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INTRODUCTION |
Integrins are heterodimeric transmembrane receptors that
mediate adhesion of cells to extracellular matrix proteins and to other
cells. Consequently, integrins have a pivotal role in numerous developmental, physiological, and pathological processes. The integrin
family contains at least 18
and
subunits that combine to
produce 24 known heterodimer combinations with distinct cell expression
patterns and overlapping ligand specificities (1). Binding of ligand to
the integrin extracellular domain induces a conformational change that
is propagated to the cytoplasmic domain and initiates downstream
signaling events (1). The ability to self-regulate adhesion to complex
extracellular matrices is of particular importance to hematopoietic
cells. Hematopoietic cells circulate in a non-adhesive state until such
time as they encounter pro-inflammatory or thrombotic signals, after
which they arrest from the circulation by means of firm adhesion to the
vascular walls and, in leukocytes, migrate to the site of inflammation.
The ability of hematopoietic cells to regulate their adhesion-dependent extravasation is mediated in part by
the integrin
v
3 (2).
The
v
3 integrin plays a vital role in the
adhesion of many cell types to the extracellular matrix and to other
cells. Although it is termed the vitronectin
(Vn)1 receptor, it is in fact
a promiscuous receptor that recognizes a number of extracellular
ligands including fibronectin (Fn), tenascin, and osteopontin (1, 3).
However, promiscuous may be an unfortunate term because recent studies
show that differential signaling by distinct
v
3 ligands may in fact result in unique cellular responses (4). We have previously shown that when
v
3 is expressed in the K562 cell line,
these cells exhibit differential adhesion to Vn and Fn (4).
v
3-mediated adhesion to Vn requires tyrosine phosphorylation at Tyr-747 within the
3
cytoplasmic tail and is dependent upon PKC activation, whereas
v
3-mediated adhesion to Fn is
constitutive, requiring neither of these events (4, 5). In addition,
v
3-mediated adhesion of LNCaP cells to
osteopontin, but not Vn, activates PI3K. However, this signaling pathway may be cell type-specific as PC3 cells activate PI3K upon adhesion to both osteopontin and Vn (6).
In this study we sought to establish the signaling events that might
necessitate
3-tyrosine phosphorylation in
v
3-mediated adhesion by comparing actin
cytoskeletal organization and signaling events after attachment to
distinct ligands. We show that
v
3-mediated adhesion to Fn is
constitutive and independent of
3-tyrosine phosphorylation because cells expressing
v
3 Y747F,Y759F
(K
v
3Y747F,Y759F) firmly adhere to Fn,
equivalent to cells expressing wild type
v
3 (K
v
3),
even in the absence of PKC activation. In contrast, firm adhesion to Vn
requires both cellular PKC activation as well as
3-tyrosine phosphorylation. Coincident with cell
adhesion is a reorganization of the actin cytoskeleton into stress
fibers. On a Fn substrate, K
v
3 exhibited
spontaneous stress fiber formation, whereas
K
v
3 attached to Vn required PKC
activation to achieve this cellular phenotype. Mutation of the
3 cytoplasmic tyrosine residues had no effect on
K
v
3Y747F,Y759F organization of stress fibers when attached to Fn but eliminated PKC-dependent
stress fiber formation upon attachment to Vn. Because Rho activity is believed to mediate actin stress fiber assembly, we examined Rho activity in K
v
3 and
K
v
3Y747F,Y759F cells attached to Vn and Fn. We show that the constitutive nature of the adhesion to Fn by
K
v
3 and
K
v
3Y747F,Y759F is due in part to direct
activation of Rho. Interestingly, whereas apparently unrelated to the
tyrosine phosphorylation of
3, adhesion to Vn, but not
Fn, results in increased PI3K activity. The PI3K inhibitor wortmannin
inhibits the PKC-dependent adhesion of
K
v
3 to Vn, whereas adhesion to Fn is
unaffected by the presence of wortmannin. These results indicate that
v
3-mediated organization of the actin
cytoskeleton, a requisite event in cell adhesion, occurs via unique,
ligand-dependent mechanisms.
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EXPERIMENTAL PROCEDURES |
Cells and Materials--
K562 cells were stably transfected with
cDNA encoding wild type (K
v
3),
Y747F,Y759F (K
v
3Y747F,Y759F)
3 or wild type (K
V
5)
5 together with
V and maintained as
previously described (7, 8). The anti-
3 monoclonal
antibodies 7G2 and LIBS-1 were gifts of Eric J. Brown and Mark H. Ginsberg, respectively. Vitronectin and fibronectin were prepared as
previously described (7). Purified GST-FnIII-10 and III10/RGE (RGD
mutated to RGE) were gifts of Denise C. Hocking (9). All reagents
unless otherwise noted were purchased from Sigma. Anti-p85 rabbit
polyclonal was purchased from Upstate Biotechnology (Lake Placid, NY).
Anti-Rho mouse monoclonal antibody was purchased from Cytoskeleton Inc (Denver, CO).
Flow Cytometry--
3 surface expression in
stably transfected K562 cells was monitored using flow cytometry.
