Detection of Integrin alpha IIbbeta 3 Clustering in Living Cells*

Charito BuensucesoDagger , Maddalena de VirgilioDagger , and Sanford J. ShattilDagger §

From the Departments of Dagger  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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In platelets, bidirectional signaling across integrin alpha IIbbeta 3 regulates fibrinogen binding, cytoskeletal reorganization, cell aggregation, and spreading. Because these responses may be influenced by the clustering of alpha IIbbeta 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 beta -galactosidase mutants were fused to the C terminus of individual alpha IIb subunits, and the chimeras were stably expressed with beta 3 in Chinese hamster ovary cells. Clustering of alpha IIbbeta 3 should bring the mutants into proximity and reconstitute beta -galactosidase activity. In the second method, alpha IIb was fused to either a green fluorescent protein (GFP) or Renilla luciferase and transiently expressed with beta 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-alpha IIbbeta 3 antibodies. Significantly, they also detected clustering upon soluble fibrinogen binding to alpha IIbbeta 3. In contrast, no clustering was observed following direct activation of alpha IIbbeta 3 by MnCl2 or an anti-alpha IIbbeta 3-activating antibody Fab in the absence of fibrinogen. Intracellular events also influenced alpha IIbbeta 3 clustering. For example, a cell-permeable, bivalent FK506-binding protein (FKBP) ligand stimulated clustering when added to cells expressing an alpha IIb(FKBP)2 chimera complexed with beta 3. Furthermore, alpha IIbbeta 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 alpha IIbbeta 3 interaction with ligands. These studies in living cells establish that alpha IIbbeta 3 clustering is modulated by fibrinogen and actin dynamics. More broadly, they should facilitate investigations of the mechanisms and consequences of integrin clustering.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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 alpha beta 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 alpha IIbbeta 3 mediates platelet adhesion and aggregation in hemostasis by engaging fibrinogen or von Willebrand factor (22, 23). As with other integrins, clustering may modulate alpha IIbbeta 3 function. Thus, in platelets and a CHO cell model system, the binding of multivalent but not monovalent ligands to alpha IIbbeta 3 leads to activation of the integrin-associated tyrosine kinases, Src and Syk (21, 24, 25). These kinases also become activated when an alpha IIb(FKBP) chimeric subunit is expressed with beta 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 alpha IIbbeta 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, beta -galactosidase (beta -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 alpha IIbbeta 3 clustering can be detected in living cells and is modulated by extracellular and intracellular factors.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Construction of Recombinant Integrins-- For beta -gal complementation assays, a pWZL plasmid containing the alpha deletion mutant (Delta alpha ) of beta -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 Delta alpha . Similarly, the beta -gal omega deletion mutant (Delta omega ) 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 alpha 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 alpha IIb/(FKBP)2/Delta alpha beta -gal/HA and the other alpha IIb/(FKBP)2/Delta omega beta -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 alpha IIb/(FKBP)2/HA or alpha 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 beta 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 beta -gal complementation assays, CHO cells stably expressing recombinant integrins were obtained by co-transfecting the appropriate pair of alpha IIb/beta -gal plasmids along with pCDM8/beta 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-alpha IIbbeta 3 antibody D57 (a gift from Mark Ginsberg, Scripps), and single cell clones expressing alpha IIbbeta 3 were isolated by fluorescence-activated cell sorter (FACS) sorting (31). Preliminary studies with the beta -gal complementation assay showed that dynamic integrin clustering was best detected at relatively low levels of alpha IIbbeta 3 expression. Consequently, at least two independent clones expressing low but easily detectable levels of alpha IIbbeta 3 were isolated and used in the studies reported below.

Characterization of Recombinant alpha IIbbeta 3-- Surface expression of alpha IIbbeta 3 was analyzed by flow cytometry using antibody D57. The affinity/avidity state of alpha IIbbeta 3 was assessed with PAC-1, a fibrinogen-mimetic monoclonal antibody (31, 32). alpha IIbbeta 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 alpha IIbbeta 3 Clustering-- CHO cells transiently or stably expressing the appropriate alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 (10, 34). The effects of cytochalasin D or latrunculin A (Calbiochem) on alpha IIbbeta 3 clustering were assessed by pre-incubating cells with the compound or vehicle for 10 min at room temperature.

alpha IIbbeta 3 clustering was detected in stably transfected cells by measuring beta -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.

