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PubMed1999

Laminin and beta1 integrin distribution in the early stages of human kidney development.

Laminin, an extracellular matrix molecule (EMM) widely expressed in the basal laminae, interacts with specific membrane receptors among which the integrin molecules are the best known. During embryo development laminin is the first synthesized EMM and plays a significant role in the morphogenesis of organs in which epithelial-mesenchymal interactions and branching take place. The present study describes the distribution of laminin and of beta1 integrin receptors during the very early stages of human kidney development. The observations were carried out on paraffin sections of human embryos ranging between the 4th and the 7th gestational week. Laminin was detected within the basement membranes of mesonephric duct, vesicles, glomerular vessels and celomic epithelium. The metanephric anlage reacted with anti-laminin immunoglobulins in the basement membrane underlying the ampullae and in few blastemic cap cells. Low levels of beta1 integrin reactivity were found in both the mesonephric and metanephric structures. This study provides for the first time data about the distribution of laminin and beta1 integrin in the early stages of human renal organogenesis suggesting a key role for these molecules in the epithelial-mesenchymal interactions necessary for kidney development.

Adult↗
PubMed2001

OSP/claudin-11 forms a complex with a novel member of the tetraspanin super family and beta1 integrin and regulates proliferation and migration of oligodendrocytes.

Oligodendrocyte-specific protein (OSP)/claudin-11 is a major component of central nervous system myelin and forms tight junctions (TJs) within myelin sheaths. TJs are essential for forming a paracellular barrier and have been implicated in the regulation of growth and differentiation via signal transduction pathways. We have identified an OSP/claudin-11-associated protein (OAP)1, using a yeast two-hybrid screen. OAP-1 is a novel member of the tetraspanin superfamily, and it is widely expressed in several cell types, including oligodendrocytes. OAP-1, OSP/claudin-11, and beta1 integrin form a complex as indicated by coimmunoprecipitation and confocal immunocytochemistry. Overexpression of OSP/claudin-11 or OAP-1 induced proliferation in an oligodendrocyte cell line. Anti-OAP-1, anti-OSP/claudin-11, and anti-beta1 integrin antibodies inhibited migration of primary oligodendrocytes, and migration was impaired in OSP/claudin-11-deficient primary oligodendrocytes. These data suggest a role for OSP/claudin-11, OAP-1, and beta1 integrin complex in regulating proliferation and migration of oligodendrocytes, a process essential for normal myelination and repair.

Amino Acid Sequence↗
PubMed2006

Interferon-gamma inhibits collagen phagocytosis in human fibroblasts by inducing subcortical actin assembly and reducing ability of beta1 integrin to bind to collagen.

OBJECTIVE: We investigated the possible roles of interferon-gamma (IFN-gamma) in modulation of extracellular and intracellular routes of collagen digestion by human fibroblasts. METHODS: Human gingival fibroblasts were treated with IFN-gamma, after which matrix metalloproteinase-1 (MMP-1) activation was determined. Following the IFN-gamma treatment, cells were further incubated with either activating antibody for beta1 integrin or actin monomer-sequestering agent latrunculin B before incubation with collagen-coated fluorescent beads. Thereafter, the binding and internalization of the beads were assessed. RESULTS: IFN-gamma had no significant effect on MMP-1 activation, however, it reduced the binding of collagen-coated beads in the minimum affinity range and, subsequently, internalization of the beads. The inhibitory effects of IFN-gamma were partially reversed by adding either the beta1 integrin activating antibody or latrunculin B. CONCLUSIONS: Although IFN-gamma does not appreciably moderate the extracellular route of collagen digestion by human fibroblasts, the reduced level of collagen phagocytosis by IFN-gamma in the cells may contribute to fibrosis in inflamed connective tissues. Further, IFN-gamma may decrease the binding of collagen and following phagocytosis in cells by inducing a subcortical actin assembly and reducing the ability of beta1 integrin to bind to collagen.

Actins↗
PubMed1997

A phospholipase D and protein kinase C inhibitor blocks the spreading of murine mammary adenocarcinoma cells altering f-actin and beta1-integrin point contact distribution.

