The eighth FIII domain of human fibronectin promotes integrin alpha5beta1 binding via stabilization of the ninth FIII domain

Binding of the extracellular matrix molecule fibronectin to the integrin receptor alpha(5)beta(1) elicits downstream signaling pathways that modulate cell function. Fibronectin-alpha(5)beta(1) interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin alpha(5)beta(1) to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin alpha(5)beta(1)-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs.     

NEDD9 stabilizes focal adhesions, increases binding to the extra-cellular matrix and differentially effects 2D versus 3D cell migration

The speed of cell migration on 2-dimensional (2D) surfaces is determined by the rate of assembly and disassembly of clustered integrin receptors known as focal adhesions. Different modes of cell migration that have been described in 3D environments are distinguished by their dependence on integrin-mediated interactions with the extra-cellular matrix. In particular, the mesenchymal invasion mode is the most dependent on focal adhesion dynamics. The focal adhesion protein NEDD9 is a key signalling intermediary in mesenchymal cell migration, however whether NEDD9 plays a role in regulating focal adhesion dynamics has not previously been reported. As NEDD9 effects on 2D migration speed appear to depend on the cell type examined, in the present study we have used mouse embryo fibroblasts (MEFs) from mice in which the NEDD9 gene has been depleted (NEDD9 -/- MEFs). This allows comparison with effects of other focal adhesion proteins that have previously been demonstrated using MEFs. We show that focal adhesion disassembly rates are increased in the absence of NEDD9 expression and this is correlated with increased paxillin phosphorylation at focal adhesions. NEDD9-/- MEFs have increased rates of migration on 2D surfaces, but conversely, migration of these cells is significantly reduced in 3D collagen gels. Importantly we show that myosin light chain kinase is activated in 3D in the absence of NEDD9 and is conversely inhibited in 2D cultures. Measurement of adhesion strength reveals that NEDD9-/- MEFs have decreased adhesion to fibronectin, despite upregulated α5β1 fibronectin receptor expression. We find that β1 integrin activation is significantly suppressed in the NEDD9-/-, suggesting that in the absence of NEDD9 there is decreased integrin receptor activation. Collectively our data suggest that NEDD9 may promote 3D cell migration by slowing focal adhesion disassembly, promoting integrin receptor activation and increasing adhesion force to the ECM.     

Effect of dexamethasone on human osteoblasts in culture: involvement of β1 integrin and integrin-linked kinase

Adhesive interactions play a critical role in cell biology, influencing vital processes from proliferation to cell death. Integrins regulate cell-ECM (extracellular matrix) adhesion and must associate with phosphorylating proteins such as ILK (integrin-linked kinase). Dysregulation of ILK expression is associated with anchorage-independent growth, cell survival and inhibition of apoptosis. Glucocorticoids influence differentiation and adhesion of osteoblasts and can affect bone protein synthesis. The objective of this study was to analyse the effect of DEX (dexamethasone) on the biology of osteoblasts, together with its influence on the expression of ILK and β1 integrin. For this, primary cultures of human osteoblasts were exposed to DEX at 10-9 M (physiological dose) and 10-6 M (pharmacological dose) for 24 and 48 h. Cell viability, apoptosis and cell adhesion were analysed, as well as protein expression of β1 integrin and ILK. It was observed that cell viability and adhesion were reduced in the cultures evaluated. In comparison with the control cultures, there was slightly less apoptosis in the cultures exposed to the physiological dose and considerably more apoptosis in those exposed to the pharmacological dose. In all treated cultures, protein expression of ILK was slightly higher than in the control cultures, whereas that of β1 integrin was significantly lower. Both proteins under study were co-localized at the cell periphery in all cultures. Our results suggest that DEX causes osteoblast anoikis, probably due to decreased β1 integrin expression, which might have had a direct influence upon ILK, reducing its activation and preventing it from playing its characteristic anti-apoptotic role.     

Activation of beta1 integrins induces cell-cell adhesion

Integrins are highly regulated receptors that can function in both cell-substrate and cell-cell adhesion. We have found that the activating anti-beta1 mAb, 12G10, can specifically and rapidly induce both cell-substrate and cell-cell adhesion of HT-1080 human fibrosarcoma and other cell types. Binding of mAb 12G10 induced clustering of cell-surface integrins, and the preferential localization of beta1 integrins expressing the 12G10 epitope at cell-cell adhesion sites. Fab fragments of mAb 12G10 induced HT-1080 cell-cell adhesion as effectively as did intact antibodies, suggesting that integrin clustering was not due to direct antibody crosslinking. Latrunculin B, an inhibitor of F-actin polymerization, inhibited cell-cell adhesion but not the clustering of integrins. Results from a novel, two-color cell-cell adhesion assay suggested that nonactivated cells can bind to activated cells and that integrin activation-induced HT-1080 cell-cell adhesion minimally requires the interaction of activated alpha2beta1 with nonactivated alpha3beta1. These findings suggest that HT-1080 cell-cell adhesion induced by integrin activation require a signaling process involving integrin clustering and the subsequent organization of the cytoskeleton. Integrin activation could therefore play a key role in cell-cell adhesion.     

