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INTRODUCTION |
Integrins mediate cell adhesion and signaling during many
developmental, physiological, and pathological processes (1-4). The
3 integrin family includes
IIb
3, often referred to as the fibrinogen
receptor, and
V
3, the vitronectin
receptor.
IIb
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).
V
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
V
3 overlaps that for
IIb
3, but includes others, such as
osteopontin, matrix metalloproteinase-2, and adenovirus penton base,
which do not interact with
IIb
3 (6,
8-10). In the adult organism,
V
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 
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
IIb
3 because
platelets must interact productively with fibrinogen or von Willebrand
factor only in vascular wounds. Studies of
IIb
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
IIb
3 function (23).
On the other hand, the significance of inside-out signaling and, in
particular, affinity modulation for
V
3
has been less certain. The ligand binding function of
V
3 has usually been assessed by cell
adhesion assays, and these have clearly shown that activation of
certain cells leads to
V
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
IIb
3
in platelets and
V
3 in cultured human
endothelial cells. Notably, prothrombin binding to
V
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
V
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
V
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
V
3 function in vivo.
The purpose of these studies was to determine whether
V
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
V
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
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
IIb
3 to activated
V
3, enabling a direct assessment of
V
3 affinity state. These studies
establish that
V
3 is subject to affinity
regulation, with direct implications for the
anchorage-dependent functions of
V
3 and for adenovirus-mediated gene delivery.
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EXPERIMENTAL PROCEDURES |
Preparation of Soluble
V
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
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, PAC1
containing NcoI and AgeI sites
was amplified by polymerase chain reaction with
-For
(5'-GGCGCGGGAGATCTCCATGGGATGTTTTGATGACCCAAACTCCA) and
-Rev
(5'-GGCGCATGACCGGTACACTCATTCCTGTTGAAGCTCTTG) and cloned into
pMT/BiP/V5-HisB. Nineteen µg of WOW-1 fd and PAC1
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
-chain
(BIOSOURCE International, Camarillo, CA). After
reduction, WOW-1 Fab migrated as an ~33-kDa fd chain and an ~25-kDa
-chain. There was no evidence of fd or
-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
V and
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
V
3 expression by single-cell fluorescence-activated cell sorting using the
V
3-specific monoclonal antibody LM609
(35). CHO cells stably expressing wild-type human
IIb
3 and
V
3(D723R) were described previously (34,
36). M21-L is a clone of the human melanoma cell line M21 that lacks the
V subunit (37).
V
3-M21-L cells were produced by transient transfection of M21-L cells with 2 µg each of
V/pcDNA3 and
3/pCDM8 using Superfect
(QIAGEN Inc.). CS-1 is a hamster melanoma cell line that does not
express
V
3 or
V
5 because it does not synthesize the
3 or
5 subunits.
V
3-CS-1 cells stably expressing hamster
V and human
3 were obtained by
transfection of CS-1 cells with human
3 (38). JY is an
immortalized human B-lymphoblastoid cell line that expresses
V
3, but not
V
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
V
3 (LM609),
IIb
3 (D57) (34), or
V
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
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
V integrin
antagonist (Peninsula Laboratories, Inc., Belmont, CA); 5 µM Integrilin, a selective
IIb
3 antagonist (42); or a 100 µg/ml
concentration of the function-blocking antibody LM609 or P1F6. In some
experiments, ligand binding and
V
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-
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
V
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
V
3,
V
3-CS-1
cells were transfected with a mammalian expression plasmid encoding Tac-
1, Tac-
3, or Tac-
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
V
3 expression in
the Tac-positive cells.
Binding of WOW-1 Fab to purified
V
3
receptors from human placenta and
IIb
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
V
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
V
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.
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RESULTS |
Interaction of a Novel Monovalent Ligand with Integrin
V
3--
To document and study the
significance of affinity modulation of
V
3, we set out to develop a monovalent
reporter ligand analogous to the activation-dependent
anti-
IIb
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
V
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
V
3 and, to a lesser extent, to purified
V
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
IIb
3 at antibody concentrations as high
as 2 µM, even though the parent antibody, PAC1 Fab, bound
half-maximally to
IIb
3 at 50 nM. These results indicate that the reengineering of PAC1 Fab converted it from an activation-dependent
IIb
3 antibody into an antibody that
reacts with activated
V
3.
To determine if WOW-1 Fab reacted preferentially with activated
V
3 in cells, Fab fragment binding was
compared with that of multivalent penton base using CHO cells stably
transfected with human
V
3
(
V
3-CHO cells). Flow cytometric analysis
showed that the surface of these cells expressed large amounts of
V
3, modest amounts of
V
5, and no detectable
IIb
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
V
3-CHO cells. However,
direct activation of
V
3 by
anti-
3 antibody AP5 caused a significant increase in the
binding of both ligands (p < 0.01) (Fig.
1B).

