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J. Biol. Chem., Vol. 277, Issue 28, 25715-25721, July 12, 2002

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Relationships between Rap1b, Affinity Modulation of Integrin _IIb3, and the Actin Cytoskeleton^*

Alessandra Bertoni, Seiji Tadokoro, Koji Eto, Nisar Pampori, Leslie V. Parise^§, Gilbert C. White^¶, and Sanford J. Shattil^**

From the Departments of  Cell Biology and  Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 and the Departments of ^§ Pharmacology and ^¶ Medicine, University of North Carolina, Chapel Hill, North Carolina 27599

Received for publication, March 22, 2002, and in revised form, April 29, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The affinity of integrin _IIb3 for fibrinogen is controlled by inside-out signals that are triggered by agonists like thrombin. Agonist treatment of platelets also activates Rap1b, a small GTPase known to promote integrin-dependent adhesion of other cells. Therefore, we investigated the role of Rap1b in _IIb3 function by viral transduction of GFP-Rap1 chimeras into murine megakaryocytes, which exhibit inside-out signaling similar to platelets. Expression of constitutively active GFP-Rap1b (V12) had no effect on unstimulated megakaryocytes, but it greatly augmented fibrinogen binding to _IIb3 induced by a PAR4 thrombin receptor agonist (p < 0.01). The Rap1b effect was cell-autonomous and was prevented by pre-treating cells with cytochalasin D or latrunculin A to inhibit actin polymerization. Rap1b-dependent fibrinogen binding to megakaryocytes was blocked by POW-2, a novel monovalent antibody Fab fragment specific for high affinity murine _IIb3. In contrast to GFP-Rap1b (V12), expression of GFP-Rap1GAP, which deactivates endogenous Rap1, inhibited agonist-induced fibrinogen binding (p < 0.01), as did dominant-negative GFP-Rap1b (N17) (p < 0.05). None of these treatments affected surface expression of _IIb3. These studies establish that Rap1b can augment agonist-induced ligand binding to _IIb3 through effects on integrin affinity, possibly by modulating _IIb3 interactions with the actin cytoskeleton.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The _IIb3 integrin is a receptor for adhesive ligands such as fibrinogen and von Willebrand factor, and ligand binding to _IIb3 is required for platelet aggregation and spreading in hemostasis. Ligand binding is regulated by positive and negative "inside-out" signals that converge on _IIb3 to control the integrin activation state through modulation of receptor affinity or avidity (1). Affinity modulation, the dominant mode of regulation in platelets, implies a change in the conformation of the _IIb3 heterodimer to increase access of ligand binding sites, while avidity modulation implies lateral movements of heterodimers in the plane of the plasma membrane, culminating in integrin clustering (2-4). Positive inside-out signals can be initiated by agonist occupancy of several different classes of excitatory receptors that couple to heterotrimeric G proteins and tyrosine kinases (5-10). _IIb3 activation can be negatively regulated by prostacyclin or nitric oxide, whose effects are mediated through cyclic AMP and cyclic GMP, respectively (5).

The signaling molecules directly responsible for regulation of _IIb3 downstream of excitatory receptors are incompletely characterized. Nonetheless, isoforms of protein kinase C and PI¹ 3-kinase as well as cytoplasmic free Ca²⁺ have been identified as key signaling intermediates (1, 5). In addition, _IIb3 function in platelets appears to be regulated in some way by the actin cytoskeleton because inhibition of actin polymerization by low micromolar concentrations of cytochalasin D or latrunculin A increases agonist-dependent fibrinogen binding, whereas higher concentrations of these agents partially inhibit fibrinogen binding (11). Therefore, protein kinase C, PI 3-kinase, and Ca²⁺ may introduce post-translational modifications in signaling/cytoskeletal proteins that associate with and regulate _IIb3 (1, 12). However, major gaps remain in the identification and characterization of integrin regulatory proteins in platelets.

The Ras family GTPase, Rap1b, may be one such integrin regulatory protein. Like other Ras family members, it cycles from an inactive, GDP-bound form to an active, GTP-bound form, with cycling regulated by one or more guanine nucleotide exchange factors (GEFs), guanine nucleotide dissociation inhibitors (GDIs), and GTPase-activating proteins (GAPs) (13). Rap1b is highly expressed in platelets, is rapidly activated in response to agonists such as thrombin, ADP, or epinephrine and partitions along with _IIb3 to the Triton-insoluble core actin cytoskeleton of aggregated platelets (14-16). Rap1b activation in response to thrombin depends initially on Ca²⁺ fluxes into the platelet cytoplasm and subsequently on protein kinase C (17). Although the function of Rap1b in platelets is unknown, it has been implicated in promoting ₁ and ₂ integrin-dependent adhesion of fibroblastic and hematopoietic cell lines and murine thymocytes (18-22). The evidence for this is based largely on overexpression of constitutively active and dominant-negative forms of Rap1, but additional support for Rap1 involvement comes from studies of mouse embryonic fibroblasts deficient in the Rap1 GEF, C3G, which exhibit defective integrin-dependent adhesion that is correctable by expression of a constitutively active Rap1 mutant (23). Because, cell adhesion by integrins is dependent on a combination of factors, including inside-out signaling, ligand binding, and post-ligand binding events, the precise mechanism(s) whereby Rap1 promotes adhesion may vary with the integrin and the cell type.

