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Blood, 1 March 2006, Vol. 107, No. 5, pp. 1925-1932. Prepublished online as a Blood First Edition Paper on November 17, 2005; DOI 10.1182/blood-2005-10-3964. This Article Abstract Full Text (PDF) Supplemental Table and Figures All Versions of this Article: 2005-10-3964v1 107/5/1925    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Nakamura, F. Articles by Ylänne, J. Search for Related Content PubMed PubMed Citation Articles by Nakamura, F. Articles by Ylänne, J. Related Collections Hemostasis, Thrombosis, and Vascular Biology Cell Adhesion and Motility Cytoskeleton Free Research Articles Related Article in Blood Online Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY The structure of the GPIb–filamin A complex Fumihiko Nakamura, Regina Pudas, Outi Heikkinen, Perttu Permi, Ilkka Kilpeläinen, Adam D. Munday, John H. Hartwig, Thomas P. Stossel, and Jari Ylänne From the Hematology Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA; Biocenter Oulu and Department of Biochemistry, University of Oulu, Finland; Laboratory of Organic Chemistry, Department of Chemistry, and Institute of Biotechnology, University of Helsinki, Finland; Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia; and Department of Biological and Environmental Science, University of Jyväskylä, Finland.    Abstract Top Abstract Introduction Materials and methods Results Discussion References   Filamin A (FLNa), a dimeric actin cross-linking and scaffold protein with numerous intracellular binding partners, anchors the platelet adhesion glycoprotein (GP) Ib-IX-V receptor to actin cytoskeleton. We mapped the GPIb binding site to a single domain of FLNa and resolved the structure of this domain and its interaction complex with the corresponding GPIb cytoplasmic domain. This is the first atomic structure of this class of membrane glycoprotein–cytoskeleton connection. GPIb binds in a groove formed between the C and D strands of FLNa domain 17. The interaction is strikingly similar to that between the 7 integrin tail and a different FLNa domain, potentially defining a conserved motif for FLNa binding. Nevertheless, the structures also reveal specificity of the interfaces, which explains different regulatory mechanisms. To verify the topology of GPIb-FLNa interaction we also purified the native complex from platelets and showed that GPIb interacts with the C-terminus of FLNa, which is in accordance with our biochemical and structural data.    Introduction Top Abstract Introduction Materials and methods Results Discussion References   Filamin A (FLNa), previously called actin-binding protein or ABP-280, is the major nonmuscle isoform of a family of extended dimeric proteins. FLNa subunits have a conserved actin-binding domain at their N-termini linked to 24 tandem filamin type immunoglobulin-like domains interrupted by 2 flexible hinges and dimerizing through the self-association of the ultimate C-terminal domain. Because of this unique structure, FLNa molecules can induce actin filaments into a 3-dimensional gel that represents a characteristic cortical actin structure.1 Additional functions of FLNa are to anchor cell-surface receptors to the actin cytoskeleton and to scaffold cytoplasmic signaling proteins. Altogether, more than 50 interaction partners of FLN are known.2,3 Human melanoma cells that do not express FLNa have impaired locomotion and unstable membranes that spasmodically bleb. Expression of FLNa restores the translational locomotion and reduces membrane blebbing.4 Using these cell lines, impairments in functions ascribed to FLNa's binding to its many partners have been documented.2 Furthermore, the importance of FLNa during development has become apparent from genetic analysis of a wide range of human diseases, the versatility of which highlights distinct FLNa-partner interactions.5,6 The glycoprotein (GP) Ib is a component of the platelet von Willebrand factor (VWF) receptor identified as the first FLNa-binding partner 2 decades ago. The GPIb-V-IX receptor consists of 4 transmembrane subunits: GPIb, disulfide-linked to GPIb, and the noncovalently associated GPIX and GPV components, in ratios of 2:2:2:1.7 The cytoplasmic tails of GPIb are attached to actin filaments by FLNa.2,8,9 Previous work showed that the GPIb cytoplasmic domain interacted with C-terminal domains of FLNa (domains 17-19)10 and delimited the GPIb binding site for FLNa to a short span