Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 QUICK SEARCH:   [advanced]


     


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pampori, N.
Right arrow Articles by Shattil, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pampori, N.
Right arrow Articles by Shattil, S. J.
Social Bookmarking
 Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 274, Issue 31, 21609-21616, July 30, 1999


Mechanisms and Consequences of Affinity Modulation of Integrin alpha Vbeta 3 Detected with a Novel Patch-engineered Monovalent Ligand*

Nisar PamporiDagger §, Takaaki HatoDagger §, Dwayne G. Stupack, Sallouha AidoudiDagger , David A. ChereshDagger , Glen R. Nemerow, and Sanford J. ShattilDagger parallel **

From the Departments of Dagger  Vascular Biology,  Immunology, and parallel  Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrin alpha Vbeta 3 mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which alpha Vbeta 3 is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post-receptor events is unknown. Affinity regulation of the related integrin, alpha IIbbeta 3, has been established using a monovalent ligand-mimetic antibody, PAC1 Fab. To determine the role of affinity modulation of alpha Vbeta 3, a novel monovalent ligand-mimetic antibody (WOW-1) was created by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single alpha V integrin-binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated alpha Vbeta 3, but not to alpha IIbbeta 3, in receptor and cell binding assays. alpha Vbeta 3 affinity varied with the cell type. Unstimulated B-lymphoblastoid cells bound WOW-1 Fab poorly (apparent Kd = 2.4 µM), but acute stimulation with phorbol 12-myristate 13-acetate increased receptor affinity >30-fold (Kd = 80 nM), with no change in receptor number. In contrast, alpha Vbeta 3 in melanoma cells was constitutively active, but ligand binding could be suppressed by overexpression of beta 3 cytoplasmic tails. Up-regulation of alpha Vbeta 3 affinity had functional consequences in that it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that alpha Vbeta 3 is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrins mediate cell adhesion and signaling during many developmental, physiological, and pathological processes (1-4). The beta 3 integrin family includes alpha IIbbeta 3, often referred to as the fibrinogen receptor, and alpha Vbeta 3, the vitronectin receptor. alpha IIbbeta 3 is confined to megakaryocytes and platelets and is required for platelet aggregation through interactions with Arg-Gly-Asp (RGD)-containing adhesive ligands, including fibrinogen and von Willebrand factor (5). alpha Vbeta 3 is more widely expressed in proliferating endothelial cells, arterial smooth muscle cells, osteoclasts, platelets, and certain subpopulations of leukocytes and tumor cells (6, 7). The list of cognate ligands for alpha Vbeta 3 overlaps that for alpha IIbbeta 3, but includes others, such as osteopontin, matrix metalloproteinase-2, and adenovirus penton base, which do not interact with alpha IIbbeta 3 (6, 8-10). In the adult organism, alpha Vbeta 3 has been implicated in processes ranging from wound healing to tumor angiogenesis (11), arterial restenosis (12), osteoporosis (13), tumor progression (14), and adenovirus internalization (8).

One fundamental function of integrins is ligand binding, which in many cases is rapidly regulated by a process variously referred to as "integrin activation," "inside-out signaling," or "affinity/avidity modulation" (15-19). Integrin activation encompasses at least two events: 1) modulation of receptor affinity through conformational changes in the alpha beta heterodimer and 2) modulation of receptor avidity through facilitation of lateral diffusion and/or clustering of heterodimers (5, 18, 20, 21). The importance of rapid regulated changes in integrin affinity/avidity is easy to appreciate for alpha IIbbeta 3 because platelets must interact productively with fibrinogen or von Willebrand factor only in vascular wounds. Studies of alpha IIbbeta 3 activation have been facilitated by the use of soluble ligands, most notably a multivalent ligand-mimetic antibody called PAC1 and its monovalent Fab fragment, which contain an R(G/Y)D tract in H-CDR31 (5, 22). Evidence acquired using these monovalent and multivalent forms of PAC1 indicates that changes in affinity and avidity play complementary roles in alpha IIbbeta 3 function (23).

On the other hand, the significance of inside-out signaling and, in particular, affinity modulation for alpha Vbeta 3 has been less certain. The ligand binding function of alpha Vbeta 3 has usually been assessed by cell adhesion assays, and these have clearly shown that activation of certain cells leads to alpha Vbeta 3-mediated adhesion (24-28). However, adhesion assays can be strongly influenced by post-ligand binding events, including changes in cell shape, that can obscure the precise contributions of affinity or avidity modulation to the overall response. Recently, Byzova and Plow (28, 29) showed that soluble prothrombin, a coagulation protein, can bind directly in an RGD-dependent fashion to alpha IIbbeta 3 in platelets and alpha Vbeta 3 in cultured human endothelial cells. Notably, prothrombin binding to alpha Vbeta 3 could be increased by MnCl2, a general activator of integrins, and by phorbol 12-myristate 13-acetate, an activator of protein kinase C. Taken together, these results indicate that alpha Vbeta 3 has the potential to be regulated at the level of ligand binding. However, the specific mechanisms, relative contributions, and biological consequences of affinity and avidity modulation of alpha Vbeta 3 remain to be established. The distinction between affinity and avidity regulation is not academic because the underlying mechanisms and effects on cell function are likely to be different (5, 18, 19). Furthermore, mechanistic insights into the regulation of ligand binding could facilitate current efforts aimed at developing drugs that inhibit or stimulate alpha Vbeta 3 function in vivo.

The purpose of these studies was to determine whether alpha Vbeta 3 is subject to affinity modulation and, if so, to explore the potential pathophysiological implications of such regulation. To accomplish this task, we characterized the binding of soluble monovalent and multivalent ligands to alpha Vbeta 3 in several cell types, reasoning that a monovalent ligand would be sensitive to affinity modulation and a multivalent ligand would be sensitive to both affinity and avidity modulation (18, 23, 30). Penton base, a coat protein from adenovirus type 2, was selected as a multivalent ligand because each of its five subunits contains a 50-amino acid RGD tract that mediates virus internalization through alpha V integrins (8). A novel monovalent ligand called WOW-1 Fab was created by replacing the H-CDR3 of PAC1 Fab with a single integrin-binding domain of penton base. This switched the selectivity of the Fab fragment from activated alpha IIbbeta 3 to activated alpha Vbeta 3, enabling a direct assessment of alpha Vbeta 3 affinity state. These studies establish that alpha Vbeta 3 is subject to affinity regulation, with direct implications for the anchorage-dependent functions of alpha Vbeta 3 and for adenovirus-mediated gene delivery.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation of Soluble alpha Vbeta 3 Ligands-- Recombinant penton base from adenovirus type 2 was baculovirus-expressed in Trichoplusia Tn 5B1-4 insect cells and purified as described previously (8). The purified protein migrated as a single ~325-kDa band on native polyacrylamide gels and an ~80-kDa band on SDS-polyacrylamide gels. Penton base was conjugated to Alexa-488 to form Alexa-penton base (aPB)1 according to the manufacturer's instructions (Molecular Probes, Inc., Eugene, OR). Purified human fibrinogen was obtained from Enzyme Research Laboratories (South Bend, IN) and labeled with fluorescein isothiocyanate (31).

WOW-1 Fab was created by replacing the 19-amino acid H-CDR3 of antibody PAC1 Fab (22) with the 50-amino acid alpha V integrin-binding domain from adenovirus type 2 penton base (32) by splice-overlap polymerase chain reaction using oligonucleotides PB-For (5'-ACACAGCCATATATTACTGTGCCAGAGCGGAAGAGAACTCCAACGCG), PB-Rev (5'-ACTGAGGTTCCTTGACCCCACGCAGCGGGGGCGGCAGCTTCTGC), Pac1-For (5'-GCGCGGGAGATCTCAGGTGCAGCTGAAGCAGTCAGGA), and Pac1-Rev (5'-GGCGCATGACCGGTACAATCCCTGGGCACAATTTTCTTG). The resulting WOW-1 fd DNA fragment was digested with BglII/AgeI and cloned into a Drosophila expression vector, pMT/BiP/V5-HisB (Invitrogen, Carlsbad, CA), which contains the Drosophila metallothionein promoter and BiP secretion signal and places a His6 tag at the C terminus of fd DNA. Similarly, PAC1kappa containing NcoI and AgeI sites was amplified by polymerase chain reaction with kappa -For (5'-GGCGCGGGAGATCTCCATGGGATGTTTTGATGACCCAAACTCCA) and kappa -Rev (5'-GGCGCATGACCGGTACACTCATTCCTGTTGAAGCTCTTG) and cloned into pMT/BiP/V5-HisB. Nineteen µg of WOW-1 fd and PAC1kappa DNA in pMT/BiP/V5-HisB were cotransfected with 1 µg of selection vector (pCoHYGRO, Invitrogen) into Drosophila melanogaster S2 cells by calcium phosphate precipitation. Stable S2 cell lines were selected with hygromycin B and screened for secretion of WOW-1 Fab after a 36-72-h induction with 500 µM CuSO4.

WOW-1 Fab was purified from 250-1000 ml of serum-free medium by column chromatography on Ni2+-nitrilotriacetic acid (QIAGEN Inc., Chatsworth, CA). Typical yields were 2-5 mg/liter with a purity of >= 90% as estimated on SDS gels stained with silver or Coomassie Blue. WOW-1 Fab migrated as a single ~58-kDa band on nonreduced SDS gels and reacted on Western blots with a monoclonal antibody specific for a linear epitope in the integrin-binding domain of penton base (33) and with affinity-purified goat anti-mouse kappa -chain (BIOSOURCE International, Camarillo, CA). After reduction, WOW-1 Fab migrated as an ~33-kDa fd chain and an ~25-kDa kappa -chain. There was no evidence of fd or kappa -homodimers. As with PAC1 Fab (22), the relative migration of WOW-1 Fab on a Sephadex G-200 column indicated that it was monomeric and therefore monovalent in aqueous solution.