Untransfected K562 cells or K
v
3 or
K
v
3Y747F,Y759F cells were incubated with
mAb anti-
3 AP3 in IMDM for 1 h at 4 °C. Cells
were then washed in IMDM and incubated with fluorescein
isothiocyanate-labeled goat anti-mouse IgG for 30 min at 4 °C. To
quantify binding site equality of
3 by 7G2 and LIBS-1,
K
v
3 or
K
v
3Y747F,Y759F cells were stimulated with or without GRGDS peptide (1 mM) or wortmannin (10 µM) in IMDM for 20 min at room temperature. Cells were
washed in IMDM and incubated with either 7G2 or LIBS-1
anti-
3 antibodies for 1 h at 4 °C. Cells were
washed in IMDM and incubated with fluorescein isothiocyanate-labeled
goat anti-mouse IgG for 30 min at 4 °C. Flow cytometry was carried
out using a Coulter Epics XL flow cytometer (Coulter, Miami, FL). Data
are expressed as mean channel fluorescence for at least three separate experiments.
Cell Adhesion Assays--
96-Well microtiter plates (Immulon II,
Dynatech, Chantilly, VA) were coated with fibronectin (10 µg/ml),
vitronectin (1 µg/ml), 7G2 anti-
3 (0.5 µg/ml),
LIBS-1 anti-
3 (0.5 µg/ml), or casein (50 µg/ml) in
PBS overnight at 4 °C. Wells were washed twice in PBS and
post-coated with 1.0% casein in PBS for 30 min at room temperature.
K
v
3 or
K
v
3Y747F,Y759F cells were added at 1 × 105 cells/well in Hank's-buffered saline solution
containing 1.0 mM Ca2+ and
Mg2+ with or without PMA (10 ng/ml) and C3 exoenzyme (10 µg/ml) after treatment with saponin (50 µg/ml) for 3 min,
piceatannol (50 µg/ml), or wortmannin (10 µM) and
allowed to adhere for 1 h at 37 °C. Wells were rinsed 3 times
in Hank's-buffered saline solution without Ca2+ or
Mg2+, and adherent cells were fixed in 3.7% formaldehyde
for 1 h at 4 °C and stained with 0.05% crystal violet for 30 min at room temperature. Crystal violet was dispersed in methanol and
quantified by absorbance at 570 nm using an Emax microplate reader
(Molecular Dynamics, Sunnyvale, CA).
3 Tyrosine Phosphorylation--
6-well tissue
culture treated plates were coated with Vn (1 µg/ml), Fn (10 µg/ml), anti-
3 7G2 (0.5 µg/ml), or
anti-
3 LIBS-1 (0.5 µg/ml) in PBS overnight at 4 °C.
K
v
3 or
K
v
3Y747F,Y759F cells were plated at
2 × 106 cells/well in serum-free IMDM media
containing 75 µM sodium orthovanadate with or without PMA
(10 ng/ml), bisindolylmaleimide (10 µM), piceatannol (50 µg/ml), or wortmannin (10 µM). The cells were lysed in
PBS buffer containing 1% Nonidet P-40, sodium orthovanadate (1 mM), aprotinin (10 µg/ml), leupeptin (10 mg/ml), and
phenylmethylsulfonyl fluoride (1 mM). Cell debris was
removed by centrifugation at 12,000 × g for 10 min at
4 °C. The lysates were precleared for 1 h at 4 °C with
gelatin-Sepharose and immunoprecipitated with goat anti-mouse-Sepharose
beads (ICN, Costa Mesa, CA) coated with anti-
3 1A2 and
anti-
V 3F12 monoclonal antibodies for 2 h at 4 °C. Samples were divided equally to determine
3-tyrosine phosphorylation and total
3.
Samples were separated on 7.5% SDS-PAGE gels and transferred to
polyvinylidene difluoride membranes (Millipore, Bedford, MA). The
membranes were blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) and 3% v/v bovine serum albumin at room temperature for 1 h. Membranes were then incubated with mAb 4G10 (Upstate Biotechnology)
for phosphotyrosine or mAb 7G2 for total
3. The
membranes were then incubated with peroxidase-coupled goat anti-mouse
IgG2b (Caltag Laboratories, Burlington, CA) for phosphotyrosine
3 and peroxidase-coupled goat anti-mouse IgG (Sigma) for
total
3. Proteins were detected with enhanced
chemiluminescence (ECL, Amersham Biosciences).
Fluorescent Microscopy--
Acid-washed glass coverslips were
coated overnight with Vn (1 µg/ml) or Fn (5 µg/ml).
K
v
3 or
K
v
3Y747F,Y759F cells were permitted to
adhere to coverslips for 1 h at 37 °C in the presence or
absence of PMA (10 ng/ml). Cells were fixed in 3.7% formaldehyde, permeabilized in ice-cold PBS containing 0.01% Nonidet P-40, and stained with 0.7 µM rhodamine-phalloidin for 15 min at
37 °C. Fluorescence was visualized on a Nikon Eclipse E800
fluorescent microscope (Nikon, Melville, NY), and images were retained
digitally with a SpotCamII digital camera and software (Diagnostic
Instruments, Sterling Heights, MI).
Rho Activity Assay--
6-well tissue-coated plates were coated
with Vn (1 µg/ml), Fn (10 µg/ml), recombinant Fn III-10 (10 µg/ml), Fn III-10RGE (10 µg/ml), 7G2 (0.5 µg/ml), or LIBS-1 (0.5 µg/ml), and K
v
3 or
K
v
3Y747F,Y759F cells were plated at
2 × 106 cells/well in the presence or absence of PMA
(10 ng/ml), C3 exoenzyme (10 µg/ml) after treatment with saponin (50 µg/ml) for 3 min, piceatannol (50 µg/ml), or wortmannin (10 µM) and allowed to adhere for 1 h at 37 °C. Rho
activity was assayed as previously described (10). Briefly,
cells were lysed in 0.5 ml of ice-cold PBS buffer containing 1%
Nonidet P-40, leupeptin (10 µg/ml), aprotinin (10 µg/ml),
phenylmethylsulfonyl fluoride (1 mM), and sodium
orthovanadate (1 mM). Cell lysates were then clarified by
centrifugation at 12,000 × g for 15 min at 4 °C.