of residues 557 to 575.11 The GPIb-FLNa interaction is essential for the platelet adhesion to VWF that binds extracellular domain of the GPIb-V-IX receptor, for maintaining normal platelet integrity and shape, and for normal signal transduction reactions involved in platelet activation.12,13 The absence of, or mutations in, VWF or in the GPIb-V-IX receptor are responsible for von Willebrand disease14 or Bernard-Soulier syndrome,15 respectively, disorders characterized by either bleeding or increased thrombosis. Additional roles of the GPIb-V-IX receptor in vascular biology have been recognized due to the identification of novel adhesive ligands for the receptor such as P-selectin, M2 integrin, the coagulation factors thrombin and factors XI and XII, and the major extracellular matrix protein, collagen.16 Rearrangements of the GPIb-IX-V receptor of platelets mediate the clearance by M2 on Kupffer cells in the liver of platelets exposed to cold temperatures, a phenomenon that greatly affects clinical platelet transfusion technology.17 Because of its physiologic importance, we characterized the GPIb-FLNa interaction in detail. Here we report mapping of the GPIb binding site to the FLNa domain 17 (IgFLNa17), nuclear magnetic resonance (NMR) structure of this domain, and a crystal structure of the GPIb peptide–IgFLNa17 complex. Collectively, our data define the atomic structure of the GPIb-FLNa interface. Recently, the crystal structure of the integrin 7–FLNa complex has also been solved45 and the 2 structures reveal not only marked similarity, which potentially provides a motif for FLNa-partner complexes, but also specificity, which explains the regulatory mechanism for integrin 7 binding. Although previous reports suggested that the complex is tight, isothermal titration calorimetry analysis of IgFLNa17 and GPIb556-577 peptide demonstrates that the tightness of the complex is explained only by engaging both sites on FLNa molecules with the corresponding alpha chains of a single VWF receptor. Finally, we report the electron micrographs of the native GPIb-FLNa complex from human platelets, the first visualization of a native complex of a transmembrane receptor with a cytoskeletal protein. The micrographs show that VWF receptor globules localize to a central position of the "V"-shaped filamentous FLNa dimer in accordance with the mapping data and suggest that complex is stable. Based on these data, we propose that the higher order (or topological) structure determines the tight-binding avidity of FLNa to the GPIb-IX-V receptor.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   Reagents The antibodies, peptides, proteins, and DNA constructs used in this study are described in the supplemental experimental procedures (available on the Blood website; see the Supplemental Materials link at the top of the online article). In vitro binding assay Binding of recombinant proteins to the GPIb was tested by mixing GST-GPIb515-610 or GST with purified recombinant human FLNa fragments or whole protein in 400 µL binding buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% [vol/vol] Tween 20, 1 mM EGTA) in the presence or absence of synthetic GPIb556-577 peptide. After incubation at room temperature for 1 hour, 10 µL glutathione-Sepharose beads were added and further incubated for 30 minutes. The beads were washed 4 times with binding buffer and proteins were solubilized in sodium dodecyl sulfate (SDS) sample buffer. Proteins were then resolved by SDS–polyacrylamide gel electrophoresis (PAGE; 9.0% or 15.0% [wt/vol]) and stained with Coomassie brilliant blue. To test FLNa interaction with the synthetic GPIb556-577 peptide (peptide precoupled to NHS-Sepharose 4B), the same process was repeated with the 5 µL beads per assay. In vivo binding assay Green fluorescent protein (GFP)–FLNa and its mutants were transiently expressed in CHO-GPIb/IX cells.18 After transfection (72 hours) the cells were rinsed with phosphate-buffered saline (PBS) and lysed in PBS containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM EGTA, 1 mM EDTA, 1 µg mL-1 pepstatin A, 1 µg mL-1 aprotinin, and 1 µg mL-1 leupeptin. Cell lysates were centrifuged at 15 000g for 10 minutes. Lysates were precleaned with GammaBind Plus Sepharose (Amersham Biosciences, Piscataway, NJ) and incubated with 2 µg goat polyclonal anti-GPIb antibody for 1 hour at 4°C followed by 30 minutes incubation with 20 µL GammaBind Plus Sepharose (50% slurry) at 4°C. Samples were washed 4 times with 500 µL ice-cold PBS containing 1% Triton X-100, 5 mM EGTA, and 1 mM EDTA, and resuspended in 30 µL 2 x sample buffer and heated for 7 minutes at 95°C. Proteins were resolved on 9% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk in TTBS (20 mM Tris-HCl [pH 7.4], 140 mM NaCl, 0.05% Tween 20) for 1 hour and incubated with the primary monoclonal antibodies (mAbs) anti-GFP or anti-GPIb for 2 hours. Immunoreactive bands were detected by enhanced chemiluminescence (Pierce, Rockford, IL). NMR NMR spectra were measured from 15N- and 15N/13C-labeled IgFLNa17 (residues 1863-1956) protein with Varian Unity INOVA 500 MHz (relaxation data at 23°C) and 800 MHz (all other spectra at 20 °C) spectrometers (Varian, Palo Alto, CA). All spectra were processed with VNMR (Varian) and analyzed with Sparky 3.106 (T. D. Goddard and D. G. Kneller, University of California, San Francisco). The structure calculations were made with the program CYANA 2.019,20 and further refined with AMBER 8.0 (D. A. Case et al, University of California, San Francisco, 2004). Details of the techniques are given in the supplemental experimental procedures. Crystallization A roughly equimolar mixture of IgFLNa17 and GPIb556-577 peptide was crystallized in 1.75 M ammonium phosphate (pH 8.2), and the crystals were refined by microseeding. The dataset was collected at the European Synchrotron Radiation Facility (ESRF) beamline ID23-1 (Grenoble, France). The data were processed with the XDS program21 (http://www.mpimf-heidelberg.mpg.de/~kabsch/xds/) and solved by molecular replacement with the Phaser program22 (http://www-structmed.cimr.cam.ac.uk/phaser/). Arp/Warp 6.1.123 (http://www.embl-hamburg.de/ARP/) and O24 (http://alpha2.bmc.uu.se/alwyn/) were used for the model building, and Refmac525 (http://www.ccp4.ac.uk/) for the refinement. Further details are given in the supplemental experimental procedures. Coordinates The coordinates for the final 20 NMR structures, the crystallographic coordinates, and structure factors have been deposited in the Protein Data Bank (accession nos. 2AAV and 2BP3),43 and the resonance assignments in the BioMagResBank (accession no. 6730).44 Preparation of FLNa-GPIb complex from human platelets Human platelets (2.5 x 1010 cells) were prepared from freshly drawn human blood as previously described26 and lysed with 20 mL 20 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1% Triton X-100, 10 mM EGTA, 100 µg mL-1 DNAseI, 2 mM PMSF, 10 µg mL-1 aprotinin, and 10 µg mL-1 leupeptin. After centrifugation at 20 000g for 30 minutes at 4°C, the supernatant fluid (20 mL) was loaded onto HitrapQ (5 mL; Amersham Biosciences) column. The column was washed with 50 mL 10 mM Tris-HCl [pH 7.4], 250 mM NaCl, 0.01% Triton X-100, and 0.5 mM EGTA, and the FLNa-GPIb complex was eluted with a 100 mL linear gradient of 100 to 600 mM NaCl in 10 mM Tris-HCl (pH 7.4), 0.01% Triton X-100, and 0.5 mM EGTA at a flow rate of 2 mL min-1. Fractions containing the FLNa-GPIb complex (eluting around 480 mM NaCl) were pooled and absorbed on a mAb3-14–Sepharose (0.6 mL). After washing the column with 10 mL HNEC (10 mM HEPES-NaOH [pH 7.2], 200 mM NaCl, 0.1 mM EGTA, and 1.0% CHAPS) solution, the FLNa-GPIb complex was eluted with 4.5 mL GST-IgFLNa3 (5 mg mL-1 HNEC solution). The eluate was passed through glutathione-Sepharose (3 mL) to remove GST-IgFLNa3, and the FLNa-GPIb complex was absorbed on WGA-agarose (1 mL; Vector Laboratories, Burlingame, CA). After washing the column with 10 mL HNEC solution, the FLNa-GPIb complex was eluted with 6 mL 2.5% (wt/vol) N-acetylglucosamine in HNEC solution. The complex was concentrated to 0.2 mg mL-1 using an Amicon Ultra-15 (Millipore, Billerica, MA) with a molecular-weight cutoff of 50 000. For electron microscopy, 60 mM (final concentration) octyl -D-glucopyranoside was added. Electron microscopy The structure of recombinant FLNa molecules and GPIb-FLNa complexes isolated from human platelets was determined in rotary shadowed specimens. Samples were applied to freshly cleaved mica, dried under vacuum, and shadowed with 1.0 nm of platinum at a 5° angle with rotation followed by 4 nm of carbon at 90° without rotation (Cressington CFE-60 apparatus, Cressington, Watford, United Kingdom). Metal replicas were floated in distilled water, picked up on 200 mesh carbon grids, and examined and photographed in a JEOL JEM-1200 EX electron microscope at an accelerating voltage of 80 kV as previously described.27 Electron micrographs were digitized on an Epson 1680 scanner at 1200 dpi and printed from Adobe Photoshop version 7.0 (Adobe Systems, San Jose, CA).    