alpha IIbbeta 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
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ABSTRACT
INTRODUCTION
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Systems to Detect alpha IIbbeta 3 Clustering-- Two methods potentially capable of detecting integrin clustering were evaluated in CHO cells. In the first, weakly complementing beta -gal deletion mutants (Delta alpha and Delta omega ) were fused to the C termini of separate alpha IIb subunits (Fig. 1A). Epitope tags were included to distinguish between chimeric subunits. The chimeric alpha IIb subunits were stably expressed along with beta 3, with the goal of achieving simultaneous surface expression of two distinct alpha IIbbeta 3 species, alpha IIb/Delta alpha beta -galbeta 3 and alpha IIb/Delta omega beta -galbeta 3. In some cases, additional stable cell lines were developed in which tandem FKBP repeats were interposed between alpha IIb and beta -gal, resulting in integrins alpha IIb/(FKBP)2/Delta alpha beta -galbeta 3 and alpha IIb/(FKBP)2/Delta omega beta -galbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 heterodimers into larger oligomers should bring the Delta alpha and Delta omega beta -gal mutants into proximity such that beta -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 alpha IIbbeta 3 clustering by beta -galactosidase complementation. A, the alpha IIb integrin subunit constructs used in establishing stable CHO cell lines. Shown are alpha IIb, the Delta alpha beta -gal mutant, the Delta omega beta -gal mutant, tandem FKBP domains, and epitope tags. The hashed line denotes the transmembrane domain (TM) of alpha IIb, with the extracellular domain to the left and intracellular domain to the right. B, co-expression of alpha IIb chimeras and beta 3 in CHO cells. CHO cells were stably transfected with the indicated integrin constructs. Cell lysates were subjected to immunoprecipitation (IP) with an anti-alpha IIbbeta 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 alpha IIbbeta 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 alpha IIb/Delta alpha beta -gal and alpha IIb/Delta omega beta -gal were ~140 and ~118 kDa, respectively. The molecular masses of reduced alpha IIb/(FKBP)2/Delta alpha beta -gal and alpha IIb/(FKBP)2/Delta omega beta -gal were ~165 and ~142 kDa. C, rationale for detection of alpha IIbbeta 3 clustering by beta -gal complementation. When alpha IIbbeta 3 is clustered, for example by fibrinogen, the Delta alpha and Delta omega beta -gal mutants are brought into close proximity, reconstituting enzyme activity and light emission in the presence of a beta -gal substrate.

In the second method, alpha IIb (or alpha IIb/(FKBP)2) was fused to GFP or Renilla luciferase and transiently expressed with beta 3 in CHO cells (Fig. 2A). Expression of these alpha IIbbeta 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 beta -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 beta -gal and BRET reporter domains can be fused to alpha IIb without disrupting expression of a functional alpha IIbbeta 3 complex.


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Fig. 2.   Monitoring alpha IIbbeta 3 clustering by BRET. A, the alpha IIb constructs used in transient transfections of CHO cells. Shown are alpha IIb, GFP, Renilla luciferase, tandem FKBP domains, and the epitope tag. B, expression of alpha IIb/GFP and alpha IIb/Rluc with beta 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 alpha IIb or HA. Untransfected CHO cells served as a negative control. The doublet consists of pro-alpha IIb (upper band) and alpha IIb (lower band). The apparent molecular masses of non-reduced alpha IIb/Rluc and alpha IIb/GFP were ~170 and ~160 kDa, respectively, whereas reduced alpha IIb/(FKBP)2/Rluc and alpha IIb/(FKBP)2/GFP were ~84 and ~76 kDa. C, rationale for detection of alpha IIbbeta 3 clustering by BRET. When alpha IIbbeta 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.

Detection of alpha IIbbeta 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 alpha IIbbeta 3, we set out to determine whether clustering could be detected under such conditions. Anti-alpha IIbbeta 3 IgG antibodies are bivalent and at appropriate concentrations should mediate apposition of two alpha IIbbeta 3 complexes. Indeed, when CHO cells expressing alpha IIb/Delta alpha beta -galbeta 3 and alpha IIb/Delta omega beta -galbeta 3 were examined by the beta -gal complementation assay, a significant increase in beta -gal signal was detected in response to 10 µg/ml D57, an IgG1 anti-alpha IIbbeta 3 monoclonal antibody (p < 0.05) (Fig. 3A). However, no increase in beta -gal signal was observed at higher concentrations of D57, a result that would be expected if the pool of alpha IIbbeta 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 beta -gal signal, which is consistent with formation of even larger alpha IIbbeta 3 oligomers (Fig. 3A). Similar results were obtained in the presence of D57 and secondary antibodies when alpha IIbbeta 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 beta -gal complementation for the detection of relatively small alpha IIbbeta 3 oligomers (Fig. 4A). The interpretation that the beta -gal and BRET responses reflected antibody-mediated clustering of alpha IIbbeta 3 was supported by the fact that no increase in beta -gal or BRET signal was obtained when cells expressed only one of the two complementary alpha IIb reporter constructs or when the reporter constructs were each expressed in separate cells (not shown).