Mammalian Cells and DNA Transfections-- cDNAs encoding full-length human alpha V and beta 3 were subcloned into pcDNA3 and pCDM8, respectively, and 2 µg of each were transfected into CHO-K1 cells to obtain transient and stable transfectants as described (34). Stable transfectants surviving antibiotic selection were further screened for high alpha Vbeta 3 expression by single-cell fluorescence-activated cell sorting using the alpha Vbeta 3-specific monoclonal antibody LM609 (35). CHO cells stably expressing wild-type human alpha IIbbeta 3 and alpha Vbeta 3(D723R) were described previously (34, 36). M21-L is a clone of the human melanoma cell line M21 that lacks the alpha V subunit (37). alpha Vbeta 3-M21-L cells were produced by transient transfection of M21-L cells with 2 µg each of alpha V/pcDNA3 and beta 3/pCDM8 using Superfect (QIAGEN Inc.). CS-1 is a hamster melanoma cell line that does not express alpha Vbeta 3 or alpha Vbeta 5 because it does not synthesize the beta 3 or beta 5 subunits. alpha Vbeta 3-CS-1 cells stably expressing hamster alpha V and human beta 3 were obtained by transfection of CS-1 cells with human beta 3 (38). JY is an immortalized human B-lymphoblastoid cell line that expresses alpha Vbeta 3, but not alpha Vbeta 5 (24, 39).

Analysis of Cell-surface Integrin Expression-- Cells were suspended in incubation buffer (137 mM NaCl, 2.7 mM KCl, 3.3 mM NaH2PO4, 3.8 mM HEPES, 1 mM MgCl2, 5.5 mM glucose, and 1 mg/ml bovine serum albumin, pH 7.4) and incubated for 30 min on ice with a monoclonal antibody (10 µg/ml) specific for alpha Vbeta 3 (LM609), alpha IIbbeta 3 (D57) (34), or alpha Vbeta 5 (P1F6) (40). After washing, the cells were incubated for another 30 min on ice with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (H + L chain-specific; BIOSOURCE International), washed again, and analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) (23). As a negative control, samples were incubated with the secondary antibody alone.

Ligand Binding Assays-- Binding of aPB, WOW-1 Fab, and fluorescein isothiocyanate-fibrinogen to cells was assessed by flow cytometry. Typically, cells were cultured overnight in low serum medium (e.g. 0.5% fetal bovine serum) and resuspended in incubation buffer at 1-1.5 × 107 cells/ml, and 4-6 × 105 cells were incubated with one of these ligands for 30 min at room temperature in a final volume of 50 µl. As indicated, some samples were also incubated in the presence of one or more of the following reagents: antibody AP5 ascites (1:50) to activate beta 3 integrins (41); 0.25 mM MnCl2 to activate integrins (18); 2 mM RGDS or 5 mM EDTA to specifically block ligand binding to integrins; 50 µM cRGDfV, a selective alpha V integrin antagonist (Peninsula Laboratories, Inc., Belmont, CA); 5 µM Integrilin, a selective alpha IIbbeta 3 antagonist (42); or a 100 µg/ml concentration of the function-blocking antibody LM609 or P1F6. In some experiments, ligand binding and alpha Vbeta 3 expression were measured simultaneously by incubation of cells with ligands in the presence of biotin-SSA6 (7 µg/ml), a non-function-blocking anti-beta 3 monoclonal antibody (22). After 30 min at room temperature, cells were washed and incubated with phycoerythrin-streptavidin (1:25 final dilution; Molecular Probes, Inc.) for 20 min on ice. In the case of WOW-1 Fab, an Alexa-conjugated anti-His6 monoclonal antibody (Accurate Chemical and Scientific Corp., Westbury, NY) was added at this stage (50 µg/ml). Cells were washed and resuspended in 0.5 ml of incubation buffer containing 2 µg/ml propidium iodide (Sigma). Ligand binding (FL1 channel) was analyzed immediately on the gated subset of live cells (propidium iodide-negative, FL3) that were strongly positive for alpha Vbeta 3 expression (FL2). Binding isotherms were subjected to nonlinear least-squares regression analysis using an equation for one-site binding (Prism 2.0 software, GraphPAD Software for Science, San Diego, CA). Two-tailed p values for paired samples were obtained by Student's t test.

To examine the effects of overexpression of isolated integrin cytoplasmic tails on ligand binding to alpha Vbeta 3, alpha Vbeta 3-CS-1 cells were transfected with a mammalian expression plasmid encoding Tac-beta 1, Tac-beta 3, or Tac-alpha 5, using Fugene-6 transfection reagent (Roche Molecular Biochemicals) (43, 44). Forty-eight h after transfection, cells were suspended in incubation buffer at 1.5 × 106/ml and incubated for 30 min at room temperature with 150 nM aPB or 425 nM WOW-1 Fab in the presence or absence of 5 mM EDTA. After washing, cells were incubated for an additional 30 min on ice with 2.5 µg/ml biotinylated anti-Tac monoclonal antibody (7G7B6), followed by incubation with phycoerythrin-conjugated anti-mouse IgG and (when WOW-1 Fab was present) 50 µg/ml Alexa-conjugated anti-His6 monoclonal antibody. Ligand binding was analyzed on the gated subset of live cells strongly positive for Tac expression. In parallel tubes, cells were co-stained with SSA6 and anti-Tac antibody to quantitate alpha Vbeta 3 expression in the Tac-positive cells.

Binding of WOW-1 Fab to purified alpha Vbeta 3 receptors from human placenta and alpha IIbbeta 3 from human platelets was measured by enzyme-linked immunosorbent assay in the presence of 50 µM CaCl2, MgCl2, and MnCl2. Nonspecific binding was determined in the presence of 2 mM RGDS (22).

Cell Adhesion Assays-- Immulon-2 microtiter wells (Dynex Laboratories, Chantilly, VA) were coated with unlabeled penton base (1-100 ng/well) overnight at 4 °C, followed by blocking with 20 mg/ml bovine serum albumin. CHO cells stably expressing alpha Vbeta 3 were labeled with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein/acetoxymethyl ester (Molecular Probes, Inc.), and cell adhesion to immobilized penton base was quantitated by cytofluorometry at 485/530 nm (23).

Adenovirus-mediated Gene Delivery-- CS-1 and alpha Vbeta 3-CS-1 cells (105 cells) were suspended for 5 min at room temperature in 100 µl of incubation buffer. In some cases, 2.5 mM MnCl2 was also present to induce maximal integrin activation. Then, replication-deficient adenovirus type 5 encoding green fluorescent protein (GFP) was added to the cell suspension at a multiplicity of infection (m.o.i.) of 50 or 500 (45). After 1 h at 37 °C, virus not internalized was digested by incubation of the cells with 0.03% trypsin and 0.35 mM EDTA for 5 min at 37 °C. After 72 h, GFP expression was quantitated by flow cytometry.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interaction of a Novel Monovalent Ligand with Integrin alpha Vbeta 3-- To document and study the significance of affinity modulation of alpha Vbeta 3, we set out to develop a monovalent reporter ligand analogous to the activation-dependent anti-alpha IIbbeta 3 antibody PAC1 Fab. We reasoned that swapping the 50-amino acid RGD motif from adenovirus penton base with the H-CDR3 of PAC1 Fab might convert the antibody into a suitable alpha Vbeta 3 ligand. To test this idea, preliminary binding studies were conducted with the new antibody, designated WOW-1 Fab, using purified integrins in the presence of 50 µM MnCl2, which activates integrins by a direct effect on the extracellular domain (18). WOW-1 Fab bound to purified alpha Vbeta 3 and, to a lesser extent, to purified alpha Vbeta 5. Binding was half-maximal at 40 nM Fab fragment and was blocked by >95% by 2 mM RGDS or 5 mM EDTA. In contrast, there was no detectable binding of WOW-1 Fab to purified alpha IIbbeta 3 at antibody concentrations as high as 2 µM, even though the parent antibody, PAC1 Fab, bound half-maximally to alpha IIbbeta 3 at 50 nM. These results indicate that the reengineering of PAC1 Fab converted it from an activation-dependent alpha IIbbeta 3 antibody into an antibody that reacts with activated alpha Vbeta 3.

To determine if WOW-1 Fab reacted preferentially with activated alpha Vbeta 3 in cells, Fab fragment binding was compared with that of multivalent penton base using CHO cells stably transfected with human alpha Vbeta 3 (alpha Vbeta 3-CHO cells). Flow cytometric analysis showed that the surface of these cells expressed large amounts of alpha Vbeta 3, modest amounts of alpha Vbeta 5, and no detectable alpha IIbbeta 3 (Fig. 1A). When aPB or WOW-1 Fab was incubated with the cells over a range of ligand concentrations (5-1000 nM) and for various periods of time at room temperature, specific ligand binding, defined as that inhibitable by 2 mM RGDS or 5 mM EDTA, reached a steady state by 30 min, and nonspecific binding accounted for <= 15% of total binding. Therefore, all subsequent binding studies were carried out under these conditions. aPB and WOW-1 Fab bound specifically but at low levels to unstimulated alpha Vbeta 3-CHO cells. However, direct activation of alpha Vbeta 3 by anti-beta 3 antibody AP5 caused a significant increase in the binding of both ligands (p < 0.01) (Fig. 1B).


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 1.   Binding of soluble Alexa-penton base and WOW-1 Fab to CHO cells expressing alpha Vbeta 3. In A, alpha Vbeta 3-CHO cells or parental CHO cells were incubated with primary antibodies specific for alpha Vbeta 3 (LM609), alpha IIbbeta 3 (D57), or alpha Vbeta 5 (P1F6), and antibody binding was detected with fluorescein isothiocyanate-labeled secondary antibody as described under "Experimental Procedures." Cells stained with secondary antibody (2°Ab) only were used as a negative control. For comparison, antibody binding to parental CHO cells was also studied. In B, the alpha Vbeta 3-CHO cells were incubated with either 75 nM aPB or 106 nM WOW-1 Fab for 30 min at room temperature in the absence or presence of a 1:50 dilution of antibody AP5 ascites to activate alpha Vbeta 3 or 5 mM EDTA to inhibit specific ligand binding. Then, binding of aPB and WOW-1 Fab was measured by flow cytometry as described under "Experimental Procedures." The data represent specific ligand binding, defined as that inhibited by EDTA, and are presented as the means ± S.E. of three independent experiments. Similar results were obtained if alpha Vbeta 3 was stimulated with the purified Fab fragment of another activating antibody (LIBS6) instead of antibody AP5 ascites. Asterisks indicate that ligand binding was significantly greater in the presence than in the absence of antibody AP5 (p < 0.01).

