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• W2022087377 abstract "“Activation” of integrins is involved in the dramatic transition of leukocytes and platelets from suspension to adhesion. The integrin αvβ3 is not known to take part in this sort of transition, even though it shares its β subunit with αIIbβ3, the activable integrin on platelets. In the context of a constitutively adhered cell, changes in activation state may be more subtle in their effects, but nonetheless important in regulating cell behavior. We hypothesized that αvβ3 can undergo conformational changes analogous to those associated with αIIbβ3 activation. Accordingly, we examined αvβ3 on the surface of M21 cells (a human melanoma cell line) and found that, like αIIbβ3, it can undergo conformational changes upon binding of a ligand analog and can be activated for ligand binding and migration by a monoclonal antibody directed against β3. Modulation of the binding of this activating antibody, AP5, ligand binding, and antibody-mediated activation all are associated with a discrete cation-binding site shared in both αIIbβ3 and αvβ3. Based on a measured Ki, this site has an apparent Kd for calcium of approximately 20 μM. At physiological levels of calcium, about 40% of the total αvβ3 on a cell's surface is in a conformation detected by AP5. The data suggest a model for both αvβ3 and αIIbβ3 function in which the molecule can exist in either of (at least) two conformational states, one stabilized either by AP5 or ligand binding, refractory to calcium binding, and enhanced for ligand recognition, the other stabilized by calcium binding and refractory to AP5 and ligand binding. Functional analysis suggests that AP5 activates αvβ3 by preventing occupation of this calcium site, and that the activated form of αvβ3 differs functionally from the basal form. The active form is more conducive to migration and the basal to tight adhesion. “Activation” of integrins is involved in the dramatic transition of leukocytes and platelets from suspension to adhesion. The integrin αvβ3 is not known to take part in this sort of transition, even though it shares its β subunit with αIIbβ3, the activable integrin on platelets. In the context of a constitutively adhered cell, changes in activation state may be more subtle in their effects, but nonetheless important in regulating cell behavior. We hypothesized that αvβ3 can undergo conformational changes analogous to those associated with αIIbβ3 activation. Accordingly, we examined αvβ3 on the surface of M21 cells (a human melanoma cell line) and found that, like αIIbβ3, it can undergo conformational changes upon binding of a ligand analog and can be activated for ligand binding and migration by a monoclonal antibody directed against β3. Modulation of the binding of this activating antibody, AP5, ligand binding, and antibody-mediated activation all are associated with a discrete cation-binding site shared in both αIIbβ3 and αvβ3. Based on a measured Ki, this site has an apparent Kd for calcium of approximately 20 μM. At physiological levels of calcium, about 40% of the total αvβ3 on a cell's surface is in a conformation detected by AP5. The data suggest a model for both αvβ3 and αIIbβ3 function in which the molecule can exist in either of (at least) two conformational states, one stabilized either by AP5 or ligand binding, refractory to calcium binding, and enhanced for ligand recognition, the other stabilized by calcium binding and refractory to AP5 and ligand binding. Functional analysis suggests that AP5 activates αvβ3 by preventing occupation of this calcium site, and that the activated form of αvβ3 differs functionally from the basal form. The active form is more conducive to migration and the basal to tight adhesion. INTRODUCTIONIntegrins comprise a large family of heterodimeric cell-surface receptors involved in cell-matrix and cell-cell interactions. These receptors mediate adhesion and modulate the cell's responses to various adhesive ligands (for review, see (1.Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1175) Google Scholar)). The integrin's role in modulating information flow recently has become a field of growing interest. Several potentially significant biochemical changes are known now to be regulated by adhesion events involving integrins, including changes in intracellular pH, changes and oscillations in intracellular free calcium, and phosphorylation on tyrosine of a number of proteins (1.Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1175) Google Scholar, 2.Lipfert L. Haimovich B. Schaller M.D. Cobb B.S. Parsons J.T. Brugge J.S. J. Cell Biol. 1992; 119: 905-912Crossref PubMed Scopus (626) Google Scholar, 3.Pelletier A.J. Bodary S.C. Levinson A.D. Mol. Biol. Cell. 1992; 3: 989-998Crossref PubMed Scopus (90) Google Scholar, 4.Kornberg L. Earp H.S. Parsons J.T. Schaller M. Juliano R.L. J. Biol. Chem. 1992; 267: 23439-23442Abstract Full Text PDF PubMed Google Scholar, 5.Haimovich B. Lipfert L. Brugge J.S. Shattil S.J. J. Biol. Chem. 1993; 268: 15868-15877Abstract Full Text PDF PubMed Google Scholar, 6.Shattil S.J. Thromb. Haemostasis. 1993; 70: 224-228Crossref PubMed Scopus (39) Google Scholar) (for review, see (7.Schwartz M.A. Cancer Res. 1993; 53: 1503-1506PubMed Google Scholar)).Integrins bind divalent cations, which are required for integrin function(8.Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar). In addition to this basic requirement of divalent cations, specific divalent cations have been suggested to regulate integrins. For example, manganese (Mn2+) appears to enhance the adhesive properties of many integrins (9.Smith J.W. Piotrowicz R.S. Mathis D. J. Biol. Chem. 1994; 269: 960-967Abstract Full Text PDF PubMed Google Scholar, 10.Crowe D.T. Chiu H. Fong S. Weissman I.L. J. Biol. Chem. 1994; 269: 14411-14418Abstract Full Text PDF PubMed Google Scholar, 11.Felding-Habermann B. Cheresh D.A. Curr. Opin. Cell Biol. 1993; 5: 864-868Crossref PubMed Scopus (352) Google Scholar) although the mechanism of this enhancement is not clear. A model has been proposed by Smith and co-workers (9.Smith J.W. Piotrowicz R.S. Mathis D. J. Biol. Chem. 1994; 269: 960-967Abstract Full Text PDF PubMed Google Scholar, 12.D'Souza S.E. Haas T.A. Piotrowicz R.S. Byers-Ward V. McGrath D.E. Soule H.R. Cierniewski C. Plow E.F. Smith J.W. Cell. 1994; 79: 659-667Abstract Full Text PDF PubMed Scopus (204) Google Scholar) that a cation must be displaced from the integrin αvβ3 as ligand binds. In this model, a calcium or other divalent cation is bound to the integrin and is necessary for proper function, but must be displaced as ligand binds.Several integrins are conformationally complex molecules. That is, they can exist in different conformational states, or activation states, that affect ligand recognition. Examples of this included αIIbβ3 and some β2 and β1 integrins on leukocytes(13.Altieri D.C. Wiltse W.L. Edgington T.S. J. Immunol. 1990; 145: 662-670PubMed Google Scholar, 14.Sims P.J. Ginsberg M.H. Plow E.F. Shattil S.J. J. Biol. Chem. 1991; 266: 7345-7352Abstract Full Text PDF PubMed Google Scholar, 15.O'Toole T.E. Loftus J.C. Du X.P. Glass A.A. Ruggeri Z.M. Shattil S.J. Plow E.F. Ginsberg M.H. Cell Regul. 1990; 1: 883-893Crossref PubMed Scopus (224) Google Scholar, 16.Ginsberg M.H. Du X. Plow E.F. Curr. Opin. Cell Biol. 1992; 4: 766-771Crossref PubMed Scopus (387) Google Scholar). In the case of αIIbβ3, binding of ligand analogs, such as RGD-containing peptides, induces a conformational change that can be detected by a change in the Stokes radius of the receptor(17.Parise L.V. Helgerson S.L. Steiner B. Nannizzi L. Phillips D.R. J. Biol. Chem. 1987; 262: 12597-12602Abstract Full Text PDF PubMed Google Scholar). This change occurs concomitantly with the presentation of “LIBS,” or ligand-induced binding sites, which are epitopes recognized by monoclonal antibodies known as anti-LIBS antibodies(18.Frelinger III, A.L. Du X. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1991; 266: 17106-17111Abstract Full Text PDF PubMed Google Scholar, 19.Du X. Gu M. Weisel J.W. Nagaswami C. Bennett J.S. Bowditch R. Ginsberg M.H. J. Biol. Chem. 1993; 268: 23087-23092Abstract Full Text PDF PubMed Google Scholar). “Activation” of αIIbβ3 results in a conformational change that can be detected by changes in fluorescence resonance energy transfer (14.Sims P.J. Ginsberg M.H. Plow E.F. Shattil S.J. J. Biol. Chem. 1991; 266: 7345-7352Abstract Full Text PDF PubMed Google Scholar) and occurs concomitantly with increased efficiency of ligand binding and changes in ligand specificity (reviewed in (16.Ginsberg M.H. Du X. Plow E.F. Curr. Opin. Cell Biol. 1992; 4: 766-771Crossref PubMed Scopus (387) Google Scholar)). It is important to recognize that the so-called “resting” or “inactive” state of αIIbβ3 is a functional integrin(3.Pelletier A.J. Bodary S.C. Levinson A.D. Mol. Biol. Cell. 1992; 3: 989-998Crossref PubMed Scopus (90) Google Scholar, 20.Pelletier A.J. Kunicki T.J. Ruggeri Z.M. Quaranta V. J. Biol. Chem. 1995; 270: 18133-18140Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21.Savage B. Shattil S.J. Ruggeri Z.M. J. Biol. Chem. 1992; 267: 11300-11306Abstract Full Text PDF PubMed Google Scholar). Therefore, we refer to it as the “basal” state. The binding of some anti-LIBS antibodies results in changes in αIIbβ3 consistent with activation; that is, the antibodies stimulate binding of ligand ((19.Du X. Gu M. Weisel J.W. Nagaswami C. Bennett J.S. Bowditch R. Ginsberg M.H. J. Biol. Chem. 1993; 268: 23087-23092Abstract Full Text PDF PubMed Google Scholar) and references therein).We recently showed for the case of αIIbβ3 that the conformational state can affect not only ligand recognition, but “outside-in” signals as well(20.Pelletier A.J. Kunicki T.J. Ruggeri Z.M. Quaranta V. J. Biol. Chem. 1995; 270: 18133-18140Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We have hypothesized that integrins in general exist on the cell surface as subpopulations in different conformation, or activation, states with specific ligand-binding and signaling functions. Implicit in this hypothesis is the assumption that integrins on constitutively adhered cells can undergo changes in conformation, or activation state, similar to those seen for αIIbβ3. Although the role of integrin activation in the case of integrins that mediate the conversion from suspension to adhesion in blood cells is obvious, the effect of integrin activation in the context of an adherent cell might be less dramatic.To explore this possibility, we have taken advantage of a conformation-sensitive antibody we have described previously. This antibody, AP5, binds a linear epitope corresponding to the first 6 amino acids of the β3 subunit(22.Honda S. Tomiyama Y. Pelletier A.J. Annis D. Honda Y. Orchekowski R. Ruggeri Z. Kunicki T.J. J. Biol. Chem. 1995; 270: 11947-11954Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). It is an anti-LIBS for αIIbβ3, that is its epitope is presented following binding of soluble ligand analogs. It also is an activating antibody; it stimulates the binding of soluble fibrinogen either to platelets or to a cell line expressing αIIbβ3(20.Pelletier A.J. Kunicki T.J. Ruggeri Z.M. Quaranta V. J. Biol. Chem. 1995; 270: 18133-18140Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 22.Honda S. Tomiyama Y. Pelletier A.J. Annis D. Honda Y. Orchekowski R. Ruggeri Z. Kunicki T.J. J. Biol. Chem. 1995; 270: 11947-11954Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Since the presentation of LIBS following ligand binding is a measure of a conformational change, we tested whether this conformational change occurred in αvβ3 on the surface of the human melanoma cell line M21 and found that it did. AP5 also activates αvβ3, as measured by increased the efficiency of cell adhesion to various ligands. Moreover, pretreatment with AP5 resulted in an increase in M21 cell migration. We found that a discrete calcium-binding site residing both in αvβ3 and αIIbβ3 was involved in regulating the conformational changes associated with LIBS presentation and activation. Calcium inhibits the presentation of the AP5 epitope on αvβ3 with a Ki of about 20 μM. Functional analysis suggests a model wherein AP5 activates αvβ3 by preventing occupation of an inhibitory calcium-binding site. Further, the binding characteristics of the active and basal receptors were investigated in intact cells. The active receptor promoted efficient, but weak, interactions with matrix and favored migration. The basal receptor, in contrast, promoted stronger adhesion and was less conducive to migration. This result indicates that activation does not represent a simple “on/off” switch of the integrin, but rather a change in function and a resulting change in cell behavior.MATERIALS AND METHODSCell Lines and MediaM21 is a human melanoma cell line that expresses αvβ3(11.Felding-Habermann B. Cheresh D.A. Curr. Opin. Cell Biol. 1993; 5: 864-868Crossref PubMed Scopus (352) Google Scholar) and no αIIbβ3(11.Felding-Habermann B. Cheresh D.A. Curr. Opin. Cell Biol. 1993; 5: 864-868Crossref PubMed Scopus (352) Google Scholar). 