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Fig. 3.   Detection of alpha IIbbeta 3 clustering by beta -galactosidase complementation. A, a CHO cell line co-expressing alpha IIb/Delta alpha beta -galbeta 3 and alpha IIb/Delta omega beta -galbeta 3 was incubated with either 10 µg/ml primary anti-alpha IIbbeta 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 beta -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 alpha IIb/(FKBP)2/Delta alpha beta -galbeta 3 and alpha IIb/(FKBP)2/Delta omega beta -galbeta 3 was incubated for 30 min with 1 µM AP1510 or an equivalent volume of vehicle as a control. Then beta -gal activity was measured. Data are the means ± S.E. of three separate experiments.


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Fig. 4.   Detection of alpha IIbbeta 3 clustering by BRET. A, CHO cells transiently transfected with alpha IIb/GFPbeta 3 and alpha IIb/Rlucbeta 3 were incubated with primary antibody D57 and/or a secondary antibody as in Fig. 3. B, CHO cells transfected with alpha IIb/(FKBP)2/GFPbeta 3 and alpha IIb/(FKBP)2/Rlucbeta 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.

To determine whether clustering of alpha IIbbeta 3 could be detected if triggered from within the cell, CHO cells expressing alpha IIbbeta 3 with tandem FKBP repeats fused to the alpha IIb chimeras were evaluated. Addition of the FKBP ligand, AP1510, at a saturating concentration of 1 µM should cause rapid clustering of these alpha IIbbeta 3 species into oligomers larger than dimers (9). Indeed, the addition of AP1510 triggered beta -gal complementation (Fig. 3B) and BRET (Fig. 4B). Thus, the beta -gal complementation and BRET assays are sufficiently sensitive to detect alpha IIbbeta 3 clustering whether this process is initiated from outside or inside living cells.

Modulation of alpha IIbbeta 3 Clustering by Fibrinogen and the Actin Cytoskeleton-- Fibrinogen mediates platelet aggregation by bridging alpha IIbbeta 3 receptors on adjacent cells. However, fibrinogen binding also triggers outside-in signaling, which may require clustering of alpha IIbbeta 3 complexes within the same cell (9, 21, 24). Because fibrinogen is a dimer with several potential sites for interaction with alpha IIbbeta 3 (37, 38), a single fibrinogen molecule might be able to support both cell aggregation and alpha IIbbeta 3 clustering. Alternatively, individual fibrinogen molecules may promote each response separately. To determine whether fibrinogen binding can trigger alpha IIbbeta 3 clustering, beta -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 alpha IIbbeta 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 alpha IIbbeta 3 (D119A)) (Fig. 6A) (40). As might be expected for interaction with a multivalent ligand, the extent of alpha IIbbeta 3 clustering was reduced at concentrations of fibrinogen >=  250 µg/ml (Fig. 6B). These results demonstrate that fibrinogen binding to alpha IIbbeta 3 causes integrin clustering. Furthermore, this response may require ligand multivalency because another bivalent anti-alpha IIbbeta 3 ligand, antibody D57, also induced alpha IIbbeta 3 clustering (Fig. 3A), but a monovalent ligand, anti-LIBS6 Fab, did not (Fig. 5A).


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Fig. 5.   Fibrinogen binding induces alpha IIbbeta 3 clustering. A, CHO cells stably co-expressing alpha IIb/Delta alpha beta -galbeta 3 and alpha IIb/Delta omega beta -galbeta 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 beta -gal complementation assay. B, CHO cells were transiently transfected with alpha IIb/GFPbeta 3 and alpha IIb/Rlucbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 clustering induced by fibrinogen. A, CHO cell lines were established that stably co-expressed alpha IIb/Delta alpha beta -galbeta 3 and alpha IIb/Delta omega beta -galbeta 3 (shaded bar). Others were established that co-expressed alpha IIb/Delta alpha beta -galbeta 3 (D119A) and alpha IIb/Delta omega beta -galbeta 3 (D119A) (black bar). Then, the effect of anti-LIBS6 Fab-induced fibrinogen binding on integrin clustering was examined by beta -gal complementation. Baseline refers to beta -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 alpha IIb/Delta alpha beta -galbeta 3 and alpha IIb/Delta omega beta -galbeta 3 were incubated for 30 min with fibrinogen and 150 µg/ml anti-LIBS6 Fab. Integrin clustering was then assessed by beta -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.

