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J. Biol. Chem., Vol. 277, Issue 3, 1919-1923, January 18, 2002

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Calcium Integrin-binding Protein Activates Platelet Integrin _IIb3^*

Shigeru Tsuboi

From the Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, California 92037

Received for publication, November 6, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

_IIb3, a platelet-specific integrin, plays a critical role in platelet aggregation. The affinity of _IIb3 for its ligands such as fibrinogen and von Willebrand factor is tightly regulated in an uncharacterized intracellular process termed inside-out signaling. Calcium integrin-binding protein (CIB) has been identified as a protein interacting with the cytoplasmic tail of the _IIb subunit of _IIb3, but its physiological role has not been defined. In the present study, I demonstrate that CIB activates _IIb3 both in vitro and in vivo. CIB interacts directly with the _IIb cytoplasmic tail, thereby increasing the affinity of _IIb3 for fibrinogen in an in vitro fibrinogen-binding assay. The interaction of CIB with the _IIb cytoplasmic tail is enhanced in a Ca²⁺-dependent manner. A physiological agonist, ADP, stimulates platelets, activating _IIb3. When the interaction of CIB with the _IIb cytoplasmic tail is blocked in native platelets by a permeable competing peptide, _IIb3 activation is not detected even in the presence of ADP. This result indicates that direct interaction of CIB with the _IIb cytoplasmic tail converts _IIb3 from a resting to an active conformation. This suggests that CIB plays an important role in one of the pathways that modulate the affinity of _IIb3 for its ligand.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Integrins are transmembrane receptors that mediate cell adhesion and cell migration. Integrins are heterodimeric integral membrane proteins composed of noncovalently associated  and  subunits (1-3). _IIb3, a platelet-specific integrin, is essential for hemostasis by virtue of its role in mediating platelet aggregation and spreading on vascular matrices at sites of injury in the vascular wall (1, 2, 4-6). _IIb3 is maintained in a resting state on circulating resting platelets. Once activation molecules such as ADP, thrombin, and collagen are generated at these injured sites, they activate nearby circulating platelets, converting _IIb3 from a resting to an active conformation in an intracellular process termed inside-out signaling, although its precise mechanisms have been unknown. Active _IIb3 binds to specific plasma proteins in solution such as fibrinogen and von Willebrand factor and links platelets together in an aggregate to form a plug (3, 6-8).

Considerable effort has been directed toward understanding how the affinity of _IIb3 for its ligand is modulated. It is proposed that the _IIb3 affinity is modulated through the cytoplasmic tails of both the _IIb and ₃ subunits (9-11). Truncation of the _IIb cytoplasmic tail induced constitutive activation of _IIb3 in transfected Chinese hamster ovary cells (9). ₃-endonexin, which associates specifically with the ₃ cytoplasmic tail, was shown to increase the affinity of _IIb3 for its ligands in transfected Chinese hamster ovary cells (10). In addition, it is reported that some other proteins interact with the cytoplasmic tails of _IIb3 (11). However, any molecules that regulate the _IIb3 affinity for its ligands by interacting with the _IIb cytoplasmic tail have not been identified. And also, the precise mechanisms that positively and negatively regulate this process in platelets where _IIb3 is endogenously expressed have been undefined.