Based on these considerations, the present study was carried out to determine whether Rap1b is an effector of inside-out signaling to _IIb3. Because platelets are not amenable to genetic manipulation, we used Sindbis virus vectors to express specific GFP-tagged chimeric proteins in primary, mature murine megakaryocytes. Megakaryocytes are nucleated cells that function primarily to produce platelets, and like platelets they exhibit an inside-out signaling pathway from excitatory receptors to _IIb3 (24-26). The results establish that Rap1b, in concert with platelet agonists, can promote fibrinogen binding to _IIb3. Furthermore, they indicate that Rap1b functions to modulate _IIb3 affinity, possibly through effects on the actin cytoskeleton.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of Sindbis Virus Vectors-- Enhanced green fluorescent protein (GFP) was amplified by PCR using pEGFP-C1 as a template (CLONTECH, Palo Alto, CA) and cloned into the MluI/SphI sites of the Sindbis expression vector, pSinRep5 (Invitrogen, Carlsbad, CA). Full-length human Rap1b (V12) or Rap1b (N17) cDNAs in pGBT9 and pCGN, respectively, were PCR-amplified to generate XbaI/SphI fragments and directionally cloned into pSinRep5. Then a XbaI/XbaI GFP cassette was subcloned in-frame into this plasmid to create GFP-Rap1b (V12) or GFP-Rap1b (N17) fusions. GFP-Rap1GAP in pSinRep5 was generated by cloning PCR-amplified human Rap1GAP (a generous gift from Alan Hall, London) as a MluI/MluI fragment, followed by a 5' in-frame insertion of the GFP cassette. Insert orientations were verified by colony PCR, and all coding sequences were verified in the Sindbis vectors by automated DNA sequencing.

To produce Sindbis viruses encoding mRNA for the GFP chimeras, the pSinRep5 plasmids and a helper plasmid (DH26S) were linearized and used as a template to synthesize in vitro capped and polyadenylated mRNA using an SP6 RNA-polymerase kit (Ambion, Austin, TX). Expression and helper mRNAs were then co-transfected in a 1:1 molar ratio by electroporation into BHK cells, which were cultured for 24 h to allow virion production. Supernatants containing virions were collected, centrifuged at 2000 × g for 10 min at 4 °C, and stored in liquid nitrogen in 1-ml aliquots. Viral titers were evaluated by transducing BHK cells and assessing GFP expression 18 h later by flow cytometry. Only viral preparations capable of inducing GFP expression in more then 50% of BHK cells at a 1:3000 dilution of viral supernatant were used for subsequent megakaryocyte experiments.

Characterization of GFP-tagged Proteins-- GFP, GFP-Rap1b, and GFP-Rap1GAP proteins were characterized by incubating NIH 3T3 cells with a 1:3 dilution of recombinant virus in 1% Dulbecco's modified Eagle's medium/fetal calf serum for 1 h at 37 °C. After 10-fold dilution in the same medium without virus, cells were cultured another 6 h, and expression of recombinant proteins was assessed in Nonidet P-40 detergent-solublized cell lysates by Western blotting (26). Blots were probed with a monoclonal antibody to GFP (1:500 dilution; CLONTECH) and an horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:3000 dilution; Bio-Rad Laboratories). Immunoreactive bands were detected by enhanced chemiluminescence using SuperSignal WestPico reagent (Pierce).

GTP loading of Rap1b was detected by a pull-down assay (14). NIH 3T3 cells were lysed at 4 °C in RIPA buffer containing 75 mM NaCl, 1% Nonidet P-40, 1% deoxycholic acid, 0.2% sodium dodecyl sulfate, 2.5 mM MgCl₂, 1 mM sodium orthovanadate, 1 mM phenylmethylsulphonyl fluoride, 2 µM leupeptin, 2 µM aprotinin, and 50 mM Tris/HCl, pH 7.4. After clarification, 500 µg of protein in 500 µl were incubated with 30 µl of a 50% slurry of glutathione-Sepharose beads precoupled to either GST or the GST-Rap binding domain of RalGDS. After three washes with RIPA buffer, the presence of GFP or GFP-Rap1b proteins in the pellets, and the amounts of recombinant or endogenous Rap1 in the starting material were examined by Western blotting with an antibody to either GFP or Rap1 (Santa Cruz Biotechnology, Santa Cruz, CA).