Spreading is a critical process involved in motility and growth of tumor cells during the metastatic cascade. Focal adhesion kinase, src-proteins and PKC have been reported to participate in the regulation of cytoskeleton organization in both normal and transformed cells during spreading. The role of other signaling enzymes such as PLD and PAP has not been studied during spreading in tumor cells. We now show that the spreading of murine mammary adenocarcinoma LM3 cells was significantly reduced by n-butanol, a PLD and PKC inhibitor, with a maximal inhibition of 54% (p < 0.001) in both the presence and absence of serum, as measured by phase-contrast microscopy. PMA only stimulated cell spreading over the control in the absence of serum and n-butanol inhibition was completely reversed by PMA treatment in both conditions. PA, the product of PLD activity, stimulated LM3 cell spreading and the same effect was observed with staurosporine. Spreading was enhanced when cells were seeded on collagen-IV- or fibronectin-coated surfaces and n-butanol could inhibit both integrin-derived signals. Cell spreading inhibition correlated with the absence of f-actin bundles and fewer beta1-integrin point contacts as determined by double immunofluorescence microscopy. In addition, n-butanol inhibited the proliferation of LM3 cells in the presence of serum (p < 0.01). These results suggest that beta1-integrin and f-actin/point contact assembly, involved in spreading and proliferation, require the participation of PLD-PKC regulatory pathways in LM3 cells.

1-Butanol↗
PubMedDate not supplied

TGF-beta2, tenascin, and integrin beta1 expression in superior limbic keratoconjunctivitis.

PURPOSE: To determine the pathophysiological etiology of superior limbic keratoconjunctivitis (SLK), we compared the superior limbic conjunctivae of SLK patients and normal controls. METHODS: Frozen sections of conjunctival specimens from five SLK patients and two controls were examined by immunohistochemical methods. Transforming growth factor (TGF)-beta2, integrin beta1, and tenascin (TN) were chosen for analysis because their expression is known to be affected by mechanical stress or injury. The staining pattern was observed by confocal laser scanning microscopy. RESULTS: Prominent positive TGF-beta2 staining on the surface region and heterogeneous staining in the suprabasal region were observed in the SLK specimens. TN expression was markedly up-regulated in the subepithelial stroma. In addition, suprabasal expression of integrin beta1 was observed. CONCLUSIONS: Up-regulation of TGF-beta2 and TN suggested that an increased amount of mechanical stress existed in the conjunctivae of the SLK patients. In addition, deposition of TN and suprabasal expression of integrin beta1 suggested that chronic minor injury contributed to the pathogenesis of SLK.

Aged↗
PubMed2003

Effect of stretching on gene expression of beta1 integrin and focal adhesion kinase and on chondrogenesis through cell-extracellular matrix interactions.

Differentiation of skeletal tissues, such as bone, ligament and cartilage, is regulated by complex interaction between genetic and epigenetic factors. In the present study, we attempted to elucidate the possible role of cell-extracellular matrix (ECM) adhesion on the inhibitory regulation in chondrogenesis responding to the tension force. The midpalatal suture cartilages in rats were expanded by orthopedic force. In situ hybridization for type I and II collagens, immunohistochemical analysis for fibronectin, alpha5 and beta1 integrins, paxillin, and vinculin, and cytochemical staining for actin were used to demonstrate the phenotypic change of chondrocytes. Immunohistochemical analysis for phosphorylation and nuclear translocation of extracellular signal-regulated kinase (ERK)-1/2 was performed. The role of the cell-ECM adhesion in the response of the chondroprogenitor cells to mechanical stress and the regulation of gene expression of focal adhesion kinase (FAK) and integrins were analyzed by using an in vitro system. A fibrous suture tissue replaced the midpalatal suture cartilage by the expansive force application for 14 days. The active osteoblasts that line the surface of bone matrix in the newly formed suture tissue strongly expressed the type I collagen gene, whereas they did not express the type II collagen gene. Although the numbers of precartilaginous cells expressing alpha5 and beta1 integrin increased, the immunoreactivity of alpha5 integrin in each cell was maintained at the same level throughout the experimental period. During the early response of midpalatal suture cartilage cells to expansive stimulation, formation of stress fibers, reorganization of focal adhesion contacts immunoreactive to a vinculin-specific antibody, and phosphorylation and nuclear translocation of ERK-1/2 were observed. In vitro experiments were in agreement with the results from the in vivo study, i.e. the inhibited expression of type II collagen and upregulation in integrin expression. The arginine-glycine-aspartic acid-containing peptide completely rescued chondrogenesis from tension-mediated inhibition. Thus, we conclude that stretching activates gene expression of beta1 integrin and FAK and inhibits chondrogenesis through cell-ECM interactions of chondroprogenitor cells.