Calcium integrin-binding protein (CIB)¹ was cloned and shown to interact with the cytoplasmic tail of the  subunit of _IIb3 in 1997 (12). CIB, consisting of 191 amino acids, is N-terminally myristoylated and has two Ca²⁺ binding sites, known as EF-hands I and II. CIB has also been identified as a binding partner for other proteins (13), but its function has been undefined. In this study, I provide evidence showing that CIB plays an important role in activation of _IIb3 in platelets.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells Lines, Transfection, Plasmids, Antibodies, and Reagents-- Human embryonic kidney 293 cells and human erythroleukemia (HEL) cells from the American Type Culture Collection were maintained in high glucose Dulbecco's modified Eagle's medium and RPMI medium, respectively. Both media were supplemented with 10% fetal calf serum, 1 mM L-glutamine, and antibiotics. Transfection was carried out using SuperFect (Qiagen). The cDNAs encoding various fragments of CIB were generated by PCR from human peripheral blood leukocyte cDNA library (OriGene Technologies, Rockville, IL) and the plasmid pJG4-5 containing a full-length CIB cDNA. Epitope tag constructs were made by the N-terminal addition of a Myc tag in the pcDNA3 vector (thus creating Myc-CIB) and the C-terminal addition of a FLAG tag in the pCMV-5 (thus creating CIB-FLAG). A polyclonal antibody to CIB was prepared in this study. The synthetic peptide of CIB, NH₂-EFQHVISRSPDFASSFKIVL-COOH (residues 172-191) (AnaSpec Inc., San Jose, CA) was used for immunization of rabbits. The antibody was purified by affinity chromatography using the synthetic peptide coupled to Sepharose beads. The monoclonal antibodies specific to the ₃ subunit (clone 1) and an active conformation of _IIb3 (PAC-1) were obtained from Transduction Laboratories (San Diego, CA) and Becton Dickinson Immunocytometry Systems, respectively. The monoclonal antibodies to Myc tag (9E10) and GST (B-14) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and a monoclonal antibody to FLAG tag (M2) was obtained from Sigma. Glutathione-Sepharose and concanavalin A-Sepharose 4B were purchased from Amersham Biosciences, Inc. Anti-Myc-Sepharose was purchased from Santa Cruz Biotechnology.

Yeast Two-hybrid Assay-- A cDNA encoding the cytoplasmic tail of the _IIb subunit (residues 989-1008) was generated by PCR from human peripheral blood leukocyte cDNA library and cloned into pGilda, a yeast expression vector (CLONTECH). The cDNAs encoding various fragments of CIB were amplified from the plasmid pJG4-5 containing a full-length CIB cDNA and cloned into pJG4-5 (Invitrogen). A yeast two-hybrid assay was performed as described (14). Briefly, EGY48 yeast strain was transformed by a LiCl method with pGlida-_IIb cytoplasmic tail, pJG4-5, containing various CIB cDNA fragments and pSH18-34, a reporter plasmid for -galactosidase assay. Transformants were assayed for Leu prototrophy, and a filter assay was performed for -galactosidase measurement.

In Vitro Binding Assay-- The _IIb cytoplasmic tail and CD40 cytoplasmic tail (negative control) were expressed in Escherichia coli (BL21DE) as glutathione S-transferase (GST) fusion proteins (GST-_IIb cytoplasmic tail and GST-CD40) and were purified from bacterial lysates by batch elution from glutathione-Sepharose. GST fusion proteins were dialyzed against TBS. CIB (full-length; residues 1-191) tagged at the N terminus with Myc was prepared from 293 cell transfectants using anti-Myc (9E10)-conjugated Sepharose (Santa Cruz Biotechnology). For testing direct interaction of CIB with the _IIb cytoplasmic tail in vitro, 10 µg of Myc-CIB was immobilized on anti-Myc-Sepharose. Two µg of GST-_IIb cytoplasmic tail was incubated with Myc-CIB-immobilized Sepharose. After washing the resin three times with TBS containing 0.05% Triton X-100, bound proteins were eluted and subjected to SDS-PAGE followed by immunoblotting using a monoclonal antibody for GST. For testing the interaction of CIB with the _IIb3 and the effect of calcium ion on it, the interaction of GST-CIB with _IIb3 was examined. Ten µg of concanavalin A-concentrated extracts (_IIb3) was incubated with anti-3 monoclonal antibody to ₃ (clone 1; Transduction Laboratories), and _IIb3 was immobilized on anti-mouse IgG-Sepharose. After incubation with 2.5 µg of purified GST fusion proteins prepared as described above and washing the resins three times with TBS containing 0.05% Triton X-100, GST fusion proteins were eluted and subjected to SDS-PAGE followed by immunoblotting using a monoclonal antibody for GST. For preparation of _IIb3, platelets were washed and lysed in 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 20 mM Tris-HCl, pH 7.4, containing 50 mM n-octyl -D-glucopyranoside and proteinase inhibitors. _IIb3 was enriched from the lysate by batch elution from concanavalin A-Sepharose 4B (15).

