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• W1571638637 abstract "Cell surface integrins can adopt distinct conformations in response to ligand binding and intracellular signals. Several integrins including αvβ3 can bind to multiple ligands. The binding of αvβ3 to fibronectin and vitronectin was used as a model to determine whether the same or distinct forms of the receptor were utilized in strong binding to the two different ligands. A spinning-disc device was used to measure the relative strength of the αvβ3-ligand bonds. The initial binding reaction for both ligands occurred in the absence of metabolic energy and resulted in a strong adhesion to fibronectin but a weak adhesion to vitronectin. Increases in the strength of the αvβ3-vitronectin bond required phosphorylation of the β3 cytoplasmic domain, intracellular signals, and the binding of cytoskeletal proteins to cytoplasmic domains of β3 controlled by Tyr-747 and Tyr-759. In contrast, αvβ3-mediated adhesion to fibronectin was unaffected by phorbol 12-myristate 13-acetate, mutations of Tyr-747 and Tyr-759 to phenylalanine, or availability of metabolic energy. This suggests that strong adhesion to fibronectin used the initial binding conformation, whereas strong binding to vitronectin required signaling-induced changes in the conformation of αvβ3. Cell surface integrins can adopt distinct conformations in response to ligand binding and intracellular signals. Several integrins including αvβ3 can bind to multiple ligands. The binding of αvβ3 to fibronectin and vitronectin was used as a model to determine whether the same or distinct forms of the receptor were utilized in strong binding to the two different ligands. A spinning-disc device was used to measure the relative strength of the αvβ3-ligand bonds. The initial binding reaction for both ligands occurred in the absence of metabolic energy and resulted in a strong adhesion to fibronectin but a weak adhesion to vitronectin. Increases in the strength of the αvβ3-vitronectin bond required phosphorylation of the β3 cytoplasmic domain, intracellular signals, and the binding of cytoskeletal proteins to cytoplasmic domains of β3 controlled by Tyr-747 and Tyr-759. In contrast, αvβ3-mediated adhesion to fibronectin was unaffected by phorbol 12-myristate 13-acetate, mutations of Tyr-747 and Tyr-759 to phenylalanine, or availability of metabolic energy. This suggests that strong adhesion to fibronectin used the initial binding conformation, whereas strong binding to vitronectin required signaling-induced changes in the conformation of αvβ3. phorbol 12-myristate 13-acetate bovine serum albumin Integrins are a family of heterodimeric cell surface receptors that mediate adhesion to extracellular matrix ligands and cell surface ligands (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9025) Google Scholar). Many integrins appear to have a single ligand, usually fibronectin or laminin, but a significant group of integrins bind to multiple ligands (2Sonnenberg A. Curr. Top. Microbiol. Immunol. 1993; 184: 7-35Crossref PubMed Scopus (176) Google Scholar). αVβ3, originally described as the vitronectin receptor, is one of the most promiscuous integrins. It binds to a variety of plasma and extracellular matrix proteins including vitronectin, fibrinogen, fibronectin, von Willebrand's factor, thrombospondin, osteopontin and bone sialic protein 1 (2Sonnenberg A. Curr. Top. Microbiol. Immunol. 1993; 184: 7-35Crossref PubMed Scopus (176) Google Scholar, 3Denis C. Williams J.A. Lu X. Meyer D. Baruch D. Blood. 1993; 82: 3622-3630Crossref PubMed Google Scholar, 4Krutzsch H.C. Choe B.J. Sipes J.M. Guo N. Roberts D.D. J. Biol. Chem. 1999; 274: 24080-24086Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). It has also been reported to serve as a receptor for adenovirus, foot and mouth disease virus, coxsackievirus A9, and hantaviruses (5Neff S. Sa-Carvalho D. Rieder E. Mason P.W. Blystone S.D. Brown E.J. Baxt B. J. Virol. 1998; 72: 3587-3594Crossref PubMed Google Scholar, 6Triantafilou K. Triantafilou M. Takada Y. Fernandez N. J. Virol. 2000; 74: 5856-5862Crossref PubMed Scopus (69) Google Scholar, 7Wickham T.J. Mathias P. Cheresh D.A. Nemerow G.R. Cell. 1993; 73: 309-319Abstract Full Text PDF PubMed Scopus (1959) Google Scholar). It binds to tat (a regulatory protein from HIV), fibroblast growth factor 2 and a fragment from the disintegrin ADAM-15, suggesting roles in the control of soluble factors as well (8Barillari G. Sgadari C. Fiorelli V. Samaniego F. Colombini S. Manzari V. Modesti A. Nair B.C. Cafaro A. Sturzl M. Ensoli B. Blood. 