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Fig. 1.
Binding of soluble Alexa-penton base and
WOW-1 Fab to CHO cells expressing
V 3.
In A, V 3-CHO cells or parental
CHO cells were incubated with primary antibodies specific for
V 3 (LM609),
IIb 3 (D57), or
V 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
V 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 V 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 V 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).
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To assess the selectivity of these ligands for
V
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
V integrins (Fig.
2) (46). On the other hand, a cyclic
peptide selective for
IIb
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
IIb
3 (42).
Furthermore, the
V
3 function-blocking antibody LM609 (100 µg/ml) inhibited ligand binding by >70%,
whereas the
V
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
IIb
3
receptors, but <500
V
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
V
3 and that they do
not recognize
IIb
3. Thus, WOW-1 Fab may
be a suitable reporter for changes in
V
3 affinity. Since WOW-1 Fab (and aPB) also recognizes
V
5, particular efforts were made in the
experiments that follow to utilize cells that express
V
3, but little or no
V
5.

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Fig. 2.
Effect of integrin inhibitors on binding of
aPB and WOW-1 Fab to
V 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.
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Affinity of
V
3 Can Be Regulated by
Inside-out Signals--
To determine if
V
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
V
3, but not
V
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
V
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
V
3, as measured by antibody LM609. To
determine whether the changes in WOW-1 Fab binding reflected changes in
V
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
V
3.

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Fig. 3.
V 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.
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Determinants of
V
3 Activation
State--
Additional experiments were performed to identify factors
that influence
V
3 affinity using readily
transfectable cell lines that stably express human
V
3.
V
3 on
vascular cells may encounter multiple ligands simultaneously during the
process of wound healing. Therefore, we wondered if the
affinity/avidity of
V
3 differed for
various ligands. Equilibrium binding of aPB, WOW-1 Fab, and the
adhesive ligand fibrinogen was compared in
V
3-CHO cells. As summarized in Table
I, each ligand bound specifically to
approximately the same total number of receptors in unstimulated
V
3-CHO cells. However, the
affinity/avidity of
V
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
V
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
V
3 can interact differentially with macromolecular ligands and that the affinity state of the receptor is
one determinant of such interactions.
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Table I
Binding of different ligands to V 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.
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In circulating platelets, the "basal" activation state of
IIb
3 must remain low to prevent
thrombosis. However, this requirement may not pertain to all cells that
express
V
3. Therefore, ligand binding was
studied simultaneously in
V
3-CHO cells
and in two unrelated melanoma cell lines,
V
3-M21-L and
V
3-CS-1, to assess cell type-specific
variations in the basal activation state of
V
3. To control for minor variations in
V
3 expression between the cell lines,
ligand binding was expressed on a "per receptor" basis using
anti-
3 antibody SSA6 to quantitate receptor expression. Unstimulated
V
3-M21-L cells bound
significantly more aPB than did
V
3-CHO
cells (p < 0.01). This difference was maintained even
after further activation of
V
3 with
antibody AP5 (p < 0.05) (Fig.
4). Similar results were obtained with
V
3-CS-1 cells instead of
V
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
V
3-CHO cells (Fig. 3B and
Table I), these results indicate that the basal activation state of
V
3 varies with the cell type.

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Fig. 4.
Comparison of aPB binding to
V 3-CHO
cells and
V 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.
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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
V
3. Certain point mutations or
truncations of the
3 cytoplasmic tail, such as
3(D723R), result in constitutive activation of
IIb
3 in CHO cells (34, 36). To determine
whether
V
3 is affected by such a
modification, ligand binding to
V
3(D723R) was assessed. This mutant was stably expressed in CHO cells to approximately the same level as wild-type
V
3 (Fig.
5A). However, unstimulated
V
3(D723R)-CHO cells bound significantly
more aPB than unstimulated
V
3-CHO cells
(p < 0.01), equivalent to the amount of aPB bound to
V
3-CHO cells treated with antibody AP5 (Fig. 5B). A second
V
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
3 cytoplasmic tail can be propagated to the
extracellular domains of
V
3 to influence
ligand binding affinity.

