Detection of Integrin
IIb
3
Clustering in Living Cells*
Charito
Buensuceso
,
Maddalena
de
Virgilio
, and
Sanford J.
Shattil
§¶
From the Departments of
Cell Biology and
§ Molecular and Experimental Medicine, The Scripps Research
Institute, La Jolla, California 92037
Received for publication, December 27, 2002, and in revised form, February 6, 2003
 |
ABSTRACT |
In platelets, bidirectional signaling across
integrin
IIb
3 regulates fibrinogen
binding, cytoskeletal reorganization, cell aggregation, and spreading.
Because these responses may be influenced by the clustering of
IIb
3 heterodimers into larger oligomers, we established two independent methods to detect integrin clustering and evaluate factors that regulate this process. In the first, weakly
complementing
-galactosidase mutants were fused to the C terminus of
individual
IIb subunits, and the chimeras were stably
expressed with
3 in Chinese hamster ovary cells.
Clustering of
IIb
3 should bring the
mutants into proximity and reconstitute
-galactosidase activity. In
the second method,
IIb was fused to either a green
fluorescent protein (GFP) or Renilla luciferase and
transiently expressed with
3. Here, integrin clustering
should stimulate bioluminescence resonance energy transfer between
a cell-permeable luciferase substrate and GFP. These methods
successfully detected integrin clustering induced by
anti-
IIb
3 antibodies. Significantly, they
also detected clustering upon soluble fibrinogen binding to
IIb
3. In contrast, no clustering was
observed following direct activation of
IIb
3 by MnCl2 or an
anti-
IIb
3-activating antibody Fab in the
absence of fibrinogen. Intracellular events also influenced
IIb
3 clustering. For example, a
cell-permeable, bivalent FK506-binding protein (FKBP) ligand stimulated
clustering when added to cells expressing an
IIb(FKBP)2 chimera complexed with
3. Furthermore,
IIb
3
clustering occurred in the presence of latrunculin A or cytochalasin D,
inhibitors of actin polymerization. These effects were enhanced by
fibrinogen, suggesting that actin-regulated clustering modulates
IIb
3 interaction with ligands. These
studies in living cells establish that
IIb
3 clustering is modulated by
fibrinogen and actin dynamics. More broadly, they should facilitate investigations of the mechanisms and consequences of integrin clustering.
 |
INTRODUCTION |
For most cell surface receptors, clustering into dimers or larger
oligomers is thought to represent an important regulatory event (1-4).
Receptor clustering may be triggered in several ways, for example,
through interactions with multivalent ligands, apposition of
dimerization interfaces, relief of cytoskeletal constraints, and
partitioning into membrane domains such as lipid rafts.
Integrins are a family of transmembrane 
heterodimers that
function as cell adhesion and signaling receptors (5). There is
substantial indirect evidence that lateral diffusion and clustering influences integrin functions (6, 7). When clustering of recombinant
integrins is induced by chemical dimerizers or is abrogated by
mutagenesis, their avidity for ligands is affected (8, 9). Indeed, in
such systems integrin clustering operates in concert with receptor
conformational changes and affinity modulation to determine ligand
binding (5, 10-12). When cells attach to extracellular matrices,
integrin clustering promotes the assembly of a range of actin-based
complexes, including initial adhesions, focal complexes, and focal
adhesions (13-17). These structures, particularly the larger ones, are
visible by light microscopy, and they function as signaling centers to
help regulate cell motility and gene expression (5, 18). Focal
adhesions can be visualized in real time using green fluorescent
protein (GFP)1 chimeras
containing focal adhesion targeting sequences (19). On the other hand,
smaller nascent integrin clusters containing perhaps a few heterodimers
are not visible by routine light microscopy; yet they are likely to
function as signaling centers in their own right and represent
precursors of the larger adhesion structures (9, 17). In some cases,
nascent integrin clusters may recruit functionally important signaling
molecules that do not partition to the larger focal adhesions (20, 21).
Consequently, the precise roles of integrin clusters might be better
understood if there were a means to detect their initial formation and
maturation in living cells.
Integrin
IIb
3 mediates platelet adhesion
and aggregation in hemostasis by engaging fibrinogen or von Willebrand
factor (22, 23). As with other integrins, clustering may modulate
IIb
3 function. Thus, in platelets and a
CHO cell model system, the binding of multivalent but not monovalent
ligands to
IIb
3 leads to activation of
the integrin-associated tyrosine kinases, Src and Syk (21, 24, 25).