Transduction of GFP-tagged Proteins into Megakaryocytes-- Bone marrow cells were harvested from 6 to 8-week-old BALB/c mice and cultured in the presence of thrombopoietin, IL-6, and IL-11 as described (26). After 5 days, mature, polyploid megakaryocytes were enriched by gravity sedimentation for 60 min at 37° in a 50-ml conical polypropylene tube and then applied to a discontinuous density gradient of 1-2-3% bovine serum albumin in phosphate-buffered saline, pH 7.4. After gravity sedimentation for 30 min at 37°, cells were resuspended in complete Iscove's Modified Dulbecco's medium to 2 × 10⁵/ml, and 0.5-ml aliquots were added to 100-mm dishes previously blocked with 1% bovine serum albumin in phosphate-buffered saline. Cells were infected with 2 ml of a 1:1 dilution of recombinant Sindbis virus for 1 h at 37° and then diluted with 5 ml of complete medium and incubated for 6 h in a CO₂ incubator.

Analysis of Fibrinogen Binding to Megakaryocytes-- After viral infection, megakaryocytes were collected in 50-ml polypropylene tubes and sedimented by gravity for 60 min at 37°. Cells were gently resuspended in modified Tyrode's buffer (137 mM NaCl, 2.9 mM KCl, 12 mM NaHCO₃, 0.1 mM CaCl₂, 0.1 mM MgCl₂, 0.1% bovine serum albumin, 0.1% glucose, 5 mM HEPES, pH 7.4), and incubated for 20 min at room temperature in a final volume of 50 µl in the presence of 200 µg/ml biotin-fibrinogen, 0.5 or 1 mM PAR4 thrombin receptor-activating peptide (AYPGFK) (27), and 10 µg/ml phycoerythrin-streptavidin (Molecular Probes, Eugene, OR). To assess nonspecific binding, parallel samples were incubated with either 10 mM EDTA or 20 µg/ml 1B5, a function-blocking antibody specific for murine _IIb3 (a gift from Barry Coller, New York) (28). In some cases, cells were pre-incubated for 10 min with either 10 µM cytochalasin D, latrunculin A or an equivalent volume of Me₂SO vehicle before addition of fibrinogen and agonist. Fibrinogen binding to large megakaryocytes was quantified by flow cytometry (26). Surface expression of _IIb3 was determined by flow cytometry after incubating cells with 10 µg/ml biotinylated anti-murine _IIb antibody or an isotype-matched control IgG (BD PharMingen, San Diego, CA).

