Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein.

Integrins are a complex family of divalent cation-dependent cell adhesion receptors composed of one alpha and one beta subunit noncovalently bound to one another. A subset of integrins contains the alpha v subunit in association with one of several beta subunits (e.g. beta 3, beta 5, beta 1). We have recently identified a novel integrin beta subunit, beta 6, that is present in a number of epithelial cell lines. Using a polyclonal antibody raised against the carboxyl-terminal peptide of beta 6, we have now identified the integrin heterodimer, alpha v beta 6, on the surface of two human carcinoma cell lines. Using affinity chromatography of lysates from the pancreatic carcinoma cell line, FG-2, we demonstrate that alpha v beta 6 binds to fibronectin, but not to vitronectin or collagen I. In contrast, the alpha v beta 5 integrin, which is also expressed on FG-2 cells, binds exclusively to vitronectin. Immobilized collagen I does not interact with alpha v integrins, but binds beta 1-containing integrins. Both alpha v beta 6 and alpha v beta 5 are eluted from their respective immobilized ligands by a hexa-peptide containing the sequence Arg-Gly-Asp (RGD). RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+. These results suggest that alpha v beta 6 functions as an RGD-dependent fibronectin receptor in FG-2 carcinoma cells. In agreement with this notion, cell adhesion assays show that FG-2 cell attachment to fibronectin is only partially inhibited by anti-beta 1 integrin antibodies, implying that other fibronectin receptors may be involved. Taken together with recent reports on the vitronectin receptor function of alpha v beta 5, our results suggest that the previously described carcinoma cell integrin, alpha v beta x (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), is a mixture of at least two different receptors: alpha v beta 5, mediating adhesion to vitronectin, and alpha v beta 6, mediating adhesion to fibronectin.     

Fibulin-5 binds human smooth-muscle cells through alpha5beta1 and alpha4beta1 integrins, but does not support receptor activation

Fibulin-5, an extracellular matrix glycoprotein expressed in elastin-rich tissues, regulates vascular cell behaviour and elastic fibre deposition. Recombinant full-length human fibulin-5 supported primary human aortic SMC (smooth-muscle cell) attachment through alpha5beta1 and alpha4beta1 integrins. Cells on fibulin-5 spread poorly and displayed prominent membrane ruffles but no stress fibres or focal adhesions, unlike cells on fibronectin that also binds these integrins. Cell migration and proliferation were significantly lower on fibulin-5 than on fibronectin. Treatment of cells on fibulin-5 with a beta1 integrin-activating antibody induced stress fibres, increased attachment, migration and proliferation, and stimulated signalling of epidermal growth factor receptor and platelet-derived growth factor receptors alpha and beta. Fibulin-5 also modulated fibronectin-mediated cell spreading and morphology. We have thus identified the beta1 integrins on primary SMCs that fibulin-5 interacts with, and have shown that failure of fibulin-5 to activate these receptors limits cell spreading, migration and proliferation.     

Adaptor protein-2 exhibits alpha 1 beta 1 or alpha 6 beta 1 integrin-dependent redistribution in rhabdomyosarcoma cells

Downregulation of several signaling pathways, such as those stimulated by growth factor receptors, occurs by internalization of signaling receptors through clathrin-coated pits. The first step in internalization or endocytosis is interaction with AP-2, which results in coated pit formation by assembly of clathrin to AP-2. Changes in endocytosis are reflected in the distribution of AP-2 molecules at the cell surface. Integrins are receptors which mediate attachment to the extracellular matrix and also stimulate numerous intracellular signaling pathways; however, it is not known how signaling through integrins is terminated or downregulated. Endocytosis through clathrin-coated pits offers an attractive mechanism for this. This work explores the relationship between AP-2 and beta(1) integrins. RD cells grown for 24 h on collagen or laminin exhibit a redistribution of AP-2 to the cell periphery relative to those grown on fibronectin or polylysine. The total AP-2 protein levels in the cells are unaffected. Blocking alpha(1)beta(1) integrin ligand binding on collagen prevents this redistribution fully. On laminin where alpha(1)beta(1) and alpha(6)beta(1) integrins are engaged, both receptors must be simultaneously blocked to prevent AP-2 redistribution, confirming that the redistribution depends on the specific engagement of the receptors. Immunofluorescence reveals that the majority of alpha(1)beta(1) integrins colocalize with alpha(6)beta(1) integrins in linear structures identified as focal adhesions. A separate fraction of alpha(1)beta(1) integrins colocalize with AP-2 in coated pits. Interestingly, alpha(6)beta(1) integrins are not located in coated pits, demonstrating that integrin colocalization with AP-2 is not necessary to induce redistribution of AP-2.     