To assess the selectivity of these ligands for alpha Vbeta 3 in this system, the effect of various function-blocking compounds was studied. Binding of aPB and WOW-1 Fab in the presence of antibody AP5 was inhibited >= 85% by 2 mM RGDS or 50 µM cRGDfV, a cyclic peptide selective for alpha V integrins (Fig. 2) (46). On the other hand, a cyclic peptide selective for alpha IIbbeta 3 (Integrilin) inhibited ligand binding by <20%, even at a concentration (1 µM) 100-fold higher than necessary to prevent fibrinogen or PAC1 binding to platelet alpha IIbbeta 3 (42). Furthermore, the alpha Vbeta 3 function-blocking antibody LM609 (100 µg/ml) inhibited ligand binding by >70%, whereas the alpha Vbeta 5 function-blocking antibody P1F6 had no such effect (data not shown). In addition, neither aPB nor WOW-1 Fab bound detectably to resting or thrombin-stimulated human platelets, which express >50,000 alpha IIbbeta 3 receptors, but <500 alpha Vbeta 3 receptors/cell (7). Collectively, these results indicate that a monovalent ligand, WOW-1 Fab, and a multivalent ligand, aPB, are sensitive to the activation state of alpha Vbeta 3 and that they do not recognize alpha IIbbeta 3. Thus, WOW-1 Fab may be a suitable reporter for changes in alpha Vbeta 3 affinity. Since WOW-1 Fab (and aPB) also recognizes alpha Vbeta 5, particular efforts were made in the experiments that follow to utilize cells that express alpha Vbeta 3, but little or no alpha Vbeta 5.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of integrin inhibitors on binding of aPB and WOW-1 Fab to alpha Vbeta 3-CHO cells. Ligand binding was carried out as described in the legend to Fig. 1 in the presence of antibody AP5 ascites (1:50) and an integrin inhibitor, as indicated. EDTA was present at 5 mM, RGDS at 2 mM, cRGDfV at 50 µM, and Integrilin at 1 µM. Data are plotted as a percentage of the value for the antibody AP5-treated sample in the absence of an inhibitor and represent the means ± S.E. of three experiments.

Affinity of alpha Vbeta 3 Can Be Regulated by Inside-out Signals-- To determine if alpha Vbeta 3 is susceptible to affinity modulation by inside-out signals, the binding of WOW-1 Fab to JY B-lymphoblasts was studied. These cells were selected because they express alpha Vbeta 3, but not alpha Vbeta 5, and they adhere rapidly to vitronectin in response to activation of protein kinase C by phorbol 12-myristate 13-acetate (24, 39). Incubation of JY cells for 15 min with 100 nM phorbol 12-myristate 13-acetate caused a significant increase in specific binding of aPB (2.7 ± 0.2-fold increase; p < 0.05), consistent with an increase in alpha Vbeta 3 affinity and/or avidity. Furthermore, phorbol 12-myristate 13-acetate caused a 2.4 ± 0.1-fold increase in the binding of WOW-1 Fab (p < 0.05). Neither response was increased further by activating antibody AP5 (Fig. 3A). Phorbol 12-myristate 13-acetate did not increase the surface expression of alpha Vbeta 3, as measured by antibody LM609. To determine whether the changes in WOW-1 Fab binding reflected changes in alpha Vbeta 3 affinity, ligand binding was analyzed over a range of antibody concentrations. Unstimulated JY cells exhibited a very low affinity for WOW-1 Fab (apparent Kd = 2600 ± 700 nM; mean ± S.E.) and a value for maximal binding of 24.8 ± 4.1 arbitrary fluorescence units (Fig. 3B). In marked contrast, JY cells stimulated with phorbol 12-myristate 13-acetate exhibited a >30-fold increase in binding affinity (apparent Kd = 80 ± 18 nM) with no change in maximal binding (23.5 ± 1.1 units). This effect was prevented if the cells were first depleted of metabolic energy by a 30-min preincubation with 0.2% sodium azide and 4 mg/ml 2-deoxy-D-glucose. These results establish that energy-dependent inside-out signals can regulate the ligand binding affinity of alpha Vbeta 3.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   alpha Vbeta 3 is susceptible to affinity modulation by inside-out signals. In A, JY lymphoblastoid cells were incubated in the presence of either 75 nM aPB or 425 nM WOW-1 Fab for 15 min without an agonist (No Tx), with 100 nM phorbol 12-myristate 13-acetate (PMA), or with phorbol 12-myristate 13-acetate plus antibody AP5 ascites (1:50). Then, specific ligand binding was determined by flow cytometry. Data are the means ± S.E. of three experiments. Asterisks denote a significant difference compared with the No Tx sample (p < 0.05). In B, binding of WOW-1 Fab to JY cells was examined over a range of Fab fragment concentrations. The data are plotted as specific (RGDS-inhibitable) binding and were subjected to nonlinear regression analysis for binding to a single site. Values for apparent Kd and maximal binding are presented under "Results." The curves are computer-generated best fits of the data. Goodness of fit (R2) values ranged from 0.94 to 1.00.

Determinants of alpha Vbeta 3 Activation State-- Additional experiments were performed to identify factors that influence alpha Vbeta 3 affinity using readily transfectable cell lines that stably express human alpha Vbeta 3. alpha Vbeta 3 on vascular cells may encounter multiple ligands simultaneously during the process of wound healing. Therefore, we wondered if the affinity/avidity of alpha Vbeta 3 differed for various ligands. Equilibrium binding of aPB, WOW-1 Fab, and the adhesive ligand fibrinogen was compared in alpha Vbeta 3-CHO cells. As summarized in Table I, each ligand bound specifically to approximately the same total number of receptors in unstimulated alpha Vbeta 3-CHO cells. However, the affinity/avidity of alpha Vbeta 3 for fibrinogen was ~15-fold lower than that for aPB, despite the fact that both ligands are multivalent and similar in molecular mass. Activation of alpha Vbeta 3 with antibody AP5 increased the binding affinity/avidity for both ligands, but it had no effect on maximal binding (Table I). On the other hand, despite the differences in valency between aPB and WOW-1 Fab, their binding constants were similar. Overall, these results show that alpha Vbeta 3 can interact differentially with macromolecular ligands and that the affinity state of the receptor is one determinant of such interactions.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Binding of different ligands to alpha Vbeta 3 expressed in CHO cells
Ligand binding was determined by flow cytometry, and binding isotherms were analyzed as described under "Experimental Procedures" and in the legend to Fig. 3. Data represent the combined results of three independent experiments with each ligand. Maximum binding (Bmax) is expressed in arbitrary fluorescence units. Goodness of fit (R2) values ranged from 0.93 to 1.00.

In circulating platelets, the "basal" activation state of alpha IIbbeta 3 must remain low to prevent thrombosis. However, this requirement may not pertain to all cells that express alpha Vbeta 3. Therefore, ligand binding was studied simultaneously in alpha Vbeta 3-CHO cells and in two unrelated melanoma cell lines, alpha Vbeta 3-M21-L and alpha Vbeta 3-CS-1, to assess cell type-specific variations in the basal activation state of alpha Vbeta 3. To control for minor variations in alpha Vbeta 3 expression between the cell lines, ligand binding was expressed on a "per receptor" basis using anti-beta 3 antibody SSA6 to quantitate receptor expression. Unstimulated alpha Vbeta 3-M21-L cells bound significantly more aPB than did alpha Vbeta 3-CHO cells (p < 0.01). This difference was maintained even after further activation of alpha Vbeta 3 with antibody AP5 (p < 0.05) (Fig. 4). Similar results were obtained with alpha Vbeta 3-CS-1 cells instead of alpha Vbeta 3-M21-L cells and with WOW-1 Fab instead of aPB (data not shown). Taken together with the marked differences observed in the binding of WOW-1 Fab to unstimulated JY lymphoblasts and alpha Vbeta 3-CHO cells (Fig. 3B and Table I), these results indicate that the basal activation state of alpha Vbeta 3 varies with the cell type.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4.   Comparison of aPB binding to alpha Vbeta 3-CHO cells and alpha Vbeta 3-M21-L melanoma cells. Binding of aPB (75 nM) to each cell line was carried out as described in the legend to Fig. 1. Specific aPB binding is expressed on a per receptor basis as the mean fluorescence intensity (mfi) of aPB binding divided by the mean fluorescence intensity of SSA6 binding. Each bar represents the mean ± S.E. of four experiments. Single and double asterisks denote p values of <0.01 and <0.05, respectively, for the difference between the CHO and melanoma cells.

Integrin cytoplasmic tails have been implicated in affinity/avidity modulation of several integrins (47), but there is no direct information about their role in regulating ligand binding to alpha Vbeta 3. Certain point mutations or truncations of the beta 3 cytoplasmic tail, such as beta 3(D723R), result in constitutive activation of alpha IIbbeta 3 in CHO cells (34, 36). To determine whether alpha Vbeta 3 is affected by such a modification, ligand binding to alpha Vbeta 3(D723R) was assessed. This mutant was stably expressed in CHO cells to approximately the same level as wild-type alpha Vbeta 3 (Fig. 5A). However, unstimulated alpha Vbeta 3(D723R)-CHO cells bound significantly more aPB than unstimulated alpha Vbeta 3-CHO cells (p < 0.01), equivalent to the amount of aPB bound to alpha Vbeta 3-CHO cells treated with antibody AP5 (Fig. 5B). A second alpha Vbeta 3(D723R) clone gave the same results, and similar results were obtained using WOW-1 Fab instead of aPB. Thus, a structural change in the beta 3 cytoplasmic tail can be propagated to the extracellular domains of alpha Vbeta 3 to influence ligand binding affinity.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of an activating mutation in the beta 3 integrin cytoplasmic tail on the binding of penton base to alpha Vbeta 3. In A, stable CHO cell lines expressing either alpha Vbeta 3 or alpha Vbeta 3(D723R) were stained with anti-beta 3 antibody SSA6 and phycoerythrin-streptavidin to assess surface expression of alpha Vbeta 3. In B, specific binding of aPB (75 nM) was studied as described in the legend to Fig. 1. aPB binding is expressed on a per receptor basis. Data represent the means ± S.E. of four experiments. The asterisk denotes a difference between alpha Vbeta 3 and alpha Vbeta 3(D723R) at the p < 0.01 level. For comparison, the corresponding value for aPB binding to antibody AP5-treated alpha Vbeta 3-CHO cells was 0.034 ± 0.002. 1°Ab, primary antibody; mfi, mean fluorescence intensity.

