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At least 307 records · Page 17Linked to original sources
PubMed1993

Homotypic leukocyte aggregation triggered by a monoclonal antibody specific for a novel epitope expressed by the integrin beta 1 subunit: conversion of nonresponsive cells by transfecting human integrin alpha 4 subunit cDNA.

The monoclonal antibody 33B6 was found to be specific for the beta 1 integrin subunit. Treatment of leukocytes with this antibody induced a vigorous homotypic aggregation that had similar physiologic conditions as aggregation induced by a monoclonal antibody specific for the alpha 4 subunit. Expression of a beta 1 subunit on the cell surface was not sufficient for mAb 33B6-mediated aggregation to occur, since cells of the K562 erythroleukemia line failed to respond even though they expressed the beta 1 subunit and the 33B6 epitope. However, after transfection with cDNA encoding the alpha 4 subunit, K562 cells acquired the ability to aggregate in response to mAb 33B6 binding. By contrast, mAb 33B6 blocked cell binding to the endothelial surface protein vascular cell adhesion molecule-1 and the extracellular matrix protein fibronectin. These results suggest that the beta 1 epitope defined by mAb 33B6 may play a novel role in regulating leukocyte adhesive interactions.

Animals↗
PubMed2002

Regulation of integrin function by CD47 ligands. Differential effects on alpha vbeta 3 and alpha 4beta1 integrin-mediated adhesion.

We examined the regulation of alpha4beta1 integrin function in melanoma cells and T cells by ligands of CD47. A CD47 antibody (B6H12) that inhibited alphavbeta3-mediated adhesion of melanoma cells induced by CD47-binding peptides from thrombospondin-1 directly stimulated alpha4beta1-mediated adhesion of the same cells to vascular cell adhesion molecule-1 and N-terminal regions of thrombospondin-1 or thrombospondin-2. B6H12 also stimulated alpha4beta1- as well as alpha2beta1- and alpha5beta1-mediated adhesion of CD47-expressing T cells but not of CD47-deficient T cells. alpha4beta1 and CD47 co-purified as a detergent-stable complex on a CD47 antibody affinity column. CD47-binding peptides based on C-terminal sequences of thrombospondin-1 also specifically enhanced adhesion of melanoma cells and T cells to alpha4beta1 ligands. Unexpectedly, activation of alpha4beta1 function by the thrombospondin-1 CD47-binding peptides also occurred in CD47-deficient T cells. CD47-independent activation of alpha4beta1 required the Val-Val-Met (VVM) motif of the peptides and was sensitive to inhibition by pertussis toxin. These results indicate that activation of alpha4beta1 by the CD47 antibody B6H12 and by VVM peptides occurs by different mechanisms. The antibody directly activates a CD47-alpha4beta1 complex, whereas VVM peptides may target an unidentified Gi-linked receptor that regulates alpha4beta1.

Amino Acid Sequence↗
PubMed1997

The epithelial cell-specific integrin, CD103 (alpha E integrin), defines a novel subset of alloreactive CD8+ CTL.

The interaction of CD8+CTL with epithelial layers is an important but poorly defined aspect of organ allograft rejection. We herein report that CD103 (formerly alpha E integrin), a known receptor for the epithelial cell-specific ligand E-cadherin, is expressed by a major subset of CD8 + CTL elicited in response to allogeneic renal epithelial cells (REC). In contrast, CD103 was expressed poorly on CD8 + CTL generated in the conventional manner by stimulation with allogeneic leukocytes, although expression could be dramatically up-regulated by supplementing cultures with REC or exogenous TGF-beta 1. That TGF-beta controls the expression of CD103 on CD8+ CTL was further supported by the capacity of anti-TGF-beta mAb to block the generation of such cells in anti-REC cultures. Clonal analyses of anti-REC cultures revealed that individual CD8+ CTL clones were discretely CD103+ or CD103-, nd maintained their respective phenotypes independently of the cell type used for clonal restimulation. In a mouse model of graft-vs-host disease, 16.4 +/- 2.7% of CD8 cells that infiltrated host kidneys were CD103+ (n = 4). CD8 kidney-infiltrating lymphocytes were predominantly of donor origin and displayed an activated/memory phenotype (CD62L-, CD44high), consistent with expression of CD103 on a CD8 effector subset elicited in vivo following allogeneic transplantation. Taken together, the present data demonstrate that CD103 identifies a novel CD8 effector subset and, moreover, that such cells may comprise a significant component of the response to allogeneic tissues. The potential for CD103+ CTL as an important effector mechanism in organ allograft rejection, and more generally, as a mechanistic basis for tissue-specific immune phenomena, is discussed.