Purification and characterization of mammalian integrins expressed by a rat neuronal cell line (PC12): evidence that they function as alpha/beta heterodimeric receptors for laminin and type IV collagen

Cells of the rat neuronal line, PC12, adhere well to substrates coated with laminin and type IV collagen, but attach poorly to fibronectin. Adhesion and neurite extension in response to these extracellular matrix proteins are inhibited by Fab fragments of an antiserum (anti-ECMR) that recognizes PC12 cell surface integrin subunits of Mr 120,000, 140,000, and 180,000 (Tomaselli, K. J., C. H. Damsky, and L. F. Reichardt. 1987. J. Cell Biol. 105:2347-2358). Here we extend our study of integrin structure and function in PC12 cells using integrin subunit-specific antibodies prepared against synthetic peptides corresponding to the cytoplasmic domains of the human integrin beta 1 and the fibronectin receptor alpha (alpha FN) subunits. Anti-integrin beta 1 immunoprecipitated a 120-kD beta 1 subunit and two noncovalently associated integrin alpha subunits of 140 and 180 kD from detergent extracts of surface-labeled PC12 cells. Immunodepletion studies using anti-integrin beta 1 demonstrated that these two putative alpha/beta heterodimers are identical to those recognized by the adhesion-perturbing ECMR antiserum. Anti-alpha FN immunoprecipitated fibronectin receptor heterodimers in human and rat fibroblastic cells, but not in PC12 cells. Thus, low levels of expression of the integrin alpha FN subunit can explain the poor attachment of PC12 cells to FN. The PC12 cell integrins were purified using a combination of lectin and ECMR antibody affinity chromatography. The purified integrins: (a) completely neutralize the ability of the anti-ECMR serum to inhibit PC12 cell adhesion to laminin and collagen IV; (b) have hydrodynamic properties that are very similar to those of previously characterized integrin alpha/beta heterodimeric receptors for ECM proteins; and (c) can be incorporated into phosphatidylcholine vesicles that then bind specifically to substrates coated with laminin or collagen IV but not fibronectin. Thus, the ligand-binding specificity of the liposomes containing the purified PC12 integrins closely parallels the substrate-binding preference of intact PC12 cells. These results demonstrate that mammalian integrins purified from a neuronal cell line can, when incorporated into lipid vesicles, function as receptors for laminin and type IV collagen.     

Identification of a tetrapeptide recognition sequence for the alpha 2 beta 1 integrin in collagen

The alpha 2 beta 1 integrin serves as either a specific cell surface receptor for collagen or as both a collagen and laminin receptor depending upon the cell type. Recently we established that the alpha 2 beta 1 integrin binds to a site within the alpha 1 (I)-CB3 fragment of type I collagen (Staatz, W. D., Walsh, J. J., Pexton, T., and Santoro, S. A. (1990) J. Biol. Chem. 265, 4778-4781). To define the alpha 2 beta 1 recognition sequence further we have prepared an overlapping set of synthetic peptides which completely spans the 148-amino acid alpha 1(I)-CB3 fragment and tested the peptides for ability to inhibit cell adhesion to collagen and laminin substrates. The minimal active recognition sequence defined by these experiments is a tetrapeptide of the sequence Asp-Gly-Glu-Ala (DGEA) corresponding to residues 435-438 of the type I collagen sequence. The DGEA-containing peptides effectively inhibited alpha 2 beta 1-mediated Mg2(+)-dependent adhesion of platelets, which use the alpha 2 beta 1 integrin as a collagen-specific receptor, to collagen but had no effect on alpha 5 beta 1-mediated platelet adhesion to fibronectin or alpha 6 beta 1-mediated platelet adhesion to laminin. In contrast, with T47D breast adenocarcinoma cells, which use alpha 2 beta 1 as a collagen/lamin receptor, adhesion to both collagen and laminin was inhibited by DGEA-containing peptides. Deletion of the alanine residue or substitution of alanine for either the glutamic or aspartic acid residues in DGEA-containing peptides resulted in marked loss of inhibitory activity. These results indicate that the amino acid sequence DGEA serves as a recognition site for the alpha 2 beta 1 integrin complex on platelets and other cells.     