Animals↗
PubMed2004

Analysis of swine beta1 integrin (CD29) epitopes through monoclonal antibodies developed using two immunization strategies.

This report describes the production and characterization of monoclonal antibodies (MAbs) to swine beta1 integrin subunit (CD29) using two different immunization strategies. MAb GP4B4 was developed from a mouse immunized with porcine peripheral blood mononuclear cells (PBMC), while MAbs GP1A5, GP1A6, and GP4A1 were produced by immunizing mice with a porcine CD29 recombinant protein (rpCD29) developed in our laboratory, which includes the ligand binding site. GP4B4 and UCP1D2 (specific to porcine CD29) immunoprecipitated two bands of approximately 115 and 150 kDa under reducing conditions. The molecule recognized by these two antibodies was studied using flow cytometry and was found in practically all cells studied, displaying a similar reaction pattern. Western blot assays performed with rpCD29 indicated that MAbs GP1A5, GP1A6, and GP4A1 recognized the 30-kDa band for this recombinant protein, confirming their specificity for the beta1, integrin subunit. Immunohistochemical analyses of these MAbs disclosed a morphological pattern associated with smooth muscle, epithelium, and myeloid cells, as expected in MAbs recognizing CD29. This MAb panel could be useful as a general anti-CD29 reagent and would allow further research into this important integrin in swine.

Animals↗
PubMed2006

Beta1 integrins modulate cell adhesion by regulating insulin-like growth factor-II levels in the microenvironment.

The interactions between cancer cells and the extracellular matrix (ECM) regulate cancer progression. The beta1C and beta1A integrins, two cytoplasmic variants of the beta1 integrin subfamily, are differentially expressed in prostate cancer. Using gene expression analysis, we show here that the beta1C variant, an inhibitor of cell proliferation, which is down-regulated in prostate cancer, up-regulates insulin-like growth factor-II (IGF-II) mRNA and protein levels. In contrast, beta1A does not affect IGF-II levels. We provide evidence that beta1C-mediated up-regulation of IGF-II levels increases adhesion to Laminin-1, a basement membrane protein down-regulated in prostate cancer, and that the beta1C cytoplasmic domain contains the structural motif sufficient to increase cell adhesion to Laminin-1. This autocrine mechanism that locally supports cell adhesion to Laminin-1 via IGF-II is selectively regulated by the beta1 cytoplasmic domain via activation of the growth factor receptor binding protein 2-associated binder-1/SH2-containing protein-tyrosine phosphatase 2/phosphatidylinositol 3-kinase pathway. Thus, the concurrent local loss of beta1C integrin, of its ligand Laminin-1, and of IGF-II in the tumor microenvironment may promote prostate cancer cell invasion and metastasis by reducing cancer cell adhesive properties. It is, therefore, conceivable that reexpression of beta1C will be sufficient to revert a neoplastic phenotype to a nonproliferative and highly adherent normal phenotype.

Adaptor Proteins, Signal Transducing↗
PubMed2003

ADAM12 induces actin cytoskeleton and extracellular matrix reorganization during early adipocyte differentiation by regulating beta1 integrin function.

Changes in cell shape are a morphological hallmark of differentiation. In this study we report that the expression of ADAM12, a disintegrin and metalloprotease, dramatically affects cell morphology in preadipocytes, changing them from a flattened, fibroblastic appearance to a more rounded shape. We showed that the highest levels of ADAM12 mRNA were detected in preadipocytes at the critical stage when preadipocytes become permissive for adipogenic differentiation. Furthermore, as assessed by immunostaining, ADAM12 was transiently expressed at the cell surface concomitant with the reduced activity of beta1 integrin. Co-immunoprecipitation studies indicated the formation of ADAM12/beta1 integrin complexes in these preadipocytes. Overexpression of ADAM12 at the cell surface of 3T3-L1 preadipocytes achieved by transient transfection or retroviral transduction led to the disappearance of the extensive network of actin stress fibers that are characteristic of these cells, and its reorganization into a cortical network located beneath the cell membrane. The cells became more rounded, exhibited fewer vinculin-positive focal adhesions, and adhered less efficiently to fibronectin in attachment assays. Moreover, ADAM12-expressing cells were more prone to apoptosis, which could be prevented by treating the cells with beta1-activating antibodies. A reduced and re-organized fibronectin-rich extracellular matrix accompanied these changes. In addition, beta1 integrin was more readily extracted with Triton X-100 from cells overexpressing ADAM12 than from control cells. Collectively, these results show that surface expression of ADAM12 impairs the function of beta1 integrins and, consequently, alters the organization of the actin cytoskeleton and extracellular matrix. These events may be necessary for early adipocyte differentiation.