These kinases also become activated when an
IIb(FKBP)
chimeric subunit is expressed with
3 in CHO cells and
the integrin is conditionally clustered with a cell-permeable, bivalent
FKBP ligand (9). Therefore, to shed more light on the clustering of
IIb
3 and other integrins, we sought here
to develop new means to assess this process in living cells. Two complementary techniques were utilized for this purpose,
-galactosidase (
-gal) complementation and bioluminescence
resonance energy transfer (BRET). Recently, each of these has been used
to study the proximity and clustering of other types of cell surface
receptors (26-30). The results establish that
IIb
3 clustering can be detected in living
cells and is modulated by extracellular and intracellular factors.
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EXPERIMENTAL PROCEDURES |
Construction of Recombinant Integrins--
For
-gal
complementation assays, a pWZL plasmid containing the alpha deletion
mutant (
) of
-gal (a gift from Dr. Helen Blau, Stanford
University; Ref. 27), was subjected to PCR using Platinum Pfx
polymerase (Invitrogen) to place SpeI restriction sites at the 5'- and 3'-ends of 
. Similarly, the
-gal omega deletion mutant (
) was subjected to PCR to place a
SpeI site at the 5'-end and a FLAG epitope
tag/SpeI site at the 3'-end. PCR products were digested with
SpeI and ligated into a SpeI-cut, dephosphorylated mammalian expression vector (pCF2E) containing full-length human integrin
IIb fused at its cytoplasmic
tail to tandem FK506-binding protein (FKBP) repeats and a hemagglutinin (HA) epitope tag (9). This resulted in two constructs, one encoding
IIb/(FKBP)2/
-gal/HA and the other
IIb/(FKBP)2/
-gal/FLAG. To obtain
the same expression constructs without FKBP, (FKBP)2 was
removed by deletion mutagenesis (Exsite site-directed mutagenesis kit;
Stratagene, La Jolla, CA). For BRET assays, plasmid templates encoding
IIb/(FKBP)2/HA or
IIb/HA (9)
were subjected to PCR to place 5' MluI and 3'
HindIII restriction sites. PCR products were sub-cloned into
the MluI and HindIII sites of the mammalian
expression vectors pGFPN3 (which encodes a GFP optimized for BRET) and
pRLUCN3 (which encodes Renilla luciferase (Rluc; PerkinElmer
Life Sciences). Transformed colonies were screened by colony
PCR, coding sequences were verified by DNA sequencing, and plasmids
were amplified and purified (QIAfilter Plasmid Midi Kit; Qiagen, Inc.,
Chatsworth, CA). Full-length human
3 was cloned into pCDM8 (31).
Cell Culture, Transfections, and Establishment of Stable Cell
Lines--
CHO cells were maintained in culture as described (31).
Transfections were performed at 70-80% cell confluency using
LipofectAMINE (Invitrogen) according to the manufacturer's
recommendations. For
-gal complementation assays, CHO cells stably
expressing recombinant integrins were obtained by co-transfecting the
appropriate pair of
IIb/
-gal plasmids along with
pCDM8/
3 and pCDM8/neomycin (31). After 48 h, cells were
subjected to selection with 0.8 mg/ml G418 (Invitrogen). Two weeks
later, cells were stained with anti-
IIb
3
antibody D57 (a gift from Mark Ginsberg, Scripps), and single cell
clones expressing
IIb
3 were isolated by
fluorescence-activated cell sorter (FACS) sorting (31). Preliminary
studies with the
-gal complementation assay showed that dynamic
integrin clustering was best detected at relatively low levels of
IIb
3 expression. Consequently, at least
two independent clones expressing low but easily detectable levels of
IIb
3 were isolated and used in the studies reported below.
Characterization of Recombinant
IIb
3--
Surface expression of
IIb
3 was analyzed by flow cytometry using
antibody D57. The affinity/avidity state of
IIb
3 was assessed with PAC-1, a
fibrinogen-mimetic monoclonal antibody (31, 32).
IIb
3 expression was evaluated by
immunoprecipitation and/or Western blotting. CHO cells were solubilized
for 10 min on ice in a buffer containing 0.5% Nonidet P-40, 50 mM NaCl, a protease inhibitor mixture (Complete,
Invitrogen), and 50 mM Tris-HCl, pH 7.4. After
clarification at 10,000 rpm for 10 min at 4 °C in a microcentrifuge,
400 µg of lysate protein were immunoprecipitated with D57 (or an
isotype-matched control IgG) and protein A-Sepharose (24, 33). After
washing, immunoprecipitates were subjected to electrophoresis in 7.5%
SDS-polyacrylamide gels and transferred to nitrocellulose.