1A. J. Pelletier, unpublished data. M21 cells were grown on tissue culture-treated Petri dishes (various) in RPMI 1640 (Bio-Whittaker, Walkersville, MD) supplemented with glutamine and 10% fetal calf serum and including a penicillin/streptomycin antibiotic mixture.Other SolutionsFor measurements of antibody binding, a buffer based on Dulbecco's-modified phosphate-buffered saline (DPBS) 2The abbreviations used are: DPBSDulbecco's phosphate-buffered salinePBSphosphate-buffered salineFITCfluorescein isothiocyanateMFImean fluorescence intensityOpnosteopontinVnvitronectin. was made, except that the concentration of calcium or magnesium was varied as indicated. This buffer was made based on the recipe from Bio-Whittaker and includes 900 μM CaCl2, 500 μM MgCl2, sodium and potassium supplied as phosphate, glucose, and sodium pyruvate (34 μg/ml). The buffer was made lacking divalent cations, and these were added as specified. For example, “40 μM calcium DPBS” would be DPBS made with only 40 μM CaCl2. Unless otherwise specified, all other components of DPBS are as given in the recipe. “PBS” when specified, is standard phosphate-buffered saline without added divalent cations.Flow CytometryFITC-labeled AP5 was generated by incubation of AP5 with FITC-Celite (Sigma) at a w:w ratio of 2 in the dark at room temperature for 1 h (in 100 mM sodium carbonate, pH 10). Celite was removed from suspension by centrifugation, and labeled antibody was separated from free FITC by chromatography on a PD-10 column (Pharmacia Biotech, Uppsala, Sweden). Relative incorporation was determined by comparing A290 to A493. Cells were removed from growth plates with 2 mM EDTA in PBS, washed three times with DPBS of the specified calcium concentration, and exposed to appropriate control or experimental antibodies for 30 min on ice in the same buffer. When “RGD” is specified, G4120 (23.Barker P.L. Bullens S. Bunting S. Burdick D.J. Chan K.S. Deisher T. Eigenbrot C. Gadek T.R. Gantzos R. Lipari M.T. Muir C.D. Napier M.A. Pitti R.M. Padua A. Quan C. Stanley M. Struble M. Tom J.Y.K. Burnier J.P. J. Med. Chem. 1992; 35: 2040-2048Crossref PubMed Scopus (197) Google Scholar) (kind gift of Dr. Thomas Gadek and Genentech Inc.) was included in the incubation at 40 μM. The dose of 40 μM was chosen because, by all functional criteria, this is in excess of saturation. Cells were washed twice with the specified buffer, on ice or at 4°C at all times, and finally collected in 0.5 ml of specified buffer. Cells were treated with propidium iodide (Sigma, 500 ng/ml, final) just prior to flow cytometry, which was performed on a Becton-Dickinson FACScan with “Lyses II” software. Appropriate forward- and side-scatter gates were set for these cells, and only those cells excluding propidium iodide were counted.Results were inspected to ensure that a homogeneous, monophasic distribution of fluorescence was obtained, and mean fluorescence intensity (MFI) was extracted from the histogram by the Lysys-II program.CalculationsMFI data were plotted and line-fitting was performed using “Delta-Graph II” software. This treatment of the data requires the assumption that the binding of the antibody over the time measured constitutes all forward rate and therefore can be considered a measure of “initial velocity” of antibody binding. We independently determined that there was no significant off-rate for the antibody over the time course used (not shown). However, if a substantial off-rate component were present, a linear double-reciprocal plot would not be obtained. In all cases, the R2 values of these lines were greater than 9.6 and generally greater than 9.9. Calculation of Ki for calcium was performed by comparing the