GTP-bound Rho was affinity-purified from lysates using a GST fusion
construct of the RhoA binding domain of rhotekin (GST-RBD) (kindly
provided by M. A. Schwartz, Scripps Institute). Lysates were
incubated with GST-RBD (~25 µg) beads for 1 h at 4 °C.
GTP-bound RhoA was separated using 12% SDS-PAGE gels under reducing
conditions. Total Rho was determined by loading 1/10 total
volume of cell lysate on a 12% SDS-PAGE under reducing conditions.
Proteins were transferred to polyvinylidene difluoride membranes and
blocked in TBST with 3% v/v bovine serum albumin at room temperature
for 1 h. Membranes were then incubated with murine mAb to RhoA
(Cytoskeleton Inc., Denver, CO). The membranes were then incubated with
peroxidase-coupled anti-mouse IgG, and proteins were detected with ECL.
AKT and Phospho-AKT Immunoblotting--
6-Well tissue culture
plates (Costar, Corning, NY) were coated as above.
K
v
3 or
K
v
3Y747F,Y759F cells were plated at
2 × 106 cells/well in the presence or absence of PMA
(10 ng/ml) for 1 h at 37 °C. Cells were lysed in 0.5 ml of
ice-cold PBS lysis buffer (PBS, 1% Nonidet P-40, 10 µg/ml each of
leupeptin, and aprotinin, 1 mM phenylmethylsulfonyl
fluoride), and lysates were cleared by centrifugation at 12,000 × g for 15 min at 4 °C. Lysates were divided evenly for
phospho-AKT Ser473 and total AKT and loaded onto
10% SDS-PAGE gels under reducing conditions. Proteins were transferred
to polyvinylidene difluoride membranes and blocked in TBST with 3% v/v
bovine serum albumin at room temperature for 1 h. Membranes were
incubated with sheep polyclonal antibodies to AKT (0.1 µg/ml) or to
phospho-AKT Ser-473 (0.5 µg/ml) (Upstate Biotechnology) for 2 h
at room temperature. The membranes were then incubated with
peroxidase-coupled anti-sheep IgG, and proteins were detected with ECL.
PI3K Immunoprecipitation--
K
v
3
and K
v
3Y747F,Y759F (1 × 107) were treated with or without Gly-Arg-Gly-Asp-Ser-Pro
(GRGDSP) (1 mM) in the presence or absence of
orthovanadate (0.75 µM) for 20 min at room temperature to
induce
3-tyrosine phosphorylation (11). Cells were then lysed in 0.5 ml of ice-cold PBS lysis buffer (PBS, 1% Nonidet P-40, 10 µg/ml each of leupeptin and aprotinin, 1 mM
phenylmethylsulfonyl fluoride). Cell debris was removed by
centrifugation at 12,000 × g for 10 min at 4 °C.
The lysates were precleared for 1 h at 4 °C with
gelatin-Sepharose and immunoprecipitated with goat anti-mouse-Sepharose beads (ICN, Costa Mesa, CA) coated with anti-
3 1A2 and
anti-
V 3F12 monoclonal antibodies for 2 h at
4 °C. Samples were divided equally to determine PI3K and total
3. Samples were separated on 7.5% SDS-PAGE gels and
transferred to polyvinylidene difluoride membranes (Millipore). The
membranes were blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) and 3% v/v bovine serum albumin at room temperature for 1 h. Membranes were then incubated with anti-p85 rabbit polyclonal
(Upstate Biotechnology) or mAb 7G2 for total
3. The
membranes were then incubated with peroxidase-coupled goat anti-mouse
IgG2b (Caltag Laboratories, Burlington, CA) for phosphotyrosine
3 and peroxidase-coupled goat anti-mouse IgG (Sigma) for
total
3. Proteins were detected with enhanced
chemiluminescence (ECL, Amersham Biosciences).
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RESULTS |
v
3-mediated Firm Adhesion to Distinct
Ligands and Actin Cytoskeletal Reorganization--
To investigate the
influence of the extracellular ligand on
v
3-mediated adhesion and actin
cytoskeletal organization, we examined the ability of K562 cells,
expressing wild type (K
v
3) or Y747F,Y759F
(K
v
3Y747F,Y759F)
v
3, to adhere to Vn or Fn in the presence
or absence of the cellular agonist PMA.
K
v
3 cells adhere to Vn only after
cellular activation by PMA (Fig. 1A); however,
K
v
3Y747F,Y759F cells are incapable of
strong adhesion to Vn even after cellular activation. As we have
previously reported, both K
v
3 and
K
v
3Y747F,Y759F cells are capable of
constitutive adhesion to Fn with or without cellular PKC activation by
PMA (Fig. 1A) (4). To determine whether the constitutive
adhesion to Fn is
v
3-mediated, we exposed
K562 cells stably expressing
V
5
(K
V
5) to Vn and Fn in the presence or
absence of PMA. K
V
5 cells do not adhere
to Fn even in the presence of PMA nor do untransfected K562 cells (data
not shown), indicating that constitutive adhesion of
K
v
3 and
K
v
3Y747F,Y759F cells to Fn is
v
3-mediated.