The activation state of certain integrins, such as alpha IIbbeta 3 and alpha 5beta 1, can be suppressed in a dominant-inhibitory fashion by overexpression of isolated beta 3 or beta 1 cytoplasmic tails, but not by alpha 5 tails (43, 44, 48). To determine if alpha Vbeta 3 is subject to this type of suppression, alpha Vbeta 3-CS-1 cells were transiently transfected with chimeric constructs consisting of the beta 3, beta 1, or alpha 5 cytoplasmic tails fused at their N termini to the extracellular and transmembrane domains of the Tac subunit of the interleukin-2 receptor, which was used to target the tails to the vicinity of the plasma membrane. Despite similar levels of expression of the chimeras, Tac-beta 3 and Tac-beta 1 caused a significant reduction in specific binding of aPB and WOW-1 Fab when compared with Tac-alpha 5 (p < 0.01) (Fig. 6, A and B). In contrast, none of these tail chimeras affected surface expression of alpha Vbeta 3 (Fig. 6C). Since the isolated beta  tails may bind proteins that normally interact with integrins (43), these results suggest that alpha Vbeta 3 may be regulated by direct interactions with intracellular proteins.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of overexpression of isolated integrin cytoplasmic tails on ligand binding to CS-1 melanoma cells expressing alpha Vbeta 3. As described under "Experimental Procedures," alpha Vbeta 3-CS-1 cells were transiently transfected with the Tac-alpha 5, Tac-beta 1, or Tac-beta 3 chimera. Forty-eight h after transfection, the cells were incubated for 30 min at room temperature with 150 nM aPB (A) or 425 nM WOW-1 Fab (B) in the presence or absence of 5 mM EDTA. The cells were stained with anti-Tac antibody and phycoerythrin-conjugated anti-mouse IgG in order to set a live gate on the Tac-expressing cells, and specific binding of aPB and WOW-1 Fab was measured by flow cytometry. C shows that the Tac constructs had no effect on expression levels of alpha Vbeta 3 as monitored with anti-beta 3 antibody SSA6. Data represent the means ± S.E. of three experiments. The asterisks indicate that ligand binding in the presence of Tac-beta 1 or Tac-beta 3 was significantly less than with Tac-alpha 5 (p < 0.01).

Functional Consequences of Affinity Modulation of alpha Vbeta 3-- To determine whether changes in receptor affinity affect the adhesive function of alpha Vbeta 3, the adhesion of alpha Vbeta 3-CHO cells to immobilized penton base was quantitated. Adhesion was dependent on the coating concentration of penton base and was half-maximal at 30-40 ng/well (Fig. 7). Activation of alpha Vbeta 3 by antibody AP5 led to a 7-fold leftward shift in the dose-response curve such that half-maximal adhesion now occurred at ~5 ng of penton base/well. Treatment of the cells with 1 mM MnCl2 caused an even further shift in the dose-response curve, either because it induced a more profound effect on alpha Vbeta 3 or it activated additional alpha V integrins (Fig. 7). Analysis of adherent cells by light microscopy showed that they had become fully spread by 90 min. Thus, affinity modulation of alpha Vbeta 3 promotes both cell adhesion and post-ligand binding responses, such as cell spreading.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of alpha Vbeta 3 activation on the adhesion of alpha Vbeta 3-CHO cells to penton base. As described under "Experimental Procedures," microtiter wells were coated with penton base, and the adhesion of alpha Vbeta 3-CHO cells was studied for 90 min at 37 °C either with no additive (open circle ) or with antibody AP5 ascites (1:50; ) or MnCl2 (0.25 mM; black-triangle). Some aliquots were also incubated with 50 µM cRGDfV under each of these conditions (, ×, and *, respectively) to assess whether cell adhesion was dependent on the presence of alpha V integrins. This experiment is representative of three so performed.

Adenoviruses utilize alpha V integrins to enter cells and are a common gene delivery vector. Therefore, we tested whether changes in alpha Vbeta 3 affinity could influence adenovirus-mediated gene transfer. Recombinant adenovirus containing cDNA encoding GFP was incubated with CS-1 melanoma cells at m.o.i. of 50 and 500, and subsequent cellular expression of GFP was taken as a marker for infection and gene transfer. CS-1 cells were chosen because they do not express alpha Vbeta 5, thus potentially restricting adenovirus internalization through stably expressed alpha Vbeta 3. When parental cells without alpha Vbeta 3 were incubated with virus for 60 min and monitored for infection 72 h later, they exhibited a relatively low level of GFP expression. Unstimulated alpha Vbeta 3-CS-1 cells exhibited a higher level of GFP expression, particularly at the higher m.o.i. (Fig. 8A). However, if incubation of alpha Vbeta 3-CS-1 cells with virus was carried out in the presence of 2.5 mM MnCl2 to activate alpha Vbeta 3, the cells subsequently exhibited a much greater increase in GFP expression at the lower m.o.i. (p < 0.01) (Fig. 8, A and B, black bars). MnCl2 had no effect on GFP expression in the parental CS-1 cells. Enhanced GFP expression in cells containing activated alpha Vbeta 3 was blocked if the cells were preincubated with an excess of WOW-1 Fab (1.7 µM) before the addition of virus (Fig. 8B, gray bar). Thus, adenovirus-mediated gene transfer is directly affected by affinity modulation of alpha Vbeta 3.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of alpha Vbeta 3 expression and activation on adenovirus-mediated gene delivery. In A, parental CS-1 cells (No alpha Vbeta 3) and alpha Vbeta 3-CS-1 cells were incubated for 1 h with an adenovirus vector encoding GFP at a multiplicity of infection of 50 or 500. In addition, aliquots of the alpha Vbeta 3-CS-1 cells were incubated with virus in the presence of 2.5 mM MnCl2 to induce maximal integrin activation. Viral infection and gene delivery were assessed 72 h later by quantitating cellular expression of GFP by flow cytometry. A depicts a single experiment, and B shows the means ± S.E. of three experiments conducted at a m.o.i. of 50. The gray bar shows the effect of preincubating alpha Vbeta 3-CS-1 cells with 1.7 µM WOW-1 Fab for 20 min before addition of virus. mfi, mean fluorescence intensity.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to determine if the affinity of alpha Vbeta 3 for ligands is subject to acute modulation in cells. If this were the case, it could have significant clinical implications since numerous physiological and pathological events are mediated by alpha Vbeta 3. We reasoned that the most direct way to evaluate alpha Vbeta 3 affinity was to employ a soluble, monovalent macromolecular ligand. Since no such ligand exists, we engineered one. First, we determined that a soluble multivalent ligand, adenovirus penton base, binds to alpha Vbeta 3 in an activation-dependent manner. Then, in an example of patch engineering (49), we grafted a small portion of penton base, the 50-amino acid alpha V integrin-binding domain, into the H-CDR3 of PAC1, a monovalent Fab antibody that is specific for activated alpha IIbbeta 3. The resulting monovalent Fab antibody, WOW-1, retained the activation-dependent characteristics of PAC1 and penton base and interacted with alpha Vbeta 3, but not alpha IIbbeta 3. Using WOW-1 Fab to study alpha Vbeta 3, the following major conclusions were reached. 1) The basal affinity state of alpha Vbeta 3 varies among cell types, being extremely low in lymphoid cells and higher in melanoma cell lines. 2) alpha Vbeta 3 is subject to rapid affinity modulation by inside-out signals, including those downstream of protein kinase C. 3) At least some of the cellular signals that regulate alpha Vbeta 3 affinity appear to converge at the cytoplasmic tails of the integrin. 4) Affinity modulation has direct functional consequences, both for the adhesion and signaling functions of alpha Vbeta 3 and for adenovirus-mediated gene transfer.

alpha Vbeta 3 Is Subject to Affinity Modulation-- Studies with numerous integrins have suggested that no single signaling pathway or mechanism is likely to regulate ligand binding to all integrins (5, 15-18, 50). This means that mechanisms of integrin activation relevant to alpha IIbbeta 3 do not necessarily apply to alpha Vbeta 3. Unfortunately, in the case of alpha Vbeta 3, it has been difficult to directly assess the precise contributions and mechanisms of affinity and avidity modulation because most preferred ligands, including vitronectin and osteopontin, are predominantly matrix-associated or difficult to work with in soluble form. Moreover, whereas cellular agonists can stimulate rapid alpha Vbeta 3-dependent adhesion and/or migration of platelets, leukocyte cell lines, endothelial cells, and vascular smooth muscle cells (24-28), the adhesion end point does not reveal the relative contributions of ligand binding and post-ligand binding events to the overall response.

Recently, Byzova and Plow (29) and Byzova et al. (51) showed that soluble prothrombin can interact with both alpha IIbbeta 3 and alpha Vbeta 3 in a cell activation-dependent manner. In particular, the binding of prothrombin to alpha Vbeta 3 in human umbilical vein endothelial cells was increased markedly by treatment with MnCl2 or a phorbol ester. The results with phorbol ester established that signals triggered by protein kinase C can increase the ligand binding function of alpha Vbeta 3 in these cells. They also raised new questions that have now been addressed in the present work. Does acute regulation of alpha Vbeta 3 affect the binding of soluble ligands other than prothrombin, and is it relevant to a wider variety of cell types? Does regulation occur at the level of alpha Vbeta 3 affinity, or do cells primarily control avidity? If alpha Vbeta 3 affinity is regulated, what cellular functions are affected by this process?