Immuno-reactive bands were detected by enhanced chemiluminescence, with
reaction times varying from 0.1-1 min (SuperSignal WestPico reagent; Pierce).
Detection of
IIb
3
Clustering--
CHO cells transiently or stably expressing the
appropriate
IIb
3 chimeras were harvested
by trypsinization, washed once, and resuspended to 0.5-1.0 × 106 cells/ml in Tyrode's buffer supplemented with 2 mM CaCl2 and MgCl2 (31). The cells
were then subjected to various treatments designed to assess
IIb
3 clustering, as indicated below for
each experiment. To induce clustering by antibodies, 100 µl of cells were added to U-bottom, 96-well non-tissue culture plates pre-blocked with 10 mg/ml heat-denatured bovine serum albumin. After incubation with 10 µg/ml antibody D57 for 30 min, cells were washed and
incubated another 30 min with 20 µg/ml goat anti-mouse IgG
(BIOSOURCE). To induce clustering of
IIb
3 constructs containing
(FKBP)2, cells were incubated for 30 min at room
temperature with 1 µM AP1510, a cell-permeable bivalent
FKBP ligand (Ariad Pharmaceuticals, Inc.), or an equivalent volume of
vehicle (9). The effects of fibrinogen on
IIb
3 clustering were evaluated by
incubating cells for 30 min at room temperature with purified
fibrinogen (Enzyme Research Laboratories, South Bend, IN). In some
cases, fibrinogen binding was stimulated with 0.5 mM
MnCl2 or 150 µg/ml anti-LIBS6 Fab, which directly
activate
IIb
3 (10, 34). The effects of
cytochalasin D or latrunculin A (Calbiochem) on
IIb
3 clustering were assessed by
pre-incubating cells with the compound or vehicle for 10 min at room temperature.
IIb
3 clustering was detected in stably
transfected cells by measuring
-gal complementation as
described (28, 35). Briefly, 100,000 cells in 100 µl were deposited
in solid white U-bottom microtiter wells (catalog number 3912, Costar,
Corning Life Sciences, Acton, MA). Cells were lysed by the addition of an equal volume of Gal-Screen substrate (Buffer B formulation; Tropix
PE Biosystems, Bedford, MA) and incubated for 30 min at room
temperature. Chemiluminescence was measured in a Rosys Lucy2 luminometer (Anthos Labtec Instruments, Wals, Austria) and expressed in
arbitrary units. The absolute mean baseline value for untreated cells
in the experiments reported here was 1,995 units.
IIb
3 clustering was detected in
transiently transfected cells by measuring BRET using the
BRET2 system (PerkinElmer Life Sciences) (36). Briefly,
50,000 cells in 15 µl of BRET2 buffer (phosphate-buffered
saline supplemented with 0.68 mM CaCl2, 0.5 mM MgCl2, 1.0 g/liter glucose, and 2 µg/ml
aprotinin) were added to microtiter wells. Immediately after the
addition of 50 µl of a luciferase substrate (coelenterazine; Deep
Blue C, PerkinElmer Life Sciences; final concentration of 10 µM), BRET was analyzed by luminometry using a 410 nm/80
nm bandpass filter for Rluc and a 515-nm/30-nm bandpass filter for GFP.
Results are expressed as the BRET ratio calculated as follows: emission
at 515 nm
BG515)/(emission at 410 nm
BG410, where BG515 is the emission at 515 nm
and BG410 the emission at 410 nm of a 5 µM
solution of coelenterazine prepared in BRET2 buffer (26,
29, 36). The absolute mean baseline BRET ratio for untreated cells in
the experiments reported here was 0.202.
 |
RESULTS |
Systems to Detect
IIb
3
Clustering--
Two methods potentially capable of detecting integrin
clustering were evaluated in CHO cells. In the first, weakly
complementing
-gal deletion mutants (
and 
) were fused
to the C termini of separate
IIb subunits (Fig.
1A). Epitope tags were
included to distinguish between chimeric subunits. The chimeric
IIb subunits were stably expressed along with
3, with the goal of achieving simultaneous surface
expression of two distinct
IIb
3 species,
IIb/
-gal
3 and
IIb/
-gal
3. In some cases,
additional stable cell lines were developed in which tandem FKBP
repeats were interposed between
IIb and
-gal,
resulting in integrins
IIb/(FKBP)2/
-gal
3 and
IIb/(FKBP)2/
-gal
3
(Fig. 1A). The latter constructs were designed to evaluate
the effects of conditional integrin clustering upon addition of 1 µM AP1510, a cell-permeable bivalent FKBP ligand (9).