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Fig. 1.
Effect of distinct extracellular ligands
on
v 3-mediated
adhesion and actin reorganization. A, microtiter plates
(96 wells) were coated with Vn (1 µg/ml) or Fn (10 µg/ml). 1 × 105 K v 3 or
K v 3Y747F,Y759F cells were allowed to
adhere for 1 h at 37 °C in the absence (gray bars)
or presence (black bars) of PMA and quantified as described
under "Experimental Procedures." Data bars represent the
mean absorbance ±S.D. from triplicate wells from three separate
experiments. WT, wild type. B-I, fluorescent
microscopy. K v 3 cells were allowed to
adhere to Vn in the presence (C) or absence (B)
of PMA (10 ng/ml) or Fn in the presence (G) or absence
(F) of PMA (10 ng/ml).
K v 3Y747F,Y759F cells were allowed to
adhere to Vn in the presence (E) or absence (D)
of PMA (10 ng/ml) or Fn in the presence (I) or absence
(H) of PMA (10 ng/ml). Cells were then stained with
rhodamine phalloidin to reveal actin organization as described under
"Experimental Procedures." The bar represents
5µm.
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Actin cytoskeletal reorganization plays a key role in the ability of
cells to adhere to the extracellular matrix (13, 14). Therefore, we
examined the structure of the actin cytoskeleton in
K
v
3 and
K
v
3Y747F,Y759F cells attached to Vn or Fn
with or without cellular PKC activation. Both
K
v
3 and
K
v
3Y747F,Y759F cells, when allowed to
adhere to Fn for 1 h, display a spread phenotype independent of
PKC activation via PMA. Fluorescent microscopy of these cells reveals
the formation of actin fibers (Fig. 1, F-I) within 1 h
of attachment to Fn, and thus, actin fiber formation on Fn is
independent of both
3 tyrosine phosphorylation and PKC activation via PMA. In contrast, actin fiber formation on Vn is dependent upon cellular PKC activation and
3-tyrosine
phosphorylation. No actin fibers are seen in
K
v
3 cells on Vn in the absence of PMA
stimulation (Fig. 1B). Stress fibers can be seen in
K
v
3 cells after adhesion to Vn for 1 h only in the presence of PMA (Fig. 1C). In addition,
3-tyrosine phosphorylation plays a pivotal role in firm
adhesion to Vn, as no stress fiber formation occurs within
K
v
3Y747F,Y759F cells even after
stimulation with PMA (Fig. 1, D and E).
Differential Inhibition of Adhesion to Distinct Ligands--
To
elucidate the possible signaling mechanisms underlying the differences
in
v
3-mediated adhesion to Vn or Fn we
utilized specific inhibitors to various signaling molecules.
PMA-induced firm adhesion of K
v
3 cells to
Vn was inhibited by pretreatment with the Rho inhibitor C3 exoenzyme
(100 nM), the PI3K inhibitor wortmannin (10 µM), and the Syk inhibitor piceatannol (50 µg/ml). These results indicate that Rho, PI3K, and Syk are involved in PMA-induced firm adhesion to Vn (Fig.
2A). Firm adhesion of
K
v
3 cells to Fn was also inhibited by C3
exoenzyme and piceatannol (Fig 2A); however, no inhibition
of firm adhesion to Fn was seen with wortmannin, suggesting that PI3K
activity is not required for
v
3-mediated
firm adhesion to Fn. Inhibition with C3 exoenzyme and piceatannol
reduced adhesion to both Fn and Vn to levels comparable with adhesion
to casein (50 µg/ml) (data not shown). Cells expressing K
v
3Y747F,Y759F are unable to adhere to Vn
even after cellular PKC activation by PMA.
K
v
3Y747F,Y759F cells exhibit the same inhibition pattern as K
v
3 cells on Fn in
that there is complete inhibition of adhesion after pretreatment with
C3 exoenzyme and piceatannol; however, we see no inhibition of adhesion
with wortmannin (Fig. 2B). These data suggest that
v
3-mediated adhesion to Fn, already shown
to be independent of
3-tyrosine phosphorylation, is
dependent upon Rho and Syk activity but not PI3K activity. In contrast
v
3-mediated adhesion to Vn is dependent
upon PI3K activity as well as Rho and Syk. The inhibitory effect of
wortmannin on Vn is not due to a decrease in
3-tyrosine
phosphorylation, because we show that in response to RGD,
3-tyrosine phosphorylation is inhibited fully in the
presence of piceatannol but only minimally in the presence of
wortmannin or the PKC inhibitor bisindolylmaleimide (Fig.
3). Therefore, wortmannin inhibition of
adhesion to Vn is not due to an inhibition of
3-tyrosine
phosphorylation, although piceatannol inhibition of adhesion to Vn may
in part be due to inhibition of
3-tyrosine
phosphorylation. However, this may be unlikely because piceatannol also
inhibits K
v
3 and
K
v
3Y747F,Y759F adhesion to Fn, where
3 phosphorylation is not required.

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Fig. 2.
Selective inhibition of
v 3-mediated
adhesion to distinct ligands. Microtiter plates (96 wells) were
coated with Vn (1 µg/ml) or Fn (5 µg/ml). 105
K v 3 (A) or
K v 3Y747F,Y759F (B) cells were
allowed to adhere for 1 h at 37 °C in the absence or presence
of PMA and specific inhibitors for Rho (10 µg/ml C3 exoenzyme), PI3K
(10 µM wortmannin), or Syk (50 µg/ml piceatannol) and
quantified as described under "Experimental Procedures." Data
bars represent the mean absorbance ±S.D. from triplicate wells
from three separate experiments. Unst., unstimulated,
treated with vehicle only.