Fibronectin promotes brain capillary endothelial cell survival and proliferation through alpha5beta1 and alphavbeta3 integrins via MAP kinase signalling

We showed previously that blood vessel maturation in the CNS is associated with a developmental switch in brain capillary endothelial cells (BCEC), from fibronectin signalling during angiogenesis to laminin signalling in the adult. To investigate the functional significance of this switch, we have examined the response of BCEC to different extracellular matrix (ECM) proteins. This showed that BCEC proliferation was significantly promoted by fibronectin (28.2 +/- 4.0%) and by vitronectin (14.8 +/- 2.1%) compared with uncoated glass (7.2 +/- 0.7%), while BCEC survival was significantly promoted by fibronectin (1130 +/- 131 cells), vitronectin (830 +/- 63 cells), collagen IV (703 +/- 77 cells) and laminin (680 +/- 34 cells) compared with the uncoated glass (367 +/- 48 cells). Biochemical studies showed that BCEC express a limited repertoire of integrins, including the beta1 integrins, alpha3beta1, alpha5beta1 and alpha6beta1, and the alphavbeta3 integrin. Function-blocking studies showed that the response to fibronectin was mediated equally by the alpha5beta1 and alphavbeta3 integrins. Analysis of signalling pathways revealed that fibronectin stimulated activation of the p44/p42 MAP kinase signalling pathway and pharmacological inhibitors of this pathway blocked BCEC proliferation on fibronectin. Taken together, these findings show that fibronectin exerts a strong angiogenic influence on endothelial cells (EC) in the CNS, and that this is mediated through the alpha5beta1 and alphavbeta3 integrins via MAP kinase signalling. In addition to a fundamental role in development, these findings may also have implications in pathological conditions of the CNS where fibronectin is re-expressed.     

The alpha 1 beta 1 and alpha E beta 7 integrins define a subset of dendritic cells in peripheral lymph nodes with unique adhesive and antigen uptake properties

Dendritic cells (DCs) are a heterogeneous population of APCs with critical roles in T cell activation and immune regulation. We report in this study the identification and characterization of a novel subset of DCs resident in skin-draining peripheral lymph nodes of normal mice. This subset of CD11c(high)CD40(high)CD8alpha(intermediate (int)) DCs expresses the collagen-binding integrin, alpha1beta1, and the E-cadherin-binding integrin, alphaEbeta7. Although alpha1beta1 and alphaEbeta7 are also expressed on CD11c(high)CD40(int)CD8alpha(high) lymphoid DCs, CD11c(high)CD40(high)CD8alpha(int) DCs demonstrate preferential integrin-mediated adhesion to collagen and fibronectin. This DC subset most likely acquires expression of these integrins in peripheral lymph node, as this subset is not found in the spleen or mesenteric lymph node, and recent DC migrants from the skin lack expression of alpha1beta1 and alphaEbeta7 integrins. Resident CD40(high) DCs express alpha1beta1 integrin and colocalize with collagen in lymph nodes. When compared with CD11c(high)CD40(high)CD8alpha(int) DCs lacking expression of these integrins, the alpha1beta1+alphaEbeta7+DC subset exhibits more efficient formation of Ag-independent conjugates with T cells, and a decreased ability to acquire soluble Ag. Thus, the alpha1beta1 and alphaEbeta7 integrins define a unique population of peripheral lymph node-derived DCs with altered functional properties and adhesive potential that localizes these cells to sites in lymph nodes where Ag presentation to T cells occurs.     