Adenovirus penton base is a homopentamer, and cryoelectron micrographs indicate that each of its five RGD tracts is situated at the apex of one of five 22-Å protrusions (33). Thus, penton base may be able to interact simultaneously with two or more alpha Vbeta 3 receptors. Consequently, this ligand should be sensitive to both affinity modulation (e.g. conformational change) and avidity modulation (e.g. lateral diffusion/clustering) of alpha Vbeta 3. In contrast and as shown experimentally for PAC1 Fab and alpha IIbbeta 3, a monovalent ligand is likely to be more sensitive to affinity than avidity modulation (23, 30). We found that both penton base and WOW-1 Fab bound specifically and saturably to alpha Vbeta 3 in an activation-dependent manner. This was true whether alpha Vbeta 3 was stimulated from outside the cell by an activating antibody or MnCl2 or from inside the cell by biochemical signals or by virtue of an activating mutation in the beta 3 cytoplasmic tail. Thus, the results with WOW-1 Fab establish that alpha Vbeta 3 is susceptible to affinity modulation. It is important to emphasize, however, that these results do not exclude a significant role for lateral diffusion and clustering of alpha Vbeta 3 receptors. Indeed, several other integrins have been shown to undergo diffusion and clustering within the plane of the plasma membrane (17, 20, 21, 52). Even in the case of alpha IIbbeta 3, the prototypic integrin regulated by affinity changes, receptor clustering facilitates irreversible ligand binding and is required for outside-in signaling (23).

Like penton base and WOW-1 Fab, soluble fibrinogen also bound to alpha Vbeta 3-CHO cells in an activation-dependent manner (Table I). On the other hand, Byzova and Plow (28) showed that adhesion of endothelial cells to immobilized fibrinogen via alpha Vbeta 3 did not require cell activation. This suggests that soluble fibrinogen is an activation-dependent ligand for alpha Vbeta 3, whereas solid-phase fibrinogen is an activation-independent ligand. A similar situation pertains to the interaction of fibrinogen with alpha IIbbeta 3 in platelets (53). Thus, interactions between beta 3 integrins and fibrinogen are governed by the physical state and conformation of both the receptor and the ligand. Of note, the apparent Kd for soluble fibrinogen binding to unstimulated alpha Vbeta 3-CHO cells (9200 nM) was much different from the apparent Kd for penton base or WOW-1 Fab (~500 nM), but this difference narrowed after receptor activation (Table I). Since alpha Vbeta 3 in vascular cells likely encounters multiple cognate ligands during wound healing, these results indicate that the relevant biological interaction will depend on several factors, including the concentration and physical state of the ligands and the activation state and membrane density of alpha Vbeta 3.

Regulation of Affinity Changes in alpha Vbeta 3-- Binding of penton base and WOW-1 Fab to alpha Vbeta 3 in JY lymphoblasts increased significantly following stimulation of the cells with phorbol 12-myristate 13-acetate (Fig. 3). On the other hand, substantial binding of these ligands to alpha Vbeta 3-CS-1 melanoma cells was observed even without deliberate cell stimulation, and binding was inhibited by overexpression of isolated beta 1 or beta 3 integrin cytoplasmic tails (Fig. 6). Taken together, these results suggest that 1) the basal affinity state of alpha Vbeta 3 varies among cell types; 2) intracellular signals can either increase or decrease alpha Vbeta 3 affinity; 3) one relevant pathway for activation of alpha Vbeta 3 involves protein kinase C; and 4) the cytoplasmic tails of alpha Vbeta 3 are involved in affinity regulation. The importance of the cytoplasmic tails is supported further by the observations that a point mutation in beta 3 (D723R) caused the receptor to be constitutively active in CHO cells (Fig. 5), and deletion of both the alpha V and beta 3 tails renders purified alpha Vbeta 3 competent to bind adhesive ligands (54). The alpha V and beta 3 tails are also necessary for outside-in signaling triggered by ligand binding to alpha Vbeta 3 (26, 38, 55).

Additional studies will be required to identify proximal regulators of alpha Vbeta 3 affinity and avidity, but certain integrin-binding proteins may be relevant in this regard (47). These include 1) beta 3-endonexin, a 13-kDa protein that binds selectively to the beta 3 cytoplasmic tail and, when overexpressed in CHO cells, increases the affinity state of alpha IIbbeta 3 (48); 2) activated R-Ras and Ha-Ras, which can increase and decrease, respectively, the affinity state of alpha 5beta 1 and alpha IIbbeta 3 in CHO cells through modulation of a Raf-1-dependent mitogen-activated protein kinase pathway (56, 57); 3) several cytoskeletal proteins, including talin, alpha -actinin, filamin, and skelemin, which may interact with the beta 3 tail (47, 58) (some of these may function to constrain integrin lateral diffusion in unstimulated cells, and others may promote integrin clustering in activated cells (20, 21, 59-61)); and 4) transmembrane proteins, including CD47 (integrin-associated protein) and CD87 (urinary plasminogen activator receptor), which may interact physically or functionally with alpha Vbeta 3 to modulate ligand binding or post-ligand binding events (47, 62-64).

Functional Implications of Affinity Modulation of alpha Vbeta 3-- Endothelial cells and vascular smooth muscle cells respond to inflammatory and angiogenic stimuli by up-regulating alpha Vbeta 3 expression (6, 65, 66). As demonstrated here, affinity and avidity modulation may represent additional, more rapid mechanisms to control alpha Vbeta 3 function by specifying which ligands can engage the integrin and to what extent. Once multivalent ligands become bound to alpha Vbeta 3, they may enhance cell adhesion and outside-in signaling by stimulating additional receptor conformational changes and receptor clustering (18, 19, 26, 55, 67). Thus, affinity and avidity modulation will inevitably influence events that are dependent on ligand binding to alpha Vbeta 3, including cell migration (68, 69), transdominant inhibition of other integrins (52, 64), and regulation of cell growth and survival (70-72).

Adenovirus infection involves an initial interaction between the viral fiber coat protein and a cellular receptor, CAR (73). This is followed by the interaction of penton base with alpha V integrins, which helps to promote virus internalization (8). As demonstrated here with an adenovirus vector carrying the GFP gene, both the level of alpha Vbeta 3 expression and its affinity state affected the extent of GFP transfer into CS-1 melanoma cells (Fig. 8). Thus, affinity modulation of alpha Vbeta 3 should be taken into account as a determinant of viral uptake in gene transfer protocols utilizing adenoviruses or other viruses that utilize this receptor.

The ligand-mimetic properties of WOW-1 Fab indicate that it should compete effectively with other RGD-containing ligands for binding to high-affinity conformations of alpha Vbeta 3. For example, WOW-1 Fab blocked adenovirus-mediated gene transfer through activated alpha Vbeta 3 (Fig. 8B). Unlike the parent PAC1 antibody, which recognizes only human alpha IIbbeta 3, WOW-1 Fab recognizes activated alpha Vbeta 3 in several mammalian and avian species, including the mouse.2 Accordingly, this ligand should prove useful for investigating the biological consequences of affinity regulation of alpha Vbeta 3 in a number of contexts, including wound healing, inflammation, and neoplasia. In addition, comparisons between WOW-1 Fab and penton base will provide an opportunity to characterize the relative contributions of affinity and avidity modulation to the functions of alpha Vbeta 3. One caveat in such experiments is the potential cross-reactivity of these ligands with alpha Vbeta 5. However, based on a mutational analysis of the RGD tract in penton base (74), it may be possible to engineer mutations in the H-CDR3 of WOW-1 Fab to abrogate its interactions with one of these integrins while retaining interactions with the other.

    ACKNOWLEDGEMENTS

We are grateful to David Phillips (Cor Therapeutics, Inc., South San Francisco, CA) for providing Integrilin, Thomas Kunicki (Scripps Research Institute) for antibody AP5, and Mark Ginsberg and Paul Hughes (Scripps Research Institute) for the alpha Vbeta 3(D723R)-CHO cells.

    FOOTNOTES

* This work was supported by National Institutes of Health Research Grants HL56595, HL48728, EY11431, HL54352, CA50286, and CA45726.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to this work.

** To who correspondence should be addressed: Dept. of Vascular Biology, Scripps Research Inst., 10550 North Torrey Pines Rd., VB-5, La Jolla, CA 92037. Tel.: 619-784-7148; Fax: 619-784-7422; E-mail: shattil@scripps.Edu.