Synergistic adhesive interactions and signaling mechanisms operating between platelet glycoprotein Ib/IX and integrin alpha IIbbeta 3. Studies in human platelets ans transfected Chinese hamster ovary cells

This study investigates three aspects of the adhesive interaction operating between platelet glycoprotein Ib/IX and integrin alpha(IIb)beta(3). These include the following: 1) examining the sufficiency of GPIb/IX and integrin alpha(IIb)beta(3) to mediate irreversible cell adhesion on immobilized von Willebrand factor (vWf) under flow; 2) the ability of the vWf-GPIb interaction to induce integrin alpha(IIb)beta(3) activation independent of endogenous platelet stimuli; and 3) the identification of key second messengers linking the vWf-GPIb/IX interaction to integrin alpha(IIb)beta(3) activation. By using Chinese hamster ovary cells transfected with GPIb/IX and integrin alpha(IIb)beta(3), we demonstrate that these receptors are both necessary and sufficient to mediate irreversible cell adhesion under flow, wherein GPIb/IX mediates cell tethering and rolling on immobilized vWf, and integrin alpha(IIb)beta(3) mediates cell arrest. Moreover, we demonstrate direct signaling between GPIb/IX and integrin alpha(IIb)beta(3). Studies on human platelets demonstrated that vWf binding to GPIb/IX is able to induce integrin alpha(IIb)beta(3) activation independent of endogenous platelet stimuli under both static and physiological flow conditions (150-1800 s(-)(1)). Analysis of the key second messengers linking the vWf-GPIb interaction to integrin alpha(IIb)beta(3) activation demonstrated that the first step in the activation process involves calcium release from internal stores, whereas transmembrane calcium influx is a secondary event potentiating integrin alpha(IIb)beta(3) activation.     

TCR-induced activation of LFA-1 involves signaling through Tiam1

Adhesion is pivotal for most leukocyte functions, and the β(2) integrin family of adhesion molecules plays a central role. The integrins need activation to become functional, but the molecular events resulting in adhesion have remained incompletely understood. In human T cells, activation through the TCR results in specific phosphorylation of the T758 on the β(2) chain of LFA-1. We now show that this phosphorylation leads to downstream binding of 14-3-3 proteins, followed by engagement of the guanine nucleotide exchange factor protein Tiam1 and Rac1 activation. Downregulation of the signaling molecules inhibits LFA-1 activity. Activation by the chemokine stromal cell-derived factor-1α also results in T758 phosphorylation and integrin activation. Thus, TCR and chemokine activation converges on LFA-1 phosphorylation, followed by similar downstream events affecting adhesion.     

α9β1 Integrin in melanoma cells can signal different adhesion states for migration and anchorage

Cell surface integrins are the primary receptors for cell migration on extracellular matrix, and exist in several activation states regulated in part by ectodomain conformation. The α9 integrin subunit, which pairs only with β1, has specific roles in the immune system and may regulate cell migration. Melanoma cells express abundant α9β1 integrin, and its role in cell migration was assessed. Ligands derived from Tenascin-C and ADAM12 supported α9β1 integrin-mediated cell attachment and GTP-Rac dependent migration, but not focal adhesion formation. Manganese ions induced α9β1 integrin- and Rho kinase-dependent focal adhesion and stress fibre formation, suggesting that the activation status of α9β1 integrin was altered. The effect of manganese ions in promoting focal adhesion formation was reproduced by β1 integrin activating antibody. The α9β1 integrin translocated to focal adhesions, where active β1 integrin was also detected by conformation-specific antibodies. Focal adhesion assembly was commensurate with reduced cell migration. Endogenous α9β1 integrin-mediated adhesion was sensitive to the PP1 chemical inhibitor and an inhibitor of endosomal vesicle recycling, but not inhibitors of protein kinase C or the small GTPase Rho. Our results demonstrated that although α9β1 integrin can induce and localise to focal adhesions in a high activation state, its intermediate activity state normally supports cell adhesion consistent with migration.     