1999; 94: 663-672PubMed Google Scholar, 9Nath D. Slocombe P.M. Stephens P.E. Warn A. Hutchinson G.R. Yamada K.M. Docherty A.J. Murphy G. J. Cell Sci. 1999; 112: 579-587Crossref PubMed Google Scholar, 10Rusnati M. Tanghetti E. Dell'Era P. Gualandris A. Presta M. Mol. Biol. Cell. 1997; 8: 2449-2461Crossref PubMed Scopus (134) Google Scholar). Some of these interactions are with pathogen-encoded proteins in which the binding has evolved as a function of the pathogen and may use unique binding sites on αvβ3. Most of these protein ligands encode an RGD sequence that is presumed to represent the binding site for αvβ3. It is possible that this minimal motif reproduced in each of these ligands possesses sufficient structural homology to serve as interchangeable binding domains for αvβ3. Peptide chemistry has been used to generate a variety of RGD mimetics by placing the RGD in different contexts and constraining its conformation with cross-linkers (11Dechantsreiter M.A. Planker E. Matha B. Lohof E. Holzemann G. Jonczyk A. Goodman S.L. Kessler H. J. Med. Chem. 1999; 42: 3033-3040Crossref PubMed Scopus (756) Google Scholar, 12Locardi E. Mullen D.G. Mattern R.H. Goodman M. J. Pept. Sci. 1999; 5: 491-506Crossref PubMed Scopus (22) Google Scholar, 13Pfaff M. Tangemann K. Muller B. Gurrath M. Muller G. Kessler H. Timpl R. Engel J. J. Biol. Chem. 1994; 269: 20233-20238Abstract Full Text PDF PubMed Google Scholar, 14Xiao Y. Truskey G.A. Biophys. J. 1996; 71: 2869-2884Abstract Full Text PDF PubMed Scopus (142) Google Scholar). These peptides show large differences in their affinity for αvβ3. Given these differences in binding to RGD domains of different conformations, the range of ligands for αvβ3 is even more remarkable. One important property that seems common to most integrins is the ability of intracellular signals to regulate their binding functions (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9025) Google Scholar, 15Calderwood D.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 2000; 275: 22607-22610Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). This process has been called “inside-out” signaling and can result in a change in the conformation of the extracellular domain of the integrin as determined by the binding of conformation-specific monoclonal antibodies or biophysical methods like fluorescence resonance energy transfer (16Frelinger A.L. Du X.P. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1991; 266: 17106-17111Abstract Full Text PDF PubMed Google Scholar, 17Sims P.J. Ginsberg M.H. Plow E.F. Shattil S.J. J. Biol. Chem. 1991; 266: 7345-7352Abstract Full Text PDF PubMed Google Scholar). In addition, direct analysis of the strength of the αvβ3-vitronectin bond has identified four different levels of binding strength (affinity) between αvβ3 and vitronectin (18Boettiger D. Huber F. Lynch L. Blystone S.D. Mol. Cell. Biol. 2001; 12: 1227-1237Crossref Scopus (39) Google Scholar). Thus, αvβ3 can assume two and probably more distinct conformations of the extracellular domain that would affect the ligand binding properties directly. Given the array of ligands, it would seem reasonable that different ligands might bind to different forms of the receptor. “Activated” αvβ3 for one ligand could be an “inactive” αvβ3 for another ligand. Because of the link between integrin binding and integrin signaling, this scenario has consequences for integrin-mediated signals that control basic cellular processes (19Miyamoto S. Teramoto H. Coso O.A. Gutkind J.S. Burbelo P.D. Akiyama S.K. Yamada K.M. J. Cell Biol. 1995; 131: 791-805Crossref PubMed Scopus (1106) Google Scholar). If different conformations of the receptor constitute the active strong-binding receptor on different ligands, this is likely to induce different effects in the cytoplasmic domains of the bound receptors and hence provide different possibilities for binding of the integrin cytoplasmic domains to cytoskeletal and signaling proteins. The Kαvβ3, Kαvβ3(Y747F), and Kαvβ3(Y759F) cells were derived from K562 cells by transfection with both αv and β3or β3 mutant integrins as described previously (20Blystone S.D. Williams M.P. Slater S.E. Brown E.J. J. Biol. Chem. 1997; 272: 28757-28761Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The K562-derived cells were adapted to growth as nonadherent cells in serum-free K562 medium (cytoSF4 from Kemp Biotechnologies, Frederick, MD) and maintained either in this medium or in Dulbecco's modified Eagle's medium with 10% calf serum. The AIIB2 (anti-human β1 integrin) monoclonal antibody was a gift from C. Damsky (University of California at San Francisco). LM609 (anti-human β3 integrin) was obtained from Chemicon. Secondary antibodies for fluorescence-activated cell sorter analysis were obtained from Jackson Immunoresearch. Fibronectin was obtained from Life Technologies, Inc. Vitronectin was purified from human plasma by the method described in Ref. 21Yatohga T. Masako I. Heiachiro K. Hayashi M. Cell Struct. Funct. 1988; 13: 281-292Crossref PubMed Scopus (439) Google Scholar. The spinning-disc device and the method have been described in detail and validated (22Garcia A.J. Ducheyne P. Boettiger D. Biomaterials. 