Animals↗
PubMed1999

An equilibrium model of endothelial cell adhesion via integrin-dependent and integrin-independent ligands.

Endothelial cell adhesion can be enhanced by supplementing integrin-mediated adhesion via fibronectin with the high-affinity avidin-biotin system in which biotin is covalently linked to membrane proteins and avidin binds to biotinylated surfaces (Bhat et al. J Biomed Mater Res 1998;41:377-85). An equilibrium model was extended to explain detachment of spreading cells following exposure to flow for this two ligand system. The two different receptor-ligand systems were treated as springs in parallel in which the equilibrium dissociation constant was a function of the separation distance of the cell from the surface. Flow experiments were performed to measure the endothelial cell adhesion strength as a function of the extent of biotinylation of the endothelium. Surfaces contained adsorbed fibronectin, avidin or both ligands. The contact area between the cell membrane and substrate was measured using total internal reflection fluorescence microscopy. Estimates of the unstressed dissociation constant for fibronectin and avidin were determined from data for adhesion strength and contact area of each ligand separately. Using these unstressed equilibrium constants, the model predicted, with reasonable accuracy, the strength of endothelial cell adhesion to surfaces containing fibronectin and avidin. The results indicate that as the extent of biotinylation increases, the avidin-biotin system contributes a larger fraction of the total adhesion strength but the maximum contribution of the avidin-biotin system is less than 50%. The magnitude of the affinity constant and force per bond for the avidin-biotin system are consistent with detachment by extraction of receptors from the cell. The resulting increase in the adhesion strength on surfaces with both avidin-biotin and fibronectin is due to the increase in contact area and the larger number of bonds formed.

Animals↗
PubMed2002

Two new beta3 integrin mutations in Indian patients with Glanzmann thrombasthenia: localization of mutations affecting cysteine residues in integrin beta3.

New mutations in the beta3 integrin subunit have been identified in two unrelated Glanzmann thrombasthenia patients originating from India and Bangladesh. Both patients had histories of excessive bleeding and were found to have Glanzmann thrombasthenia based on absent ADP-induced platelet aggregation. Immunoblotting of platelet lysates of Patient 1 demonstrated reduced levels of alphaIIb and an unexpected high Mr beta3 band of approximately 260,000, with little or no normal-sized beta3. Upon reduction, a weak beta3 band of normal Mr was observed. Platelet lysates of Patient 2 demonstrated undetectable levels of beta3. Sequence analyses identified homozygous mutations in the beta3 genes of both patients. Patient 1 had a C506Y missense mutation resulting in the expression of an unpaired cysteine; we propose that the Mr approximately 260,000 band is a disulfide-bonded beta3 dimer. Patient 2 had an insertion mutation resulting in a frameshift and premature termination. Both mutations affect biogenesis of platelet alphaIIbbeta3 receptors.

Adult↗
PubMed2004

Integrin beta3 regions controlling binding of murine mAb 7E3: implications for the mechanism of integrin alphaIIbbeta3 activation.

Abciximab, a derivative of the murine mAb 7E3, protects against ischemic complications of percutaneous coronary interventions by inhibiting ligand binding to the alphaIIbbeta3 receptor. In this study we identified regions on integrin beta3 that control 7E3 binding. Murine/human amino acid substitutions were created in two regions of the betaA domain that previous studies found to influence 7E3 binding: the C177-C184 loop and K125-N133. The T182N substitution and a K125Q mutation reduced 7E3 binding to human beta3 in complex with alphaIIb. The introduction of both the human C177-C184 region and human W129 into murine beta3 was necessary and sufficient to permit 7E3 binding to the human alphaIIb/murine beta3 complex. Although we cannot exclude allosteric effects, we propose that 7E3 binds between C177-C184 and W129, which are within 15 A of each other in the crystal structure and close to the beta3 metal ion-dependent adhesion site. We previously demonstrated that 7E3 binds more rapidly to activated than unactivated platelets. Because it has been proposed that alphaIIbbeta3 changes from a bent to an extended conformation upon activation, we hypothesized that 7E3 binds less well to the bent than the extended conformation. In support of this hypothesis we found that 7E3 bound less well to an alphaIIbbeta3 construct locked in a bent conformation, and unlocking the conformation restored 7E3 binding. Thus, our data are consistent with alphaIIbbeta3 existing in variably bent conformations in equilibrium with each other on unactivated platelets, and activation resulting in alphaIIbbeta3 adopting a more extended conformation.