Tetraspanin CD151 Stimulates Adhesion-dependent Activation of Ras, Rac, and Cdc42 by Facilitating Molecular Association between β1 Integrins and Small GTPases

Tetraspanin CD151 associates with laminin-binding α(3)β(1)/α(6)β(1) integrins in epithelial cells and regulates adhesion-dependent signaling events. We found here that CD151 plays a role in recruiting Ras, Rac1, and Cdc42, but not Rho, to the cell membrane region, leading to the formation of α(3)β(1)/α(6)β(1) integrin-CD151-GTPases complexes. Furthermore, cell adhesion to laminin enhanced CD151 association with β(1) integrin and, thereby, increased complex formation between the β(1) family of integrins and small GTPases, Ras, Rac1, and Cdc42. Adhesion receptor complex-associated small GTPases were activated by CD151-β(1) integrin complex-stimulating adhesion events, such as α(3)β(1)/α(6)β(1) integrin-activating cell-to-laminin adhesion and homophilic CD151 interaction-generating cell-to-cell adhesion. Additionally, FAK and Src appeared to participate in this adhesion-dependent activation of small GTPases. However, engagement of laminin-binding integrins in CD151-deficient cells or CD151-specific siRNA-transfected cells did not activate these GTPases to the level of cells expressing CD151. Small GTPases activated by engagement of CD151-β(1) integrin complexes contributed to CD151-induced cell motility and MMP-9 expression in human melanoma cells. Importantly, among the four tetraspanin proteins that associate with β(1) integrin, only CD151 exhibited the ability to facilitate complex formation between the β(1) family of integrins and small GTPases and stimulate β(1) integrin-dependent activation of small GTPases. These results suggest that CD151 links α(3)β(1)/α(6)β(1) integrins to Ras, Rac1, and Cdc42 by promoting the formation of multimolecular complexes in the membrane, which leads to the up-regulation of adhesion-dependent small GTPase activation.     

Tumor cell surface alpha 4 beta 1 integrin mediates adhesion to vascular endothelium: demonstration of an interaction with the N-terminal domains of INCAM-110/VCAM-1.

Hematogenous metastasis involves adhesive interactions between blood-borne tumor cells and the vessel wall. By the use of in vitro assays, the adhesion of human melanoma, osteosarcoma, and kidney carcinoma (but not colon carcinoma) cell lines was shown to involve the cytokine-inducible endothelial cell surface protein inducible cell adhesion molecule 110 (INCAM-110) and the alpha 4 beta 1 integrin, molecules normally involved in endothelial-leukocyte interactions. Tumor adhesion to human endothelial cell monolayers was increased 1.9- to 8.2-fold by endothelial activation with the cytokine tumor necrosis factor (TNF) and inhibited by the anti-INCAM-110 monoclonal antibody (mAb) E1/6. Each of these tumor cells expressed members of the beta 1 integrin family of adhesion molecules, and antibodies to the alpha 4 and beta 1 integrin subunits inhibited tumor-endothelial adhesion (48-87% inhibition). A cDNA encompassing the three N-terminal Ig-like domains of vascular cell adhesion molecule 1 (VCAM-1) encoded a protein recognized by the anti-INCAM-110 mAb E1/6 and, when captured onto plastic, supported melanoma cell adhesion by an alpha 4 integrin-dependent mechanism. In contrast to mAb E1/6, a second anti-INCAM-110 mAb Hu8/4 neither inhibited adhesion to activated endothelium nor bound the first three Ig-like domains of INCAM-110/VCAM-1. These data indicate that the adherence of several human tumors to activated endothelium is mediated by an interaction of alpha 4 beta 1 integrin and the N-terminal Ig-like domains of endothelial INCAM-110/VCAM-1. Tumor acquisition of the alpha 4 integrin subunit and endothelial expression of INCAM-110 may affect the frequency and distribution of metastasis.     

Focal Adhesion Kinase Modulates Cell Adhesion Strengthening via Integrin Activation

Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell–ECM forces.     

The Src family kinases Hck and Fgr are dispensable for inside-out, chemoattractant-induced signaling regulating beta 2 integrin affinity and valency in neutrophils, but are required for beta 2 integrin-mediated outside-in signaling involved in sustained adhesion