1997; 18: 1091-1098Crossref PubMed Scopus (185) Google Scholar, 23Garcia A.J. Huber F. Boettiger D. J. Biol. Chem. 1998; 273: 10988-10993Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Briefly, suspensions containing 2 × 105 cells were washed and resuspended in adhesion buffer (24 mm HEPES, 137 mm NaCl, 2.7 mm KCl, 1 mmMgCl2, and 2 mm glucose, pH 7.4). The cells were counted and diluted, and PMA1 (20 nm final concentration) was added as required. The cells were plated on 25-mm round coated coverslips mounted on the spinning-disc device in a total volume of 400 µl. After a 15-min incubation, the chamber of the spinning-disc device was filled with adhesion buffer, and the cells were spun for 5 min. After spinning, the coverslips were fixed with formalin, permeabilized with Triton X-100, and stained with ethidium homodimer (Molecular Probes). The coverslips were analyzed by counting the number of cells in 61 microscope fields (12% of the surface area) using a Nikon Optiphot fluorescent microscope with an ×10 fluor objective, Ludl XYZ stage, and Ludl filter wheel/shutter driven by ScopePro V1.0 software and a Photometrics SenSys cooled charge-coupled device camera and analyzed by Image-Pro V3.0 software. The data analysis, curve fits, and statistical calculations were made using SigmaPlot v5.0. For experiments in which the cells were cross-linked before spinning, sulfo-BSOCOES (bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, Pierce) was added to a final concentration of 4 mm after a 10-min adsorption period and incubated for 5 min before spinning. Note that there is variation in the potency of different lots of fibronectin as measured in terms of the adhesion strength produced under standard conditions for which we use TS 2/16 monoclonal antibody-activated α5β1 on K562 cells. Kαvβ3, Kαvβ3(Y747F), and Kαvβ3(Y759F) cells were incubated for 2 h in the presence of 75 µm sodium orthovanadate either in suspension or on vitronectin with or without PMA (20 nm). The cells were extracted with PBS buffer containing 1% Nonidet P-40, sodium orthovanadate (2 mm), aprotinin (10 µg/ml), phenylmethylsulfonyl fluoride (350 µg/ml), and leupeptin (10 µg/ml). The lysates were precleared overnight with gelatin-Sepharose and immunoprecipitated with PM6/13 anti-β3 monoclonal antibody (Chemicon) and goat anti-mouse IgG beads (ICN, Costa Mesa, CA). Samples were separated on 8% SDS-polyacrylamide gel electrophoresis transferred to nitrocellulose and blotted with 4G10 (UBI, Lake Placid, NY) for phosphotryrosine or rabbit polyclonal antibody to β3cytoplasmic domain (Chemicon AB1932) developed with ECL (Amersham Pharmacia Biotech) and analyzed with a Fuji LAS-1000 using ScienceLab 2.5 software. To develop a model for the analysis of αvβ3-mediated adhesion, K652 cells were stably transformed with vectors expressing αv and β3 or mutants of β3 integrin (24Blystone S.D. Lindberg F.P. Williams M.P. McHugh K.P. Brown E.J. J. Biol. Chem. 1996; 271 (E. J.): 31458-31462Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). These cells express α5β1 integrin in an inactive form and αvβ3. The adhesion of these cells to fibronectin was analyzed using the spinning-disc device (22Garcia A.J. Ducheyne P. Boettiger D. Biomaterials. 1997; 18: 1091-1098Crossref PubMed Scopus (185) Google Scholar). This device subjects cells to a hydrodynamic shear gradient that detaches the cells from the substrate. Fig.1A shows a typical profile of the proportion of cells remaining as a function of increasing shear stress for Kαvβ3 cells (K562 cells expressing wild-type αvβ3) plated for 15 min on a fibronectin substrate. The addition of the monoclonal antibody LM609 to block αvβ3-mediated adhesion produced a leftward shift in the force-detachment profile (Fig.1 B). From the sigmoid curve fits for each of these plots, a mean cell detachment shear stress can be calculated (τ50). This mean cell detachment stress will be called the “adhesion strength” for the particular cell population and experimental conditions. Fig. 1 C shows that the adhesion strength of Kαvβ3 cells to fibronectin was reduced to background levels similar to a BSA substrate by LM609 but was not affected by AIIB2, which blocks β1-mediated adhesion. LM609 had a similar adhesion-blocking effect for both the Y747F and the Y759F mutants as well as PMA-activated Kαvβ3 cells to fibronectin (data not shown). Finally, α5β1expressed on K562 cells has only been functionally activated in the presence of Mn2+ or activating antibodies even in the cases in which cross-talk between αvβ3 and α5β1 has been demonstrated. Thus, the adhesion of the Kαvβ3 cells to fibronectin was mediated only by αvβ3. To provide a basis for quantitative comparison of αvβ3-fibronectin bond strengths, we determined the relationship between detachment