ADAM Proteins↗
PubMed2006

L1, beta1 integrin, and cadherins mediate axonal regeneration in the embryonic spinal cord.

Embryonic birds and mammals are capable of axon regeneration after spinal cord injury, but this ability is lost during a discrete developmental transition. We recently showed that changes within maturing neurons, as opposed to changes solely in the spinal cord environment, significantly restrict axon regeneration during development. The developmental changes within neurons that limit axon regeneration remain unclear. One gap in knowledge is the identity of the adhesive receptors that embryonic neurons use to extend axons in the spinal cord. Here we test the roles of L1/NgCAM, beta1 integrin, and cadherins, using a coculture system in which embryonic chick brainstem neurons regenerate axons into an explant of embryonic spinal cord. By in vivo and in vitro methods, we found that brainstem neurons reduce axonal expression of L1 as they mature. Disrupting either L1 or beta1 integrin function individually in our coculture system partially inhibited growth of brainstem axons in spinal cords, while disrupting cadherin function alone had no effect. However, when all three adhesive receptors were blocked simultaneously, axon growth in the spinal cord was reduced by 90%. Using immunohistochemistry and in situ hybridization we show that during the period when neurons lose their regenerative capacity they reduce expression of mRNA for N-cadherin, and reduce axonal L1/NgCAM protein through a post-transcriptional mechanism. These data show that embryonic neurons use L1/NgCAM, beta1 integrin, and cadherin receptors for axon regeneration in the embryonic spinal cord, and raise the possibility that a reduced expression of these essential receptors may contribute to the low-regenerative capacity of older neurons.

Age Factors↗
PubMed2001

HEMCAM/CD146 downregulates cell surface expression of beta1 integrins.

HEMCAM/gicerin, an immunoglobulin superfamily protein, is involved in homophilic and heterophilic adhesion. It interacts with NOF (neurite outgrowth factor), a molecule of the laminin family. Alternative splicing leads to mRNAs coding for HEMCAM with a short (HEMCAM-s) or a long cytoplasmic tail (HEMCAM-l). To investigate the cellular function of these two variants, we stably transfected murine fibroblasts with either form of HEMCAM. Expression of each isoform of this protein in L cells delayed proliferation and modified their adhesion properties to purified extracellular matrix proteins. Expression of either HEMCAM-s or HEMCAM-l inhibited integrin-dependent adhesion and spreading of fibroblasts to laminin 1, showing that this phenomenon did not depend on the cytoplasmic region. By contrast, L-cell adhesion and spreading to fibronectin depended on the HEMCAM isoform expressed. Flow cytometry and immunoprecipitation studies revealed that the expression of HEMCAM downregulated expression of the laminin-binding integrins alpha3beta1, alpha6beta1 and alpha7beta1, and fibronectin receptor alpha5beta1 from the cell surface. Semi-quantitative PCR and northern blot experiments showed that the expression of alpha6beta1 integrin modified by HEMCAM occurred at a translation or maturation level. Thus, our data demonstrate that HEMCAM regulates fibroblast adhesion by controlling beta1 integrin expression.

Animals↗
PubMed2002

Defective Rac-mediated proliferation and survival after targeted mutation of the beta1 integrin cytodomain.

Cell matrix adhesion is required for cell proliferation and survival. Here we report that mutation by gene targeting of the cytoplasmic tail of beta1 integrin leads to defective proliferation and survival both in vivo and in vitro. Primary murine embryonic fibroblasts (MEFs) derived from mutant homozygotes display defective cell cycle coupled to impaired activation of the FAK-PI3K-Akt and Rac-JNK signaling pathways. Expression in homozygous MEFs of a constitutively active form of Rac is able to rescue proliferation, survival, and JNK activation. Moreover, although showing normal Erk phosphorylation, mutant cells fail to display Erk nuclear translocation upon fibronectin adhesion. However, expression of the constitutively activated form of Rac restores Erk nuclear localization, suggesting that adhesion-dependent Rac activation is necessary to integrate signals directed to promote MAPK activity. Altogether, our data provide the evidence for an epistatic interaction between the beta1 integrin cytoplasmic domain and Rac, and indicate that this anchorage-dependent signaling pathway is crucial for cell growth control.