ratio of the slopes of the lines obtained using the relationship: [I]÷(S2/S1−1)=Ki(Eq. 1) where S1 and S2 are the slopes of the line without and with inhibitor, respectively, and [I] is the concentration of inhibitor present. This was necessary because the units of binding, MFI, could not be related directly to concentration of antibody-antigen complex with our instrumentation. However, the ratio of the slopes is dimensionless and could be used to calculate Ki. “Without inhibitor” is 1 μM Ca2+, since this concentration resulted in maximal binding.Adhesion AssayThe assay used was that of Calof and Lander (24.Calof A.L. Lander A.D. J. Cell Biol. 1991; 115: 779-794Crossref PubMed Scopus (171) Google Scholar). Briefly, this assay was performed with cells labeled with [35S]methionine adhered to polystyrene plates coated with the appropriate ligand or control. Following adhesion, nonadhered cells were removed from the plate by centrifugation in the inverted orientation at the indicated G force. Adhered cells are fixed, the plates are exposed to a phosphorimaging plate, and the remaining 35S (a measure of adhered cells) was quantitated on a phosphorimaging scanner (Applied Biosystems). For Fig. 9, the amount of adhesion obtained on bovine serum albumin (less than 2% maximal adhesion, see Fig. 2) was subtracted to obtain corrected “integrated volume,” a direct measure of cells adhered. In Fig. 2, integrated volume is expressed as a percentage of cells adhering to poly-L-lysine. Values are averages of 6 wells ± 1 S.D. Vitronectin was obtained from Telios Pharmaceuticals (San Diego, CA), osteopontin made from a recombinant fusion protein was the kind gift of Drs. Jeffrey Smith and Dana Hu of the La Jolla Cancer Research Foundation, and fibrinogen was the kind gift of Dr. Z. Ruggeri of The Scripps Research Institute; poly-L-lysine was from Sigma. Ligands were coated at the indicated concentrations for 4 h. Plates were washed once with PBS and blocked with 2% heat-denatured bovine serum albumin (Sigma) for 2 h. Cells were pretreated with 100 μg/ml AP5 in 1 μM calcium DPBS for the indicated times on ice. Adhesion was carried out in 100 μM calcium DPBS or as indicated. Values for counts bound and background were calculated by the Applied Biosystems software.Figure 2:Adhesion of M21 cells to Vn and Opn was quantitated either with or without pretreatment with AP5 (100 μg/ml). Values are averages of 6 points ± 1 S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Migration AssayThe assay employed was a “Transwell” plate assay (Transwell plates with a pore size of 8 μ, Costar, Cambridge, MA). The lower side of the membrane was coated with Vn or Opn as indicated for 3 h at room temperature. Both sides were blocked with 1% bovine serum albumin for 1 h. Cells were removed from growth plates as above and treated with 100 μg/ml AP5 for 20 min on ice in 10 μM calcium DPBS, or mock-treated. 5 × 104 cells were added per well in the indicated buffer and centrifuged briefly to bring them in contact with the membrane. Following a 20-h incubation at 37°C, cells were fixed and stained with “Diff-quick” (Baxter Scientific), and cells were removed from the top surface of the membrane with cotton swabs. Cells that had migrated to the lower side of the membrane were counted; values presented reflect the averages from at least 15 fields ± 1 S.D.RESULTSAP5 Is an Activating, Anti-LIBS Antibody for αvβ3The human melanoma cell line, M21, which expresses high levels of αvβ3(11.Felding-Habermann B. Cheresh D.A. Curr. Opin. Cell Biol. 1993; 5: 864-868Crossref PubMed Scopus (352) Google Scholar, 25.Felding-Habermann B. Mueller B.M. Romerdahl C.A. Cheresh D.A. J. Clin. Invest. 1992; 89: 2018-2022Crossref PubMed Google Scholar) but no detectable αIIbβ3(11.Felding-Habermann B. Cheresh D.A. Curr. Opin. Cell Biol. 1993; 5: 864-868Crossref PubMed Scopus (352) Google Scholar, 25.Felding-Habermann B. Mueller B.M. Romerdahl C.A. Cheresh D.A. J. Clin. Invest. 