force and the number of receptor-ligand bonds (23Garcia A.J. Huber F. Boettiger D. J. Biol. Chem. 1998; 273: 10988-10993Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The number of αvβ3-fibronectin bonds was varied by varying the density of the substrate-adsorbed fibronectin. Fig.1 D shows that there was a linear relationship between fibronectin density and adhesion strength. A similar linear relationship has been reported for antibody-activated α5β1-mediated adhesion to fibronectin, endogenously activated α5β1 to fibronectin, and for endogenously activated αvβ3 to vitronectin (18Boettiger D. Huber F. Lynch L. Blystone S.D. Mol. Cell. Biol. 2001; 12: 1227-1237Crossref Scopus (39) Google Scholar, 23Garcia A.J. Huber F. Boettiger D. J. Biol. Chem. 1998; 273: 10988-10993Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 25Garcia A.J. Takagi J. Boettiger D. J. Biol. Chem. 1998; 273: 34710-34715Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The slope of this plot is determined by the product of the receptor density and the “binding strength constant.” The binding strength constant is a measure of the strength of the integrin-ligand bond and is analogous to the measurement of K D used in diffusion-based analysis of receptor-ligand bonds. Using force to measure bond strength is based on the proposition that the primary function of the integrin-ligand bond is adhesion of cells to other cells or to extracellular matrix and to resist mechanical force that would break this adhesion. K562 cells express about 1 × 105α5β1 receptor/cell (23Garcia A.J. Huber F. Boettiger D. J. Biol. Chem. 1998; 273: 10988-10993Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), and the Kαvβ3 cells expressed 5–8 × 104 αvβ3 receptors/cell. Maximal adhesion strength to fibronectin (160 ng/cm2) for TS2/16-activated K562 cells using α5β1 was 70 dynes/cm2, and for the Kαvβ3 cells using the αvβ3 receptors the adhesion strength to the same density of fibronectin was 90 dynes/cm2 (23Garcia A.J. Huber F. Boettiger D. J. Biol. Chem. 1998; 273: 10988-10993Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). At this stage of investigation there is no accurate measure of the number of receptor-ligand bonds per cell. With this caveat, the data suggest that the strength of the αvβ3-fibronectin bond is similar to the α5β1-fibronectin bond or up to 2-fold stronger. The αvβ3-mediated adhesion of Kαvβ3 cells to a vitronectin substrate is a multistage process (18Boettiger D. Huber F. Lynch L. Blystone S.D. Mol. Cell. Biol. 2001; 12: 1227-1237Crossref Scopus (39) Google Scholar). The first stage involves a ligand-induced fit in which a change in the conformation of αvβ3 is induced by ligand binding, which results in the phosphorylation of Tyr-747, in the cytoplasmic domain of β3 and binding of the LIBS-1 antibody, which detects occupied forms of β3 integrins (16Frelinger A.L. Du X.P. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1991; 266: 17106-17111Abstract Full Text PDF PubMed Google Scholar, 18Boettiger D. Huber F. Lynch L. Blystone S.D. Mol. Cell. Biol. 2001; 12: 1227-1237Crossref Scopus (39) Google Scholar). Although this initial αvβ3-vitronectin bond is specific, it is quite weak and has been missed using the common wash-type adhesion assays. Strong adhesion requires intracellular signals (20Blystone S.D. Williams M.P. Slater S.E. Brown E.J. J. Biol. Chem. 1997; 272: 28757-28761Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Fig. 2shows that this initial binding did not require metabolic energy. As positive controls, this treatment blocked the ability of PMA to induce increased adhesion to vitronectin and abolished the ability of IMR90 cells to activate any detectable α5β1 mediated to fibronectin. Thus, there is a measurable adhesion reaction between the Kαvβ3 cells and either a vitronectin or fibronectin substrate that is energy-independent and hence unlikely to involve intracellular signals or cytoskeletal rearrangements. A second aspect of the initial binding reaction between αvβ3 and vitronectin is that it sets up the intracellular signaling by inducing the phosphorylation of Tyr-747 in the cytoplasmic domain of β3. This was demonstrated by the addition of RGD peptides to Kαvβ3 cells in suspension that induced both the binding of LIBS-1 antibody and the phosphorylation of Tyr-747 in the cytoplasmic domain of β3 (20Blystone S.D. Williams M.P. Slater S.E. Brown E.J. J. Biol. Chem. 1997; 272: 28757-28761Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Fig. 3shows that a similar induction of β3 phosphorylation was observed when Kαvβ3 cells were plated on either vitronectin or fibronectin. Mutation of Tyr-747 to phenylalanine reduced the phosphorylation to baseline levels, whereas a reduced level of phosphorylation was observed for the Y759F mutant. This phosphorylation in response to ligand occupancy