Amino Acid Substitution↗
PubMed1998

Distinct involvement of beta3 integrin cytoplasmic domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal assembly and phosphotyrosine signaling.

We have investigated the structural requirements of the beta3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during alphav beta3-mediated cell spreading. Using CHO cells transfected with various beta3 mutants, we demonstrate a close correlation between alphav beta3-mediated cell spreading and tyrosine phosphorylation of FAK and paxillin, and highlight a distinct involvement of the NPLY747 and NITY759 motifs in these signaling processes. Deletion of the NITY759 motif alone was sufficient to completely prevent alphav beta3-dependent focal contact formation, cell spreading, and FAK/paxillin phosphorylation. The single Y759A substitution induced a strong inhibitory phenotype, while the more conservative, but still phosphorylation-defective, Y759F mutation restored wild type receptor function. Alanine substitution of the highly conserved Tyr747 completely abolished alphav beta3-dependent formation of focal adhesion plaques, cell spreading, and FAK/paxillin phosphorylation, whereas a Y747F substitution only partially restored these events. As none of these mutations affected receptor-ligand interaction, our results suggest that the structural integrity of the NITY759 motif, rather than the phosphorylation status of Tyr759 is important for beta3-mediated cytoskeleton reorganization and tyrosine phosphorylation of FAK and paxillin, while the presence of Tyr at residue 747 within the NPLY747 motif is required for optimal beta3 post-ligand binding events.

Amino Acid Sequence↗
PubMed2001

Modulation of integrin signal transduction by ILKAP, a protein phosphatase 2C associating with the integrin-linked kinase, ILK1.

ILKAP, a protein serine/threonine (S/T) phosphatase of the PP2C family, was isolated in a yeast two-hybrid screen baited with integrin-linked kinase, ILK1. Association of ILK1 and ILKAP was independent of the catalytic activity of either partner, as assayed in co-precipitation and two-hybrid experiments. Condi tional expression of ILKAP in HEK 293 cells resulted in selective inhibition of ECM- and growth factor-stimulated ILK1 activity, but did not inhibit Raf-1 kinase activity. A catalytic mutant of ILKAP, H154D, did not inhibit ILK1 kinase activity. Two cellular targets of ILK1, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase B (PKB)/AKT, were differentially affected by ILKAP-mediated inhibition of ILK1. Catalytically active, but not mutant ILKAP, strongly inhibited insulin-like growth factor-1-stimulated GSK3beta phosphorylation on Ser9, but did not affect phosphorylation of PKB on Ser473, suggesting that ILKAP selectively affects ILK-mediated GSK3beta signalling. Consistent with this, active, but not H154D mutant or the related PP2Calpha, selectively inhibited transactivation of a Tcf/Lef reporter gene, TOPFlash, in 293 cells. We propose that ILKAP regulates ILK1 activity, targeting ILK1 signalling of Wnt pathway components via modulation of GSK3beta phosphorylation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1991

Plasticity of integrin expression by nerve-derived connective tissue cells. Human Schwann cells, perineurial cells, and fibroblasts express markedly different patterns of beta 1 integrins during nerve development, neoplasia, and in vitro.