In Vivo Binding Assay-- For co-immunoprecipitation of CIB with _IIb subunit, HEL cells were transfected with the pFLAG-CMV-5-CIB only, because HEL cells express _IIb3 endogenously. Forty-eight h after transfection, cells were harvested and solubilized in a lysis buffer (150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 20 mM Tris-HCl, pH 7.4, containing 50 mM n-octyl -D-glucopyranoside and proteinase inhibitors). _IIb was immunoprecipitated with a monoclonal anti-_IIb antibody (M-148) (Santa Cruz Biotechnology). Immunoprecipitated proteins were subjected to SDS-PAGE and analyzed by immunoblotting using anti-_IIb and anti-FLAG (M2) (Sigma).

Fibrinogen-binding Assay-- Binding of fibrinogen to _IIb3 was examined by using an immunocapture assay as described (15). A monoclonal antibody to ₃ (clone 1, mouse IgG1) (Transduction Laboratories, San Diego, CA) was coated overnight onto microtiter wells (Flow laboratories, McLean, VA). The wells were washed and blocked with 3% bovine serum albumin, and then _IIb3 was captured on the wells. Unbound materials were removed by washing with TBS containing 2 mM CaCl₂ and 2 mM MgCl₂, and the wells were incubated for 2 h with GST fusion proteins and/or peptides at room temperature. The wells were washed and then incubated for 2 h with ¹²⁵I-labeled fibrinogen (300 nM) (Amersham Pharmacia Biotech) at room temperature. The wells were washed three times, and then bound radioactivity was counted.

Peptide Synthesis-- A peptide termed CA-2, corresponding to the C-terminal portion of CIB containing the amino acid sequence Arg¹⁷⁹-Lys¹⁸⁸ (NH₂-RSPDFASSFK-COOH) and its scrambled version (NH₂-SDFKSASPFR-COOH) were synthesized by AnaSpec Inc. (San Jose, CA) using a standard solid-phase Fmoc procedure. Corresponding peptides palmitoylated on the N-terminal amino acid were also synthesized in an identical manner. To label peptides with fluorescein isothiocyanate (FITC), 5 mM of peptides were incubated with 4 mg/ml FITC (Sigma) in 50 µM Na₂CO₃ (pH 9.5) at room temperature overnight. The reaction mixture was applied to Sephadex G-50 (Amersham Pharmacia Biotech) (1 × 2 cm) to remove free FITC.

Platelet Stimulation and Fluorescence-activated Cell Sorting Analysis-- Platelets were obtained from a regional blood center and healthy volunteers. Blood was collected into 0.15 (v/v) acid-citrate-dextrose (38 mM citric acid, 75 mM sodium citrate, 124 mM dextrose) plus anticoagulant and then centrifuged at 180 × g for 10 min. Platelet-rich plasma was then acidified to pH 6.5 with acid-citrate-dextrose, and PGE₁ (1 µM) was added. The platelets were pelleted through plasma by centrifugation at 750 × g for 10 min. The platelet pellet was resuspended in Tyrode's buffer (130 mM NaCl, 10 mM trisodium citrate, 9 mM NaHCO₃, 6 mM dextrose, 0.9 mM MgCl₂, 0.81 mM KH₂PO₄, 10 mM Tris-HCl, pH 7.4) adjusted to 2 × 10⁸/ml (15, 16). The platelets were then stimulated with 100 µM ADP at 37 °C under nonstirring conditions. After 15 min, binding of FITC-labeled PAC-1, an _IIb3-specific, activation-dependent monoclonal antibody (Becton Dickinson Immunocytometry Systems), was analyzed using the FACSort flow cytometer (Becton Dickinson Immunocytometry Systems).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CIB Interacts with the _IIb Cytoplasmic Tail through Its C Terminus-- To identify the region of CIB responsible for interacting with the _IIb cytoplasmic tail, yeast two-hybrid assays using deletion fragments of CIB as a bait were performed. While C-terminal deletion mutants failed to interact with the _IIb cytoplasmic tail, baits containing the C-terminal fragment were positive for the interaction, indicating that the _IIb interacting region resides in the C terminus of CIB (residues 158-191) (Fig. 1A).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   CIB interacts with the IIb cytoplasmic tail through its C terminus. A, to map the regions in CIB required for interaction, deletion mutants of CIB were analyzed for their ability to interact with the IIb cytoplasmic tail by the yeast two-hybrid assay. +, detectable activity of -galactosidase; , no detectable activity. CIB contains two EF-hand motifs (EF-I and EF-II), a calcium binding domain, and the N-terminal myristoylation site (myr). B, direct interaction of CIB with the IIb cytoplasmic tail. Bindings of GST-IIb cytoplasmic tail to Myc-tagged Siah (Drosophila seven in absentia homologous protein, a negative control) (lane 1, cont.) and Myc-tagged CIB (lane 2) were examined. C, in vivo interaction of CIB with the IIb cytoplasmic tail. HEL cells were transiently transfected with FLAG-tagged CIB (CIB-FLAG) expression vector. Lane 1, mock-transfected HEL cells; lane 2, cells transfected with a full-length CIB. Lysates were subjected to immunoprecipitation with anti-IIb antibody and were then analyzed by SDS-PAGE and immunoblotting with anti-IIb and anti-FLAG (M2) monoclonal antibodies.