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Fig. 5.
Effect of an activating mutation in the
3 integrin cytoplasmic tail on the
binding of penton base to
V 3.
In A, stable CHO cell lines expressing either
V 3 or
V 3(D723R) were stained with
anti- 3 antibody SSA6 and phycoerythrin-streptavidin to
assess surface expression of V 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
V 3 and
V 3(D723R) at the p < 0.01 level. For comparison, the corresponding value for aPB binding to
antibody AP5-treated V 3-CHO cells was
0.034 ± 0.002. 1°Ab, primary antibody;
mfi, mean fluorescence intensity.
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The activation state of certain integrins, such as
IIb
3 and
5
1, can be
suppressed in a dominant-inhibitory fashion by overexpression of
isolated
3 or
1 cytoplasmic tails, but
not by
5 tails (43, 44, 48). To determine if
V
3 is subject to this type of
suppression,
V
3-CS-1 cells were
transiently transfected with chimeric constructs consisting of the
3,
1, or
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-
3 and Tac-
1 caused a significant
reduction in specific binding of aPB and WOW-1 Fab when compared with
Tac-
5 (p < 0.01) (Fig.
6, A and B). In
contrast, none of these tail chimeras affected surface expression of
V
3 (Fig. 6C). Since the
isolated
tails may bind proteins that normally interact with
integrins (43), these results suggest that
V
3 may be regulated by direct
interactions with intracellular proteins.

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Fig. 6.
Effect of overexpression of isolated integrin
cytoplasmic tails on ligand binding to CS-1 melanoma cells
expressing
V 3.
As described under "Experimental Procedures,"
V 3-CS-1 cells were transiently
transfected with the Tac- 5, Tac- 1, or
Tac- 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 V 3 as monitored with
anti- 3 antibody SSA6. Data represent the means ± S.E. of three experiments. The asterisks indicate that
ligand binding in the presence of Tac- 1 or
Tac- 3 was significantly less than with
Tac- 5 (p < 0.01).
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Functional Consequences of Affinity Modulation of
V
3--
To determine whether changes in
receptor affinity affect the adhesive function of
V
3, the adhesion of
V
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
V
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
V
3 or it activated
additional
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
V
3
promotes both cell adhesion and post-ligand binding responses, such as
cell spreading.

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Fig. 7.
Effect of
V 3
activation on the adhesion of
V 3-CHO
cells to penton base. As described under "Experimental
Procedures," microtiter wells were coated with penton base, and the
adhesion of V 3-CHO cells was studied for
90 min at 37 °C either with no additive ( ) or with antibody AP5
ascites (1:50; ) or MnCl2 (0.25 mM; ).
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 V
integrins. This experiment is representative of three so
performed.
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Adenoviruses utilize
V integrins to enter cells and are
a common gene delivery vector. Therefore, we tested whether changes in
V
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
V
5, thus potentially
restricting adenovirus internalization through stably expressed
V
3. When parental cells without
V
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
V
3-CS-1 cells exhibited a higher level of
GFP expression, particularly at the higher m.o.i. (Fig.
8A). However, if incubation of
V
3-CS-1 cells with virus was carried out
in the presence of 2.5 mM MnCl2 to activate
V
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
V
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
V
3.

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Fig. 8.
Effect of
V 3
expression and activation on adenovirus-mediated gene delivery. In
A, parental CS-1 cells (No
V 3) and
V 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
V 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 V 3-CS-1 cells with 1.7 µM WOW-1 Fab for 20 min before addition of virus.
mfi, mean fluorescence intensity.
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DISCUSSION |
The purpose of this study was to determine if the affinity of
V
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
V
3. We reasoned
that the most direct way to evaluate
V
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
V
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
V
integrin-binding domain, into the H-CDR3 of PAC1, a monovalent Fab
antibody that is specific for activated
IIb
3. The resulting monovalent Fab
antibody, WOW-1, retained the activation-dependent
characteristics of PAC1 and penton base and interacted with
V
3, but not
IIb
3. Using WOW-1 Fab to study
V
3, the following major conclusions were
reached. 1) The basal affinity state of
V
3 varies among cell types, being
extremely low in lymphoid cells and higher in melanoma cell lines. 2)
V
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
V
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
V
3 and for adenovirus-mediated gene transfer.
V
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
IIb
3 do
not necessarily apply to
V
3.
Unfortunately, in the case of
V
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
V
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
IIb
3 and
V
3
in a cell activation-dependent manner. In particular, the binding of prothrombin to
V
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
V
3 in these cells. They also raised new
questions that have now been addressed in the present work. Does acute
regulation of
V
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
V
3 affinity, or do cells primarily
control avidity? If
V
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
V
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
V
3. In contrast
and as shown experimentally for PAC1 Fab and
IIb
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
V
3 in an activation-dependent manner. This was true whether
V
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
3 cytoplasmic
tail. Thus, the results with WOW-1 Fab establish that
V
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
V
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
IIb
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
V
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
V
3
did not require cell activation. This suggests that soluble fibrinogen is an activation-dependent ligand for
V
3, whereas solid-phase fibrinogen is an
activation-independent ligand. A similar situation pertains to the
interaction of fibrinogen with
IIb
3 in
platelets (53). Thus, interactions between
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
V
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
V
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
V
3.
Regulation of Affinity Changes in
V
3--
Binding of penton base and WOW-1
Fab to 