Expression of
IIb
3 constructs was
confirmed by immunoprecipitation and Western blotting (Fig.
1B) and analyzing surface expression using flow cytometry (not shown). In all of these cell lines, clustering of
IIb
3 heterodimers into larger oligomers
should bring the 
and 
-gal mutants into proximity such
that
-gal enzyme activity is reconstituted (Fig. 1C)
(28). This approach has been used to detect oligomerization of
epidermal growth factor receptors (27).

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Fig. 1.
Monitoring
IIb 3
clustering by -galactosidase
complementation. A, the IIb
integrin subunit constructs used in establishing stable CHO cell lines.
Shown are IIb, the  -gal mutant, the 
-gal mutant, tandem FKBP domains, and epitope tags. The
hashed line denotes the transmembrane domain
(TM) of IIb, with the extracellular domain to
the left and intracellular domain to the right.
B, co-expression of IIb chimeras and
3 in CHO cells. CHO cells were stably transfected with
the indicated integrin constructs. Cell lysates were subjected to
immunoprecipitation (IP) with an
anti- IIb 3 antibody, and
immunoprecipitates were probed with antibodies to the HA and FLAG tags
as described under "Experimental Procedures." A5 CHO cells
expressing wild type IIb 3 served as a
negative control. The immunoreactive bands have been aligned for
clarity. Based on migration of molecular weight standards, the apparent
molecular masses of reduced IIb/ -gal and
IIb/ -gal were ~140 and ~118 kDa,
respectively. The molecular masses of reduced
IIb/(FKBP)2/ -gal and
IIb/(FKBP)2/ -gal were ~165 and
~142 kDa. C, rationale for detection of
IIb 3 clustering by -gal
complementation. When IIb 3 is clustered,
for example by fibrinogen, the  and  -gal mutants are
brought into close proximity, reconstituting enzyme activity and light
emission in the presence of a -gal substrate.
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In the second method,
IIb (or
IIb/(FKBP)2) was fused to GFP or
Renilla luciferase and transiently expressed with
3 in CHO cells (Fig.
2A). Expression of these
IIb
3 constructs was confirmed by Western
blotting of cell lysates (Fig. 2B) and flow cytometry. In
this system, if integrin clustering results in appropriately oriented
GFP and Rluc moieties coming within ~80 Å of each other, BRET should
occur between a cell-permeable luciferase substrate (coelenterazine)
and GFP (Fig. 2C). This technique has been used to detect
oligomerization of G protein-coupled and insulin receptors (29, 30,
36). Though not shown, the chimeric integrins used for BRET and
-gal
assays were functional in that they bound the ligand-mimetic antibody,
PAC-1, after direct integrin activation with 0.5 mM
MnCl2 or 150 µg/ml anti-LIBS-6 Fab. Moreover, they supported specific cell adhesion and spreading on immobilized fibrinogen. Overall, these results indicate that
-gal and BRET reporter domains can be fused to
IIb without disrupting
expression of a functional
IIb
3
complex.

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Fig. 2.
Monitoring IIb 3
clustering by BRET. A, the IIb
constructs used in transient transfections of CHO cells. Shown are
IIb, GFP, Renilla luciferase, tandem FKBP
domains, and the epitope tag. B, expression of
IIb/GFP and IIb/Rluc with
3 in CHO cells. CHO cells were transiently transfected
with the indicated integrin constructs. Forty-eight hours later, cell
lysates were subjected to Western blotting with antibodies to
IIb or HA. Untransfected CHO cells served as a negative
control. The doublet consists of pro- IIb (upper band)
and IIb (lower band). The apparent molecular
masses of non-reduced IIb/Rluc and
IIb/GFP were ~170 and ~160 kDa, respectively,
whereas reduced IIb/(FKBP)2/Rluc and
IIb/(FKBP)2/GFP were ~84 and ~76 kDa.
C, rationale for detection of
IIb 3 clustering by BRET. When
IIb 3 is clustered and GFP and Rluc are
brought within ~80Å of each other, BRET occurs between the
luciferase substrate (coelenterazine) and GFP, resulting in increased
light emission at 515 nm and decreased emission at 410 nm.