To confirm that CIB interacts directly with the _IIb cytoplasmic tail, in vitro binding assays were performed. GST-_IIb cytoplasmic tail (GST-_IIb C-tail) was incubated with Myc-tagged recombinant CIB (Myc-CIB) immobilized on anti-Myc-Sepharose. As shown in Fig. 1B, Myc-CIB interacted directly with the GST-_IIb cytoplasmic tail (Fig. 1B, lane 2), whereas a negative control, Myc-tagged Siah (Drosophila seven in absentia homologous protein) (14) failed to interact with the GST-_IIb cytoplasmic tail (Fig. 1B, lane 1).

To examine whether CIB interacts with the _IIb cytoplasmic tail in cells, co-immunoprecipitation analysis of lysates from transfected cells was performed. HEL cells, a human erythroleukemic cell line that endogenously expresses _IIb3, were transiently transfected with FLAG-tagged CIB constructs, because the endogenous expression of CIB is too low to detect. CIB was tagged with FLAG at the C terminus, since the N-terminally tagged construct of CIB failed to be N-myristoylated, and N-myristoylation is critical in correct localization of CIB in cells (13). FLAG-tagged full-length CIB was co-immunoprecipitated with _IIb subunit (Fig. 1C, lane 2), indicating that CIB interacts with the _IIb cytoplasmic tail in cells.

Interaction of CIB with the _IIb Cytoplasmic Tail Is Enhanced in a Ca²⁺-dependent Manner-- Because CIB is a calcium-binding protein, we then asked whether the interaction of CIB with the _IIb cytoplasmic tail is affected by Ca²⁺. To address this question, the in vitro interaction of CIB with _IIb3 was tested for the influence of Ca²⁺ concentration. GST-CIB bound to _IIb3 in the presence of either EGTA or 100 nM of CaCl₂, which is equivalent to the Ca²⁺ concentration observed in resting platelets (17) (Fig. 2, lanes 3 and 4). In the presence of 10 µM CaCl₂, binding of GST-CIB to _IIb3 was enhanced about 3 times more than in the presence of 100 nM CaCl₂ as determined densitometric analysis (Fig. 2, lane 5). Ten µM CaCl₂ is equivalent to the Ca²⁺ concentration in ADP-stimulated platelets (17). These results suggest that the interaction of CIB with the _IIb cytoplasmic tail must be enhanced in ADP-stimulated platelets compared with resting platelets. When _IIb3 was incubated with GST-CIB in the presence of excess amounts of the GST-_IIb cytoplasmic tail, binding of GST-CIB to _IIb3 was not detected (Fig. 2, lane 6), indicating that GST-CIB binds to _IIb3 through the cytoplasmic tail of _IIb.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Interaction of CIB with the IIb cytoplasmic tail is enhanced in a Ca2+-dependent manner. IIb3 was prepared from platelet extracts using concanavalin A-Sepharose. GST fusion proteins, GST-CIB, GST-IIb cytoplasmic tail, and GST-CD40 were purified from bacterial extracts (BL21) with glutathione-Sepharose. IIb3 immobilized on anti-mouse IgG-Sepharose was incubated with the indicated GST fusion proteins in the presence of EGTA or CaCl2. After washing the resins, proteins were eluted and analyzed by immunoblotting with anti-GST monoclonal antibody. Lane 1, no nonspecific interaction of GST-CIB with anti-mouse IgG-Sepharose resin alone; lane 2, no nonspecific interaction of GST-CD40 with IIb3; lane 3, interaction of CIB with IIb3 in the absence of Ca2+; lane 4, interaction in the presence of 100 nM Ca2+; lane 5, interaction in the presence of 10 µM Ca2+; lane 6, interaction in the presence of an excess amount (20 µg) of GST-IIb cytoplasmic tail.