Development of a Recombinant Antibody Fab Fragment Specific for High Affinity Murine _IIb3-- PAC-1 Fab is a recombinant IgG₁, RGD-containing antibody Fab fragment specific for high affinity human _IIb3 (29). To determine whether Rap1b modulates _IIb3 affinity in mouse megakaryocytes, PAC-1 was re-engineered to recognize high affinity murine _IIb3. Specifically, PAC-1 Fab heavy chain cDNA was subjected to splice-overlap extension PCR such that an 11-amino acid stretch of H-CDR-3 (PSYYRGDGAGP) was replaced with a 13-amino acid stretch from kistrin (CRIPRGDMPDDRC), an integrin-binding snake venom peptide (30). When expressed as a secreted heavy chain along with the PAC-1 light chain in Drosophila S2 cells (31), the resulting Fab fragment, named POW-2, was found to be selective for high affinity murine _IIb3 (see "Results"). Serum-free S2 culture supernatant containing POW-2 Fab was concentrated 10-fold and dialyzed extensively against phosphate-buffered saline. Preliminary studies showed that the Fab in this preparation was monomeric by size exclusion chromatography on a Sephadex G-200 column (29). Furthermore, forced oligomerization of _IIb3 in a Chinese hamster ovary cell model system did not promote POW-2 Fab binding, indicating that POW-2 was not sensitive to changes in _IIb3 avidity (3). POW-2 Fab interaction with mouse platelets and megakaryocytes was analyzed by flow cytometry (29). For platelets, POW-2 binding was assessed with a secondary goat anti-mouse Ig (H+L) antibody (Fab')₂ conjugated with Alexa-488 (31). For megakaryocytes, POW-2 was used to compete for biotin-fibrinogen binding.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of GFP-Rap1 Chimeric Proteins-- The purpose of this investigation was to determine the role of Rap1b in _IIb3 function. Toward this end, RNA Sindbis virus vectors encoding either GFP, constitutively active GFP-Rap1b (V12), dominant-negative GFP-Rap1b (N17), or GFP-Rap1GAP were introduced into murine megakaryocytes, and fibrinogen binding to _IIb3 was examined. GFP was fused to the N termini of these constructs to facilitate flow cytometric identification of transduced cells. Prior to megakaryocyte studies, recombinant protein expression was verified by transducing murine NIH 3T3 cells and examining Western blots of cell lysates with an antibody to GFP. In each case, a single immunoreactive band with the appropriate electrophoretic mobility was observed (Fig. 1A). To determine whether GFP-Rap1b (V12) was active in the sense that it was loaded with GTP, lysates from virally infected cells were incubated with glutathione-Sepharose beads coated with the GST-Rap1 binding domain of RalGDS, a Rap1b effector (20). GFP-Rap1b (V12) bound to these beads, whereas GFP-Rap1b (N17) and GFP did not (Fig. 1B). Binding of GFP-Rap1b (V12) was specific because it failed to bind to beads coated with GST (not shown). GFP-Rap1GAP was functional in that it eliminated GTP-loading of endogenous Rap1 (Fig. 1C). On the basis of these results, these viral vectors were used to introduce the GFP-tagged chimeras into megakaryocytes.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Expression of GFP-Rap1 proteins in murine cells. As described under "Experimental Procedures," NIH 3T3 cells were incubated for 6 h with Sindbis viruses encoding either GFP, GFP-Rap1b (V12), GFP-Rap1b (N17), or GFP-Rap1GAP. In panel A, cells were lysed in Nonidet P-40 buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting using anti-GFP antibody as a probe. In panel B, cells were incubated with viruses encoding GFP, GFP-Rap1b (V12), or GFP-Rap1b (N17), lysed in RIPA buffer, and then subjected to a pull-down assay using glutathione-Sepharose beads coated with GST-Rap1 binding domain of RalGDS. After washing, GFP-tagged proteins retained on the beads (e.g. GTP-Rap1b) were analyzed on a Western blot using an anti-GFP antibody. In parallel, the amount of recombinant GFP proteins in 10 µg of each cell lysate (e.g. total GFP) was assessed using an antibody to GFP. In panel C, infection of cells with viruses encoding GFP or GFP-Rap1GAP was carried out in the presence of 10% fetal calf serum, the latter added to stimulate endogenous Rap1. After cell lysis, GTP-Rap1 retained on the beads and total Rap1 in 10 µg of cell lysate were assessed by Western blotting using an antibody to Rap1.

Rap1b and Inside-out _IIb3 Signaling in Megakaryocytes-- Large, mature megakaryocytes derived from bone marrow cultures were incubated for 6 h with Sindbis viruses encoding the GFP-tagged chimeras, and fibrinogen binding was then examined by flow cytometry (26). The light scattering mode of the flow cytometer was used to identify large megakaryocytes, the FL1 fluorescence channel to identify virally transduced, GFP-positive cells, the FL2 channel to quantify the binding of biotin-fibrinogen (using phycoerythrin-streptavidin), and the FL3 channel to exclude propidium iodide-positive, dead cells. After viral infection, the percentage of large megakaryocytes that expressed the recombinant GFP proteins ranged from 10 to 50% from experiment to experiment. Fig. 2 shows the fibrinogen binding data for a single experiment in the form of dot plots, where cells expressing the recombinant protein are above the horizontal lines and cells not expressing the recombinant protein are below the horizontal lines. Note that megakaryocytes that had been incubated with the control Sindbis virus encoding GFP (Fig. 2, panels A and D) bound little or no fibrinogen unless the cells were stimulated during the fibrinogen binding step with a PAR4 thrombin receptor-activating peptide (AYPGKF) (27). Both GFP-positive and GFP-negative cells appeared to respond in a similar fashion. Unstimulated megakaryocytes expressing either GFP-Rap1b (V12), which is constitutively active, or GFP-Rap1GAP, which inactivates endogenous Rap1 (18), also bound little fibrinogen (Fig. 2, panels B and C). In contrast, when stimulated with AYPGKF, megakaryocytes expressing GFP-Rap1b (V12) appeared to bind relatively more fibrinogen than non-expressing cells (Fig. 2, panel E), while megakaryocytes expressing GFP-Rap1GAP appeared to bind relatively less fibrinogen than non-expressing cells (Fig. 2, panel F).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Effect of Rap1b on fibrinogen binding to megakaryocytes. Megakaryocytes were incubated for 6 h with Sindbis viruses encoding either GFP (panels A and D), GFP-Rap1b (V12) (panels B and E) or GFP-Rap1GAP (panels C and F). Cells were then incubated for 20 min with 200 µg/ml biotin-fibrinogen in the presence or absence of 1 mM PAR4 receptor-activating peptide (AYPGKF), and fibrinogen binding was assessed by flow cytometry using phycoerythrin-strepavidin as the fluorophore. Each panel is a dot plot representing 10,000 large megakaryocytes. Blue dots below the horizontal lines represent cells not expressing the recombinant protein, and red dots above the horizontal lines are cells expressing the protein. The number in each dot plot represents the percentage of all GFP-positive events present in the upper right hand quadrant.