1992; 89: 2018-2022Crossref PubMed Google Scholar) 1 was used in these studies. AP5, directly labeled with FITC, was incubated with M21 cells at the indicated concentrations either in the presence or absence of a cyclic RGD analog, G4120 at 40 μM(23.Barker P.L. Bullens S. Bunting S. Burdick D.J. Chan K.S. Deisher T. Eigenbrot C. Gadek T.R. Gantzos R. Lipari M.T. Muir C.D. Napier M.A. Pitti R.M. Padua A. Quan C. Stanley M. Struble M. Tom J.Y.K. Burnier J.P. J. Med. Chem. 1992; 35: 2040-2048Crossref PubMed Scopus (197) Google Scholar). G4120 binds with high affinity to both αvβ3 and αIIbβ3(23.Barker P.L. Bullens S. Bunting S. Burdick D.J. Chan K.S. Deisher T. Eigenbrot C. Gadek T.R. Gantzos R. Lipari M.T. Muir C.D. Napier M.A. Pitti R.M. Padua A. Quan C. Stanley M. Struble M. Tom J.Y.K. Burnier J.P. J. Med. Chem. 1992; 35: 2040-2048Crossref PubMed Scopus (197) Google Scholar) and stimulates binding of AP5 to cells expressing αIIbβ3(20.Pelletier A.J. Kunicki T.J. Ruggeri Z.M. Quaranta V. J. Biol. Chem. 1995; 270: 18133-18140Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Following the incubation and washes, bound AP5 was quantitated by flow cytometry. Fig. 1A shows histograms of fluorescence distribution for 10,000 cells incubated either with a control antibody or 10 μg/liter AP5 with or without G4120. M21 cells comprise a single, homogeneous population of cells with respect to AP5 binding, an indication of “error.” Binding to all cells is increased dramatically by the inclusion of G4120. Fig. 1B shows mean fluorescence intensities (MFI) taken from similar histograms over the indicated concentrations of AP5, in the presence or absence of G4120. In all cases, the distribution of fluorescence within the population measured was similar to that seen in the histograms in Fig. 1A.Figure 1:A, cells were exposed to FITC-labeled AP5 at 10 μg/ml in DPBS on ice in the presence or absence of the cyclic RGD analog, G4120 (40 μM) as indicated. The control antibody was FITC-labeled anti-αIIbβ3 (CP8). Histograms represent fluorescence of 10,000 individual cells. B, MFI was calculated from similar histograms at the indicated concentrations of AP5, in the presence or absence of G4120, as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since AP5 is an activating antibody for αIIbβ3, we next wanted to determine if it could activate αvβ3 as well. αvβ3 has been reported to support adhesion to many extracellular matrix proteins, including vitronectin (Vn), fibrinogen, fibronectin, laminin, denatured collagen I, and osteopontin (Opn)(9.Smith J.W. Piotrowicz R.S. Mathis D. J. Biol. Chem. 1994; 269: 960-967Abstract Full Text PDF PubMed Google Scholar, 11.Felding-Habermann B. Cheresh D.A. Curr. Opin. Cell Biol. 1993; 5: 864-868Crossref PubMed Scopus (352) Google Scholar). However, binding studies with αvβ3 often were performed under different conditions. For example, αvβ3 will only support adhesion to fibrinogen in the presence of the divalent cation manganese (Mn2+)(11.Felding-Habermann B. Cheresh D.A. Curr. Opin. Cell Biol. 1993; 5: 864-868Crossref PubMed Scopus (352) Google Scholar). Similarly, αvβ3 binding to Opn also is stimulated by Mn2+ and inhibited by Ca2+(9.Smith J.W. Piotrowicz R.S. Mathis D. J. Biol. Chem. 1994; 269: 960-967Abstract Full Text PDF PubMed Google Scholar).We therefore thought it likely that αvβ3 binding to these substrates might be regulated by activation and the alternate cations may induce a kind of activation, as has been suggested ((9.Smith J.W. Piotrowicz R.S. Mathis D. J. Biol. Chem. 1994; 269: 960-967Abstract Full Text PDF PubMed Google Scholar) and references therein). Accordingly, we examined the ability of AP5 to stimulate adhesion of M21 cells to Vn or Opn in the absence of Mn2+ and in low Ca2+ (40 μM). Mg2+ was included at 400 μM in these experiments (see “Materials and Methods”). The assay for adhesion we employed was that of Calof and Lander(24.Calof A.L. Lander A.D. J. Cell Biol. 1991; 115: 779-794Crossref PubMed Scopus (171) Google Scholar), which includes a low speed centrifugation of the inverted plate to remove unbound or loosely adhered cells. The results, expressed as the percent of cells that remain adhered following 5 min at 100 × g, are shown in Fig. 2. AP5 increased the adhesion of cells to both substrata under these conditions. We have performed more conventional plate assays with similar results on fibrinogen as well (data not shown). Adhesion to fibrinogen, Opn, and Vn in these latter assays was inhibited completely by inclusion of 40 μM G4120 to block αvβ3. LM609 (150 μg/ml), a blocking, anti-αvβ3 antibody, inhibited 100% of the adhesion to Opn and about 85% of adhesion to Vn. This residual adhesion probably was due to αvβ5 on the surface of M21(11.Felding-Habermann B. Cheresh D.A. Curr. Opin. Cell Biol. 1993; 5: 864-868Crossref PubMed Scopus (352) Google Scholar, 25.Felding-Habermann B. Mueller B.M. Romerdahl C.A. Cheresh D.A. J. Clin. Invest. 