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 alpha IIbbeta 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 alpha IIbbeta 3. To evaluate this hypothesis, CHO cells were incubated with latrunculin A or cytochalasin D, and alpha IIbbeta 3 clustering was monitored by the beta -gal complementation assay. Latrunculin A, which inhibits actin polymerization by sequestering G actin monomers (44), caused a small but significant increase in alpha IIbbeta 3 clustering at a concentration of 3 µM (p < 0.05). Interestingly, addition of 150 µg/ml fibrinogen increased the beta -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). alpha IIbbeta 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 alpha IIbbeta 3, at least when alpha IIb is fused to beta -gal mutants. Furthermore, the enhanced beta -gal responses observed in the presence of fibrinogen suggest that the actin cytoskeleton may influence alpha IIbbeta 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 alpha IIb/Delta alpha beta -galbeta 3 and alpha IIb/Delta omega beta -galbeta 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 beta -gal complementation. Data are the means ± S.E. of three independent experiments, each performed in triplicate.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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alpha IIb and beta 3 are expressed in plasma membranes as an obligate heterodimer (46), and alpha IIbbeta 3 is usually depicted in models as a heterodimer in platelets. However, alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 under certain experimental conditions, including after fibrinogen binding (49-51). One possible structural basis for alpha IIbbeta 3 oligomerization comes from recent in vitro studies with transmembrane/cytoplasmic domain fusions of alpha IIb and beta 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 alpha IIbbeta 3 by a chemical dimerizer can promote ligand binding and outside-in signaling (9), the factors that regulate alpha IIbbeta 3 clustering remain to be characterized.

An impediment to progress in this area has been the inability to monitor alpha IIbbeta 3 clustering in living cells. To overcome this, we employed two complementary methods, beta -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 alpha IIbbeta 3 would lead to an increase in homomeric interactions between the alpha IIb cytoplasmic tails on adjacent alpha IIbbeta 3 complexes. Therefore, reporter groups were fused to the C terminus of individual alpha IIb subunits, and clustering was monitored by beta -gal complementation and BRET assays. alpha IIb tails were used here instead of beta 3 tails as fusion partners, because our previous experience indicates that alpha IIb fusions are tolerated, whereas beta 3 fusions may impair integrin function (9). The following conclusions can be drawn from these studies. 1) alpha IIbbeta 3 clustering can be detected in living cells, whether triggered from outside or inside the cell. 2) Fibrinogen binding to alpha IIbbeta 3 induces integrin clustering, whereas direct activation of alpha IIbbeta 3 in the absence of fibrinogen does not. 3) Inhibition of actin polymerization causes dose-dependent clustering of alpha IIbbeta 3, a response that is enhanced by fibrinogen.

To validate the beta -gal complementation and BRET assays, we conditionally clustered alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 from a low- to a high-affinity/avidity state (22, 39). In the present experiments, this conversion process was accomplished by activating alpha IIbbeta 3 directly with an anti-beta 3 antibody Fab (anti-LIBS6) or with MnCl2. Unlike fibrinogen, these reagents by themselves failed to induce detectable clustering of alpha IIbbeta 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 alpha IIbbeta 3. Rather, alpha IIbbeta 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 alpha IIbbeta 3 clustering.

Fibrinogen is a dimer, and each half-molecule contains several potential interaction sites with alpha IIbbeta 3 (37, 38). Consequently, when present at sub-saturating concentrations, fibrinogen might be expected to bring two alpha IIbbeta 3 complexes together in cis, an assumption consistent with the present data (Fig. 5). However, this may not represent the full extent of alpha IIbbeta 3 oligomerization induced by fibrinogen. First, the beta -gal complementation and BRET assays were capable of detecting alpha IIbbeta 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-alpha IIbbeta 3 antibody (Fig. 4A), but it did detect clustering in response to fibrinogen (Fig. 5). Finally, ligands like fibrinogen might trigger initial oligomerization of alpha IIbbeta 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 alpha IIbbeta 3 that expose or re-orient dimerization interfaces (51, 52). Recent progress in the structural analysis of beta 3 integrins provides a starting point for identifying changes in alpha IIbbeta 3 that may affect clustering (11, 12, 54).

The alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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 alpha IIb subunit and the heterologous expression of alpha IIbbeta 3. Although alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 clustering, as demonstrated here, should now permit the examination of some unresolved questions in alpha IIbbeta 3 biology. Among them is the question of to what extent is alpha IIbbeta 3 clustering regulated by talin, an integrin- and actin-binding protein that has been implicated in affinity modulation of alpha IIbbeta 3 (56) and clustering of Drosophila integrins (57). Do mutations in alpha IIb or beta 3 that promote affinity modulation also cause integrin clustering (31, 58-61)? Is alpha IIbbeta 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 beta -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; beta -gal, beta -galactosidase; BRET, bioluminescence resonance energy transfer; HA, hemagglutinin; Rluc, Renilla luciferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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