Alleles↗
PubMed2002

Specific lectin binding to beta1 integrin and fibronectin on the apical membrane of madin-darby canine kidney cells.

Although lectins have previously been used to identify specific cell types in the kidney and various other tissues, the proteins labeled were not identified. We hypothesized that fluorescently labeled lectins could provide a useful tool for direct labeling of membrane-associated glycoproteins. Protein fractions from Madin-Darby canine kidney (MDCK) cells were exposed to a panel of 16 fluorescently labeled lectins to identify suitable lectin-protein pairs. Peanut agglutinin (PNA) selectively bound a 220-240 kDa O-linked glycoprotein with a slightly acidic isoelectric point, while Sambucus nigra agglutinin (SNA) labeled a 130 kDa glycoprotein with a highly acidic isoelectric point. Both proteins were readily labeled by lectins applied to the apical surface of living confluent cells. The proteins were isolated by lectin affinity columns and identified by mass spectrometry. Peptides from the PNA-binding protein shared molecular weight and amino acid composition with fibronectin. Fragments of the SNA-binding protein showed amino-acid identity with peptides from beta1 integrin. The identities of these proteins were validated by Western blotting. Binding of PNA to a 220 kDa protein was inhibited by an anti-fibronectin antibody, and binding of a 130 kDa protein by SNA was diminished by an anti-beta1 integrin antibody. We conclude that PNA and SNA can be used as specific markers for fibronectin and beta1 integrin, respectively, in MDCK cells.

Animals↗
PubMed2000

Translocation of Yersinia entrocolitica across reconstituted intestinal epithelial monolayers is triggered by Yersinia invasin binding to beta1 integrins apically expressed on M-like cells.

Yersinia enterocolitica cross the intestinal epithelium via translocation through M cells, which are located in the follicle-associated epithelium (FAE) of Peyer's patches (PP). To investigate the molecular basis of this process, studies were performed using a recently developed in vitro model, in which the enterocyte-like cell line Caco-2 and PP lymphocytes are co-cultured in order to establish FAE-like structures including M cells. Here, we demonstrate that Y. enterocolitica does not adhere significantly to the apical membrane of differentiated enterocyte-like Caco-2 cells that express binding sites for Ulex europaeus agglutinin (UEA)-1. In contrast, Y. enterocolitica adhered to, and was internalized by, cells that lacked UEA-1 binding sites and displayed a disorganized brush border. These cells were considered to be converted to M-like cells. Further analysis revealed that part of these cells expressed beta1 integrins at their apical surface and, as revealed by comparison of wild-type and mutant strains, interacted with invasin of Y. enterocolitica. Consistently, anti-beta1 integrin antibodies significantly inhibited internalization of inv-expressing yersiniae. Experiments with Yersinia mutant strains deficient in YadA or Yop secretion revealed that these virulence factors play a minor role in this process. After internalization, yersiniae were transported within LAMP-1-negative vacuoles to, and released at, the basal surface. Internalization and transport of yersiniae was inhibited by cytochalasin D, suggesting that F-actin assembly is required for this process. These results provide direct evidence that expression of beta1 integrins at the apical surface of M cells enables interaction with the invasin of Y. enterocolitica, and thereby initiates internalization and translocation of bacteria.

Adhesins, Bacterial↗
PubMed2006

Bone morphogenetic protein-2 promotes the haptotactic migration of murine osteoblastic and osteosarcoma cells by enhancing incorporation of integrin beta1 into lipid rafts.

Cell migration is essential for both organogenesis and tumor progression. Bone morphogenetic proteins (BMPs) are reported to be critical for not only bone formation but also tumor invasion. Here, we found that treatment with recombinant human BMP-2 (rhBMP-2) enhanced the haptotactic response of murine osteoblastic MC3T3-E1 and osteosarcoma Dunn cells to various extracellular matrix (ECM) components, including fibronectin, type I collagen, and laminin-1. Function-blocking antibody against integrin alpha5beta1 partially inhibited haptotaxis to fibronectin, suggesting that the response was propagated via these integrins. rhBMP-2 slightly increased the expression level of integrin beta1, and enhanced the speed of cell spreading on fibronectin, focal adhesion formation and phosphorylation of focal adhesion kinase (FAK) at Tyr397. By means of sucrose gradient flotation, incorporation of integrin beta1 in fractions of detergent (CHAPS) resistant membrane was increased when the cells were treated with rhBMP-2. Further, treatment with methyl-beta-cyclodextrin to deplete membrane cholesterol abrogated the effect of rhBMP-2 on haptotaxis, and exogenously added cholesterol reversed this inhibitory effect. Collectively, these results provide insights into the mechanism by which BMP signaling enhances cell migration by modulating fibronectin-integrin beta1 signaling via cholesterol enriched membrane microdomains, lipid rafts.