1992; 89: 2018-2022Crossref PubMed Google Scholar). For this reason, Opn adhesion was used in most subsequent assays.AP5 Binding Is Inhibited by CalciumWe previously reported that the reactivity of AP5 toward αIIbβ3 is inhibited by calcium(22.Honda S. Tomiyama Y. Pelletier A.J. Annis D. Honda Y. Orchekowski R. Ruggeri Z. Kunicki T.J. J. Biol. Chem. 1995; 270: 11947-11954Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). We next determined if the same is true for αvβ3. Fig. 3 shows the same graph of MFI versus AP5 concentration in the presence or absence of 40 μM G4120 as is shown in Fig. 1B. That experiment was performed in DPBS, which includes 900 μM CaCl2. At the same time, a parallel experiment was performed in DPBS lacking CaCl2 (all other components of DPBS were included). Under these conditions, binding of AP5 is constitutively high and is insensitive to the addition of G4120. Since the effect of G4120 and that of low calcium are not additive, the ligand analog must act directly to overcome the effect of the calcium, rather than through an independent mechanism. Thus, the binding of calcium to αvβ3 inhibits the presentation of this ligand-induced binding site, and the ligand analog overcomes that inhibition.Figure 3:Cells were exposed to the indicated concentrations of FITC-labeled AP5 in the presence or absence of G4120 (40 μM) either in DPBS or DPBS lacking calcium. Bound AP5 was quantitated by flow cytometry and is expressed as MFI for 10,000 cells. The data for the DPBS case are exactly the same as those presented in Fig. 1B. ▪, -Ca, -G4120; ▴, +Ca, -G4120; •, -Ca, +G4120; ◊, +Ca, +G4120.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Calcium is not exerting its effect on AP5 directly. In equilibrium dialysis experiments, no binding of 45Ca to AP5 was detected at concentrations that inhibit binding of AP5 to αvβ3 (data not shown). Also, since the effect of G4120 clearly is on the receptor, we would expect low calcium and G4120 to have additive effects on the overall binding of the antibody if the effect of the calcium were on the antibody. Furthermore, results from detailed analysis of the inhibition (below) are inconsistent with calcium exerting its effect on AP5.Fig. 4 shows the effect of calcium concentration over a range from 100 nM to 2 mM on the binding of AP5. Starting with a calcium-minus “DPBS,” buffers of the indicated calcium concentration were made. The binding of AP5 is maximal at approximately 1 μM Ca2+ and is inhibited significantly at 10 μM.Figure 4:FITC-labeled AP5 (130 μg/ml) was exposed to M21 cells in a buffer based on DPBS, but made with the indicated concentrations of calcium. Washes were carried out in the same buffer as the incubations. MFI of 10,000 cells is presented. Maximal binding of AP5 occurs at approximately 1 μM calcium.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We performed a similar test of the effect of calcium on AP5 binding over a range of AP5 concentrations from 5-140 μg/ml, which is shown in Fig. 5A. These data show that even at high levels of calcium (900 μM) at which the inhibitory effect of calcium has reached a plateau, AP5 still binds in a dose-dependent manner. The shape of the curves suggests that there are two different effects of" @default.
• W2022087377 created "2016-06-24" @default.
• W2022087377 creator A5003212140 @default.
• W2022087377 creator A5042820746 @default.
• W2022087377 creator A5068090004 @default.
• W2022087377 date "1996-01-01" @default.
• W2022087377 modified "2023-10-09" @default.
• W2022087377 title "Activation of the Integrin αvβ3 Involves a Discrete Cation-binding Site That Regulates Conformation" @default.
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