Alpha 4 beta 1 integrin/VCAM-1 interaction activates alpha L beta 2 integrin-mediated adhesion to ICAM-1 in human T cells

Modulation of integrin affinity and/or avidity provides a regulatory mechanism by which leukocyte adhesion to endothelium is strengthened or weakened at different stages of emigration. In this study, we demonstrate that binding of high-affinity alpha 4 beta 1 integrins to VCAM-1 strengthens alpha L beta 2 integrin-mediated adhesion. The strength of adhesion of Jurkat cells, a human leukemia T cell line, or MnCl2-treated peripheral blood T cells to immobilized chimeric human VCAM-1/Fc, ICAM-1/Fc, or both was quantified using parallel plate flow chamber leukocyte detachment assays in which shear stress was increased incrementally (0.5-30 dynes/cm2). The strength of adhesion to VCAM-1 plus ICAM-1, or to a 40-kDa fragment of fibronectin containing the CS-1 exon plus ICAM-1, was greater than the sum of adhesion to each molecule alone. Treatment of Jurkat or blood T cells with soluble cross-linked VCAM-1/Fc or HP2/1, a mAb to alpha 4, significantly increased adhesion to ICAM-1. These treatments induced clustering of alpha L beta 2 integrins, but not the high-affinity beta 2 integrin epitope recognized by mAb 24. Up-regulated adhesion to ICAM-1 was abolished by cytochalasin D, an inhibitor of cytoskeletal rearrangement. Taken together, our data suggest that the binding of VCAM-1 or fibronectin to alpha 4 beta 1 integrins initiates a signaling pathway that increases beta 2 integrin avidity but not affinity. A role for the cytoskeleton is implicated in this process.     

The collagen-binding A-domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens

We have previously assigned an integrin alpha(2)beta(1)-recognition site in collagen I to the sequence, GFOGERGVEGPOGPA (O = Hyp), corresponding to residues 502-516 of the alpha(1)(I) chain and located in the fragment alpha(1)(I)CB3 (Knight, C. G., Morton, L. F., Onley, D. J., Peachey, A. R., Messent, A. J., Smethurst, P. A., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (1998) J. Biol. Chem. 273, 33287-33294). In this study, we show that recognition is entirely contained within the six-residue sequence GFOGER. This sequence, when in triple-helical conformation, readily supports alpha(2)beta(1)-dependent cell adhesion and exhibits divalent cation-dependent binding of isolated alpha(2)beta(1) and recombinant alpha(2) A-domain, being at least as active as the parent collagen. Replacement of E by D causes loss of recognition. The same sequence binds integrin alpha(1) A-domain and supports integrin alpha(1)beta(1)-mediated cell adhesion. Triple-helical GFOGER completely inhibits alpha(2) A-domain binding to collagens I and IV and alpha(2)beta(1)-dependent adhesion of platelets and HT 1080 cells to these collagens. It also fully inhibits alpha(1) A-domain binding to collagen I and strongly inhibits alpha(1)beta(1)-mediated adhesion of Rugli cells to this collagen but has little effect on either alpha1 A-domain binding or adhesion of Rugli cells to collagen IV. We conclude that the sequence GFOGER represents a high-affinity binding site in collagens I and IV for alpha(2)beta(1) and in collagen I for alpha(1)beta(1). Other high-affinity sites in collagen IV mediate its recognition of alpha(1)beta(1).     