indicates that the initial outside-in signaling was similar for fibronectin and vitronectin. The only identified difference in the initial binding reaction was the strong binding to fibronectin as opposed to a weak binding to vitronectin Strong αvβ3-mediated adhesion to vitronectin in the Kαvβ3 model system requires the addition of either thrombin or PMA to provide a co-stimulatory signal and produce full activation of αvβ3 (20Blystone S.D. Williams M.P. Slater S.E. Brown E.J. J. Biol. Chem. 1997; 272: 28757-28761Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The effect of PMA stimulation on the adhesion strength of Kαvβ3 cells to vitronectin and fibronectin was analyzed using the spinning disc. Fig.4shows that the addition of PMA increased the strength of the αvβ3-mediated adhesion to vitronectin by 10-fold but had no effect on the strength of the αvβ3-mediated adhesion to fibronectin. Adhesion of PMA-stimulated Kαvβ3 cells to fibronectin was blocked by LM609 showing that the adhesion remained αvβ3-dependent. Although PMA-induced inside-out signals increased the strength of the αvβ3-vitronectin bonds, its signaling was either blocked by the substitution of fibronectin for vitronectin or it had no effect on the strength of the αvβ3-fibronectin bond. Y747F and Y759F mutations in the cytoplasmic domain of β3integrin reduces the strength of the αvβ3-mediated adhesion to vitronectin for both Kαvβ3 cells and CS-1 melanoma cells (18Boettiger D. Huber F. Lynch L. Blystone S.D. Mol. Cell. Biol. 2001; 12: 1227-1237Crossref Scopus (39) Google Scholar). In the Kαvβ3 cells, these mutations seemed to control binding sites in the cytoplasmic domain of β3 that interacted with the intracellular signaling process to induce the increase in the strength of the αvβ3-vitronectin bond (18Boettiger D. Huber F. Lynch L. Blystone S.D. Mol. Cell. Biol. 2001; 12: 1227-1237Crossref Scopus (39) Google Scholar). To examine the effect of these mutations on the activation of αvβ3-mediated adhesion to vitronectin and fibronectin the relative receptor-ligand bond strengths for PMA-stimulated Kαvβ3, Kαvβ3(Y747F), and Kαvβ3(Y759F) cells were determined. The adhesion strength of each of these lines was determined for different ligand densities. So that the relative slopes would be proportional to differences in the strength of the αvβ3-ligand bonds, the plots for each of the mutant lines were normalized to the wild-type Kαvβ3 cells for surface expression of αvβ3 as measured by flow cytometry. Fig.5shows that the mutants dramatically decreased the binding strength of the αvβ3-vitronectin bond but had no significant effect on the binding strength of the αvβ3-fibronectin bond. The extracellular domains of integrins bind to their extracellular ligands. This receptor-ligand linkage is the mechanically weak link in the adhesion chain between the cell and the plastic or glass substrate (23Garcia A.J. Huber F. Boettiger D. J. Biol. Chem. 1998; 273: 10988-10993Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The application of force using the spinning disc or washing the cells or centrifugation causes the weakest connection to break. All published adhesion assays rely on this application of force to separate cells from the ligand-coated substrate and on the receptor-ligand bond being the weakest link and hence the one which determines whether the cell is removed or not. The intracellular domain of integrin is linked to the cytoskeleton through a combination of linkers that may include talin, vinculin, and α-actinin (26Schoenwaelder S.M. Burridge K. Curr. Opin. Cell Biol. 1999; 11: 274-286Crossref PubMed Scopus (651) Google Scholar). To measure the relative strength of this mechanical link, cell-impermeant cross-linkers were used to strengthen the extracellular elements in the cell-to-substrate chain. Now the application of force to the cell can be used to measure the mechanical strength of the intracellular integrin-cytoskeleton connections, which are now the weakest link. In this assay, the normal adhesion strength was measured as shown above and compared with a “cross-linked adhesion strength.” If the αvβ3-fibronectin bond was the weakest link in the adhesion chain before cross-linking, there should be an increase in the measured force required for cell detachment after cross-linking (illustrated in Fig. 6,Control). After treatment with cytochalasin D to disassemble the actin filaments, the cross-linked adhesion strength was the same as the normal adhesion strength (Fig. 6, CD). In this case, it was not the αvβ3-fibronectin bond but the link between αvβ3 and the cytoskeleton that was the weak link that was broken by the application of force. In addition, the adhesion strength was reduced by cytochalasin D, in agreement with previous analyses (27Lotz M.M. Burdsal C.A. Erickson H.P. McClay D.R. J. Cell Biol. 1989; 109: 