To validate the conclusions drawn from this single experiment, the results of five such experiments are summarized in Fig. 3, which depicts specific fibrinogen binding, defined as binding inhibited by 10 mM EDTA. Neither GFP-Rap1b (V12) nor GFP-Rap1GAP significantly influenced basal fibrinogen binding to unstimulated megakaryocytes (Fig. 3A). However, compared with megakaryocytes expressing GFP, those expressing GFP-Rap1b (V12) bound significantly more fibrinogen in response to AYPGKF (p < 0.01), whether a subsaturating (0.5 mM) or a saturating (1 mM) concentration of the agonist was employed (Fig. 3A). Identical results were obtained if 20 µg/ml 1B5, a function-blocking anti-_IIb3 antibody, was used instead of EDTA to determine specific fibrinogen binding (not shown). In contrast to the results with GFP-Rap1b (V12), cells expressing GFP-Rap1GAP bound 46% less fibrinogen than GFP-expressing cells in response to 1 mM AYPGKF (p < 0.01). These effects of GFP-Rap1b (V12) or GFP-Rap1GAP were confined to the subpopulation of megakaryocytes in each sample that had been successfully transduced (compare Fig. 3, A versus B). In addition, in three independent experiments, transduction of megakaryocytes with GFP-Rap1b (N17), which acts in a dominant-negative fashion by binding to some Rap1 GEFs (32), inhibited fibrinogen binding induced by 1 mM AYPGKF by 27 ± 8% (p < 0.05). Taken together, these results establish that Rap1b promotes agonist-induced fibrinogen binding to _IIb3, and this effect is cell-autonomous.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Effect of Rap1b on specific fibrinogen binding to megakaryocytes. Data for five independent experiments of the kind illustrated in the legend to Fig. 2 are summarized. Specific fibrinogen binding was defined as that inhibited by 10 mM EDTA. It was expressed relative to the binding observed with agonist-stimulated megakaryocytes incubated with the Sindbis/GFP virus, which was arbitrarily set at 100%. Data are the means ± S.E. Asterisks represent significant differences from the GFP sample, determined by Student's t test (p < 0.01).

The observed effects of Rap1b and Rap1GAP expression on fibrinogen binding to megakaryocytes suggest that Rap1b modulates _IIb3 affinity and/or avidity. However, similar results might be obtained if Rap1b were to modify expression levels of _IIb3 or if Sindbis virus infection, per se, were to somehow alter the responsiveness of the megakaryocytes to agonists. Therefore these potential confounding variables were investigated. The levels of _IIb3 expressed on the surface of megakaryocytes were measured with an antibody to the _IIb subunit. None of the Sindbis virus constructs affected _IIb3 expression, either before or after stimulation with AYPGKF (Fig. 4A). Furthermore, transduction of megakaryocytes with Sindbis virus encoding GFP did not affect agonist-induced fibrinogen binding when the responses of GFP-positive cells were compared with GFP-negative cells (Fig. 4B), or when cells exposed to virus were compared with mock-transfected cells (not shown). We conclude that Rap1b modulates fibrinogen binding to _IIb3 by an effect on integrin affinity and/or avidity.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Effect of Sindbis virus infection on IIb3 expression and function in megakaryocytes. In panel A, megakaryocytes were incubated for 6 h with the indicated Sindbis viruses, and surface expression of IIb3 was determined by flow cytometry, using an antibody specific for the IIb subunit. Where indicated, cells were stimulated with 1 mM PAR4 receptor-activating peptide (AYPGKF) during the binding assay. Data are presented as specific binding of the anti-IIb antibody and expressed relative to the binding observed with unstimulated, non-transduced (GFP-negative) megakaryocytes that had been incubated with the Sindbis/GFP virus. In panel B, megakaryocytes were incubated with Sindbis virus encoding GFP. Biotin-fibrinogen binding was then assessed in the presence or absence of AYPGKF and 10 mM EDTA, as indicated. Binding is expressed as mean fluorescence intensity in arbitrary units. Data represent the means ± S.E. of 3-7 experiments.