Neutrophil beta(2) integrins are activated by inside-out signaling regulating integrin affinity and valency; following ligand binding, beta(2) integrins trigger outside-in signals regulating cell functions. Addressing inside-out and outside-in signaling in hck(-/-)fgr(-/-) neutrophils, we found that Hck and Fgr do not regulate chemoattractant-induced activation of beta(2) integrin affinity. In fact, beta(2) integrin-mediated rapid adhesion, in static condition assays, and neutrophil adhesion to glass capillary tubes cocoated with ICAM-1, P-selectin, and a chemoattractant, under flow, were unaffected in hck(-/-)fgr(-/-) neutrophils. Additionally, examination of integrin affinity by soluble ICAM-1 binding assays and of beta(2) integrin clustering on the cell surface, showed that integrin activation did not require Hck and Fgr expression. However, after binding, hck(-/-)fgr(-/-) neutrophil spreading over beta(2) integrin ligands was reduced and they rapidly detached from the adhesive surface. Whether alterations in outside-in signaling affect sustained adhesion to the vascular endothelium in vivo was addressed by examining neutrophil adhesiveness to inflamed muscle venules. Intravital microscopy analysis allowed us to conclude that Hck and Fgr regulate neither the number of rolling cells nor rolling velocity in neutrophils. However, arrest of hck(-/-)fgr(-/-) neutrophils to >60 microm in diameter venules was reduced. Thus, Hck and Fgr play no role in chemoattractant-induced inside-out beta(2) integrin activation but regulate outside-in signaling-dependent sustained adhesion.     

TGF—β1短时处理降低肝癌细胞与Fn的粘附及FAK的磷酸化

In order to investigate whether TGF-beta 1 could rapidly regulate integrin induced signaling, we treated SMMC-7721 human hepatocellular carcinoma cells with human recombinant TGF-beta 1 for 10 min, and examined cell adhesion, integrin amount and FAK tyrosine phosphorylation. We used cell adhesion assay to estimate the affinity of alpha 5 beta 1 integrin with fibronectin, and analyzed the amount of integrin alpha 5 and beta 1 subunits by performing FACS analysis. Then western blot analysis was carried out to examine tyrosine phosphorylation level of FAK. Our results showed that TGF-beta 1 could rapidly attenuated cell adhesion onto Fn without changing the expression of alpha 5 beta 1 integrin, and at the meantime dephosphorylated FAK. It suggested that TGF-beta 1 rapidly regulated the activation of integrin, and stimulated FAK dephosphorylation, which might induce depolarization in SMMC-7721 hepatocellular carcinoma cells, then facilitates the detachment of tumor cells at early stages of migration.     

Tetraspanin CD9 in cell migration

Tetraspanin CD9 is associated with integrin adhesion receptors and it was reported that CD9 regulates integrin-dependent cell migration and invasion. Pro- and anti-migratory effects of CD9 have been linked to adhesion-dependent signalling pathways, including phosphorylation of FAK (focal adhesion kinase) and activation of phosphoinositide 3-kinase, p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase). In the present paper, we describe a novel mechanism whereby CD9 specifically controls localization of talin1, one of the critical regulators of integrin activation, to focal adhesions: CD9-deficiency leads to impaired localization of talin1 to focal adhesions and correlates with increased motility of breast cancer cells.     

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.     

Integrin alphavbeta6 mediates HT29-D4 cell adhesion to MMP-processed fibrinogen in the presence of Mn2+

Mn(2+) was found to induce adhesion of HT29-D4 adenoma carcinoma cells to fibrinogen (Fb). This was independent of the expression of the beta3 integrin subunit and involved endogenous alphavbeta6 but not alphavbeta5 integrin. Thus, addition of Mn(2+) led to a change in integrin alphavbeta6 specificity. Furthermore, Mn(2+) was found to strongly activate the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in the HT29-D4 cell line. As a MAPK inhibitor strongly reduced the Mn(2+)-induced cell adhesion to Fb, it is suggested that a link between MAPK activation and cell adhesion to Fb exists. Both expression and activity of matrix metalloproteinase-9 (MMP-9) were enhanced by Mn(2+) and this led to Fb processing. MMP inhibitors prevented Mn(2+)-mediated cell adhesion to Fb, leading us to suggest that Mn(2+) promoted convergent changes in integrin alphavbeta6 conformation and Fb structure through activation of ERK/MAPK and MMP-9. Finally, we found that Mn(2+) and activators of the ERK pathway cooperated in HT29-D4 cell adhesion to Fb. Such a process may be involved in bone metastasis of some cancer cells.     