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Fig. 3.
Selective inhibition of ligand induced
3-tyrosine phosphorylation
(PY). Shown is a Western blot using
anti-phosphotyrosine mAb from 3 immunoprecipitates
from1 × 106 K v 3 cells
treated with or without RGD (1 mM) and specific inhibitors
for PKC (10 µM bisindolylmaleimide), PI3K (10 µM wortmannin), or Syk (50 µg/ml piceatannol). Total
3 was determined by Western blot of half the
3 immunoprecipitation probed with mAb 7G2. Control cells
are left untreated in suspension.
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Ligand-specific Activation of Rho--
Actin cytoskeletal
reorganization into stress fibers has been shown to require Rho
activity in many cell types (14-18). Here we demonstrate that
v
3-mediated adhesion to Fn as well as
stress fiber formation requires neither PKC activation nor
3-tyrosine phosphorylation. To determine whether the
constitutive firm adhesion to Fn is mediated by Rho activation we
assayed Rho activity using a GST-RBD pull-down assay for active
GTP-bound Rho in cells that were allowed to adhere to Vn or Fn with or
without PMA (Fig. 4A). Elevated Rho activity was seen in both
K
v
3 and
K
v
3Y747F,Y759F cells that were allowed to
adhere to Fn for 1 h even in the absence of PMA stimulation. In
contrast, adhesion to Vn did not lead to an increase in Rho activity in
K
v
3 cells unless there was coincident cellular PKC activation and only minor increases in Rho activity were
seen in K
v
3Y747F,Y759F under similar
conditions. To confirm that the constitutive adhesion and Rho
activation on Fn is
v
3-mediated we show
that when K
V
5 cells are exposed to Fn
there is no significant Rho activation even in the presence of PMA
(Fig. 4A). Additionally, untransfected K562 cells exhibit no
Rho activity on either Vn or Fn (data not shown). We also show that
when PI3K is inhibited by the presence of wortmannin, Rho activation is
inhibited in K
v
3 cells adherent to Vn but
not on Fn (Fig. 4B). Control addition of C3 exoenzyme
inhibits Rho activity in cells adherent to either Vn or Fn. This
suggests that when
v
3 is bound to Fn
there is a direct activation of Rho that circumvents the need for PKC
activity,
3-tyrosine phosphorylation, and PI3K activity.
These results support a hypothesis in which external stimulation,
3-tyrosine phosphorylation, and PI3K are required for
Rho activation and subsequent
v
3-mediated
adhesion to Vn. This supports our previous work suggesting that
tyrosine-phosphorylated
3 is required for the actions of
PKC during
v
3-mediated adhesion to Vn. To
determine whether sites other than the RGD motif of Fn are responsible
for the constitutive adhesion of K
v
3 and
K
v
3Y747F,Y759F cells we employed
recombinant Fn fragment III-10 and assayed for Rho activity. We find
activated Rho in K
v
3 cells adhered to
FnIII-10 regardless of PMA activation (Fig. 4C). However,
when K
V
5 cells are adhered to FnIII-10
there is no Rho activity even in the presence of PMA. In addition when
K
v
3 and K
V
5
cells are allowed to adhere to FnIII-10RGE, in which the RGD motif has
been changed to RGE, there is no constitutive Rho activity (Fig.
4C). These results indicate that it is the RGD motif of Fn
that is required for constitutive
v
3-mediated adhesion to Fn.

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Fig. 4.
Ligand-specific Rho activation.
A, K v 3,
K v 3Y747F, Y759F, or
K V 5 cells were plated on 6-well plates
coated with either Vn (1 µg/ml) or Fn (10 µg/ml), treated with or
without PMA (10 ng/ml), and allowed to adhere for 1 h at 37 °C.
Cell lysates were incubated with GST-RBD immobilized on agarose beads.
GTP-Rho was detected by Western blotting, and 1/10 of the total
lysates were probed for Rho to demonstrate equal loading.
WT, wild type. B,
K v 3 cells were allowed to adhere to Vn or
Fn in the presence or absence of PMA (10 ng/ml), wortmannin (10 µM), or C3 exoenzyme (10 µg/ml). Cell lysates were
incubated with GST-RBD immobilized on agarose beads. GTP-Rho was
detected by Western blotting, and 1/10 of the total cell lysates
were probed for Rho to demonstrate equal loading. C,
K v 3 or K V 5
cells were adhered to Vn, Fn fragment III-10, or Fn fragment III-10RGE
in the presence or absence of PMA (10 ng/ml) for 1 h at 37 °C.
Cell lysates were incubated with GST-RBD immobilized on agarose beads.
GTP-Rho was detected by Western blotting, and 1/10 of the total
lysates were probed for Rho to demonstrate equal loading.
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PI3K Activation on Distinct Ligands--
In this study we show
that inhibition of PI3K activity by wortmannin inhibits both adhesion
to Vn and Rho activation. To investigate the role of PI3K in
v
3-mediated adhesion to Vn and Fn, we
measured phosphorylated Ser-473 AKT as an indicator of PI3K activity
and also the association of PI3K with
3 (6).
K
v
3 and
K
v
3Y747F,Y759F were adhered to Vn or Fn,
and phospho-Ser-473 levels were detected using a phospho-specific AKT
Ser-473 antibody. Phospho-Ser-473 can only be detected in
K
v
3 cells adherent to Vn (Fig.
5A), suggesting that PI3K is
active in a ligand-dependant manner only on Vn. Having demonstrated
that the inhibition of PI3K by wortmannin does not inhibit
3-tyrosine phosphorylation (Fig. 3), we investigated the
role of
3-tyrosine phosphorylation in PI3K localization. K
v
3 or
K
v
3Y747F,Y759F were treated with RGD to
induce
3-tyrosine phosphorylation and Western blotted
with anti-p85 after
3 immunoprecipitations, demonstrating p85 association specifically with tyrosine-phosphorylated
3 (Fig. 5B). Additionally, the removal of
Na3VO4, which reduces
3-tyrosine
phosphorylation, partially blocked association of p85 with
3. These results indicate that PI3K is activated in a
v
3-ligand-dependent manner
and associates with
v
3 in a
3-tyrosine phosphorylation-dependent
manner.

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Fig. 5.
Distinct
v 3
ligands differentially activate PI3K. A, PI3K is activated
on Vn but not Fn. K v 3 or
K v 3Y747F,Y759F cells were plated on
6-well plates coated with either Vn (1 µg/ml) or Fn (10 µg/ml),
treated with or without PMA (10 ng/ml), and allowed to adhere for
1 h at 37 °C. Cell lysates were analyzed for the presence of
phosphorylated AKT and total AKT by Western blotting. WT,
wild type. B, the P85 subunit of PI3K is only associated
with phosphorylated 3. K v 3
or K v 3Y747F,Y759F cells were incubated
with or without RGD peptide (1 mM) and
Na3VO4 (100 nM), and cell lysates
were immunoprecipitated with mAb 7G2 bound to Sepharose. Western
blotting was performed using a p85 mAb. Total 3 was
determined by Western blot of half of the 3
immunoprecipitation (IP) probed with mAb 7G2.
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Adhesion to Distinct anti-
3 Antibodies Mimics
Adhesion to Vn and Fn--
Various anti-
3 antibodies
have been developed that recognize specific epitopes within the
extracellular domain of
3, and some of the more useful
anti-
3 antibodies developed are the ligand-induced binding site (LIBS) antibodies (19). These LIBS antibodies recognize epitopes that are exposed within the integrin extracellular domain upon
binding to an RGD sequence within an ECM protein such as Vn or Fn.
Using this we have explored the possibility that
v
3 may in fact exhibit adhesive
properties to different anti-
3 antibodies that mimic
adhesion to distinct ECM proteins. This would enable us to eliminate
any possible contributions of other integrins expressed within our K562
system. Using multiple assays we determined that 7G2 and LIBS-1,
surprisingly both LIBS type antibodies, mimic
v
3-mediated adhesion to Vn and Fn,
respectively. As seen with adhesion to Vn or Fn (Fig.
6A), adhesion to
anti-
3 mAb 7G2 and LIBS-1 induces tyrosine
phosphorylation of
3 in
K
v
3 cells (Fig. 6B). To
further analyze 7G2 and LIBS-1, we employed adhesion assays to screen
anti-
3 antibodies as possible ligand mimetics of Vn and
Fn. Interestingly, we find that both K
v
3
and K
v
3Y747F,Y759F cells bind to LIBS-1
without the need for cellular PKC activation (Fig.
7, A and B). In
contrast, K
v
3 cells need cellular PKC activation to establish strong adhesion to 7G2, and
K
v
3Y747F,Y759F are unable to adhere to
7G2 even in the presence of cellular PKC activation (Fig. 7,
A and B). PKC-induced adhesion of
K
v
3 cells to 7G2 was inhibited by C3
exoenzyme (100 nM) and wortmannin (10 µM). As
seen with adhesion of these cells to Vn, this suggests a role for Rho
and PI3K in PMA-induced adhesion of K
v
3
cells to 7G2. In addition, adhesion of
K
v
3 cells to LIBS-1 is also inhibited by
C3 exoenzyme (100 nM) (Fig. 7A), but adhesion is unaffected by pretreatment with wortmannin (10 µM),
suggesting, as seen with adhesion to Fn, that PI3K is not required for
adhesion. Fig. 7B shows that
K
v
3Y747F,Y759F cells are incapable of
adhering to 7G2 even in the presence of PMA. Additionally,
K
v
3Y747F,Y759F cells display inhibited
adhesion to LIBS-1 in the presence of C3 exoenzyme (100 nM)
but not wortmannin (10 µM), also suggesting a role for
Rho, but not PI3K. Table I shows that
both K
v
3 and K
v
3Y747F,Y759F cells express
3 equally, and both 7G2 and LIBS-1 bind equally in an
RGD-dependent fashion, thus confirming that any lack of
adhesion to Vn or 7G2 by K
v
3Y747F,Y759F
cells is not due to a lack of expression of the mutant
3
integrin. Table I also shows that PI3K is uninvolved in the
conformation-dependent recognition of
3 by
either 7G2 or LIBS-1, as anti-
3 reactivity is unchanged
in the presence of wortmannin. In this study we show that Rho is
constitutively active on a Fn substrate but requires a cellular agonist
such as PMA and active PI3K to become active on Vn. Rho is also found
to be constitutively active in K
v
3 or
K
v
3Y747F,Y759F cells adherent to LIBS-1
(Fig. 8). However, as seen with Vn,
there is an absolute requirement for cellular PKC activation before Rho
activation occurs in K
v
3 on 7G2, and no
Rho activity was detected in
K
v
3Y747F,Y759F cells even after PMA
treatment. Taken together these data indicate that
v
3-mediated adhesion to 7G2 and LIBS-1
mimic
v
3-mediated adhesion to Vn and Fn,
respectively. This fact will enable us to further decipher signaling
events downstream of
v
3 adhesion to
specific ligands with no input from other integrins.

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Fig. 6.
Ligand and antibody induced
3-tyrosine phosphorylation.
A, Vn and Fn induce 3-tyrosine
phosphorylation (PY). K v 3 or
K v 3Y747F,Y759F cells were either kept in
suspension and incubated with PMA (10 ng/ml) or RGD (1 mM)
peptide or allowed to adhere to Vn (1 µg/ml) or Fn (10 µg/ml). Cell
lysates were immunoprecipitated with mAb 7G2 bound to Sepharose and
Western-blotted with anti-phosphotyrosine 4G10. Total 3
was determined by Western blot of half of the 3
immunoprecipitation probed with mAb 7G2. WT, wild type.
B, LIBS-1 and 7G2 binding to 3 induce
tyrosine phosphorylation. K v 3 and
K v 3Y747F,Y759F were either kept in
suspension and incubated with PMA (10 ng/ml) or allowed to adhere to
mAb 7G2 (7G2) or mAb LIBS-1 (LIBS-1), and cell lysates were
immunoprecipitated with mAb 7G2 bound to Sepharose and Western-blotted
with anti-phosphotyrosine 4G10. Total 3 was determined
by Western blot of half of the 3 immunoprecipitation
probed with mAb 7G2.
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Fig. 7.
Selective inhibition of
v 3-mediated
adhesion to anti- 3 antibodies. Microtiter
plates (96 wells) were coated with mAb 7G2 (0.5 µg/ml) or mAb LIBS-1
(0.5 µg/ml). 105 K v 3
(A) or K v 3Y747F,Y759F
(B) cells were allowed to adhere for 1 h at 37 °C in
the absence or presence of PMA (10 ng/ml) and specific inhibitors of
Rho (10 µg/ml C3 exoenzyme) or PI3K (10 µM wortmannin)
and quantified as described under "Experimental Procedures."
Data bars represent the mean absorbance ±S.D. from
triplicate wells from three separate experiments.
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Table I
PI3K inhibition does not affect 3 conformational change
K v 3, K v 3Y747F,Y759F, or
untransfected K562 cells were incubated with either 7G2 (0.5 µg/ml)
or LIBS-1 (0.5 µg/ml) in the absence (control) or presence of RGD (1 mM) or wortmannin (Wn) (10 µM). Antibody
binding was quantified as described under "Experimental
Procedures." Data are expressed as mean channel fluorescence for
three separate experiments.
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Fig. 8.
Rho becomes activated on LIBS type
anti- 3 antibodies.
K v 3 or
K v 3Y747F,Y759F were plated on 6-well
tissue culture plates coated with either mAb 7G2 (0.5 µg/ml) or mAb
LIBS-1 (0.5 µg/ml), treated with or without PMA (10 ng/ml), and
allowed to adhere for 1 h at 37 °C. Cell lysates were incubated
with GST-RBD immobilized on agarose beads. GTP-Rho was detected by
Western blotting, and 1/10 of the total lysates were probed for
Rho to demonstrate equal loading. WT, wild type.
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 |
DISCUSSION |
The ability of integrins, including
v
3, to adhere to multiple ECM proteins
has prompted the use of the term "promiscuous." However, this term
may be inaccurate because we and others show that
v
3-mediated adhesion to distinct ligands
is differentially regulated (4, 5). Hematopoietic cells and leukocytes
in particular are able to regulate their adhesive properties, a
requirement for appropriate vascular egress of leukocytes and
subsequent extravascular immune surveillance (2). Integrin activation
can be defined as the events necessary to permit firm adhesion,
including a possible enhancement of integrin affinity, avidity, and
integrin-cytoskeletal interactions. A requirement for
3-tyrosine phosphorylation during
v
3 activation has been established;
however, the mechanism and the circumstances under which this
modification contributes to the overall activation state of
v
3 have not been have not been completely
established (5, 12, 20, 21).
In this study we show that
v
3-mediated
firm adhesion to distinct ECM ligands initiates unique signaling
pathways and distinct cellular phenotypes. We show that
v
3-mediated firm adhesion to Vn requires
both cellular PKC activity and
3-tyrosine
phosphorylation. Additionally, we show that firm adhesion to Vn also
requires both PI3K and Syk activities. Furthermore, Rho activity is
only up-regulated in cells adherent to Vn when there is coincident PKC
and PI3K activities and tyrosine phosphorylation of
3.
PI3K activity is up-regulated after adhesion to Vn, but not Fn, and
PI3K translocation to
v
3 complexes occurs
only with tyrosine-phosphorylated
3. In contrast,
v
3-mediated firm adhesion to Fn results
in or in fact may be a result of constitutive Rho activation that
requires no cellular PKC activation,
3-tyrosine
phosphorylation, or PI3K activity. These data clearly outline distinct
signaling pathways that are involved in
v
3 activation by and subsequent adhesion to distinct ligands. Parallel results using ligand mimetic
anti-
3 mAbs validate the single integrin-multiple
pathway conclusion. The unique behavior of the anti-
3
mAb 7G2 indicates that the signaling pathway utilized by
v
3 adhesion to Vn defines the leukocyte
response integrin, a
3-related integrin of neutrophils originally characterized using this mAb. These mAb data support our
hypothesis that
3-tyrosine phosphorylation and the
mechanisms dependent upon this modification are likely to be a
hematopoietic cell-specific event. If this conclusion is correct, a
role exists for CD47 co-characterized with leukocyte response integrin
in
v
3 function that has not yet been
determined. The role for CD47 in
v
3-mediated adhesion to Vn is perhaps
related to the recruitment of a greater complexity of signaling
molecules than is required for
v
3-mediated adhesion to Fn or its mimetic
mAb LIBS-1.