1795-1805Crossref PubMed Scopus (341) Google Scholar). This reduction demonstrates that an intact cytoskeleton and its linkage to the ligand-bound integrin are structural requirements for strong cell adhesion. In the absence of this cytoskeletal connection, the application of force to the cell would result in the detachment of the integrin from the cell rather than the dissociation of the integrin-ligand bond. The interesting result comes from the experiment done in the presence of azide plus deoxyglucose (Fig. 6, azide/DOG). As shown above, treatment with azide plus deoxyglucose had no effect on the αvβ3-mediated adhesion strength on fibronectin. However, unlike the control, there was no difference between the normal adhesion strength and the cross-linked adhesion strength. This implies that the increased strength of the αvβ3-actin linkage in the control required metabolic energy. Also note that the adhesion strength of the azide plus deoxyglucose-treated cells was significantly higher than that for the cytochalasin D-treated cells. This implies that αvβ3 actin assembly was necessary for strong adhesion even in the metabolically blocked cells. These data demonstrate that there are two distinct binding modes for αvβ3 integrin. The first mode is represented by the binding of αvβ3 to vitronectin. This binding is more typical of what is expected for integrins; strong adhesion depends on intracellular signals to activate the binding process. The second mode is represented by the binding of αvβ3 to fibronectin. This binding seems to be independent of intracellular signals or integrin activation. Activation-independent integrin-mediated adhesion has not been described previously, but this possibility has not been explored extensively. Most integrin-mediated adhesion that has been analyzedin vitro requires metabolic energy (25Garcia A.J. Takagi J. Boettiger D. J. Biol. Chem. 1998; 273: 34710-34715Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 28Faull R.J. Kovach N.L. Harlan J.M. Ginsberg M.H. J. Cell Biol. 1993; 121: 155-162Crossref PubMed Scopus (207) Google Scholar). Energy in the form of ATP is required for kinase activity and cytoskeletal assembly. Depletion of the ATP levels with a combination of azide plus deoxyglucose would alter the kinetics of these processes and reduce the rates at which they occur. This treatment also is sufficient to block integrin activation in several cell types including the Kαvβ3 cells used for these studies. Azide plus deoxyglucose treatment has been used in this report to separate the passive signaling-independent phase of integrin binding from the active signaling-dependent phase of integrin binding (25Garcia A.J. Takagi J. Boettiger D. J. Biol. Chem. 1998; 273: 34710-34715Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). 2D. Boettiger, unpublished data. The Kαvβ3 cell system also provides a parallel validation of this rationale. Activation of αvβ3-vitronectin binding depends on the addition of the co-stimulatory factors thrombin or PMA (24Blystone S.D. Lindberg F.P. Williams M.P. McHugh K.P. Brown E.J. J. Biol. Chem. 1996; 271 (E. J.): 31458-31462Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). This provides a means of controlling the activation process experimentally. Also, the Y747F mutation in the cytoplasmic domain of β3blocks this activation process even in the presence of PMA (20Blystone S.D. Williams M.P. Slater S.E. Brown E.J. J. Biol. Chem. 1997; 272: 28757-28761Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Adhesion to vitronectin in the absence of co-stimulatory signals or in cells bearing the Y747F mutation gave the same low adhesion strength observed in the presence of azide plus deoxyglucose. Thus, the form of αvβ3 involved in the low strength binding to vitronectin and the high strength binding seem similar. The consequence of this is that the activation process is ligand-specific,i.e. activation of integrin for one ligand does not necessarily activate its binding to alternative ligands. This helps to limit the total number of integrin conformations required to accommodate the binding to multiple ligands and the regulation of binding activity. The binding strength observed in the presence of azide plus deoxyglucose is likely to represent the cell-independent binding of the αvβ3 to its ligand, which is also observed using purified αvβ3 to bind to purified ligand (29Charo I.F. Nannizzi L. Smith J.W. Cheresh D. J. Cell Biol. 1990; 111: 2795-2800Crossref PubMed Scopus (231) Google Scholar). Many analyses using purified integrin receptors and purified ligand demonstrate the ability of integrins to bind ligand in the absence of other cellular components or signals. In the case of αvβ3 binding to vitronectin, the strength of the interaction was not sufficient to mediate strong substrate adhesion and extra-sensitive adhesion assays