Strikingly selective expression patterns of beta 1, alpha 2, alpha 3, and alpha 5 integrin subunits were revealed in endoneurium, perineurium, and epineurium of fetal and adult human peripheral nerve by immunostaining with specific antibodies. The alpha 2 subunit was expressed only on Schwann cells both in fetal and adult nerve, whereas the alpha 3 epitopes were expressed exclusively in the adult tissue and were primarily present on perineurial cells. The alpha 5 epitopes were expressed only on the innermost cell layer of perineurium of fetal and adult nerve. The tumor cells within schwannomas and cutaneous neurofibromas expressed both alpha 2 and alpha 3 subunits, indicating that Schwann cells have the potential to express also the alpha 3 subunit in vivo. Cell cultures established from human fetal nerve and neurofibromas revealed expression of the alpha 2 and alpha 5 epitopes on Schwann cells, perineurial cells, and fibroblasts, whereas only Schwann cells contained the alpha 3 epitopes which were occasionally concentrated on the adjacent Schwann cells at cell-cell contacts. Our findings emphasize that nerve connective tissue cells change their profiles for expression of extracellular matrix receptors under conditions which have different regulatory control signals exerted by, for example, axons, humoral factors, or the extracellular matrix of the peripheral nerve. This plasticity may play an important role during nerve development and in neoplastic processes affecting the connective tissue compartments of peripheral nerve.

Antibodies, Monoclonal↗
PubMed2004

Mutations in and near the second calcium-binding domain of integrin alphaIIb affect the structure and function of integrin alphaIIbbeta3.

Calcium-binding domains in the alpha-subunit of integrins contain a central loop structure. To examine the importance of the loop structure, a series of alphaIIb mutants containing changes to the calcium-liganding amino acids have been constructed. Significantly, none of the mutant alphaIIbbeta3 complexes was detected on the surface of transfected cells, but mutant pro-alphaIIb was detected in cell lysates in complex with beta3. To study the importance of the regions flanking the second calcium-binding domain for ligand-binding and ligand-binding specificity, three alphaIIb/alpha5 chimaeras containing alpha5 sequences flanking or flanking and including the second calcium-binding domain were constructed. The chimaera containing both alpha5-flanking regions was not expressed on the cell surface, but FR1 and FR2, substituting either the first or second flanking region, were expressed. FR1beta3-transfected cells lost the ability to adhere to fibrinogen and to support aggregation and had minimal fibrinogen-binding ability. The heterodimer complex was less stable than the wild-type. FR2beta3-transfected cells adhered to fibrinogen and bound soluble fibrinogen with higher affinity when compared with wild-type. In addition, the heterodimer complex was more stable than wild-type. These results indicate that the conformation of the second calcium-binding domain is critical for maturation of the alphaIIbbeta3 complex and expression on the cell surface and that the surrounding sequences are critical for alphaIIbbeta3 function.

Animals↗
PubMed2005

Calcium- and magnesium-dependent interactions between calcium- and integrin-binding protein and the integrin alphaIIb cytoplasmic domain.

Calcium- and integrin-binding protein (CIB) is a small EF-hand calcium-binding protein that is involved in hemostasis through its interaction with the alphaIIb cytoplasmic domain of integrinalphaIIbbeta(3). We have previously demonstrated that CIB lacks structural stability in the absence of divalent metal ions but that it acquires a well-folded conformation upon addition of Ca(2+) or Mg(2+). Here, we have used fluorescence spectroscopy, NMR spectroscopy, and isothermal titration calorimetry to demonstrate that both Ca(2+)-bound CIB (Ca(2+)-CIB) and the Mg(2+)-bound protein (Mg(2+)-CIB) bind with high affinity and through a similar mechanism to alphaIIb cytoplasmic domain peptides, but that metal-free CIB (apo-CIB) binds in a different manner. The interactions are thermodynamically distinct for Ca(2+)-CIB and Mg(2+)-CIB, but involve hydrophobic interactions in each case. Since the Mg(2+) concentration inside the cell is sufficient to saturate CIB at all times, our results imply that CIB would be capable of binding to the alphaIIb cytoplasmic domain independent of an intracellular Ca(2+) stimulus in vivo. This raises the question of whether CIB can act as a Ca(2+) sensor in alphaIIbbeta(3) signaling or if other regulatory mechanisms such as fibrinogen-induced conformational changes in alphaIIbbeta(3), post-translational modifications, or the binding of other accessory proteins mediate the interactions between CIB and alphaIIbbeta(3). Differences in NMR spectra do suggest, however, that Ca(2+)-binding to the Mg(2+)- CIB-alphaIIb complex induces subtle structural changes that could further modulate the activity of alphaIIbbeta(3).

Calcium↗