2 N. Pampori, D. G. Stupack, and S. J. Shattil, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: H-CDR3, heavy chain hypervariable region 3; aPB, Alexa-penton base; CHO, Chinese hamster ovary; GFP, green fluorescent protein; m.o.i., multiplicity of infection.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Hynes, R. O. (1992) Cell 69, 11-25[CrossRef][Medline] [Order article via Infotrieve]
2. Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995) Annu. Rev. Cell Dev. Biol. 11, 549-599[CrossRef][Medline] [Order article via Infotrieve]
3. Hynes, R. O. (1996) Dev. Biol. 180, 402-412[CrossRef][Medline] [Order article via Infotrieve]
4. Aplin, A. E., Howe, A., Alahari, S. K., and Juliano, R. L. (1998) Pharmacol. Rev. 50, 197-263[Abstract/Free Full Text]
5. Shattil, S. J., Kashiwagi, H., and Pampori, N. (1998) Blood 91, 2645-2657[Free Full Text]
6. Felding-Habermann, B., and Cheresh, D. A. (1993) Curr. Opin. Cell Biol. 5, 864-868[CrossRef][Medline] [Order article via Infotrieve]
7. Coller, B. S., Cheresh, D. A., Asch, E., and Seligsohn, U. (1991) Blood 77, 75-83[Abstract/Free Full Text]
8. Wickham, T. J., Mathias, P., Cheresh, D. A., and Nemerow, G. R. (1993) Cell 73, 309-319[CrossRef][Medline] [Order article via Infotrieve]
9. Hu, D. D., Hoyer, J. R., and Smith, J. W. (1995) J. Biol. Chem. 270, 9917-9925[Abstract/Free Full Text]
10. Brooks, P. C., Strömblad, S., Sanders, L. C., Von Schalscha, T. L., Aimes, R. T., Stetler-Stevenson, W. G., Quigley, J. P., and Cheresh, D. A. (1996) Cell 85, 683-693[CrossRef][Medline] [Order article via Infotrieve]
11. Brooks, P. C., Clark, R. A. F., and Cheresh, D. A. (1994) Science 264, 569-571[Abstract/Free Full Text]
12. Schwartz, S. M. (1997) J. Clin. Invest. 99, 2814-2816[Medline] [Order article via Infotrieve]
13. Engleman, V. W., Nickols, G. A., Ross, F. P., Horton, M. A., Griggs, D. W., Settle, S. L., Ruminski, P. G., and Teitelbaum, S. L. (1997) J. Clin. Invest. 99, 2284-2292[Medline] [Order article via Infotrieve]
14. Brooks, P. C., Strömblad, S., Klemke, R., Visscher, D., Sarkar, F. H., and Cheresh, D. A. (1995) J. Clin. Invest. 96, 1815-1822
15. Diamond, M. S., and Springer, T. A. (1994) Curr. Biol. 4, 506-517[CrossRef][Medline] [Order article via Infotrieve]
16. Brown, E., and Hogg, N. (1996) Immunol. Lett. 54, 189-193[CrossRef][Medline] [Order article via Infotrieve]
17. van Kooyk, Y., and Figdor, C. G. (1997) Biochem. Soc. Trans. 25, 515-520[Medline] [Order article via Infotrieve]
18. Bazzoni, G., and Hemler, M. E. (1998) Trends Biochem. Sci. 23, 30-34[CrossRef][Medline] [Order article via Infotrieve]
19. Dustin, M. L. (1998) Cell Adhes. Commun. 6, 255-262[Medline] [Order article via Infotrieve]
20. Kucik, D. F., Dustin, M. L., Miller, J. M., and Brown, E. J. (1996) J. Clin. Invest. 97, 2139-2144[Medline] [Order article via Infotrieve]
21. Yauch, R. L., Felsenfeld, D. P., Kraeft, S. K., Chen, L. B., Sheetz, M. P., and Hemler, M. E. (1997) J. Exp. Med. 186, 1347-1355[Abstract/Free Full Text]
22. Abrams, C., Deng, Y. J., Steiner, B., O'Toole, J., and Shattil, S. J. (1994) J. Biol. Chem. 269, 18781-18788[Abstract/Free Full Text]
23. Hato, T., Pampori, N., and Shattil, S. J. (1998) J. Cell Biol. 141, 1685-1695[Abstract/Free Full Text]
24. Stupack, D. G., Shen, C., and Wilkins, J. A. (1992) Exp. Cell Res. 203, 443-448[CrossRef][Medline] [Order article via Infotrieve]
25. Bennett, J. S., Chan, C., Vilaire, G., Mousa, S. A., and DeGrado, W. F. (1997) J. Biol. Chem. 272, 8137-8140[Abstract/Free Full Text]
26. Blystone, S. D., Williams, M. P., Slater, S. E., and Brown, E. J. (1997) J. Biol. Chem. 272, 28757-28761[Abstract/Free Full Text]
27. Sadhu, C., Masinovsky, B., and Staunton, D. E. (1998) J. Immunol. 160, 5622-5628[Abstract/Free Full Text]
28. Byzova, T. V., and Plow, E. F. (1998) J. Cell Biol. 143, 2081-2092[Abstract/Free Full Text]
29. Byzova, T. V., and Plow, E. F. (1997) J. Biol. Chem. 272, 27183-27188[Abstract/Free Full Text]
30. Neri, D., Montigiani, S., and Kirkham, P. M. (1996) Trends Biotechnol. 14, 465-470[CrossRef][Medline] [Order article via Infotrieve]
31. Shattil, S. J., Cunningham, M., and Hoxie, J. A. (1987) Blood. 70, 307-315[Abstract/Free Full Text]
32. Mathias, P., Wickham, T., Moore, M., and Nemerow, G. (1994) J. Virol. 68, 6811-6814[Abstract/Free Full Text]
33. Stewart, P. L., Chiu, C. Y., Huang, S., Muir, T., Zhao, Y., Chait, B., Mathias, P., and Nemerow, G. R. (1997) EMBO J. 16, 1189-1198[CrossRef][Medline] [Order article via Infotrieve]
34. O'Toole, T. E., Katagiri, Y., Faull, R. J., Peter, K., Tamura, R., Quaranta, V., Loftus, J. C., Shattil, S. J., and Ginsberg, M. H. (1994) J. Cell Biol. 124, 1047-1059[Abstract/Free Full Text]
35. Cheresh, D. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6471-6475[Abstract/Free Full Text]
36. Hughes, P. E., Diaz-Gonzalez, F., Leong, L., Wu, C. Y., McDonald, J. A., Shattil, S. J., and Ginsberg, M. H. (1996) J. Biol. Chem. 271, 6571-6574[Abstract/Free Full Text]
37. Cheresh, D. A., and Spiro, R. C. (1987) J. Biol. Chem. 262, 17703-17711[Abstract/Free Full Text]
38. Filardo, E. J., Brooks, P. C., Deming, S. L., Damsky, C., and Cheresh, D. A. (1995) J. Cell Biol. 130, 441-450[Abstract/Free Full Text]
39. Rothlein, R., and Springer, T. A. (1986) J. Exp. Med. 163, 1132-1149[Abstract/Free Full Text]
40. Lin, E. C. K., Ratnikov, B. I., Tsai, P. M., Carron, C. P., Myers, D. M., Barbas, C. F., III, and Smith, J. W. (1997) J. Biol. Chem. 272, 23912-23920[Abstract/Free Full Text]
41. Pelletier, A. J., Kunicki, T., Ruggeri, Z. M., and Quaranta, V. (1995) J. Biol. Chem. 270, 18133-18140[Abstract/Free Full Text]
42. Scarborough, R. M., Naughton, M. A., Teng, W., Rose, J. W., Phillips, D. R., Nannizzi, L., Arfsten, A., Campbell, A. M., and Charo, I. F. (1993) J. Biol. Chem. 268, 1066-1073[Abstract/Free Full Text]
43. LaFlamme, S. E., Thomas, L. A., Yamada, S. S., and Yamada, K. M. (1994) J. Cell Biol. 126, 1287-1298[Abstract/Free Full Text]
44. Chen, Y.-P., O'Toole, T. E., Shipley, T., Forsyth, J., LaFlamme, S. E., Yamada, K. M., Shattil, S. J., and Ginsberg, M. H. (1994) J. Biol. Chem. 269, 18307-18310[Abstract/Free Full Text]
45. Huang, S., Stupack, D., Mathias, P., Wang, Y., and Nemerow, G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8156-8161[Abstract/Free Full Text]
46. Brooks, P. C., Montgomery, A. M. P., Rosenfeld, M., Reisfeld, R. A., Hu, T., Klier, G., and Cheresh, D. A. (1994) Cell 79, 1157-1164[CrossRef][Medline] [Order article via Infotrieve]
47. Hemler, M. E. (1998) Curr. Opin. Cell Biol. 10, 578-585[CrossRef][Medline] [Order article via Infotrieve]
48. Kashiwagi, H., Schwartz, M. A., Eigenthaler, M. A., Davis, K. A., Ginsberg, M. H., and Shattil, S. J. (1997) J. Cell Biol. 137, 1433-1443[Abstract/Free Full Text]
49. Smith, G. (1998) Trends Biochem. Sci. 23, 457-460[CrossRef][Medline] [Order article via Infotrieve]
50. Zhong, C., Chrzanowska-Wodnicka, M., Brown, J., Shaub, A., Belkin, A. M., and Burridge, K. (1998) J. Cell Biol. 141, 539-551[Abstract/Free Full Text]
51. Byzova, T. V., Rabbani, R., D'Souza, S., and Plow, E. F. (1998) Thromb. Haemostasis 80, 726-734[Medline] [Order article via Infotrieve]
52. Blystone, S. D., Graham, I. L., Lindberg, F. P., and Brown, E. J. (1994) J. Cell Biol. 127, 1129-1137[Abstract/Free Full Text]
53. Savage, B., Shattil, S. J., and Ruggeri, Z. M. (1992) J. Biol. Chem. 267, 11300-11306[Abstract/Free Full Text]
54. Mehta, R. J., Diefenbach, B., Brown, A., Cullen, E., Jonczyk, A., Gussow, D., Luckenbach, G. A., and Goodman, S. L. (1998) Biochem. J. 330, 861-869
55. Chellaiah, M., Fitzgerald, C., Filardo, E. J., Cheresh, D. A., and Hruska, K. A. (1996) Endocrinology 137, 2432-2440[Abstract]
56. Zhang, Z., Vuori, K., Wang, H.-G., Reed, J. C., and Ruoshlati, E. (1996) Cell 85, 61-69[CrossRef][Medline] [Order article via Infotrieve]
57. Hughes, P. E., Renshaw, M. W., Pfaff, M., Forsyth, J., Keivens, V. M., Schwartz, M. A., and Ginsberg, M. H. (1996) Cell 88, 521-530
58. Reddy, K. B., Gascard, P., Price, M. G., Negrescu, E. V., and Fox, J. E. B. (1998) J. Biol. Chem. 273, 35039-35047[Abstract/Free Full Text]
59. Qi, W., Loh, E., Vilaire, G., and Bennett, J. S. (1998) J. Biol. Chem. 273, 15271-15278[Abstract/Free Full Text]
60. Burridge, K., and Chrzanowska-Wodnicka, M. (1996) Annu. Rev. Cell Dev. Biol. 12, 463-519[CrossRef][Medline] [Order article via Infotrieve]
61. Sampath, R., Gallagher, P. J., and Pavalko, F. M. (1998) J. Biol. Chem. 273, 33588-33594[Abstract/Free Full Text]
62. Lindberg, F. P., Gresham, H. D., Schwarz, E., and Brown, E. J. (1993) J. Cell Biol. 123, 485-496[Abstract/Free Full Text]
63. Wei, Y., Lukashev, M., Simon, D. I., Bodary, S. C., Rosenberg, S., Doyle, M. V., and Chapman, H. A. (1996) Science 273, 1551-1555[Abstract]
64. Porter, J. C., and Hogg, N. (1998) Trends Cell Biol. 8, 390-396 [CrossRef][Medline] [Order article via Infotrieve]
65. Swerlick, R. A., Brown, E. J., Xu, Y., Lee, K. H., Manos, S., and Lawley, T. J. (1992) J. Invest. Dermatol. 99, 715-772[CrossRef][Medline] [Order article via Infotrieve]
66. Stromblad, S., and Cheresh, D. A. (1996) Trends Cell Biol. 6, 462-468 [CrossRef][Medline] [Order article via Infotrieve]
67. Miyamoto, S., Akiyama, S. K., and Yamada, K. M. (1995) Science 267, 883-885[Abstract/Free Full Text]
68. Bilato, C., Curto, K. A., Monticone, R. E., Pauly, R. R., White, A. J., and Crow, M. T. (1997) J. Clin. Invest. 100, 693-704[Medline] [Order article via Infotrieve]
69. Weerasinghe, D., McHugh, K. P., Ross, F. P., Brown, E. J., Gisler, R. H., and Imhof, B. A. (1998) J. Cell Biol. 142, 595-607[Abstract/Free Full Text]
70. Schneller, M., Vuori, K., and Ruoslahti, E. (1997) EMBO J. 16, 5600-5607[CrossRef][Medline] [Order article via Infotrieve]
71. Scatena, M., Almeida, M., Chaisson, M. L., Fausto, N., Nicosia, R. F., and Giachelli, C. M. (1998) J. Cell Biol. 141, 1083-1093[Abstract/Free Full Text]
72. Ruegg, C., Yilmaz, A., Bieler, G., Bamat, J., Chaubert, P., and Lejeune, F. J. (1998) Nat. Med. 4, 408-414[CrossRef][Medline] [Order article via Infotrieve]
73. Leon, R. P., Hedlund, T., Meech, S. J., Li, S., Schaack, J., Hunger, S. P., Duke, R. C., and DeGregori, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13159-13164[Abstract/Free Full Text]
74. Wickham, T. J., Carrion, M. E., and Kovesdi, I. (1995) Gene Ther. 2, 750-756[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
JEMHome page
D. M. Beauvais, B. J. Ell, A. R. McWhorter, and A. C. Rapraeger
Syndecan-1 regulates {alpha}v{beta}3 and {alpha}v{beta}5 integrin activation during angiogenesis and is blocked by synstatin, a novel peptide inhibitor
J. Exp. Med., March 16, 2009; 206(3): 691 - 705.
[Abstract] [Full Text] [PDF]