were necessary to detect this adhesion (18Boettiger D. Huber F. Lynch L. Blystone S.D. Mol. Cell. Biol. 2001; 12: 1227-1237Crossref Scopus (39) Google Scholar, 20Blystone S.D. Williams M.P. Slater S.E. Brown E.J. J. Biol. Chem. 1997; 272: 28757-28761Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In contrast, αvβ3binding to fibronectin was able to mediate strong adhesion under the same conditions, implying that this interaction has increased its ability to withstand mechanical force. This state for αvβ3 seems to be an intrinsic property of the receptor. Within the cell, this intrinsic binding is modulated by intracellular signals and attachment of integrin to the cytoskeleton. This is observed for αvβ3 binding to vitronectin in which metabolic energy, phosphorylation, assembled actin, and tyrosines at positions at 747 and 759 in the cytoplasmic domain of β3 were required to produce strong adhesion. This arm of the process is cell-dependent and hence likely to be also cell type-dependent. Although the experiments described here show only the ability of these cellular processes to increase the integrin-ligand bond strength, it is possible that cellular processes can decrease the integrin-ligand bond strength. This is seen for αiibβ3, which can bind fibrinogen as a purified molecule but seems to be restrained by the actin cytoskeleton from binding fibrinogen in nonstimulated platelets (30Bennett J.S. Zigmond S. Vilaire G. Cunningham M.E. Bednar B. J. Biol. Chem. 1999; 274: 25301-25307Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The interpretation of the data relies on an understanding of the analytic methods and the specificity of these analyses. The “bond strength” is directly related to the initial slope of the adhesion strength (mean cell detachment force) plotted as a function of ligand density. These plots followed the expected relationship for bimolecular binding with an initial linear rise to a plateau. The plateau was achieved only in the case of the αvβ3-vitronectin bond, because limitations in fibronectin coating do not allow it to reach saturation levels. For analysis of the differences between αvβ3-fibronectin and αvβ3-vitronectin bonds and the effects of mutants or PMA stimulation, the data compare relative bond strengths. The linearity of the data for the binding of αvβ3 to fibronectin argues against both inhomogeneities in the fibronectin coating and cooperative binding. Because the Kαvβ3 cells express both αvβ3 and α5β1 as potential fibronectin receptors, it is important to distinguish between the adhesion mediated by each receptor. In the experiments described, we believe that the α5β1 integrin is inactive and does not contribute for several reasons: (a) specific antibodies to αvβ3 reduced the adhesion of the Kαvβ3 (and β3 mutant) cells to fibronectin to the same level as BSA; (b) antibodies to β1 integrin had no effect even on the PMA-stimulated cells; and (c) although α5β1 on these cells can be passively activated by either Mn2+ or activating monoclonal antibodies (28Faull R.J. Kovach N.L. Harlan J.M. Ginsberg M.H. J. Cell Biol. 1993; 121: 155-162Crossref PubMed Scopus (207) Google Scholar), there are no other known mechanisms of activation, and these elements are not used for this report. Cross-talk between αvβ3 and α5β1 has been described in these cells, but the assays of cell adhesion, cell migration, and α5β1-mediated phagocytosis described require the addition of Mn2+ (31Blystone S.D. Slater S.E. Williams M.P. Crow M.T. Brown E.J. J. Cell Biol. 1999; 145: 889-897Crossref PubMed Scopus (106) Google Scholar, 32Blystone S.D. Lindberg F.P. LaFlamme S.E. Brown E.J. J. Cell Biol. 1995; 130: 745-754Crossref PubMed Scopus (96) Google Scholar). In those studies the presence of the β3 cytoplasmic domain suppressed (not activated) α5β1 function. Extensive analysis of cytoplasmic domain mutants of β3has lead to the conclusion that this domain is critical for the regulation of ligand binding (33Hughes P.E. O'Toole T.E. Ylanne J. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1995; 270: 12411-12417Crossref PubMed Scopus (171) Google Scholar, 34O'Toole T.E. Ylanne J. Culley B.M. J. Biol. Chem. 1995; 270: 8553-8558Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 35Schaffner-Reckinger E. Gouon V. Melchior C. Plancon S. Kieffer N. J. Biol. Chem. 1998; 273: 12623-12632Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). This has been extended using the spinning-disc analysis showing that Tyr-747 and Tyr-759 make specific quantitative contributions to the αvβ3 bond strength (18Boettiger D. Huber F. Lynch L. Blystone S.D. Mol. Cell. Biol. 2001; 12: 1227-1237Crossref Scopus (39) Google Scholar). The role of the cytoplasmic domain in the regulation of ligand binding and bond strength is consistent with models in which integrin function is controlled through links between integrin and the cytoskeleton (26Schoenwaelder S.M. Burridge K. Curr. Opin. Cell Biol. 1999; 11: 274-286Crossref PubMed Scopus (651) Google Scholar, 36Horwitz A.F. Duggan K. Buck C.A. Beckerle M.C. Burridge K. Nature. 