Heparin II domain of fibronectin uses alpha4beta1 integrin to control focal adhesion and stress fiber formation, independent of syndecan-4

Co-signaling events between integrins and cell surface proteoglycans play a critical role in the organization of the cytoskeleton and adhesion forces of cells. These processes, which appear to be responsible for maintaining intraocular pressure in the human eye, involve a novel cooperative co-signaling pathway between alpha5beta1 and alpha4beta1 integrins and are independent of heparan sulfate proteoglycans. Human trabecular meshwork cells isolated from the eye were plated on type III 7-10 repeats of fibronectin (alpha5beta1 ligand) in the absence or presence of the heparin (Hep) II domain of fibronectin. In the absence of the Hep II domain, cells had a bipolar morphology with few focal adhesions and stress fibers. The addition of the Hep II domain increased cell spreading and the numbers of focal adhesions and stress fibers. Cell spreading and stress fiber formation were not mediated by heparan sulfate proteoglycans because treatment with chlorate, heparinase, or soluble heparin did not prevent Hep II domain-mediated cell spreading. Cell spreading and stress fiber formation were mediated by alpha4beta1 integrin because soluble anti-alpha4 integrin antibodies inhibited Hep II domain-mediated cell spreading and soluble vascular cell adhesion molecule-1 (alpha4beta1 ligand)-induced cell spreading. This is the first demonstration of the Hep II domain mediating cell spreading and stress fiber formation through alpha4beta1 integrin. This novel pathway demonstrates a cooperative, rather than antagonistic, role between alpha5beta1 and alpha4beta1 integrins and suggests that interactions between the Hep II domain and alpha4beta1 integrin could modulate the strength of cytoskeleton-mediated processes in the trabecular meshwork of the human eye.     

Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 integrins in cell--collagen interactions: identification of conformation dependent alpha 1 beta 1 binding sites in collagen type I.

Integrins can mediate the attachment of cells to collagen type I. In the present study we have investigated the possible differences in collagen type I recognition sites for the alpha 1 beta 1 and alpha 2 beta 1 integrins. Different cyanogen bromide (CB) fragments of the alpha 1 (I) collagen chain were used in cell attachment experiments with three rat cell types, defined with regard to expression of collagen binding integrins. Primary rat hepatocytes expressed alpha 1 beta 1, primary rat cardiac fibroblasts alpha 1 beta 1 and alpha 2 beta 1, and Rat-1 cells only alpha 2 beta 1. All three cell types expressed alpha 3 beta 1 but this integrin did not bind to collagen--Sepharose or to immobilized collagen type I in a radioreceptor assay. Hepatocytes and cardiac fibroblasts attached to substrata coated with alpha 1(I)CB3 and alpha 1(I)CB8; Rat-1 cells attached to alpha 1(I)CB3 but only poorly to alpha 1(I)CB8-coated substrata. Cardiac fibroblasts and Rat-1 cells spread and formed beta 1-integrin-containing focal adhesions when grown on substrata coated with native collagen or alpha 1(I)CB3; focal adhesions were also detected in cardiac fibroblasts cultured on alpha 1(I)CB8. The rat alpha 1 specific monoclonal antibody 3A3 completely inhibited hepatocyte attachment to alpha 1(I)CB3 and alpha 1(I)CB8, as well as the attachment of cardiac fibroblasts to alpha 1(I)CB8, but only partially inhibited the attachment of cardiac fibroblasts to alpha 1(I)CB3. 3A3 IgG did not inhibit the attachment of Rat-1 cells to collagen type I or to alpha 1(I)CB3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Integrin alpha 2 beta 1 promotes activation of protein phosphatase 2A and dephosphorylation of Akt and glycogen synthase kinase 3 beta

Serine/threonine kinase Akt is a downstream effector protein of phosphatidylinositol-3-kinase (PI-3K). Many integrins can function as positive modulators of the PI-3K/Akt pathway. Integrin alpha 2 beta 1 is a collagen receptor that has been shown to induce specific signals distinct from those activated by other integrins. Here, we found that, in contrast what was found for cells adherent to fibronectin, alpha 2 beta 1-mediated cell adhesion to collagen leads to dephosphorylation of Akt and glycogen synthase kinase 3 beta (GSK3 beta) and concomitantly to the induction of protein serine/threonine phosphatase 2A (PP2A) activity. PP2A activation can be inhibited by mutation in the alpha 2 cytoplasmic domain and by a function-blocking anti-alpha 2 antibody. Akt can be coprecipitated with PP2A, and coexpression of Akt with PP2Ac (catalytic subunit) inhibits Akt kinase activity. Integrin alpha 2 beta 1-related activation of PP2A is dependent on Cdc42. These results indicate that cell adhesion to collagen modulates Akt activity via the alpha 2 beta 1-induced activation of PP2A.     