3T3 Cells↗
PubMed1996

Stimulation of glioma-cell migration by laminin and inhibition by anti-alpha3 and anti-beta1 integrin antibodies.

An induction of laminin in the confrontation zone between tumor cells and normal brain tissue has been observed in our model systems in vivo and in vitro. In order to study the effects of ECM components on glioma-cell migration and invasion, we have used 2 lacZ-transfected glioma cell lines, AN1/lacZ and U-251 /lacZ. Cell migration from multicellular spheroids was studied using different types of media: DMEM with 10% serum, Ultra Culture medium, and filtrated DMEM with serum in which the protein fraction > 100 kDa had been removed by ultrafiltration. Laminin, fibronectin and collagen type-IV were individually added to the different media, and cell migration from the spheroids was studied. The results show that cell migration in both cell lines, was stimulated by laminin and fibronectin. Collagen type-IV stimulated only cell migration of U-251/lacZ cells. Scanning electron microscopy revealed an extensive change in cell shape as a result of laminin stimulation. Flowcytometric studies showed that both AN1/lacZ and U-251/lacZ strongly express the alpha3 beta1 integrin receptor, which can bind to several ECM components (laminin, fibronectin, collagen). Immunofluorescence microscopy demonstrated that the same integrin sub-units were expressed in multicellular spheroids. When monoclonal antibodies to alpha3 and beta1 were added to the laminin-stimulated cultures, cell migration was significantly reduced. This indicates that the alpha3 beta1 integrin receptor plays an important role during glioma-cell migration.

Antigens, CD↗
PubMed2000

Fetal and adult hematopoietic stem cells require beta1 integrin function for colonizing fetal liver, spleen, and bone marrow.

Homing of hematopoietic stem cells (HSCs) into hematopoietic organs is a prerequisite for the establishment of hematopoiesis during embryogenesis and after bone marrow transplantation. We show that beta1 integrin-deficient HSCs from the para-aortic splanchnopleura and the fetal blood had hematolymphoid differentiation potential in vitro and in fetal organ cultures but were unable to seed fetal and adult hematopoietic tissues. Adult beta1 integrin null HSCs isolated from mice carrying loxP-tagged beta1 integrin alleles and ablated for beta1 integrin expression by retroviral cre transduction failed to engraft irradiated recipient mice. Moreover, absence of beta1 integrin resulted in sequestration of HSCs in the circulation and their reduced adhesion to endothelioma cells. These findings define beta1 integrin as an essential adhesion receptor for the homing of HSCs.

Aging↗
PubMed2002

A cell-free electrochemiluminescence assay for measuring beta1-integrin-ligand interactions.

We have developed a cell-free assay for binding of solubilized beta1 integrins to their physiologically relevant ligands using an electrochemiluminescent detection method. The method utilizes ruthenium-conjugated monoclonal antibodies for detection of either purified integrins or, more conveniently, integrin-expressing cell lysates, which are captured on beads coated with extracellular matrix or vascular ligand proteins. For the interaction of alpha1beta1 integrin with collagen IV, a signal of 10-fold over background was generated with samples containing only 10 ng (0.05 pmol) of integrin. This interaction is cation-dependent and can be inhibited by blocking antibodies to the alpha1 subunit. The method was extended to studies of ligand binding by integrins alpha2beta1, alpha4beta1, alpha5beta1, and alpha6beta1. For each integrin-ligand pair, the specificity of the interaction was verified with neutralizing antibodies against the specific integrin. The specific binding signal correlated with the activating ability of the labeled antibody used for detection, although the ability of divalent cations (Mn2+, Mg2+, Ca2+) to support integrin-ligand binding varied dramatically among the various integrin-ligand pairs. The assay provides a simple method for investigating integrin-ligand interactions without avidity and/or signaling effects which can complicate conventional cell-based assay methods.

Animals↗