Home page
J. Immunol.Home page
H. S. Lee, C. Moon, H. W. Lee, E.-M. Park, M.-S. Cho, and J. L. Kang
Src Tyrosine Kinases Mediate Activations of NF-{kappa}B and Integrin Signal during Lipopolysaccharide-Induced Acute Lung Injury
J. Immunol., November 15, 2007; 179(10): 7001 - 7011.
[Abstract] [Full Text] [PDF]


Home page
Arterioscler. Thromb. Vasc. Bio.Home page
Y. Wu, V. Rizzo, Y. Liu, I. M. Sainz, N. G. Schmuckler, and R. W. Colman
Kininostatin Associates With Membrane Rafts and Inhibits {alpha}v{beta}3 Integrin Activation in Human Umbilical Vein Endothelial Cells
Arterioscler. Thromb. Vasc. Biol., September 1, 2007; 27(9): 1968 - 1975.
[Abstract] [Full Text] [PDF]


Home page
J. Cell Sci.Home page
G. N. M. Hajj, M. H. Lopes, A. F. Mercadante, S. S. Veiga, R. B. da Silveira, T. G. Santos, K. C. B. Ribeiro, M. A. Juliano, S. G. Jacchieri, S. M. Zanata, et al.
Cellular prion protein interaction with vitronectin supports axonal growth and is compensated by integrins
J. Cell Sci., June 1, 2007; 120(11): 1915 - 1926.
[Abstract] [Full Text] [PDF]


Home page
Mol. Biol. CellHome page
A. W. Orr, M. H. Ginsberg, S. J. Shattil, H. Deckmyn, and M. A. Schwartz
Matrix-specific Suppression of Integrin Activation in Shear Stress Signaling
Mol. Biol. Cell, November 1, 2006; 17(11): 4686 - 4697.
[Abstract] [Full Text] [PDF]


Home page
JEMHome page
G. H. Mahabeleshwar, W. Feng, D. R. Phillips, and T. V. Byzova
Integrin signaling is critical for pathological angiogenesis
J. Exp. Med., October 30, 2006; 203(11): 2495 - 2507.
[Abstract] [Full Text] [PDF]


Home page
JCBHome page
K. Gaus, S. Le Lay, N. Balasubramanian, and M. A. Schwartz
Integrin-mediated adhesion regulates membrane order
J. Cell Biol., August 28, 2006; 174(5): 725 - 734.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
L. Gao, Y. Feng, R. Bowers, M. Becker-Hapak, J. Gardner, L. Council, G. Linette, H. Zhao, and L. A. Cornelius
Ras-Associated Protein-1 Regulates Extracellular Signal-Regulated Kinase Activation and Migration in Melanoma Cells: Two Processes Important to Melanoma Tumorigenesis and Metastasis
Cancer Res., August 15, 2006; 66(16): 7880 - 7888.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
J. Murtagh, H. Lu, and E. L. Schwartz
Taxotere-Induced Inhibition of Human Endothelial Cell Migration Is a Result of Heat Shock Protein 90 Degradation
Cancer Res., August 15, 2006; 66(16): 8192 - 8199.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
C. M. Borza, A. Pozzi, D.-B. Borza, V. Pedchenko, T. Hellmark, B. G. Hudson, and R. Zent
Integrin {alpha}3beta1, a Novel Receptor for {alpha}3(IV) Noncollagenous Domain and a Trans-dominant Inhibitor for Integrin {alpha}vbeta3
J. Biol. Chem., July 28, 2006; 281(30): 20932 - 20939.
[Abstract] [Full Text] [PDF]


Home page
Mol. Pharmacol.Home page
H. Lu, J. Murtagh, and E. L. Schwartz
The Microtubule Binding Drug Laulimalide Inhibits Vascular Endothelial Growth Factor-Induced Human Endothelial Cell Migration and Is Synergistic when Combined with Docetaxel (Taxotere)
Mol. Pharmacol., April 1, 2006; 69(4): 1207 - 1215.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
T. A. Bunch, T. L. Helsten, T. L. Kendall, N. Shirahatti, D. Mahadevan, S. J. Shattil, and D. L. Brower
Amino Acid Changes in Drosophila {alpha}PS2betaPS Integrins That Affect Ligand Affinity
J. Biol. Chem., February 24, 2006; 281(8): 5050 - 5057.
[Abstract] [Full Text] [PDF]


Home page
JCBHome page
W. Yuan, T. M. Leisner, A. W. McFadden, Z. Wang, M. K. Larson, S. Clark, C. Boudignon-Proudhon, S. C.-T. Lam, and L. V. Parise
CIB1 is an endogenous inhibitor of agonist-induced integrin {alpha}IIb{beta}3 activation
J. Cell Biol., January 17, 2006; 172(2): 169 - 175.
[Abstract] [Full Text] [PDF]


Home page
JCBHome page
C. Cluzel, F. Saltel, J. Lussi, F. Paulhe, B. A. Imhof, and B. Wehrle-Haller
The mechanisms and dynamics of {alpha}v{beta}3 integrin clustering in living cells
J. Cell Biol., October 24, 2005; 171(2): 383 - 392.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
A. Katsumi, T. Naoe, T. Matsushita, K. Kaibuchi, and M. A. Schwartz
Integrin Activation and Matrix Binding Mediate Cellular Responses to Mechanical Stretch
J. Biol. Chem., April 29, 2005; 280(17): 16546 - 16549.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
B. Felding-Habermann, R. A. Lerner, A. Lillo, S. Zhuang, M. R. Weber, S. Arrues, C. Gao, S. Mao, A. Saven, and K. D. Janda
Combinatorial antibody libraries from cancer patients yield ligand-mimetic Arg-Gly-Asp-containing immunoglobulins that inhibit breast cancer metastasis
PNAS, December 7, 2004; 101(49): 17210 - 17215.
[Abstract] [Full Text] [PDF]


Home page
JCBHome page
D. M. Beauvais, B. J. Burbach, and A. C. Rapraeger
The syndecan-1 ectodomain regulates {alpha}v{beta}3 integrin activity in human mammary carcinoma cells
J. Cell Biol., October 11, 2004; 167(1): 171 - 181.
[Abstract] [Full Text] [PDF]


Home page
BloodHome page
S. J. Shattil and P. J. Newman
Integrins: dynamic scaffolds for adhesion and signaling in platelets
Blood, September 15, 2004; 104(6): 1606 - 1615.
[Abstract] [Full Text] [PDF]


Home page
BloodHome page
J. S. Bennett
Dotting the I of an I-domain
Blood, July 15, 2004; 104(2): 299 - 300.
[Full Text] [PDF]


Home page
CirculationHome page
M. M. Sadeghi, S. Krassilnikova, J. Zhang, A. A. Gharaei, H. R. Fassaei, L. Esmailzadeh, A. Kooshkabadi, S. Edwards, P. Yalamanchili, T. D. Harris, et al.
Detection of Injury-Induced Vascular Remodeling by Targeting Activated {alpha}v{beta}3 Integrin In Vivo
Circulation, July 6, 2004; 110(1): 84 - 90.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
P. H. Weinreb, K. J. Simon, P. Rayhorn, W. J. Yang, D. R. Leone, B. M. Dolinski, B. R. Pearse, Y. Yokota, H. Kawakatsu, A. Atakilit, et al.
Function-blocking Integrin {alpha}v{beta}6 Monoclonal Antibodies: DISTINCT LIGAND-MIMETIC AND NONLIGAND-MIMETIC CLASSES
J. Biol. Chem., April 23, 2004; 279(17): 17875 - 17887.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
R. I. Litvinov, G. Vilaire, H. Shuman, J. S. Bennett, and J. W. Weisel
Quantitative Analysis of Platelet {alpha}v{beta}3 Binding to Osteopontin Using Laser Tweezers
J. Biol. Chem., December 19, 2003; 278(51): 51285 - 51290.
[Abstract] [Full Text] [PDF]