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes

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.     

Monkey rotavirus binding to alpha2beta1 integrin requires the alpha2 I domain and is facilitated by the homologous beta1 subunit

Rotaviruses utilize integrins during virus-cell interactions that lead to infection. Cell binding and infection by simian rotavirus SA11 were inhibited by antibodies (Abs) to the inserted (I) domain of the alpha2 integrin subunit. To determine directly which integrins or other proteins bind rotaviruses, cell surface proteins precipitated by rotaviruses were compared with those precipitated by anti-alpha2beta1 Abs. Two proteins precipitated by SA11 and rhesus rotavirus RRV from MA104 and Caco-2 cells migrated indistinguishably from alpha2beta1 integrin, and SA11 precipitated beta1 from alpha2beta1-transfected CHO cells. These viruses specifically precipitated two MA104 cell proteins only, but an additional 160- to 165-kDa protein was precipitated by SA11 from Caco-2 cells. The role of the alpha2 I domain in rotavirus binding, infection, and growth was examined using CHO cell lines expressing wild-type or mutated human alpha2 or alpha2beta1. Infectious SA11 and RRV, but not human rotavirus Wa, specifically bound CHO cell-expressed human alpha2beta1 and, to a lesser extent, human alpha2 combined with hamster beta1. Binding was inhibited by anti-alpha2 I domain monoclonal Abs (MAbs), but not by non-I domain MAbs to alpha2, and required the presence of the alpha2 I domain. Amino acid residues 151, 221, and 254 in the metal ion-dependent adhesion site of the alpha2 I domain that are necessary for type I collagen binding to alpha2beta1 were not essential for rotavirus binding. Rotavirus-alpha2beta1 binding led to increased virus infection and RRV growth. SA11 and RRV require the alpha2 I domain for binding to alpha2beta1, and their binding to this integrin is distinguishable from that of collagen.     

Urokinase receptors are required for alpha 5 beta 1 integrin-mediated signaling in tumor cells

Up-regulation of urokinase receptors is common during tumor progression and thought to promote invasion and metastasis. Urokinase receptors bind urokinase and a set of beta1 integrins, but it remains unclear to what degree urokinase receptor/integrin binding is important to beta1 integrin signaling. Using site-directed mutagenesis, single amino acid mutants of the urokinase receptor were identified that fail to associate with either alpha3beta1 (D262A) or alpha5beta1 (H249A) but associate normally with urokinase. To study the effects of these mutations on beta1 integrin function, endogenous urokinase receptors were first stably silenced in tumor cell lines HT1080 and H1299, and then wild type or mutant receptors were expressed. Knockdown of urokinase receptors resulted in markedly reduced fibronectin and alpha5beta1-dependent ERK activation and metalloproteinase MMP-9 expression. Re-expression of wild type or D262A mutant receptors but not the alpha5beta1 binding-deficient H249A mutant reconstituted fibronectin responses. Because urokinase receptor.alpha5beta1 complexes bind in the fibronectin heparin-binding domain (Type III 12-14) whereas alpha5beta1 primarily binds in the RGD-containing domain (Type III 7-10), signaling pathways leading to ERK and MMP-9 responses were dissected. Binding to III 7-10 led to Src/focal adhesion kinase activation, whereas binding to III 7-14 caused Rac 1 activation. Tumor cells engaging fibronectin required both Type III 7-10- and 12-14-initiated signals to activate ERK and up-regulate MMP-9. Thus urokinase receptor binding to alpha5beta1 is required for maximal responses to fibronectin and tumor cell invasion, and this operates through an enhanced Src/Rac/ERK signaling pathway.     