Mechanistic Aspects of Rap1b Function in Inside-out Signaling-- To begin to investigate the mechanism by which Rap1b modulates fibrinogen binding to megakaryocytes, we developed a novel reagent capable of reporting on changes in the affinity of murine _IIb3. PAC-1 is a murine IgM monoclonal antibody specific for activated human _IIb3 (33). Because it is multimeric, PAC-1 IgM is sensitive to changes in both _IIb3 affinity and avidity. In contrast, and as might be predicted, a recombinant, monomeric, and monovalent Fab fragment of PAC-1 is sensitive only to changes in _IIb3 affinity (3, 29). Because PAC-1 Fab is specific for human _IIb3, we re-engineered it to recognize high affinity murine _IIb3. By removing an 11-amino acid segment from the H-CDR3 of PAC-1 Fab and replacing it with a 13-amino acid RGD-containing segment from the disintegrin, kistrin, a new recombinant Fab fragment called POW-2 was created (Fig. 5A). Recombinant POW-2 bound to _IIb3 on agonist-activated mouse (or human) platelets. Binding was specific for high affinity _IIb3 because minimal binding was observed to unstimulated platelets, to stimulated platelets incubated with EDTA, kistrin or antibody 1B5 to block fibrinogen binding to _IIb3 or to stimulated platelets incubated with dibutyryl cyclic AMP to inhibit platelet activation (Fig. 5B). Although kistrin recognizes _V3 and ₅₁ in addition to _IIb3, POW-2 only recognized _IIb3 in murine cells (not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Characterization of POW-2 Fab and its effect on fibrinogen binding to megakaryocytes. Panel A shows the amino acid sequences within the H-CDR3 regions of PAC-1 and POW-2 Fabs. The bold letters represent the swapped sequences that converted PAC-1 Fab into POW-2 Fab. Panel B validates the binding specificity of POW-2 Fab for IIb3 using murine platelets and flow cytometry, as described under "Experimental Procedures." Incubation of platelets with 130 µg/ml POW-2 Fab was carried out for 30 min in the presence of an agonist and inhibitors, as indicated. The agonist was 1 mM AYPGKF, EDTA was used at 10 mM, kistrin at 5 µM, 1B5 at 20 µg/ml, and dibutyryl cyclic AMP (db-cAMP) at 1 mM. Panel C shows the effect of 130 µg/ml POW-2 Fab on the specific binding of 50 µg/ml biotin-fibrinogen to megakaryocytes. Where indicated, 0.5 mg/ml of unlabeled fibrinogen was used instead of POW-2 Fab as a competitor. Data represent the means ± S.E. of three experiments.

Therefore, POW-2 Fab was used to compete with biotin-fibrinogen for binding to AYPGKF-stimulated mouse megakaryocytes, with the rationale that it would compete successfully only for binding to high affinity _IIb3. POW-2 Fab was used at 130 µg/ml, a concentration that in preliminary studies inhibited the specific binding of 50 µg/ml biotin-fibrinogen to AYPGKF-stimulated mouse platelets by >95%. As shown in Fig. 5C, POW-2 Fab inhibited agonist-dependent fibrinogen binding to GFP-expressing megakaryocytes by 75% and to GFP-Rap1b (V12)-expressing megakaryocytes by 82%. In fact, POW-2 was almost as good a competitor of biotin-fibrinogen as was an excess of unlabeled fibrinogen (Fig. 5C). This substantial blockade of fibrinogen binding by POW-2 Fab suggests that AYPGKF and Rap1b (V12) modulate the affinity of _IIb3.

Because _IIb3 interacts with and may be regulated by components of the platelet actin cytoskeleton (11, 34), we examined whether inhibition of actin polymerization affected Rap1b (V12)-dependent fibrinogen binding to stimulated megakaryocytes. Pre-incubation of GFP-expressing megakaryocytes with 10 µM cytochalasin D blocked fibrinogen binding induced by AYPGKF by 40%, consistent with previous studies of this concentration of cytochalasins in platelets (11, 35, 36). More importantly, cytochalasin D completely blocked the increment in fibrinogen binding caused by GFP-Rap1b (V12) (Fig. 6). Similar results were obtained if actin polymerization was blocked with 10 µM latrunculin A instead of cytochalasin D (not shown). Thus, Rap1b may regulate _IIb3 affinity and fibrinogen binding through effects on the actin cytoskeleton.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Effect of cytochalasin D on specific fibrinogen binding to megakaryocytes. Megakaryocytes were transduced with Sindbis/GFP or Sindbis/GFP-Rap1b (V12) viruses. Cells were then incubated for 10 min with 10 µM cytochalasin D (CD) or Me2SO vehicle as a control, and fibrinogen binding was determined by flow cytometry. The data represent specific fibrinogen binding to transduced megakaryocytes and is expressed relative to binding observed with agonist-stimulated cells transduced with the Sindbis/GFP virus. Data are the means ± S.E. of three experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, recombinant human Rap1b constructs were expressed in primary murine megakaryocytes to assess the potential role of this GTPase in inside-out _IIb3 signaling. Megakaryocytes were chosen because they respond to platelet agonists by engaging fibrinogen via _IIb3, but unlike their anucleate platelet progeny, they are amenable to genetic manipulation (25, 26, 37). The major new findings are the following. 1) Expression of constitutively active Rap1b (V12) augments fibrinogen binding to _IIb3 when megakaryocytes are stimulated through the PAR4 thrombin receptor. In contrast, expression of Rap1GAP, which inactivates endogenous Rap1, or expression of Rap1b (N17), a dominant-negative construct, has the opposite effect. 2) Modulation of fibrinogen binding by Rap1b appears to be due primarily to effects on _IIb3 affinity. 3) Regulation of _IIb3 activation state by Rap1b may depend on the actin cytoskeleton, because no Rap1b effect was observed if actin polymerization was blocked by cytochalasin D or latrunculin A. These results establish a role for Rap1b in affinity modulation of _IIb3, and they raise important new questions about the identities and mechanisms of action of the relevant Rap1b regulators and effectors in megakaryocytes and platelets.