Home page
Arterioscler. Thromb. Vasc. Bio.Home page
G.A. Stouffer and S.S. Smyth
Effects of Thrombin on Interactions Between {beta}3-Integrins and Extracellular Matrix in Platelets and Vascular Cells
Arterioscler. Thromb. Vasc. Biol., November 1, 2003; 23(11): 1971 - 1978.
[Abstract] [Full Text] [PDF]


Home page
ScienceHome page
S. Tadokoro, S. J. Shattil, K. Eto, V. Tai, R. C. Liddington, J. M. de Pereda, M. H. Ginsberg, and D. A. Calderwood
Talin Binding to Integrin {beta} Tails: A Final Common Step in Integrin Activation
Science, October 3, 2003; 302(5642): 103 - 106.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
S. De, J. Chen, N. V. Narizhneva, W. Heston, J. Brainard, E. H. Sage, and T. V. Byzova
Molecular Pathway for Cancer Metastasis to Bone
J. Biol. Chem., October 3, 2003; 278(40): 39044 - 39050.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
M. Rolli, E. Fransvea, J. Pilch, A. Saven, and B. Felding-Habermann
Activated integrin {alpha}v{beta}3 cooperates with metalloproteinase MMP-9 in regulating migration of metastatic breast cancer cells
PNAS, August 5, 2003; 100(16): 9482 - 9487.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
D. P. Ly, K. M. Zazzali, and S. A. Corbett
De Novo Expression of the Integrin {alpha}5{beta}1 Regulates {alpha}v{beta}3-mediated Adhesion and Migration on Fibrinogen
J. Biol. Chem., June 6, 2003; 278(24): 21878 - 21885.
[Abstract] [Full Text] [PDF]


Home page
JCBHome page
R.-P. Czekay, K. Aertgeerts, S. A. Curriden, and D. J. Loskutoff
Plasminogen activator inhibitor-1 detaches cells from extracellular matrices by inactivating integrins
J. Cell Biol., March 3, 2003; 160(5): 781 - 791.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
B. Butler, M. P. Williams, and S. D. Blystone
Ligand-dependent Activation of Integrin alpha vbeta 3
J. Biol. Chem., February 7, 2003; 278(7): 5264 - 5270.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
S.-J. Leu, S. C.-T. Lam, and L. F. Lau
Pro-angiogenic Activities of CYR61 (CCN1) Mediated through Integrins alpha vbeta 3 and alpha 6beta 1 in Human Umbilical Vein Endothelial Cells
J. Biol. Chem., November 22, 2002; 277(48): 46248 - 46255.
[Abstract] [Full Text] [PDF]


Home page
Circ. Res.Home page
J. Y.-J. Shyy and S. Chien
Role of Integrins in Endothelial Mechanosensing of Shear Stress
Circ. Res., November 1, 2002; 91(9): 769 - 775.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
K. Eto, R. Murphy, S. W. Kerrigan, A. Bertoni, H. Stuhlmann, T. Nakano, A. D. Leavitt, and S. J. Shattil
Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling
PNAS, October 1, 2002; 99(20): 12819 - 12824.
[Abstract] [Full Text] [PDF]


Home page
J. Cell Sci.Home page
R. Faccio, M. Grano, S. Colucci, A. Villa, G. Giannelli, V. Quaranta, and A. Zallone
Localization and possible role of two different alpha v beta 3 integrin conformations in resting and resorbing osteoclasts
J. Cell Sci., July 15, 2002; 115(14): 2919 - 2929.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
A. Bertoni, S. Tadokoro, K. Eto, N. Pampori, L. V. Parise, G. C. White, and S. J. Shattil
Relationships between Rap1b, Affinity Modulation of Integrin alpha IIbbeta 3, and the Actin Cytoskeleton
J. Biol. Chem., July 5, 2002; 277(28): 25715 - 25721.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
J. Pilch, R. Habermann, and B. Felding-Habermann
Unique Ability of Integrin alpha vbeta 3 to Support Tumor Cell Arrest under Dynamic Flow Conditions
J. Biol. Chem., June 7, 2002; 277(24): 21930 - 21938.
[Abstract] [Full Text] [PDF]


Home page
CirculationHome page
R. M. Scarborough, M. Lele, M. Sajid, N. Wajih, and G. A. Stouffer
Eptifibatide and 7E3, but Not Tirofiban, Inhibit {alpha}v{beta}3 Integrin-Mediated Binding of Smooth Muscle Cells to Thrombospondin and Prothrombin Response
Circulation, February 12, 2002; 105 (6): e46 - e46.
[Full Text] [PDF]


Home page
J. Cell Sci.Home page
J. C. Adams
Regulation of protrusive and contractile cell-matrix contacts
J. Cell Sci., January 15, 2002; 115(2): 257 - 265.
[Abstract] [Full Text] [PDF]


Home page
J. Immunol.Home page
M. L. Lupher Jr., E. A. S. Harris, C. R. Beals, L. Sui, R. C. Liddington, and D. E. Staunton
Cellular Activation of Leukocyte Function-Associated Antigen-1 and Its Affinity Are Regulated at the I Domain Allosteric Site
J. Immunol., August 1, 2001; 167(3): 1431 - 1439.
[Abstract] [Full Text] [PDF]


Home page
CirculationHome page
M. Lele, M. Sajid, N. Wajih, and G. A. Stouffer
Eptifibatide and 7E3, but Not Tirofiban, Inhibit {alpha}v{beta}3 Integrin-Mediated Binding of Smooth Muscle Cells to Thrombospondin and Prothrombin
Circulation, July 31, 2001; 104(5): 582 - 587.
[Abstract] [Full Text] [PDF]


Home page
J. Leukoc. Biol.Home page
N. Hogg and B. Leitinger
Shape and shift changes related to the function of leukocyte integrins LFA-1 and Mac-1
J. Leukoc. Biol., June 1, 2001; 69(6): 893 - 898.
[Abstract] [Full Text] [PDF]


Home page
Mol. Biol. CellHome page
D. Boettiger, F. Huber, L. Lynch, and S. Blystone
Activation of {alpha}v{beta}3-Vitronectin Binding Is a Multistage Process in which Increases in Bond Strength Are Dependent on Y747 and Y759 in the Cytoplasmic Domain of {beta}3
Mol. Biol. Cell, May 1, 2001; 12(5): 1227 - 1237.
[Abstract] [Full Text]


Home page
Proc. Natl. Acad. Sci. USAHome page
B. Felding-Habermann, T. E. O'Toole, J. W. Smith, E. Fransvea, Z. M. Ruggeri, M. H. Ginsberg, P. E. Hughes, N. Pampori, S. J. Shattil, A. Saven, et al.
Integrin activation controls metastasis in human breast cancer
PNAS, February 13, 2001; 98(4): 1853 - 1858.
[Abstract] [Full Text] [PDF]


Home page
BloodHome page
S. Honda, Y. Tomiyama, N. Pampori, H. Kashiwagi, T. Kiyoi, S. Kosugi, S. Tadokoro, Y. Kurata, S. J. Shattil, and Y. Matsuzawa
Ligand binding to integrin {alpha}v{beta}3 requires tyrosine 178 in the {alpha}v subunit
Blood, January 1, 2001; 97(1): 175 - 182.
[Abstract] [Full Text] [PDF]


Home page
J. Virol.Home page
T. Jackson, D. Sheppard, M. Denyer, W. Blakemore, and A. M. Q. King
The Epithelial Integrin alpha vbeta 6 Is a Receptor for Foot-and-Mouth Disease Virus
J. Virol., June 1, 2000; 74(11): 4949 - 4956.
[Abstract] [Full Text]


Home page
JCBHome page
M. Shiraga, A. Ritchie, S. Aidoudi, V. Baron, D. Wilcox, G. White, B. Ybarrondo, G. Murphy, A. Leavitt, and S. Shattil
Primary Megakaryocytes Reveal a Role for Transcription Factor NF-E2 in Integrin {alpha}IIb{beta}3 Signaling
J. Cell Biol., December 27, 1999; 147(7): 1419 - 1430.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
O. Helluin, C. Chan, G. Vilaire, S. Mousa, W. F. DeGrado, and J. S. Bennett
The Activation State of alpha vbeta 3 Regulates Platelet and Lymphocyte Adhesion to Intact and Thrombin-cleaved Osteopontin
J. Biol. Chem., June 9, 2000; 275(24): 18337 - 18343.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
D.-Q. Zheng, A. S. Woodard, G. Tallini, and L. R. Languino
Substrate Specificity of alpha vbeta 3 Integrin-mediated Cell Migration and Phosphatidylinositol 3-Kinase/AKT Pathway Activation
J. Biol. Chem., August 4, 2000; 275(32): 24565 - 24574.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
S. Pasco, J.-C. Monboisse, and N. Kieffer
The alpha 3(IV)185-206 Peptide from Noncollagenous Domain 1 of Type IV Collagen Interacts with a Novel Binding Site on the beta 3 Subunit of Integrin alpha vbeta 3 and Stimulates Focal Adhesion Kinase and Phosphatidylinositol 3-Kinase Phosphorylation
J. Biol. Chem., October 13, 2000; 275(42): 32999 - 33007.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
J. Wang, H. Chen, and E. J. Brown
L-plastin Peptide Activation of alpha vbeta 3-mediated Adhesion Requires Integrin Conformational Change and Actin Filament Disassembly
J. Biol. Chem., April 20, 2001; 276(17): 14474 - 14481.
[Abstract] [Full Text] [PDF]


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pampori, N.
Right arrow Articles by Shattil, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pampori, N.
Right arrow Articles by Shattil, S. J.
Social Bookmarking
 Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement