* Department of Vascular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La
Jolla, California; and § Ehime University School of Medicine, Ehime, Japan
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Abstract |
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Integrin IIb
3 mediates platelet aggregation
and "outside-in" signaling. It is regulated by changes in
receptor conformation and affinity and/or by lateral
diffusion and receptor clustering. To document the relative contributions of conformation and clustering to
IIb
3 function,
IIb was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified
IIb subunits were expressed with
3 in CHO cells, and
the heterodimers could be clustered into morphologically detectable oligomers upon addition of AP1510, a
membrane-permeable, bivalent FKBP ligand. Integrin clustering by AP1510 caused binding of fibrinogen and
a multivalent (but not monovalent) fibrinogen-mimetic
antibody. However, ligand binding due to clustering
was only 25-50% of that observed when
IIb
3 affinity
was increased by an activating antibody or an activating
mutation. The effects of integrin clustering and affinity modulation were additive, and clustering promoted irreversible ligand binding. Clustering of
IIb
3 also promoted cell adhesion to fibrinogen or von Willebrand
factor, but not as effectively as affinity modulation.
However, clustering was sufficient to trigger fibrinogen-independent tyrosine phosphorylation of pp72Syk
and fibrinogen-dependent phosphorylation of
pp125FAK, even in non-adherent cells. Thus, receptor
clustering and affinity modulation play complementary
roles in
IIb
3 function. Affinity modulation is the predominant regulator of ligand binding and cell adhesion,
but clustering increases these responses further and
triggers protein tyrosine phosphorylation, even in the
absence of affinity modulation. Both affinity modulation and clustering may be needed for optimal function
of
IIb
3 in platelets.
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Introduction |
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INTEGRINS are type I transmembrane heterodimers
that mediate cell adhesion and signaling in a highly
regulated manner (Clark and Brugge, 1995
). Several
modes of integrin regulation have been demonstrated or
postulated, including control of expression on the cell surface by coordinate subunit biosynthesis and recycling (Bennett, 1990
; Bretscher, 1992
), modulation of receptor
affinity by conformational changes in the heterodimer
(Sims et al., 1991
; Shattil et al., 1998
), and modulation of
receptor avidity by lateral diffusion of heterodimers to
form higher order multimers or clusters (Detmers et al.,
1987
; van Kooyk et al., 1994
; Kucik et al., 1996
; Bazzoni
and Hemler, 1998
). The latter process may be promoted by interactions of integrins with multivalent, extracellular
ligands (Peerschke, 1995b
; Simmons et al., 1997
), and with
components of the dynamic actin cytoskeleton (Sastry and
Horwitz, 1993
; Fox et al., 1996
; Kucik et al., 1996
). Integrin
function must sometimes be regulated acutely over seconds to minutes to enable the kinds of rapid changes in
cell adhesion and migration that are required during immune responses, inflammation, and hemostasis. Several integrins in blood cells are targets of this type of activation or "inside-out" signaling, including
4
1,
L
2, and
M
2 in
leukocytes, and
IIb
3 and
V
3 in platelets (Bennett et al.,
1997
; Bazzoni and Hemler, 1998
; Shattil et al., 1998
). It
seems likely that some combination of conformational
change and receptor clustering is involved in activating the
ligand-binding function of these integrins. However, evidence to support one or the other mechanism has been
largely indirect, and it has been difficult to determine the
relative contributions of each in intact cells. The distinction between integrin affinity and avidity modulation is
not academic because the underlying mechanisms may be
different, with implications for therapeutic strategies to
block or promote integrin functions in pathological conditions (Coller, 1997
; Bazzoni and Hemler, 1998
).
One of the best-studied integrins from the standpoint of
acute regulation is IIb
3, which interacts with Arg-Gly-Asp-containing ligands, such as fibrinogen and von Willebrand factor (vWf),1 to effect platelet aggregation and
spreading on vascular surfaces. Platelet agonists, such as
thrombin and ADP, cause rapid changes in the adhesive
function of
IIb
3, as evidenced by increases in the binding
of soluble fibrinogen, vWf, and ligand-mimetic mAbs, including PAC1 (Shattil et al., 1985
). Antagonists, such as
prostacyclin and nitric oxide can inhibit and, under some
conditions reverse these acute changes (Graber and Hawiger, 1982
; Freedman et al., 1997
). Coupled with evidence
from fluorescence resonance energy transfer studies showing that the
IIb and
3 subunits undergo changes in relative orientation during platelet activation (Sims et al., 1991
),
a current hypothesis is that ligand binding to
IIb
3 is controlled, at least in part, by changes in heterodimer conformation that affect access of the ligand to recognition sites in the receptor (Loftus and Liddington, 1997
; Shattil et al., 1998
). On the other hand, it is entirely possible that clustering of
IIb
3 also occurs during the process of platelet activation. Indeed, clustering of
IIb
3 on the platelet surface has
been detected by electron microscopy after ligand binding
(Isenberg et al., 1987
; Simmons et al., 1997
). Were clustering
to occur directly in response to platelet agonists, it could enhance ligand binding through chelate and rebinding effects.
Furthermore, "outside-in" signaling through
IIb
3, manifested by activation of specific protein tyrosine kinases, lipid
kinases, and cytoskeletal reorganization (Fox et al., 1993
;
Banfic et al., 1998
; Shattil et al., 1998
), seems to require the
binding of multivalent ligands (Huang et al., 1993
), indirectly
suggesting a functional role for oligomerization of
IIb
3.
Since platelets are not amenable to genetic manipulations ex vivo, heterologous expression systems have been
used to study the structure and function of IIb
3 (O'Toole
et al., 1994
; Loh et al., 1996
). For example, human
IIb
3
expressed in CHO cells binds little or no fibrinogen or
PAC1 and is therefore, in a constitutively low affinity/
avidity state, just as it is in resting platelets. However, the
affinity of
IIb
3 can be increased by incubation of the cells
with particular "LIBS" mAb Fab fragments that bind to
the
or
integrin subunit and induce a conformational
change in the extracellular portion of the receptor to expose ligand binding sites (O'Toole et al., 1994
). Under
these experimental conditions, monovalent LIBS Fab fragments by themselves would not be expected to induce receptor clustering. In the present study, we have used new
modifications of this experimental system to establish the
separate contributions of affinity modulation and receptor
clustering to the functions of
IIb
3. Specifically, single or
tandem repeats of the FK506-binding protein, FKBP12 (FKBP), have been fused to the cytoplasmic tail of
IIb to
conditionally cluster heterodimers into oligomers from inside the cell using AP1510, a synthetic, bivalent, and membrane-permeable FKBP dimerizer (Amara et al., 1997
).
The results establish that affinity modulation and receptor
clustering can play complementary roles in the adhesive and signaling functions of this prototypic integrin. Whereas
affinity modulation is the predominant mechanism for regulating ligand binding to
IIb
3, receptor clustering facilitates this process and promotes outside-in signaling, even
in the absence of affinity modulation.
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Materials and Methods |
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Plasmid Constructions and Expression of Recombinant
Forms of IIb
3 in CHO Cells
A pCDM8 template containing full-length IIb (O'Toole et al., 1994
) was
subjected to PCR with Pfu polymerase (Stratagene, La Jolla, CA) to place
XbaI and SpeI restriction sites at the 5' and 3' ends of
IIb, respectively.
The PCR product was cut with XbaI and SpeI and ligated into an XbaI-cut,
CMV-based mammalian expression vector, pCF1E (ARIAD Pharmaceutical, Inc., Cambridge, MA). Plasmids with inserts in the correct orientation were amplified and purified for CHO cell transfections (Maxi-Prep;
QIAGEN Inc., Chatsworth, CA). The resulting
IIb(FKBP)/pCF1E plasmid encoded
IIb fused in-frame to FKBP, which in turn was fused in-frame to a hemagglutinin epitope tag (see Fig. 1). To construct
IIb fused
to two tandem FKBP repeats (
IIb(FKBP)2), a single FKBP was removed
from pCF1E with XbaI/SpeI and ligated into SpeI-cut
IIb(FKBP)/pCF1E.
The remaining
IIb and
3 cDNAs depicted in Fig. 1 were in pCDM8
(O'Toole et al., 1994
). cDNA coding full-length human Syk was in EMCV
(Gao et al., 1997
). Plasmid inserts were analyzed by automated sequencing to confirm authenticity.
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cDNAs were transfected into CHO-K1 cells with lipofectamine according to the manufacturer's instructions (GIBCO BRL, Gaithersburg, MD).
Typically, 0.5-2 µg of each plasmid was used, supplemented when necessary with empty vector DNA (pCDNA3; Invitrogen, San Diego, CA) for a
total of 4 µg per dish. Cells were maintained for 48 h for transient expression or subjected to antibiotic selection for stable expression. Stable cell lines
were selected further for high integrin expression by single cell FACS®
sorting using an IIb
3-specific murine mAb, D57 (O'Toole et al., 1994
).
Characterization of Recombinant Integrins in CHO Cells
Cell surface expression of recombinant IIb
3 was assessed by flow cytometry using biotin-D57 and FITC-streptavidin (Leong et al., 1995
).
IIb expression was also evaluated by Western blotting. 48 h after transfection,
the cells were lysed in 66 mM Tris-HCl, pH 7.4, containing 2% SDS and
30 µg of protein were electrophoresed in SDS-7.5% polyacrylamide gels
under nonreducing conditions, transferred to nitrocellulose, and then subjected to Western blotting with murine mAb B1B5 specific for
IIb or antibody 12CA5 specific for the hemagglutinin epitope tag (Abrams et al.,
1992
). After addition of affinity-purified, HRP-conjugated goat anti-
mouse IgG (Biosource International, Camarillo, CA), blots were developed for 0.1-1 min by enhanced chemiluminescence (ECL; Amersham,
Arlington Heights, IL).
Confocal Microscopy
To establish whether AP1510 could induce clustering of IIb(FKBP)2
3
that was detectable morphologically, cells stably expressing
IIb(FKBP)2
3
were incubated in the presence of 1,000 nM AP1510 (or 0.5% EtOH as a
vehicle control) for 30 min at room temperature. Then, analogous to the
method used by Yauch and co-workers to detect antibody-induced integrin clustering (Yauch et al., 1997
), the cells were incubated with 10%
goat serum for 30 min at room temperature, followed by 10 µM FITC-D57 or unlabeled D57 for 30 min on ice. After washing, the sample containing unlabeled D57 was incubated for 30 min with FITC-labeled goat
anti-mouse heavy and light chains (1:500; Biosource International) to deliberately cluster the integrin as a positive control. All samples were fixed in 4% paraformaldehyde, resuspended in mounting medium (Fluorosave; Calbiochem-Novabiochem, San Diego, CA), and analyzed on glass slides
with an MRC 1024 laser scanning confocal imaging system (Bio-Rad Laboratories, Hercules, CA).
Measurements of Ligand Binding Due to Clustering and
Affinity Modulation of IIb
3
Ligand binding to IIb
3 in CHO cells was assessed by flow cytometry using a saturating amount of the fibrinogen-mimetic, murine monoclonal
IgM
antibody, PAC1 (Kashiwagi et al., 1997
). CHO cells were resuspended to 107 cells/ml in Tyrode's buffer supplemented with 2 mM CaCl2 and MgCl2 (O'Toole et al., 1994
). For most experiments, 4 × 105 cells were
added to tubes containing a final concentration of 0.4% PAC1 ascites or
40 nM purified PAC1 in a final vol of 50 µl, and then incubated for 30 min
at room temperature. In some experiments, monovalent recombinant
PAC1 Fab produced in insect cells and purified by nickel-agarose chromatography was used instead of PAC1 IgM at a final concentration of 30 nM
(Abrams et al., 1994
). As indicated for each experiment, cell incubations
were also carried out in the presence of one or more of the following reagents: 10-5,000 nM AP1510 (or vehicle buffer) to cluster
IIb(FKBP)2
3
or
IIb(FKBP)
3 (Amara et al., 1997
), 150 µg/ml anti-LIBS6 Fab to convert
IIb
3 into a high affinity form through conformational changes (Du
et al., 1993
; Kashiwagi et al., 1997
), and 10 µM integrilin, an
IIb
3 antagonist to specifically block PAC1 binding (Scarborough et al., 1993
). Preliminary experiments with AP1510 and anti-LIBS6 Fab indicated that a 10-
30-min incubation of cells with these reagents was sufficient to achieve
their maximum effects. Cells were then washed and incubated on ice for
30 min with biotin-D57, followed by phycoerythrin-streptavidin and either
FITC-labeled goat anti-mouse µ heavy chain antibody (to label PAC1
IgM) or FITC-labeled goat anti-mouse heavy and light chain antibody (to label PAC1 Fab) (both from Biosource International). Samples were diluted with 0.5 ml Tyrode's buffer containing 2 µg/ml propidium iodide
(Sigma Chemical Co., St. Louis, MO) and analyzed on a FACSCalibur®
flow cytometer (Becton Dickinson Co., Mountain View, CA). After electronic compensation, PAC1 binding (FL1 channel) was analyzed on the
gated subset of live cells (propidium iodide-negative, FL3) that was positive for
IIb
3 expression (FL2). To control for variations in integrin expression from transfection to transfection, PAC1 binding, measured as
mean fluorescence intensity in arbitrary units, was expressed relative to
the levels of
IIb
3, measured simultaneously with biotin-D57.
Adhesion of CHO Cells to Fibrinogen and vWF
Immulon-2 microtiter wells (Dynex Laboratories, Chantilly, VA) were
coated with purified fibrinogen (Enzyme Research Laboratories, South
Bend, IN) or vWf (Ruggeri et al., 1983) overnight at 4°C at coating concentrations ranging from 0.01-2 µg/well, followed by blocking with 20 mg/ml
BSA. CHO cells stably expressing
IIb(FKBP)2
3 were labeled for 30 min
at 37°C with 2 µM BCECF-AM (Molecular Probes, Eugene, OR). After
washing, the cells were resuspended to 106/ml, incubated for 30 min in the
presence of 1,000 nM AP1510 and/or 150 µg/ml anti-LIBS6 Fab, and then
100-µl aliquots were added to the coated microtitre wells for 90 min at
37°C. After washing three times with 150 µl of PBS, cell adhesion was
quantitated by cytofluorimetry (Leng et al., 1998
).
Protein Tyrosine Phosphorylation in CHO Cells
Stable cell lines expressing IIb(FKBP)2
3 were transiently transfected
with EMCV-Syk and placed into complete DME with 10% FBS. 24 h after transfection, the amount of serum was reduced to 0.5%, and 48 h after
transfection, the cells were resuspended to 3 × 106/ml in DME and slowly
rotated at 37°C for 45 min in the presence of 20 µM cycloheximide. Then
cells were incubated for 10 min with one or more of the following: 1,000 nM AP1510 to stimulate receptor clustering, 150 µg/ml anti-LIBS6 Fab to
increase integrin affinity, or 250 µg/ml fibrinogen to achieve ligand binding.
As a positive tyrosine phosphorylation control, one aliquot of cells was allowed to attach for 60 min to a dish coated with
IIb
3 antibody D57. Cells
were lysed in RIPA buffer containing 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 158 mM NaCl, 10 mM Tris, pH 7.4, 1 mM Na2EGTA,
0.5 mM leupeptin, 0.25 mg/ml Pefabloc, 5 µg/ml aprotinin, 20 mM NaF, 3 mM
-glycerophosphate, 10 mM sodium pyrophosphate, and 5 mM sodium vanadate. After clarification, 200 µg of protein were immunoprecipitated
with rabbit antiserum specific for Syk or FAK (Gao et al., 1997
). Immunoprecipitates were subjected to Western blotting with anti-phosphotyrosine
mAbs, 4G10, and PY20 (Upstate Biotechnology Inc., Lake Placid, NY
and Transduction Laboratories, Lexington, KY, respectively), followed by
stripping and reprobing with mAb 4D10 to Syk or antiserum to FAK
(Gao et al., 1997
). Immunoreactive bands detected by ECL were quantitated by calibrated densitometry using a flatbed scanner, Power Center Pro 240 computer, and NIH Image software.
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Results |
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Heterologous Expression of IIb
3 Containing
Dimerization Motifs
The purpose of these studies was to evaluate the possible
contributions of integrin clustering and affinity modulation to the adhesive and signaling functions of IIb
3. A
CHO cell model system, previously used to study factors
that influence the ligand-binding affinity of human
IIb
3
(O'Toole et al., 1994
; Hughes et al., 1996
; Zhang et al.,
1996
; Kashiwagi et al., 1997
), has now been adapted to
study integrin clustering. Full-length
IIb was engineered to contain one or two FKBP repeats and a hemagglutinin
epitope tag at the extreme COOH terminus of the cytoplasmic tail (Fig. 1). In theory, a protein containing a single FKBP may dimerize upon addition of a membrane-permeable, bivalent FKBP ligand, such as AP1510, and a
protein with two or more FKBP repeats may form larger
oligomers (Amara et al., 1997
; Yap et al., 1997
; Yang et al.,
1998
). Consequently, we reasoned that if
IIb(FKBP) or
IIb(FKBP)2 were successfully coexpressed on the surface
of CHO cells along with
3, then
IIb
3 heterodimers might
be converted into a dimer of dimers ((
IIb
3)2) or into
even larger oligomers in response to AP1510.
After transient or stable expression in CHO cells, both
IIb(FKBP)
3 and
IIb(FKBP)2
3 were found to be expressed to the same extent as wild-type
IIb
3, as determined by the binding of D57, an
IIb
3-specific antibody
(Fig. 2 A). In addition, Western blotting of the cell lysates
with an antibody specific for the extracellular domain of
IIb (B1B5) showed that
IIb(FKBP) and
IIb(FKBP)2 exhibited the predicted slower electrophoretic mobilities
compared with the smaller wild-type
IIb subunit (Fig. 2
B). The
IIb(FKBP) and
IIb(FKBP)2 fusion proteins also
reacted on Western blots with an antibody to the COOH-terminal epitope tag, further suggesting that they represented full-length proteins (Fig. 2 B). Thus, the fusion of
one or two FKBP repeats to the cytoplasmic tail of
IIb does not interfere with the transient or stable expression
of this subunit in CHO cells to form an
IIb
3 complex.
Therefore, in the following studies transient and stable cell
lines were used as indicated, depending on the experimental protocol.
|
Conditional Clustering of IIb
3 in CHO Cells
Large integrin oligomers might be detectable in CHO cells at
the level of light microscopy (van Kooyk et al., 1994; Yauch et al., 1997
). To determine if clusters of
IIb(FKBP)2
3 could be detected morphologically, CHO cells stably expressing
this integrin were incubated for 30 min at room temperature with 1,000 nM AP1510 (or vehicle buffer as a control),
stained with FITC-D57 on ice, fixed, and then examined
by confocal microscopy. D57 staining was entirely surface
associated, and cells that had been treated with buffer instead of AP1510 exhibited a finely patchy distribution of
IIb(FKBP)2
3 (Fig. 3, A-C). In contrast,
IIb(FKBP)2
3 in cells treated with AP1510 exhibited a coarse patchiness
(Fig. 3, E-G), similar to that observed when the D57 antibody was deliberately cross-linked with a secondary antibody
before cell fixation (Fig. 3 H). The same results with AP1510
were obtained with another independent
IIb(FKBP)2
3
clone; in contrast, AP1510 caused no discernible clustering
of wild-type
IIb
3 in CHO cells (not shown). These results
are consistent with the conclusion that oligomerization of
IIb(FKBP)2
3 can be induced conditionally from within
the cell using AP1510, enabling us to conduct a detailed
study of the functional consequences of integrin clustering.
|
Receptor Clustering in the Regulation of Ligand
Binding to IIb
3
Activation of IIb
3 is required for the binding of soluble,
macromolecular Arg-Gly-Asp-containing ligands, such as
fibrinogen, vWf, and fibrinogen-mimetic antibodies, such
as PAC1. To evaluate the contribution of clustering to
IIb
3 activation, flow cytometry was used to quantitate
the specific binding of PAC1 to transiently transfected
CHO cells. Specific binding was defined as that inhibitable by 10 µM integrilin, an
IIb
3-selective antagonist, and it
was expressed relative to the amount of
IIb
3 on the cell
surface, determined simultaneously with antibody D57. In
cells expressing
IIb(FKBP)2
3, there was little binding of
PAC1, indicating that, like
IIb
3, this integrin is in a constitutive low affinity/avidity state. AP1510 caused a dose-dependent increase in PAC1 binding to
IIb(FKBP)2
3
cells (Fig. 4, closed circles), without affecting the levels of
surface expression of this integrin. However, PAC1 binding due to AP1510 appeared modest compared with binding in response to upregulation of integrin affinity by an
activating antibody Fab fragment, anti-LIBS6 Fab (Fig. 4,
open circles).
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To evaluate possible mechanistic differences between
integrin clustering and affinity modulation in the control
of ligand binding, additional experiments were performed
with cells expressing IIb(FKBP)2
3. First, we considered
the possibility that AP1510 caused PAC1 binding by increasing integrin affinity rather than (or in addition to)
avidity. However, AP1510 failed to stimulate the binding
of a monovalent PAC1 Fab fragment to
IIb(FKBP)2
3, although this form of PAC1 bound normally in response to
anti-LIBS6 Fab (Fig. 5). Since a monovalent ligand might
be expected to be sensitive to affinity modulation but less
sensitive than a multivalent ligand to avidity modulation,
this result suggests that AP1510 was indeed working by
clustering the integrin. Second, PAC1 binding in response
to AP1510 was completely prevented if the cells were preincubated for 30 min with 4 mg/ml of 2-deoxy-D-glucose
and 0.2% sodium azide to deplete metabolic ATP (two
separate experiments). Since oligomerization by AP1510
should be energy independent, this suggests that metabolic
energy is needed to maintain the receptor in a proper conformation, even when ligand binding is triggered by receptor clustering. Third, the effect of a specific point mutation
(
3(S752P)) or a truncation (
3(
724)) of the
3 cytoplasmic tail were studied because both have been shown to
disrupt affinity modulation of
IIb
3 in platelets and CHO
cells (Chen et al., 1992
, 1994
; O'Toole et al., 1994
; Wang
et al., 1997
). Whereas
3(S752P) abolished PAC1 binding
in response to AP1510,
3(
724) had no such effect (Fig.
6). Thus the
3 cytoplasmic tail plays a role in ligand binding triggered by integrin clustering, but there must be differences in the structural features of
3 required for affinity and avidity modulation.
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|
Thus far, the results support the validity of this model
system to study integrin clustering, and they suggest that
both clustering and affinity modulation can regulate ligand
binding to IIb
3. Further studies were performed to determine the relative contributions of clustering and affinity
modulation to ligand binding under conditions in which
the effects of AP1510 and anti-LIB6 Fab were maximal
(1,000 nM and 150 µg/ml, respectively). CHO cells were transiently transfected with either
IIb
3,
IIb(FKBP)
3,
IIb(FKBP)2
3, or
IIb/
6A
3 (a constitutive, high affinity
mutant [O'Toole et al., 1994
]), and ligand binding was
evaluated 48 h later. Whereas AP1510 had no effect on
PAC1 binding to wild-type
IIb
3, it increased binding to
both
IIb(FKBP)
3 and
IIb(FKBP)2
3 such that specific PAC1 binding was increased approximately twofold (P < 0.001) (Fig. 7). However, PAC1 binding induced by AP1510
amounted to only 50% of the binding observed with the
high affinity
IIb/
6A
3 chimera, and only 25% of the binding induced by anti-LIBS6 Fab (Fig. 7). Nevertheless, the
PAC1 binding caused by clustering was statistically significant (P < 0.03) and approximately additive to the binding caused by affinity modulation (Fig. 7).
|
Fibrinogen and PAC1 binding to activated platelets is
initially reversible by the addition of EDTA, but binding
becomes progressively irreversible over 15-60 min (Peerschke, 1995a; Fox et al., 1996
). In CHO cells that expressed
IIb(FKBP)2
3 and were treated with both anti-LIBS6 Fab
and AP1510 to achieve maximal PAC1 binding, the added
component of ligand binding resulting from AP1510 was fully reversible at 10 min but irreversible at 30 min (Fig. 8). Similar results were obtained when FITC-fibrinogen was
used instead of PAC1 to monitor ligand binding (not
shown). This series of experiments demonstrates that affinity modulation is the predominant regulator of ligand
binding to
IIb
3. However, receptor clustering plays an
additive role in promoting eventual irreversible binding of
the ligand.
|
IIb
3 Clustering in the Regulation of Cell Adhesion
Activation of platelets by agonists leads to increased cell
adhesion to the IIb
3 ligands, fibrinogen, and vWf (Savage et al., 1992
). To determine the relative contributions of
IIb
3 clustering and affinity modulation to cell adhesion,
CHO cells that stably expressed
IIb(FKBP)2
3 were
loaded with BCECF as a fluorescent marker and incubated
for 90 min in microtiter wells coated with fibrinogen or vWf.
Cell adhesion was dependent on the coating concentration
of fibrinogen (Fig. 9, left panel ) and vWf (Fig. 9, right
panel ), as well as on the presence of
IIb(FKBP)2
3, since
it was blocked by 10 µM integrilin. AP1510 (1,000 nM) increased the extent of cell adhesion, but only very modestly
and only at the higher coating concentrations of fibrinogen and vWf. On the other hand, increasing integrin affinity
with anti-LIBS6 Fab (150 µg/ml) caused a more marked
increase in cell adhesion, even at the lower ligand concentrations (Fig. 9, left and right panels). Thus under these assay conditions, receptor clustering is not as effective as affinity modulation in regulating cell adhesion via
IIb
3.
|
IIb
3 Clustering in the Regulation of
Outside-In Signaling
In platelets and CHO cell transfectants, fibrinogen binding
to IIb
3 leads to tyrosine phosphorylation and activation
of Syk and FAK. The binding of soluble fibrinogen is sufficient to activate Syk, but additional post-ligand binding
events, such as cytoskeletal reorganization, are necessary
for activation of FAK (Huang et al., 1993
; Clark et al.,
1994
; Gao et al., 1997
). To study the role of integrin clustering in these events, CHO cells stably expressing
IIb(FKBP)2
3
were transiently-transfected with human Syk, and tyrosine
phosphorylation of Syk and endogenous hamster FAK
was examined. Fig. 10 A shows the raw data for a single experiment and Fig. 10 B shows a summary of three separate experiments. Cells maintained in suspension for 10 min in the absence or presence of fibrinogen exhibited a
low level of tyrosine phosphorylation of Syk and FAK.
However, addition of 1,000 nM AP1510 caused an average
2.8-fold increase in tyrosine phosphorylation of Syk, even
in the absence of fibrinogen (P < 0.05), and this response was greater still in the presence of fibrinogen (5.4-fold;
P < 0.02). In contrast, in the absence of fibrinogen integrin clustering by AP1510 did not stimulate an increase in
FAK tyrosine phosphorylation, but increased FAK phosphorylation was observed in the presence of fibrinogen
(3.5-fold; P < 0.001). Thus, integrin clustering can trigger
tyrosine phosphorylation of Syk even in the absence of fibrinogen binding, whereas phosphorylation of FAK requires both receptor clustering and fibrinogen binding. Affinity modulation by anti-LIBS6 is not necessary in either
case.
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![]() |
Discussion |
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In this study, engraftment of one or two FKBP repeats
onto the COOH terminus of the IIb subunit enabled us to
cluster integrin
IIb
3 in a conditional fashion by treating
CHO cells with a synthetic, bivalent FKBP ligand, AP1510.
This permitted us for the first time to conduct a detailed
comparison of the functional effects of receptor clustering,
initiated from within the cell, with the effects of increasing
integrin affinity through conformational changes. The major conclusions of this work are: (a) Conformational changes
play a predominant role in
IIb
3 activation in CHO cells, as monitored by ligand binding and cell adhesion assays.
(b) Clustering causes a modest increment in reversible and
ultimately irreversible binding of multivalent ligands to
IIb
3, and this binding is additive to that caused by affinity modulation. (c) Ligand binding resulting from receptor
clustering is dependent on cellular metabolic energy and is
sensitive to some, but not all, of the mutations or deletions
in the
3 cytoplasmic tail that block affinity modulation of
the receptor. (d) Integrin clustering promotes ligand-independent tyrosine phosphorylation of Syk, and ligand-
dependent phosphorylation of FAK, even when cells are
maintained in suspension and even in the absence of deliberate affinity modulation. Thus, by being able to manipulate integrin clustering and affinity separately and in a controlled manner, we conclude that these two processes play
complementary roles in the function of
IIb
3.
Integrin Clustering and Inside-Out Signaling
Inside-out signaling is responsible for acute regulation of
the ligand binding function of integrins. In the case of integrins that normally engage soluble adhesive ligands in
vivo, such as IIb
3, inside-out signaling can be monitored
directly using labeled ligands or ligand-mimetic antibodies, such as PAC1. Alternatively, it can be assessed by cell
adhesion assays. Although physiologically relevant, cell
adhesion is a more indirect measure of integrin activation
because it can be influenced by factors, such as actin polymerization, cell spreading, and focal adhesion turnover,
that may affect the overall strength of the adhesion process through mechanisms other than regulation of ligand
binding (Burridge and Chrzanowska-Wodnicka, 1996
; Yamada and Geiger, 1997
; Hall, 1998
). Thus, when possible,
it is preferable to monitor inside-out signaling by ligand
binding assays, as in the current study.
Ligand binding to integrins is thought to be regulated by
a combination of affinity and avidity modulation (van
Kooyk and Figdor, 1993; Diamond and Springer, 1994
;
Bazzoni and Hemler, 1998
). In the case of
IIb
3, platelet
activation is believed to cause a modification of the conformation or orientation of the integrin cytoplasmic tails
that is transmitted across the membrane, leading to increased access of the ligand to binding sites in the receptor
(Loftus and Liddington, 1997
; Shattil et al., 1998
). However, cell activation appears to promote ligand binding to
certain
1 and
2 integrins by also stimulating the lateral
diffusion and clustering of these receptors (van Kooyk and
Figdor, 1993
; Kucik et al., 1996
; Bazzoni and Hemler,
1998
; Shattil et al., 1998
), and the same might be true for
IIb
3. Several experimental approaches have been used to
cluster integrins, including treatment of cells with multivalent antibodies or chemical cross-linkers, incubation of cells
with ligand-coated beads, and promotion of cell spreading
(Kornberg et al., 1991
; Dorahy et al., 1995
; Hotchin and
Hall, 1995
; Miyamoto et al., 1995
). Whereas each of these
has provided important information about outside-in signaling, none is entirely suitable for studies of soluble
ligand binding to
IIb
3. The use here of AP1510 to cluster
IIb(FKBP)
3 or
IIb(FKBP)2
3, while CHO cells were
maintained in suspension demonstrates unambiguously
that affinity and avidity modulation can complement one
another with respect to the control of ligand binding.
Given the wide variety of soluble, matrix- and cell-associated ligands that integrins must contend with, it is likely
that the relative contributions of affinity and avidity modulation will vary with the integrin and the cell type.
Fortunately, the fusion of single or tandem FKBP repeats to the IIb cytoplasmic tail did not interfere with
IIb
3
expression or function in CHO cells. Perhaps this means
that the very COOH terminus of the
subunit is dispensable for the integrin functions that were assessed. On the
other hand, direct attachment of FKBP to the
3 tail interferes with energy-dependent affinity modulation of
IIb
3,
possibly by disrupting necessary interactions of the
3 tail
with regulatory proteins (Hato, T., and Shattil, S.J., unpublished observations). We ascribe any functional effects
of AP1510 on
IIb(FKBP)
3 and
IIb(FKBP)2
3 to receptor clustering. Although the evidence for this is strong, it is
largely indirect. First, AP1510 only affected those forms of
IIb
3 that contained FKBP repeats (Figs. 6 and 7). Second, confocal microscopy showed that AP1510 treatment
was associated with the appearance of coarse patches of
integrin staining in the surface membrane (Fig. 3). Finally,
AP1510 caused binding of a multivalent but not monovalent form of PAC1, precisely what might be expected in
the case of integrin clustering (Fig. 5). Interestingly, the
effect of AP1510 on PAC1 binding to
IIb(FKBP)
3 and
IIb(FKBP)2
3 was nearly equivalent (Fig. 7). Assuming
that AP1510 can only dimerize
IIb(FKBP)
3, this suggests
that formation of a dimer of dimers, e.g., (
IIb(FKBP)
3)2, may be sufficient to initiate some ligand binding to
IIb
3.
What are the biological implications of IIb
3 clustering
during inside-out signaling? Ligand binding resulting from
clustering of
IIb(FKBP)2
3 required a normal
3 cytoplasmic tail since a tail point mutation (S752P) disrupted this
process (Fig. 6). This same mutation disrupts energy-
dependent affinity modulation of
IIb
3 in CHO cells and
in human platelets, where it is responsible for a bleeding
diathesis (Chen et al., 1992
, 1994
). In contrast, another
3
cytoplasmic modification, a truncation starting at residue
Arg 724, did not disrupt ligand binding induced by clustering of
IIb(FKBP)2
3, although it does abolish affinity
modulation of
IIb
3 in CHO cells and platelets (O'Toole
et al., 1994
; Wang et al., 1997
). This suggests that there are
differences in the structural elements of the
3 tail that are
needed for affinity and avidity modulation. A growing
number of proteins have been shown to interact directly
with the cytoplasmic tails of
or
integrin subunits, at
least in vitro, and overexpression of some of them, including calreticulin (
tails) (Coppolino et al., 1997
), cytohesin-1 (
2 tail) (Kolanus et al., 1996
), and
3-endonexin (
3 tail) (Kashiwagi et al., 1997
) can affect ligand binding
and cell adhesion. When overexpressed in CHO cells, several other signaling proteins, including H-ras (Hughes et
al., 1996
), R-ras (Zhang et al., 1996
), and CD98 (Fenczik et
al., 1997
) have been shown to modulate the ligand binding
properties of
IIb
3; however, it is not known if any of
these proteins interact directly with the integrin. The relative effects of potential regulatory molecules such as these
on receptor clustering and receptor affinity remain to be
determined.