GFP-Rap1b and GFP-Rap1GAP constructs were introduced into mature, bone marrow-derived mouse megakaryocytes using Sindbis viruses. In this system, the recombinant proteins were expressed rapidly, and cell integrity was preserved long enough for functional studies of _IIb3 to be carried out. Indeed, viral transduction, per se, had no detrimental effect on _IIb3 surface expression or on agonist-induced fibrinogen binding (Fig. 4). In addition, N-terminal fusion of GFP to these constructs did not adversely affect their function, as exemplified by the ability of Rap1b (V12) to bind GTP and the ability of GFP-Rap1GAP to reduce GTP loading of endogenous Rap1 (Fig. 1). Because expression of GFP alone did not interfere with agonist-induced fibrinogen binding to megakaryocytes, the opposing effects on fibrinogen binding of GFP-Rap1b (V12) and GFP-Rap1GAP provide strong evidence that _IIb3 activation can be modulated by Rap1b (Figs. 2 and 3). Furthermore, although the recombinant proteins used here were human in origin, the high degree of sequence conservation between human and murine Rap1b (e.g. 85% overall amino acid identity; 95% in the switch regions)² indicates that our results are unlikely to be complicated by species differences.

One caveat in overexpression work with Rap1b (V12) and Rap1b (N17) is that the results obtained may not necessarily reflect the function of endogenous Rap1b. For example, dominant-negative Rap1b (N17) may exert functions in addition to the expected one of titrating Rap1 GEFs, and it may not titrate all Rap1 GEFs (32). Indeed, this may explain why GFP-Rap1GAP was a better inhibitor of agonist-induced fibrinogen binding to megakaryocytes than GFP-Rap1b (N17) (e.g. 46% versus 27%). Nonetheless, the present study demonstrates unambiguously that Rap1b can promote inside-out signaling to _IIb3 in primary megakaryocytes, thus providing a strong rationale to further evaluate the functional relationships between Rap1b and _IIb3 in platelets. The rapid activation of Rap1b in platelets stimulated with thrombin, ADP, or epinephrine is consistent with a role in inside-out signaling (14-17). Rap1b is a substrate for protein kinase A in platelets (38). While the significance of phosphorylation to Rap1b function is unclear, the phosphorylation of one or more proteins by protein kinase A inhibits agonist-induced ligand-binding to _IIb3 in platelets (39). Perhaps Rap1b is one of the relevant protein kinase A substrates in this context.

This work provides new insights into the role of Rap1b in integrin function. Previous studies in fibroblasts, various hematopoietic cell lines and murine thymocytes have demonstrated that overexpression of Rap1 or its GEFs and GAPs affects cell adhesion, aggregation, and phagocytosis dependent on ₁ or ₂ integrins (18-22, 40). Because all of these responses involve integrin ligation as well as post-ligand binding events, these observed effects of Rap1 do not necessarily pinpoint which phase is targeted by the GTPase. However, in one study employing a B lymphocyte cell line, Rap1 (V12) increased the expression of an activation epitope on _L2 and the binding of a soluble ICAM-1/IgG fusion protein to the cells, consistent with affinity modulation of _L2 by Rap1 (19). On the other hand, work in transgenic thymocytes concluded that Rap1 was sufficient to modulate the clustering and avidity, rather than the affinity, of ₁ and ₂ integrins (22).