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Detection of
IIb
3 Clustering Induced
by Extracellular or Intracellular Means--
Because integrin
clustering may be triggered by the binding of ligands to the
extracellular or intracellular portions of
IIb
3, we set out to determine whether
clustering could be detected under such conditions.
Anti-
IIb
3 IgG antibodies are bivalent and
at appropriate concentrations should mediate apposition of two
IIb
3 complexes. Indeed, when CHO cells
expressing
IIb/
-gal
3 and
IIb/
-gal
3 were examined by the
-gal complementation assay, a significant increase in
-gal signal
was detected in response to 10 µg/ml D57, an IgG1
anti-
IIb
3 monoclonal antibody
(p < 0.05) (Fig.
3A). However, no increase in
-gal signal was observed at higher concentrations of D57, a result
that would be expected if the pool of
IIb
3 available for cross-linking had
become saturated with D57 (not shown). At 10 µg/ml D57, the addition
of a second antibody to cross-link the primary antibody caused a
further increase in
-gal signal, which is consistent with formation
of even larger
IIb
3 oligomers (Fig.
3A). Similar results were obtained in the presence of D57
and secondary antibodies when
IIb
3
clustering was analyzed by BRET. In this case, however, no response was
seen with D57 alone, suggesting that BRET in the present configuration may be less sensitive than
-gal complementation for the detection of
relatively small
IIb
3 oligomers (Fig.
4A). The interpretation that
the
-gal and BRET responses reflected antibody-mediated clustering
of
IIb
3 was supported by the fact that no
increase in
-gal or BRET signal was obtained when cells expressed
only one of the two complementary
IIb reporter
constructs or when the reporter constructs were each expressed in
separate cells (not shown).

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Fig. 3.
Detection of
IIb 3 clustering by -galactosidase
complementation. A, a CHO cell line co-expressing
IIb/ -gal 3 and
IIb/ -gal 3 was incubated with
either 10 µg/ml primary anti- IIb 3
antibody (Ab) D57, 20 µg/ml anti-mouse secondary antibody,
or D57 followed by secondary antibody. After washing, integrin
clustering was monitored by measuring -gal activity as described
under "Experimental Procedures." Activity is depicted as a
percentage above baseline, defined as enzyme activity in the presence
of buffer instead of antibodies. B, a CHO cell line
co-expressing IIb/(FKBP)2/
-gal 3 and
IIb/(FKBP)2/ -gal 3
was incubated for 30 min with 1 µM AP1510 or an
equivalent volume of vehicle as a control. Then -gal activity was
measured. Data are the means ± S.E. of three separate
experiments.
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Fig. 4.
Detection of
IIb 3
clustering by BRET. A, CHO cells transiently
transfected with IIb/GFP 3 and IIb/Rluc 3 were incubated with
primary antibody D57 and/or a secondary antibody as in Fig. 3.
B, CHO cells transfected with
IIb/(FKBP)2/GFP 3 and
IIb/(FKBP)2/Rluc 3 were
incubated with AP1510 as in Fig. 3. The BRET ratio was then determined,
as described under "Experimental Procedures." Data are
depicted as a percentage above baseline, defined as the BRET ratio in
the presence of buffer. Data are the means ± S.E. of three
experiments.
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To determine whether clustering of
IIb
3
could be detected if triggered from within the cell, CHO cells
expressing
IIb
3 with tandem FKBP repeats
fused to the
IIb chimeras were evaluated. Addition of
the FKBP ligand, AP1510, at a saturating concentration of 1 µM should cause rapid clustering of these
IIb
3 species into oligomers larger than
dimers (9). Indeed, the addition of AP1510 triggered
-gal
complementation (Fig. 3B) and BRET (Fig. 4B).
Thus, the
-gal complementation and BRET assays are sufficiently sensitive to detect
IIb
3 clustering
whether this process is initiated from outside or inside living cells.
Modulation of
IIb
3 Clustering by
Fibrinogen and the Actin Cytoskeleton--
Fibrinogen mediates
platelet aggregation by bridging
IIb
3
receptors on adjacent cells. However, fibrinogen binding also triggers
outside-in signaling, which may require clustering of
IIb
3 complexes within the same cell (9,
21, 24). Because fibrinogen is a dimer with several potential sites for
interaction with
IIb
3 (37, 38), a single
fibrinogen molecule might be able to support both cell aggregation and
IIb
3 clustering. Alternatively, individual fibrinogen molecules may promote each response
separately. To determine whether fibrinogen binding can trigger
IIb
3 clustering,
-gal complementation
and BRET assays were performed at a sub-saturating concentration of
fibrinogen (150 µg/ml) (39). Ligand binding was induced with 0.5 mM MnCl2 or anti-LIBS6 Fab. Under these
conditions, fibrinogen induced
IIb
3
clustering (Fig. 5). This response was fibrinogen-dependent because it was inhibited by EDTA and
was not observed with MnCl2 or anti-LIBS6 Fab in the
absence of fibrinogen. Moreover, no significant
fibrinogen-dependent clustering was observed in cells
expressing an integrin mutant that is incapable of binding fibrinogen
IIb
3 (D119A)) (Fig.