The extent of PAC1 binding observed in response to integrin clustering was only a fraction of that observed with
affinity modulation. Yet ligand binding resulting from
clustering and affinity modulation was additive (Fig. 7).
Clustering also facilitated CHO cell adhesion to immobilized fibrinogen and vWf, but once again, this effect was
relatively minor compared with the effect of affinity modulation and it was apparent only at the higher coating concentrations of the ligands (Fig. 9). On the other hand, if
clustering of IIb
3 were to cause increased ligand binding in platelets as it does in CHO cells, it could affect the ultimate size of a platelet aggregate and hence the delicate
balance between adequate and inadequate hemostasis or
the difference between partial and total arterial occlusion
by a platelet-rich thrombus. Conceivably, the effects of integrin clustering on ligand binding may be even more pronounced in platelets than in the CHO cell model system because receptor density may be higher in platelets and
clustering may be stimulated by agonists that trigger integrin interactions with multivalent, polymerizing ligands on
both sides of the plasma membrane (Hartwig, 1992
; Fox
et al., 1996
; Simmons and Albrecht, 1996
). Furthermore,
any increase in adhesive strength promoted by
IIb
3 clustering in vivo might help platelets resist detachment from
sites of vascular injury in response to hemodynamic forces (Savage et al., 1996
).
Integrin Clustering and Outside-In Signaling
A potential limitation of the chemical dimerization approach used here is that it may not reflect or trigger the
types of interactions between IIb
3, cytoskeletal proteins,
and signaling molecules that take place normally during
outside-in signaling. For example, in platelets, the binding
of fibrinogen to
IIb
3 is sufficient to trigger tyrosine phosphorylation and activation of Syk, whereas tyrosine phosphorylation of FAK requires additional post-ligand binding
events that occur during platelet aggregation or spreading
(Haimovich et al., 1993
; Huang et al., 1993
). In this regard,
clustering of
IIb(FKBP)2
3 in CHO cells by AP1510 caused
significant tyrosine phosphorylation of Syk, even when the
cells were maintained in suspension without fibrinogen
(Fig. 10). Since integrin-dependent tyrosine phosphorylation of Syk correlates with induction of Syk kinase activity
in both platelets and CHO cells (Clark et al., 1994
; Gao et
al., 1997
), these results suggest that the binding of multivalent fibrinogen to
IIb
3 triggers Syk activation, at least in
part, by inducing integrin clustering.
In contrast to the results for Syk, clustering of IIb(FKBP)2
3
by AP1510 was not sufficient to cause tyrosine phosphorylation of FAK in cells maintained in suspension. However,
fibrinogen binding together with receptor clustering were
sufficient to induce the response (Fig. 10). These results
highlight the apparent differences in coupling mechanisms
between
IIb
3 and Syk and
IIb
3 and FAK (Gao et al.,
1997
). At the same time, they demonstrate unambiguously that conditional clustering of
IIb
3 in CHO cells can recapitulate a pattern of outside-in signaling that is characteristic of platelets. In nucleated cells, integrin and growth
factor signaling pathways collaborate to regulate gene expression, cell adhesion, and motility (Schwartz et al., 1995
;
Juliano, 1996
; Sastry and Horwitz, 1996
; Yamada and Geiger, 1997
). One hallmark of integrated signaling networks
is tight control of enzyme activity and protein subcellular localization though regulated protein-protein interactions
(Pawson and Scott, 1997
). Chemical inducers of dimerization can be used to promote controlled homodimerization
and heterodimerization of proteins in vivo as well as ex
vivo (Rivera et al., 1996
; Spencer et al., 1996
; Clackson,
1997
; Yang et al., 1998
). Consequently, they should prove
useful in evaluating diverse aspects of integrin signaling.
![]() |
Footnotes |
---|
Received for publication 20 March 1998 and in revised form 14 May 1998.
These studies were supported, in part by grants from the National Institutes of Health (HL56595, HL57900).The authors are grateful to J. Amara and V. Rivera (ARIAD Pharmaceuticals, Inc., Cambridge, MA) for supplying pCF1E and AP1510; and to
several colleagues for their gifts of other reagents: J. Brugge (Harvard
Medical School, Boston, MA [EMCV-Syk]); M. Ginsberg (Scripps Research Institute [antibodies D57 and No. 2308, IIb and
3 pCDM8 plasmids]); D. Phillips (Cor Therapeutics, Inc., South San Francisco, CA [Integrilin]); and Z. Ruggeri (Scripps Research Institute [von Willebrand
factor]).
![]() |
Abbreviations used in this paper |
---|
ECL, enhanced chemiluminescence; FKBP, FK506 binding protein, or FKBP12; vWf, von Willebrand factor.
![]() |
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