beta(1) integrins is partially inhibited by each alpha-subunit-specific antagonist and completely inhibited by combining the antagonists. beta(1) integrins recognize both the second and third type 1 repeats, and each type 1 repeat shows pan-beta(1) specificity and divalent cation dependence for promoting cell adhesion. Adhesion to the type 2 repeats is less sensitive to alpha-subunit antagonists, but a beta(1) blocking antibody and two disintegrins inhibit adhesion to immobilized type 2 repeats. beta(1) integrin expression is necessary for cell adhesion to the type 1 or type 2 repeats, and beta(1) integrins bind in a divalent cation-dependent manner to a type 1 repeat affinity column. The widely used TSP1 function blocking antibody A4.1 binds to a site in the third type 2 repeat. A4.1 proximally inhibits beta(1) integrin-dependent adhesion to the type 2 repeats and indirectly inhibits integrin-dependent adhesion mediated by the TSP1 type 1 repeats. Although antibody A4.1 is also an antagonist of CD36 binding to TSP1, these data suggest that some biological activities of A4.1 result from antagonism of these novel beta(1) integrin binding sites.

Antibodies, Monoclonal↗