6A) (40). As might be expected for interaction with a multivalent ligand, the extent of
IIb
3 clustering was reduced at
concentrations of fibrinogen
250 µg/ml (Fig. 6B).
These results demonstrate that fibrinogen binding to
IIb
3 causes integrin clustering.
Furthermore, this response may require ligand multivalency because
another bivalent anti-
IIb
3 ligand,
antibody D57, also induced
IIb
3
clustering (Fig. 3A), but a monovalent ligand, anti-LIBS6
Fab, did not (Fig. 5A).

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Fig. 5.
Fibrinogen binding induces
IIb 3
clustering. A, CHO cells stably co-expressing
IIb/ -gal 3 and IIb/
-gal 3 were incubated for 30 min with 150 mg/ml
anti-LIBS6 Fab in the absence or presence of 150 µg/ml fibrinogen.
Then, integrin clustering was monitored by the -gal complementation
assay. B, CHO cells were transiently transfected with
IIb/GFP 3 and IIb/Rluc 3
and, 24 h later, incubated for 30 min with 1 mM
MnCl2 in the absence or presence of 150 µg/ml fibrinogen.
Where indicated, 10 mM EDTA was added to block fibrinogen
binding to IIb 3. Then integrin clustering
was monitored by BRET. Data are the means ± S.E. of three
experiments.
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Fig. 6.
Characteristics of
IIb 3
clustering induced by fibrinogen. A, CHO cell lines
were established that stably co-expressed IIb/
-gal 3 and IIb/
-gal 3 (shaded bar). Others were
established that co-expressed IIb/
-gal 3 (D119A) and IIb/
-gal 3 (D119A) (black bar).
Then, the effect of anti-LIBS6 Fab-induced fibrinogen binding on
integrin clustering was examined by -gal complementation. Baseline
refers to -gal activity in the presence of anti-LIBS6 Fab alone.
Data are the means ± S.E. of five experiments, each conducted
with an independent pair of CHO cell clones. B, CHO cells
expressing IIb/ -gal 3 and
IIb/ -gal 3 were incubated for 30 min with fibrinogen and 150 µg/ml anti-LIBS6 Fab. Integrin clustering
was then assessed by -gal complementation. Enzyme activity is
depicted as a percentage above baseline, defined as enzyme activity in
the presence of buffer instead of fibrinogen. Data are the means ± S.E. of triplicate measurements from an experiment that is
representative of two so performed.
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Integrins in unstimulated platelets are maintained in a low
affinity/avidity state and are tethered to components of the actin cytoskeleton (41, 42). Inhibitors of actin polymerization, such as
latrunculin A or cytochalasin D, promote fibrinogen binding to
platelets, an effect that has been attributed to perturbation of actin
dynamics, relief of cytoskeletal constraints, and
IIb
3 activation (43). Were these
relationships to be preserved in CHO cells, we would hypothesize that
the inhibition of actin polymerization would promote lateral mobility
and clustering of
IIb
3. To evaluate this
hypothesis, CHO cells were incubated with latrunculin A or cytochalasin
D, and
IIb
3 clustering was monitored by the
-gal complementation assay. Latrunculin A, which inhibits actin
polymerization by sequestering G actin monomers (44), caused a small
but significant increase in
IIb
3
clustering at a concentration of 3 µM (p < 0.05). Interestingly, addition of 150 µg/ml fibrinogen increased the
-gal signal further, an effect observed over a range of
latrunculin A concentrations and without the need for MnCl2
or anti-LIBS6 Fab (Fig. 7A).
IIb
3 clustering and its enhancement by
fibrinogen were also observed at micromolar concentrations
concentrations of cytochalasin D, which inhibits actin
polymerization by binding to barbed actin filament ends (45) (Fig.
7B). Although the dose-response curves for latrunculin A and
cytochalasin D differed, these results indicate that inhibition of
actin polymerization can influence the oligomerization state of
IIb
3, at least when
IIb is
fused to
-gal mutants. Furthermore, the enhanced
-gal responses
observed in the presence of fibrinogen suggest that the actin
cytoskeleton may influence
IIb
3/fibrinogen interactions, in part, by
regulating integrin clustering.

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Fig. 7.
Effect of latrunculin A and cytochalasin D on
integrin clustering. CHO cells stably expressing
IIb/ -gal 3 and
IIb/ -gal 3 were incubated for 10 min with latrunculin A (A) or cytochalasin D (B)
in the absence or presence of 150 µg/ml fibrinogen. Integrin
clustering was assessed by -gal complementation. Data are the
means ± S.E. of three independent experiments, each performed in
triplicate.
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 |
DISCUSSION |
IIb and
3 are expressed in plasma
membranes as an obligate heterodimer (46), and
IIb
3 is usually depicted in models as a
heterodimer in platelets. However,
IIb
3
heterodimers probably exist in equilibrium with larger oligomers, and
this equilibrium may be regulated with implications for integrin
signaling. Indeed, when platelets are stimulated to bind fibrinogen in
response to ADP, clusters of
IIb
3 can be
visualized on the platelet surface by electron microscopy (47, 48). In
addition, electron microscopic and biochemical techniques have detected
oligomers of detergent-solubilized
IIb
3
under certain experimental conditions, including after fibrinogen
binding (49-51). One possible structural basis for
IIb
3 oligomerization comes from recent
in vitro studies with transmembrane/cytoplasmic domain
fusions of
IIb and
3, which indicate that
homomeric contacts can occur between transmembrane domains (52).
Despite these observations and experimental evidence in CHO cells that
forced clustering of
IIb
3 by a chemical
dimerizer can promote ligand binding and outside-in signaling (9), the
factors that regulate
IIb
3 clustering
remain to be characterized.
An impediment to progress in this area has been the inability to
monitor
IIb
3 clustering in living cells.
To overcome this, we employed two complementary methods,
-galactosidase complementation and BRET, each of which is
capable of reporting the proximity between two proteins (28, 30). We
reasoned that any stimulus that caused oligomerization of
IIb
3 would lead to an increase in
homomeric interactions between the
IIb cytoplasmic tails
on adjacent
IIb
3 complexes. Therefore,
reporter groups were fused to the C terminus of individual
IIb subunits, and clustering was monitored by
-gal
complementation and BRET assays.
IIb tails were used
here instead of
3 tails as fusion partners, because our
previous experience indicates that
IIb fusions are
tolerated, whereas
3 fusions may impair integrin
function (9). The following conclusions can be drawn from these
studies. 1)
IIb
3 clustering can be
detected in living cells, whether triggered from outside or inside the
cell. 2) Fibrinogen binding to
IIb
3
induces integrin clustering, whereas direct activation of
IIb
3 in the absence of fibrinogen does
not. 3) Inhibition of actin polymerization causes
dose-dependent clustering of
IIb
3, a response that is enhanced by fibrinogen.
To validate the
-gal complementation and BRET assays, we
conditionally clustered
IIb
3 using
anti-integrin antibodies or the FKBP dimerization system (Figs. 3 and
4). The results indicated that integrin clustering by these means can
be detected, but they do not establish whether clustering occurs
under more biologically relevant conditions. To address the latter, we
evaluated the binding of fibrinogen to
IIb
3, an event that mediates platelet
aggregation in vitro and thrombus formation in
vivo (23, 39). The binding of soluble fibrinogen to platelets
requires the agonist-induced conversion of
IIb
3 from a low- to a
high-affinity/avidity state (22, 39). In the present experiments, this
conversion process was accomplished by activating
IIb
3 directly with an anti-
3 antibody
Fab (anti-LIBS6) or with MnCl2. Unlike fibrinogen, these reagents by themselves failed to induce detectable clustering of
IIb
3 (Fig. 5). Thus, neither the binding
of a monovalent ligand like anti-LIBS6 Fab nor the direct activation of
the receptor is sufficient to induce clustering of
IIb
3. Rather,
IIb
3 clustering in living cells appears
to require the binding of a multivalent ligand like fibrinogen. It
remains to be determined whether other physiological multivalent
ligands, such as the von Willebrand factor, also induce
IIb
3 clustering.