In megakaryocytes, Rap1b increased fibrinogen binding to _IIb3, but only when the cells were stimulated with an agonist (Figs. 2 and 3). This suggests that signals from Rap1b are not sufficient to activate _IIb3, but rather they may be required to achieve maximal _IIb3 activation in response to agonist-triggered signals. Furthermore, the results with POW-2, a monomeric and monovalent ligand-mimetic Fab specific for high affinity murine _IIb3, establish that Rap1b functions, in large part, by modulating _IIb3 affinity in megakaryocytes (Fig. 5). Overall, these results indicate that Rap1 is capable of regulating the activation state of ₁, ₂, and ₃ integrins. However, the precise mechanism of regulation, e.g. affinity versus avidity modulation, appears to depend on the integrin and the cell type. Future studies should consider the possibility that Rap1 may also influence post-ligand binding events, such as changes in cell shape or polarity (13).

How is Rap1b activated in megakaryocytes and platelets, and which Rap1b effector(s) modulate _IIb3 affinity? Addressing these questions is made complicated by the plethora of Rap1 GEFs and GAPs in various cells and tissues, some of which are not specific for Rap1 (13, 41-43). Furthermore, the effector functions of many of the known proteins that bind selectively to GTP-Rap1 have yet to be completely characterized (13, 44). Most importantly, relatively little information is available about the repertoire of Rap1 regulators and effectors in platelets and megakaryocytes. Because Rap1b (V12) augmented the fibrinogen binding response to a PAR4 thrombin receptor agonist, the relevant Rap1 GEFs or GAPs may themselves be regulated by signaling molecules activated downstream of one or more heterotrimeric G proteins. In this context, the products of phospholipase C-mediated phospholipid hydrolysis, Ca²⁺ and diacylglycerol, have been implicated in agonist-dependent Rap1b activation in platelets (17). Indeed, platelets and megakaryocytes contain a Rap1 GEF (CalDAG-GEFI) likely to be activated by Ca²⁺ and diacylglycerol (41, 45).³ Potential Rap1 effectors identified in platelets include RalGDS, p110 PI 3-kinase, and Raf-1 (13, 46-48).

Integrins are coupled to actin filaments through actin-binding proteins, such as -actinin, filamin, and talin (12). Consequently, a conceptual link is often made between changes in the actin cytoskeleton and changes in integrin clustering or avidity (4, 22, 49). We found that inhibition of actin polymerization by 10 µM latrunculin A or cytochalasin D blocked Rap1b-dependent fibrinogen binding to megakaryocytes (Fig. 6). Because fibrinogen binding is controlled primarily by changes in _IIb3 affinity (Fig. 5) (3), we propose that Rap1b may regulate _IIb3 affinity through an effect on actin dynamics or organization. A causal link between changes in actin and changes integrin affinity has not been established unambiguously. However, this idea is consistent with the recent observation that relatively low concentrations of cytochalasin D or latrunculin A, which may release integrins from cytoskeletal constraints, increase agonist-dependent fibrinogen binding to platelets (11). In this regard, at least two Rap1b effectors in platelets, RalGDS and p110 PI 3-kinase, have been proposed to influence actin filament organization (46, 47, 50). Studies in cell lines have demonstrated that Ras family members in addition to Rap1, such as H-Ras and R-Ras, can influence ligand binding to integrins, although the net effects vary considerably among cell types (51-53). Given the suitability of primary megakaryocytes for the molecular analysis of _IIb3 signaling, the experimental system employed here should prove useful for identifying the physiological regulators and effectors of Rap1b responsible for modulating _IIb3 affinity and for determining the roles of other Ras family members in _IIb3 function.

	ACKNOWLEDGEMENTS

We thank Barry Coller, Alan Hall, and Mark Larson for reagents, and Mark Ginsberg and Martin Schwartz for critical review of the manuscript.

	FOOTNOTES

^* These studies were supported by research grants from the National Institutes of Health (to L. V. P., G. C. W., and S. J. S.) and by a postdoctoral fellowship from the American Heart Association (to K. E.). This work was presented in part at the Annual Meeting of the American Society of Hematology, December, 2002, Orlando, FL and published in abstract form (Bertoni, A., Tadokoro, S., Eto, K., Pampori, N., Parise, L., White, G. C., and Shattil, S. J. (2001) Blood 98, 752a).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: The Dept. of Cell Biology, The Scripps Research Institute, 10550 North 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, May 6, 2002, DOI 10.1074/jbc.M202791200

² G. C. White, unpublished observations.

³ R. Murphy, K. Eto, S. Kerrigan, A. Bertoni, S. Shattil, and A. Leavitt, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: PI, phosphatidylinositol; GFP, enhanced green fluorescent protein; RIPA, radioimmune precipitation assay buffer; GST, glutathione S-transferase; IL, interleukin; GEF, guanine nucleotide exchange factors; GAP, GTPase-activating proteins.

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