CIB Activates _IIb3 in Vitro-- Binding of physiological ligands to _IIb3 expressed on agonist-stimulated platelets is an initial and critical step in platelet aggregation. The observation of a direct interaction of CIB with the cytoplasmic tail of _IIb in cells raised the possibility that CIB modulates the affinity of _IIb3 for its ligand in platelets. To test this possibility, the effects of CIB on the affinity of _IIb3 for fibrinogen, one of its physiological ligands, were first examined in an in vitro immunocapture assay using ¹²⁵I-labeled fibrinogen. Binding of fibrinogen to _IIb3 dramatically increased in a dose-dependent manner following the addition of GST-CIB, whereas a negative control, GST-CD40, did not increase binding (Fig. 3A). Binding was efficiently inhibited by NH₂-GRGDSP-COOH RGD peptide but not by NH₂-GRGESP-COOH RGE peptide (Invitrogen), indicating that fibrinogen binds to a physiological binding site of _IIb3 in this assay (Fig. 3A). The increase in the fibrinogen binding to _IIb3 by GST-CIB is 77-84% of the fibrinogen binding to activated _IIb3 from ADP-stimulated platelets (Fig. 3A, open bar). This result suggests that the interaction of GST-CIB with the _IIb cytoplasmic tail triggers a structural change in the extracellular domain of _IIb3 so that fibrinogen can bind to it and that the structural change of _IIb3 by GST-CIB is nearly equivalent to that of activated _IIb3 prepared from agonist-stimulated platelets.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   CIB activates IIb3 in vitro. CIB activates IIb3 by interacting with the IIb cytoplasmic tail. A, GST-CIB interacts with the IIb cytoplasmic tail, increasing the IIb3 affinity for fibrinogen. Binding of fibrinogen to immunocaptured IIb3 was measured using 125I-labeled fibrinogen. RGD, GRGDSP peptide for inhibition of the binding of fibrinogen to IIb3; RGE, GRDESP peptide for use as an inactive control in the binding of fibrinogen to IIb3; +cont., IIb3 was incubated with GST-CD40 as a negative control. Fibrinogen binding to activated IIb3 prepared from ADP-stimulated platelets was measured (open bar). B, a peptide designated CA-2 corresponding to the C terminus of CIB (residues 179-188) inhibited the interaction of CIB with the IIb cytoplasmic tail in vitro. Binding of GST-CD40 (open bar, as a negative control) and GST-IIb cytoplasmic tail (closed bars) to Myc-CIB was measured by an enzyme-linked immunosorbent assay using anti-GST in the presence of CA-2. A scrambled version of CA-2 (cont., scCA-2) was used as a control. C, blocking of the interaction of CIB with the IIb cytoplasmic tail inhibits fibrinogen binding to IIb3 in vitro. Binding of fibrinogen to IIb3 was measured in the presence of CA-2. Scrambled CA-2 was also used as a control. In A-C, all of the results were expressed as the mean ± S.E. The analysis was performed three times independently, and one representative result is shown.

To confirm that the increase in fibrinogen binding to _IIb3 is due to direct interaction of CIB with the _IIb cytoplasmic tail, the interaction was inhibited with a synthetic peptide. To do so, several different peptides were synthesized based on the amino acid sequence of the region of CIB (residues 158-191) that interacts with the _IIb cytoplasmic tail (Fig. 1A). Among those, a peptide designated CA-2 (NH₂-RSPDFASSFK-COOH), corresponding to the C terminus of CIB (residues 179-188), efficiently inhibited interaction of CIB with the GST-_IIb cytoplasmic tail in an in vitro binding assay (Fig. 3B). Binding of fibrinogen to _IIb3 was reduced in the presence of CA-2 (Fig. 3C), while a control peptide, which is a scrambled version of CA-2 (scCA-2), had no effect on the binding (Fig. 3C), indicating that the increase in fibrinogen binding to _IIb3 is a consequence of the direct interaction of CIB with the _IIb cytoplasmic tail.

CIB Activates _IIb3 in Vivo-- When platelets are stimulated with physiological agonists such as ADP and thrombin, _IIb3 is activated, increasing its affinity for fibrinogen. The next question is whether CIB plays a role in increasing the affinity of _IIb3 for fibrinogen in platelets where _IIb3 is endogenously expressed. To address this question, _IIb3 activation was examined while the interaction of CIB with the _IIb cytoplasmic tail was inhibited in intact platelets. To inhibit this interaction, CA-2 was introduced directly into platelets, since platelets are not amenable to direct genetic manipulation. Charged peptides such as CA-2 can be introduced into cells by palmitoylation, which overcomes the inability of a charged peptide to penetrate the plasma membrane (15, 16). When platelets were stimulated with ADP, _IIb3 was activated as determined by high affinity binding of PAC-1, a monoclonal antibody specific for an active conformation of _IIb3 (Fig. 4, A and B). However, no significant PAC-1 binding was detected even in the presence of ADP, when platelets were preincubated with palmitoylated CA-2 (pCA-2) to block the interaction of CIB with the _IIb cytoplasmic tail (Fig. 4C). By contrast, when platelets were preincubated with a palmitoylated control peptide (pscCA-2), PAC-1-binding was detected at a level identical to that achieved without pCA-2 and in the presence of ADP (Fig. 4D). When platelets were preincubated with nonpalmitoylated CA-2 and palmitic acid, PAC-1-binding was also detected (Fig. 4E). To confirm that the palmitoylated peptides enter the platelets, the cell permeability of FITC-labeled peptide with and without palmitate was examined. Platelets were incubated for 1-2 min with 100 µM FITC-labeled CA-2 or FITC-labeled pCA-2, washed extensively, and then analyzed by flow cytometry. Fig. 4F showed that only the palmitoylated FITC-labeled CA-2 (closed histogram) had significant cellular association, indicating that pCA-2 is cell-permeable. These results indicate that pCA-2 inhibits _IIb3 activation by blocking the interaction of CIB with _IIb in ADP-stimulated platelets. This finding is consistent with the findings shown above that inhibition of CIB interaction with the _IIb cytoplasmic tail reduced the affinity of _IIb3 for fibrinogen in vitro (Fig. 3C). No changes in the expression level of CIB after ADP stimulation were observed (Fig. 4G). These results, taken together demonstrate that direct interaction of CIB with the _IIb cytoplasmic tail converts _IIb3 from a resting to an active conformation in native platelets.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   CIB activates IIb3 in vivo. Blocking of the interaction of CIB with the IIb cytoplasmic tail inhibits IIb3 activation in ADP-stimulated platelets. A, IIb3 was PAC-1-negative on resting platelets. B, when platelets were stimulated with 100 µM ADP, IIb3 became PAC-1-positive. C-E, platelets were preincubated with 100 µM pCA-2 (C), a scrambled version of pCA-2 (pscCA-2) as a control (D), or nonpalmitoylated CA-2 (npCA-2) plus palmitic acid (E) for 30 min and then stimulated with 100 µM ADP. After 15 min, FITC-labeled PAC-1 binding was analyzed by flow cytometry. The analysis was performed three times independently, and one representative result is shown. The numbers indicate positive cells in percentages. F, cell permeability of palmitoylated CA-2 (pCA-2). Platelets were incubated for 1-2 min with FITC-labeled peptides and analyzed by flow cytometry for the cellular association of palmitoylated FITC-labeled CA-2 (closed histogram) or unmodified FITC-labeled CA-2 (open histogram). G, total lysates prepared from resting and ADP-stimulated platelets were analyzed by immunoblotting with anti-CIB polyclonal antibody.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

When platelets are stimulated with physiological agonists such as ADP, _IIb3 is converted from a resting to an active conformation (Fig. 4, A and B). _IIb3 activation is induced by the interaction of CIB with the _IIb cytoplasmic tail (Figs. 3 and 4, C-E). No changes in the expression level of CIB were detected after ADP stimulation (Fig. 4G). These observations suggest that inside-out signaling enhances the interaction of CIB with the _IIb cytoplasmic tail, inducing a conformational change in the extracellular domain of _IIb3 expressed on agonist-stimulated platelets.

It is possible that an increase in the intracellular Ca²⁺ concentration resulting from agonist stimulation may account for the increase in the affinity of CIB for the _IIb cytoplasmic tail in platelets, since the interaction of CIB with the _IIb3 cytoplasmic tail was enhanced in a Ca²⁺-dependent manner (Fig. 2). Shock et al. also reported that binding of CIB to the _IIb cytoplasmic domain peptide is enhanced in a Ca²⁺-dependent manner (18).

Another factor that may increase the affinity of CIB for the _IIb cytoplasmic tail is a change in its subcellular localization dependent upon Ca²⁺ concentration. CIB is a member of the calcium-myristoyl-switch protein family (19, 20). The members of this family include recoverin, GCAP, and frequenin and others, and all have both a myristoyl tag and Ca²⁺ binding sites (EF-hands) (20). Structural analyses reveal that the myristoyl tag is sequestered inside the protein in the absence of Ca²⁺ and that binding of Ca²⁺ to the protein leads to the extrusion of its myristoyl tag, making it available to interact with membrane targets (19). An increase in intracellular Ca²⁺ concentration following agonist stimulation in platelets may make CIB available to interact with the _IIb cytoplasmic tail by recruiting CIB to the membrane. This would result in an increase in the affinity of CIB for the _IIb cytoplasmic tail, thereby activating _IIb3.

In contrast to the results shown in Fig. 3, Vallar et al. (21) recently showed that CIB did not stimulate PAC-1-binding to inactive _IIb3, indicating that CIB does not activate _IIb3. Such a discrepancy probably comes from the differences in assay methods. First, Vallar et al. (21) performed the entire _IIb3-CIB binding assay in a liquid phase, while in the present study, CIB was added to _IIb3 immunocaptured onto wells. Immunocaptured _IIb3 may be more concentrated on the surface of the wells, resulting in an increase in assay sensitivity. Second, in the study of Vallar et al. (21), _IIb3 activation was evaluated by the amounts of the active form of _IIb3 immunoprecipitated with PAC-1. By contrast, binding of fibrinogen to _IIb3 was measured in Fig. 3. Considering the efficiency of immunoprecipitation with PAC-1, measurement of fibrinogen binding is more sensitive, quantitative, and physiologically relevant than their assay method. Finally, in this study, the _IIb3-activating activity of CIB was confirmed in an experiment using intact platelets (Fig. 4).

One highly plausible mechanism for the _IIb3 affinity modulation for its ligand is proposed (6, 15). The cytoplasmic tails of the _IIb and ₃ interact with each other possibly through a salt bridge, maintaining _IIb3 in a latent state. Inside-out signaling induces the dissociation of the interaction between the cytoplasmic tails of _IIb and ₃, thereby converting a conversion of the extracellular domain of _IIb3 from the latent to an active conformation. CIB may act as an positive regulator to dissociate this interaction by interacting with the _IIb cytoplasmic tail. Future studies will be important to determine how the interaction of CIB with the _IIb cytoplasmic tail is regulated.

In conclusion, the results of this study demonstrate that CIB is a key player in the modulation of _IIb3 affinity for its ligand in platelets.

	ACKNOWLEDGEMENTS

I thank Drs. Minoru Fukuda, Erkki Ruoslahti, and Robert Liddington for critical comments and Dr. S. Matsuzawa for technical advice.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HD39187 (to S. T.).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 may be addressed: Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, CA 92037. Tel.: 858-646-3100 (Ext. 3684); Fax: 858-646-3193; E-mail: stsuboi@burnham.org.