Opposing roles of integrin alpha6Abeta1 and dystroglycan in laminin-mediated extracellular signal-regulated kinase activation

Laminin-integrin interactions can in some settings activate the extracellular signal-regulated kinases (ERKs) but the control mechanisms are poorly understood. Herein, we studied ERK activation in response to two laminins isoforms (-1 and -10/11) in two epithelial cell lines. Both cell lines expressed beta1-containing integrins and dystroglycan but lacked integrin alpha6beta4. Antibody perturbation assays showed that both cell lines bound to laminin-10/11 via the alpha3beta1and alpha6beta1 integrins. Although laminin-10/11 was a stronger adhesion complex than laminin-1 for both cell lines, both laminins activated ERK in only one of the two cell lines. The ERK activation was mediated by integrin alpha6beta1 and not by alpha3beta1 or dystroglycan. Instead, we found that dystroglycan-binding domains of both laminin-1 and -10/11 suppressed integrin alpha6beta1-mediated ERK activation. Moreover, the responding cell line expressed the two integrin alpha6 splice variants, alpha6A and alpha6B, whereas the nonresponding cell line expressed only alpha6B. Furthermore, ERK activation was seen in cells transfected with the integrin alpha6A subunit, but not in alpha6B-transfected cells. We conclude that laminin-1 and -10/11 share the ability to induce ERK activation, that this is regulated by integrin alpha6Abeta1, and suggest a novel role for dystroglycan-binding laminin domains as suppressors of this activation.     

TNF-alpha potentiates C5a-stimulated eosinophil adhesion to human bronchial epithelial cells: a role for alpha 5 beta 1 integrin.

Cooperative action of inflammatory mediators and adhesion molecules orchestrates eosinophil recruitment during allergic inflammation in the airways. This study investigated the mechanisms involved in increasing eosinophil adhesion to human bronchial epithelial cells (HBEC) following priming and activation of eosinophils with TNF-alpha and complement protein C5a, respectively. Under primed conditions, eosinophil adhesion increased 3-fold from basal (16%), and the effect was significantly greater (p < 0.05) than the increase following stimulation with C5a alone (2-fold). Eosinophil contact with HBEC was essential for priming. In contrast to C5a, adhesion of eotaxin-stimulated eosinophils to HBEC was not primed with TNF-alpha nor IL-5, a known eosinophil-priming agent. Priming caused activation of alpha(M)beta(2) integrin; mAb against either the common beta(2) integrin subunit or its ICAM-1 ligand reduced the primed component of adhesion. Using mAbs against beta(1) or alpha(5), but not alpha(4) integrin subunit, together with anti-beta(2) integrin mAb, reduced stimulated adhesion to basal levels. Cross-linking alpha(5)beta(1) integrin increased alpha(M)beta(2) integrin-dependent adhesion of eosinophils. There are no known adhesion molecule ligands of alpha(5)beta(1) integrin expressed on HBEC; however, fibronectin, the major matrix protein ligand for alpha(5)beta(1) integrin, was detected in association with HBEC monolayers. A mAb against fibronectin, in combination with anti-beta(2) integrin mAb, reduced adhesion to basal levels. In conclusion, alpha(5)beta(1) integrin may provide a contact-dependent costimulus for eosinophil priming that, together with TNF-alpha, potentiated C5a activation of alpha(M)beta(2) integrin and increased eosinophil adhesion to ICAM-1. Fibronectin, associated with HBEC, may act as a ligand for alpha(5)beta(1) integrin. Dual regulation of eosinophil priming may prevent inappropriate activation of eosinophils in the circulation.     

Synergistic activity of the ninth and tenth FIII domains of human fibronectin depends upon structural stability

The ninth and tenth FIII domains (FIII9-10) of human fibronectin act in synergy to promote cell adhesion via the interaction with integrin receptors. Here we describe the functional and structural properties of a set of recombinant FIII9-10 mutants containing various alanine substitutions within the key synergistic site, DRVPHSRN in FIII9, either alone or in combination with another substitution (Leu(1408) to Pro), on the opposite face of FIII9, that increases stability and the functional capacity of FIII9-10. We show that the introduction of mutations into the synergistic sequence of FIII9-10 has a negative effect on the adhesion of baby hamster kidney fibroblasts and results in reduced ability of these ligands to recognize integrin alpha(5)beta(1). Conformational stability of the FIII9 domain in the synergy site mutants is likewise reduced in comparison with native FIII9. The Leu(1408) to Pro substitution in mutant FIII9-10 proteins carrying substitutions in the synergy site results in a substantial recovery of the adhesive activity of the mutants and affinity to alpha(5)beta(1). In keeping with the enhancement of functional activity, the Leu(1408) to Pro substitution in the FIII9-10 synergy site mutants also causes a significant increase in conformational stability of FIII9. These observations imply a strong positive correlation between the biological activity and conformational stability of the assessed FIII9-10 mutants and suggest that a Leu(1408) to Pro substitution restores the biological activity of the mutants via their ability to restore their conformational stability. We conclude that domain stability may be a major determinant of the synergistic potential of FIII9. Our data underscore the value of using more than one approach in such structure-function studies and the requirement for validating the global structural integrity of protein ligands in which sequences that disrupt function have been perturbed.     

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.