Fibrinogen is a dimer, and each half-molecule contains several
potential interaction sites with
IIb
3
(37, 38). Consequently, when present at sub-saturating concentrations,
fibrinogen might be expected to bring two
IIb
3 complexes together in cis, an assumption consistent with the present data (Fig. 5). However, this may
not represent the full extent of
IIb
3
oligomerization induced by fibrinogen. First, the
-gal
complementation and BRET assays were capable of detecting
IIb
3 clustering by fibrinogen, but
neither assay allows precise assessment of the extent of clustering. Second, the BRET assay was not sensitive enough to detect clustering by
a bivalent anti-
IIb
3 antibody (Fig.
4A), but it did detect clustering in response to fibrinogen
(Fig. 5). Finally, ligands like fibrinogen might trigger initial
oligomerization of
IIb
3 through direct
binding, a process that likely contributes to the biphasic clustering
response observed in Fig. 6B. However, further oligomerization may be promoted by fibrinogen/fibrinogen interactions (53) and post-ligand binding changes in
IIb
3 that expose or re-orient
dimerization interfaces (51, 52). Recent progress in the structural
analysis of
3 integrins provides a starting point for
identifying changes in
IIb
3 that may
affect clustering (11, 12, 54).
The
IIb
3 clustering that we observed in
CHO cells in response to latrunculin A or cytochalasin D may be
relevant to recent platelet studies demonstrating increased
ADP-dependent fibrinogen binding in response to these same
compounds (43). Those results were interpreted to reflect basal
constraints on the fibrinogen binding function of
IIb
3 imposed by the actin cytoskeleton
and release of these constraints when actin polymerization and filament turnover are prevented. Our results are compatible with this
interpretation in the sense that
IIb
3
clustering due to actin polymerization inhibitors increased further in
the presence of fibrinogen (Fig. 7). This suggests that actin dynamics
may affect fibrinogen binding, at least in part, by regulating
IIb
3 clustering. Indeed, single particle
tracking studies indicate that the actin cytoskeleton exerts prominent
effects on integrin lateral diffusion, a possible antecedent to
actin-regulated integrin clustering (6).
The methods used here to detect
IIb
3
clustering have certain limitations for extrapolation of the results to
platelets or to other integrins. First, they require the fusion of
bulky reporter groups to the
IIb subunit and the
heterologous expression of
IIb
3. Although
IIb
3 appeared to function normally under
these conditions and support fibrinogen-dependent cell
adhesion and spreading, anomalous behavior could occur in other
experimental settings. Second, as already mentioned, these methods can
detect integrin oligomerization but provide no quantitative information as to its extent. Some steady-state
IIb
3
oligomerization might take place in resting cells, particularly at high
receptor densities, but such an event could be difficult to discern by
these methods, whose strength is to report changes in receptor
oligomerization. Third, integrin activation and clustering may occur
preferentially at certain sites in the cell (55), spatial information
that is not readily attainable by these methods. Nonetheless, the
ability to detect apparently even minor changes in
IIb
3 clustering, as demonstrated here,
should now permit the examination of some unresolved questions in
IIb
3 biology. Among them is the question of to what extent is
IIb
3 clustering
regulated by talin, an integrin- and actin-binding protein that has
been implicated in affinity modulation of
IIb
3 (56) and clustering of
Drosophila integrins (57). Do mutations in
IIb or
3 that promote affinity modulation
also cause integrin clustering (31, 58-61)? Is
IIb
3 ligation, perhaps even by some
monovalent ligands, sufficient to induce certain outside-in signaling
responses, or is integrin clustering always required?
 |
ACKNOWLEDGEMENTS |
We are grateful to Helen Blau for the
-galactosidase plasmids, Allan Atkinson for the BRET plasmids, Mark
Ginsberg for antibodies D57 and anti-LIBS6 Fab, and Victor Rivera for AP1510.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL-56595 and HL-57900 and presented in part at the annual meeting of the American Society of Hematology, December 6-10, 2002, Philadelphia, PA, (Buensuceso, C., de Virgilio, M., and Shattil, S. J.
(2002) Blood 100, 50a (abstr.)).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Cell
Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., VB-5, La Jolla, CA 92037. Tel.: 858-784-7148; Fax: 858-784-7422; E-mail: shattil@scripps.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M213234200
 |
ABBREVIATIONS |
The abbreviations used are:
GFP, green
fluorescent protein;
CHO, Chinese hamster ovary;
FKBP, FK506-binding
protein;
-gal,
-galactosidase;
BRET, bioluminescence resonance
energy transfer;
HA, hemagglutinin;
Rluc, Renilla
luciferase.
 |
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