1986; 320: 531-533Crossref PubMed Scopus (826) Google Scholar). Experiments in which fibronectin-coated beads were placed on cells demonstrated that α5β1on the cell surface bound fibronectin on the beads and that the α5β1-fibronectin complexes became linked to the cytoskeleton and were transported toward the nuclear region, as visualized by analysis of the movement of the beads. Using a laser trap to restrain bead movement demonstrated that this application of force produced a strengthening of the bead-cytoskeleton linkage and a progressive increase in the force necessary to restrain the movement of the bead (37Choquet D. Felsenfield D.P. Sheetz M.P. Cell. 1997; 88: 39-48Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar). This was interpreted in terms of the increase in the α5β1-cytoskeletal linkage. However, if the weakest link in the connection from the bead to the fibronectin to α5β1 integrin to cytoskeleton were the α5β1-fibronectin link, as required by the adhesion assays, then strengthening the α5β1-cytoskeletal link alone without also increasing the α5β1-fibronectin bond would not lead to the observed strengthening of the whole chain. This suggests that mechanical tension increased the integrin-ligand bond strength as well. The experiments with the spinning disc are analogous to the laser trap results except that the force is applied to the cell containing the receptor rather than the bead containing the ligand. We have advanced the analysis one step further by developing a means to distinguish between the receptor-ligand and the receptor-cytoskeletal linkages. This is done through the use of cell-impermeant chemical cross-linkers that can cross-link integrins and their ligands (18Boettiger D. Huber F. Lynch L. Blystone S.D. Mol. Cell. Biol. 2001; 12: 1227-1237Crossref Scopus (39) Google Scholar, 38Enomoto-Iwamoto M. Menko A.S. Philp N. Boettiger D. Cell Adhes. Commun. 1993; 1: 191-202Crossref PubMed Scopus (23) Google Scholar,39Garcia A.J. Boettiger D. Biomaterials. 1999; 20: 2427-2433Crossref PubMed Scopus (176) Google Scholar). Because this strengthens the weak link, we can distinguish changes in the strength of the receptor-ligand bond from the receptor-cytoskeleton linkage. For a range of αvβ3-vitronectin bond strengths, the strength of the αvβ3-cytoskeleton link remains approximately twice the αvβ3-fibronectin bond strength (18Boettiger D. Huber F. Lynch L. Blystone S.D. Mol. Cell. Biol. 2001; 12: 1227-1237Crossref Scopus (39) Google Scholar). On a fibronectin substrate, the αvβ3-cytoskeleton linkage was also about twice the αvβ3-fibronectin bond strength, in agreement with other integrin-ligand pairs. In contrast, for ATP-depleted cells plated on fibronectin, both the αvβ3-fibronectin and the αvβ3-cytoskeleton links were similar in strength, indicating that the increase in strength of the cytoskeletal connection was energy-dependent. Thus, this recapitulates the laser trap experiments and reinforces the hypothesis that integrin-ligand bond strength is controlled through connections between integrin and the cytoskeleton (26Schoenwaelder S.M. Burridge K. Curr. Opin. Cell Biol. 1999; 11: 274-286Crossref PubMed Scopus (651) Google Scholar). This leads to a model in which αvβ3 binds to fibronectin and produces a strong initial bond that does not alter the pre-existing αvβ3-cytoskeletal connections and does not respond to intracellular signals that strengthen this linkage. In contrast, the binding of αvβ3 to vitronectin induces a rearrangement of the cytoskeleton, resulting in an initial detachment of αvβ3 from the cytoskeleton leaving a bond strength similar to what has been observed after the treatment with cytochalasin D (23Garcia A.J. Huber F. Boettiger D. J. Biol. Chem. 1998; 273: 10988-10993Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Intracellular signals can now increase the strength of the αvβ3-cytoskeletal linkages, form a complex, and increase the αvβ3-vitronectin bond strength. It is likely that these different modes will lead to differences in the intracellular signals that are generated. This would provide a new means for the regulation of signals using the same receptor to convey two distinct signals depending on the particular extracellular ligand that is bound. We thank Joel Bennett and Richard Assoian for critical reading of the manuscript and Carolyn Damsky for donation of AIIB2 antibody." @default.
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• W1571638637 title "Distinct Ligand-binding Modes for Integrin αvβ3-Mediated Adhesion to Fibronectinversus Vitronectin" @default.
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