The ECM is composed of multiple proteins that serve as ligands for
integrins expressed on a variety of cells. The ability of specific
integrins to adhere to distinct ligands in a regulated manner may be a
fundamental part of the regulated adhesion required for hematopoietic
cell function. Many reports have shown distinct phenotypes resulting
from different integrins recognizing a single substrate (1). Here we
show that distinct phenotypes can also result from a single integrin
recognizing different substrates. This would allow hematopoietic cells
to use distinct ligands for discrete steps in the processes of
arresting from the vasculature, transendothelial migration, and
migration within the ECM to the site of inflammation. Although
v
3-mediated adhesion to Fn or Vn can both
ultimately result in firm adhesion, the activation of distinct
signaling pathways would allow for the initiation of additional
cellular functions specific to an immediate cellular challenge such as
transendothelial migration.
The ability of cells to firmly adhere to multiple ECM proteins requires
regulated integrin ligation and initiation of specific subsequent
signaling pathways that ultimately lead to an actin cytoskeletal
reorganization that supports firm adhesion, and integrin activation is
a critical step in this process. Integrin activation can be defined as
the process by which integrin ligation leads to actin cytoskeletal
reorganization via Rho activity and consequent firm adhesion. By this
definition we can utilize active GTP-bound Rho as a biochemical marker
of an activated integrin complex. Here we show that distinct ligands
can initiate two modes of integrin activation that lead to Rho
activation and firm adhesion employing different signaling components
downstream of integrin ligation. Integrin activation on Vn is highly
regulated and requires
3 tyrosine phosphorylation,
cellular PKC activity, PI3K activity, and Syk activity, which
ultimately lead to Rho activation and stress fiber formation. In
contrast, adhesion to Fn does not require
3-tyrosine
phosphorylation, cellular PKC activity, or PI3K activity; however, firm
adhesion to both ligands requires Rho activity. Although the Syk
inhibitor piceatannol blocks RGD-induced phosphorylation of
3, it is unlikely that this is the mechanism whereby
piceatannol inhibits
v
3-mediated adhesion
to Fn, because this event is independent of
3-tyrosine
phosphorylation and yet inhibited by piceatannol. In contrast to
another report, we find that wortmannin has no effect on RGD-induced
3-tyrosine phosphorylation, and therefore, its
inhibitory effects are likely due to inhibition of the increase in PI3K
activity and/or the translocation of PI3K to the integrin complex (22).
Furthermore, we do not find an effect of wortmannin on the RGD-induced
conformational change of
v
3 (23). This correlates with our previous reports showing the requirement for tyrosine phosphorylation of
3 for Vn adhesion, shown
here to be PI3K-dependent, is unrelated to the conformation
change in the receptor after ligand binding.
Although the need for ligand-dependent pathways of
cytoskeletal reorganization remains to be determined, our data suggest that these signaling pathways converge at the level of a RhoGEF. One
potential scenario involves PKC-dependent activation of Src causing Pyk2 activation and translocation to the integrin complex. This
simplistic path provides neither a role for PI3K activity or its
translocation to the integrin unless it functions to recruit the RhoGEF
or Syk. It is also possible that PI3K is required for the activation of
Src. The requirement of Syk activity in both pathways probably
implicates Vav as the RhoGEF responsible for Rho activation. In cells
adherent to Fn or LIBS-1, Rho activation may result from direct
v
3 association with and auto-activation of Syk as previously described (24). This determination will require
additional reagents specific for active Vav. Of equal interest is the
ability of Fn ligation of
v
3 to
up-regulate Rho without the need for
3-tyrosine
phosphorylation, PI3K activity, or PKC activity. It has been reported
that
3-tyrosine phosphorylation can be a negative
regulator of Fn adhesion; thus, it seems likely that the recruitment of
a
v
3 binding partner in a
3-tyrosine phosphorylation-dependent manner
is responsible for the initiation of these distinct signaling pathways.
The nature of the proximal binding partner is most likely that of an
adapter molecule, although we cannot rule out that PI3K may bind
directly to tyrosine-phosphorylated
3 integrins.
Identification of this proximal binding partner will permit further
discrimination of the signaling events initiated by
v
3 after attachment to Vn or Fn.
Furthermore, the Rho activation, firm adhesion, and stress fiber
formation seen in K
v
3Y747F,Y759F cells
adherent to fibronectin argues that these cytoplasmic tyrosine mutations selectively target signaling pathways and do not grossly disturb the functional structure of the receptor.
Our description of distinct signaling pathways resulting from different
ligand recognition by the same integrin provides an additional level of
ligand discrimination and integrin regulation. Whether this is unique
to hematopoietic cell types remains to be determined; however, these
cell types are the most in need of self-regulated adhesion. Related
phenomena such as tumor metastasis may utilize similar signaling
pathways. The characterization of ligand-mimetic mAb, which also
initiate unique signaling, will advance future study of this integrin
behavior in cell types with complex integrin expression patterns.