The role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells

We have examined cultures of neonatal human foreskin keratinocytes (HFKs) to determine the ligands and functions of integrins alpha 2 beta 1, and alpha 3 beta 1 in normal epidermal stratification and adhesion to the basement membrane zone (BMZ) in skin. We used three assay systems, HFK adhesion to purified extracellular matrix (ECM) ligands and endogenous secreted ECM, localization of integrins in focal adhesions (FAs), and inhibition of HFK adhesion with mAbs to conclude: (a) A new anti-alpha 3 beta 1 mAb, P1F2, localized alpha 3 beta 1 in FAs on purified laminin greater than fibronectin/collagen, indicating that laminin was the best exogeneous ligand for alpha 3 beta 1. However, in long term culture, alpha 3 beta 1 preferentially codistributed in and around FAs with secreted laminin-containing ECM, in preference to exogenous laminin. Anti-alpha 3 beta 1, mAb P1B5, detached prolonged cultures of HFKs from culture plates or from partially purified HFK ECM indicating that interaction of alpha 3 beta 1 with the secreted laminin-containing ECM was primarily responsible for HFK adhesion in long term culture. (b) In FA assays, alpha 2 beta 1 localized in FAs conincident with initial HFK adhesion to exogenous collagen, but not laminin or fibronectin. However, in inhibition assays, anti-alpha 2 beta 1 inhibited initial HFK adhesion to both laminin and collagen. Thus, alpha 2 beta 1 contributes to initial HFK adhesion to laminin but alpha 3 beta 1 is primarily responsible for long-term HFK adhesion to secreted laminin-containing ECM. (c) Serum or Ca2(+)-induced aggregation of HFKs resulted in relocation of alpha 2 beta 1 and alpha 3 beta 1 from FAs to cell-cell contacts. Further, cell-cell adhesion was inhibited by anti-alpha 3 beta 1 (P1B5) and a new anti-beta 1 mAb (P4C10). Thus, interaction of alpha 3 beta 1 with either ECM or membrane coreceptors at cell-cell contacts may facilitate Ca2(+)-induced HFK aggregation. (d) It is suggested that interaction of alpha 3 beta 1 with a secreted, laminin-containing ECM in cultured HFKs, duplicates the role of alpha 3 beta 1 in basal cell adhesion to the BMZ in skin. Further, relocation of alpha 2 beta 1 and alpha 3 beta 1 to cell-cell contacts may result in detachment of cells from the BMZ and increased cell-cell adhesion in the suprabasal cells contributing to stratification of the skin.     

Generation of a minimal alpha5beta1 integrin-Fc fragment

The tertiary structure of the integrin heterodimer is currently unknown, although several predictive models have been generated. Detailed structural studies of integrins have been consistently hampered for several reasons, including the small amounts of purified protein available, the large size and conformational flexibility of integrins, and the presence of transmembrane domains and N-linked glycosylation sites in both receptor subunits. As a first step toward obtaining crystals of an integrin receptor, we have expressed a minimized dimer. By using the Fc dimerization and mammalian cell expression system designed and optimized by Stephens et al. (Stephens, P. E., Ortlepp, S., Perkins, V. C., Robinson, M. K., and Kirby, H. (2000) Cell. Adhes. Commun. 7, 377-390), a series of recombinant soluble human alpha(5)beta(1) integrin truncations have been expressed as Fc fusion proteins. These proteins were examined for their ligand-binding properties and for their expression of anti-integrin antibody epitopes. The shortest functional alpha(5)-subunit truncation contained the N-terminal 613 residues, whereas the shortest beta(1)-subunit was a fragment containing residues 121-455. Each of these minimally truncated integrins displayed the antibody binding characteristics of alpha(5)beta(1) purified from human placenta and bound ligand with the same apparent affinity as the native receptor.     

Collagen IV regulates Caco-2 migration and ERK activation via alpha1beta1- and alpha2beta1-integrin-dependent Src kinase activation

Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.