Published, JBC Papers in Press, November 9, 2001, DOI 10.1074/jbc.M110643200

	ABBREVIATIONS

The abbreviations used are: CIB, calcium integrin-binding protein; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; HEL, human erythoroleukemia; TBS, Tris-buffered saline; pCA-2, palmitoylated CA-2.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				W. Yuan, T. M. Leisner, A. W. McFadden, Z. Wang, M. K. Larson, S. Clark, C. Boudignon-Proudhon, S. C.-T. Lam, and L. V. Parise CIB1 is an endogenous inhibitor of agonist-induced integrin {alpha}IIb{beta}3 activation J. Cell Biol., January 17, 2006; 172(2): 169 - 175. [Abstract] [Full Text] [PDF]

				A. P. Yamniuk and H. J. Vogel Calcium- and magnesium-dependent interactions between calcium- and integrin-binding protein and the integrin {alpha}IIb cytoplasmic domain Protein Sci., June 1, 2005; 14(6): 1429 - 1437. [Abstract] [Full Text] [PDF]

				C. J. Blamey, C. Ceccarelli, U. P. Naik, and B. J. Bahnson The crystal structure of calcium- and integrin-binding protein 1: Insights into redox regulated functions Protein Sci., May 1, 2005; 14(5): 1214 - 1221. [Abstract] [Full Text] [PDF]

				H. R. Gentry, A. U. Singer, L. Betts, C. Yang, J. D. Ferrara, J. Sondek, and L. V. Parise Structural and Biochemical Characterization of CIB1 Delineates a New Family of EF-hand-containing Proteins J. Biol. Chem., March 4, 2005; 280(9): 8407 - 8415. [Abstract] [Full Text] [PDF]

				S. J. Shattil and P. J. Newman Integrins: dynamic scaffolds for adhesion and signaling in platelets Blood, September 15, 2004; 104(6): 1606 - 1615. [Abstract] [Full Text] [PDF]

				G. J. Zwartz, A. Chigaev, D. C. Dwyer, T. D. Foutz, B. S. Edwards, and L. A. Sklar Real-time Analysis of Very Late Antigen-4 Affinity Modulation by Shear J. Biol. Chem., September 10, 2004; 279(37): 38277 - 38286. [Abstract] [Full Text] [PDF]

				D. N. Saunders, S. L. Hird, S. L. Withington, S. L. Dunwoodie, M. J. Henderson, C. Biben, R. L. Sutherland, C. J. Ormandy, and C. K. W. Watts Edd, the Murine Hyperplastic Disc Gene, Is Essential for Yolk Sac Vascularization and Chorioallantoic Fusion Mol. Cell. Biol., August 15, 2004; 24(16): 7225 - 7234. [Abstract] [Full Text] [PDF]

				O. Vinogradova, J. Vaynberg, X. Kong, T. A. Haas, E. F. Plow, and J. Qin Membrane-mediated structural transitions at the cytoplasmic face during integrin activation PNAS, March 23, 2004; 101(12): 4094 - 4099. [Abstract] [Full Text] [PDF]

				D. A. Calderwood Integrin activation J. Cell Sci., March 1, 2004; 117(5): 657 - 666. [Abstract] [Full Text] [PDF]

				G.A. Stouffer and S.S. Smyth Effects of Thrombin on Interactions Between {beta}3-Integrins and Extracellular Matrix in Platelets and Vascular Cells Arterioscler. Thromb. Vasc. Biol., November 1, 2003; 23(11): 1971 - 1978. [Abstract] [Full Text] [PDF]

				U. P. Naik and M. U. Naik Association of CIB with GPIIb/IIIa during outside-in signaling is required for platelet spreading on fibrinogen Blood, August 15, 2003; 102(4): 1355 - 1362. [Abstract] [Full Text] [PDF]

				W. T. Barry, C. Boudignon-Proudhon, D. D. Shock, A. McFadden, J. M. Weiss, J. Sondek, and L. V. Parise Molecular Basis of CIB Binding to the Integrin alpha IIb Cytoplasmic Domain J. Biol. Chem., August 2, 2002; 277(32): 28877 - 28883. [Abstract] [Full Text] [PDF]