Results Top Abstract Introduction Materials and methods Results Discussion References   The cytoplasmic domain of GPIb interacts with FLNa domain 17 in vitro To characterize the interaction of GPIb and FLNa in more detail, we performed in vitro reconstitution and competition assays using recombinant protein and synthetic peptides. A GST fusion protein encompassing residues 515 to 610 of the cytoplasmic tail of GPIb bound to FLNa. A synthetic 22-mer peptide (hereafter designated GPIb556-577), including the previously defined FLNa-binding site11,28 (Figure 1A), completely inhibited this binding (Figure 1B) and also efficiently bound full-length FLNa when immobilized on Sepharose beads (Figure 1C). Figure 1D shows that GST-fusion proteins representing IgFLNa17 and 19, but not 18, bound to GPIb556-577 beads. Untagged IgFLNa constructs also bound GPIb515-610, but IgFLNa17 bound with a higher apparent affinity than IgFLNa19 (Figure 1E). Isothermal titration calorimety demonstrated that GPIb556-577 has affinity to IgFLNa17 (Kd = 11 µM; Figure S1). Furthermore, IgFLNa19 did not bind GPIb515-610 in the presence of a 1:1 molar mixture with IgFLNa17 (Figure 1F). Most important, IgFLNa17, but not IgFLNa19, competed with full-length FLNa for binding to GST-GPIb515-610 (Figure 1G). Taken together, the results indicate that IgFLNa17 is the major binding site for GPIb. View larger version (52K): [in this window] [in a new window]   Figure 1.. Identification of protein domains responsible for GPIb/FLNa interactions. (A) Amino acid sequence of the synthetic 22-mer peptide (GPIb556-577) that binds FLNa and was used for crystallography. (B) Binding of purified recombinant human FLNa (2µg 400µL-1) to GST-GPIb515-610 (5µg 400µL-1) is inhibited by the 22-mer peptide, GPIb556-577, in a dose-dependent manner. (C) Full-length FLNa (2µg 400µL-1) binds the GPIb556-577 peptide immobilized on Sepharose 4B but not Sepharose 4B alone. (D) In vitro binding of GST-IgFLNa17, -18, and -19 (2 µg each) to the GPIb556-577 peptide beads. Top and bottom rows indicate bound and input proteins to GPIb556-577, respectively. (E) Tag-free IgFLNa17 binds GPIb556-577 peptide with higher affinity than IgFLNa19. Increasing amount of purified IgFLNa17 or -19 were incubated with GPIb556-577 peptide beads. (F) IgFLNa17 is the major binding site for GPIb556-577 peptide. Various amounts of IgFLNa17 and 19 at 1:1 molar ratio were incubated with GPIb556-577 peptide beads in the pull-down experiment shown. (G) FLNa17 but not FLNa19 interferes the binding of full-length FLNa to GPIb peptide beads. Various amounts of FLNa17 or -19 were mixed with 1 µg purified full-length FLNa and then incubated with GPIb556-577 peptide beads. (B-G) Bound proteins were resolved by SDS-PAGE, and stained with Coomassie blue.   The structure of FLNa domain 17 determined by NMR The structure of free IgFLNa17 was solved by high-resolution solution state NMR spectroscopy (Table 1). A family of 20 structures is presented in Figure 2A. The structure consists of a -sandwich of relatively straight 3- and 4-strand -sheets (Figure 2A) resembling other FLN-type immunoglobulin-like domains (Table S1). In all, the solution structure of the domain is well defined (Table 1), but it contains 2 relatively loose loops close to the domain N-terminus (loops BC, DE; Figure 2A). In the case of loop BC this can be explained by a structural flexibility, which is visible in the absence of long-range nuclear Overhauser effects (NOEs), and is revealed by the relaxation time measurements (Figure S2B). The absence of long-range NOEs in the protruding loop DE leaves its structure ill-defined (Figure S2A). The titration of a 15N-labeled IgFLNa17 sample with the GPIb556-577 peptide showed that the peptide interaction with IgFLNa17 was in slow exchange in the NMR time scale. Thus, the binding could not be followed directly by observing chemical shift changes in the 1H-15N HSQC spectrum of the IgFLNa17 (data not shown), and would have required a full assignment for the spectra of the complex form. This was not pursued further, as parallel crystallization efforts for the complex form proved to be successful. View this table: [in this window] [in a new window]   Table 1.. NMR restraints   View larger version (52K): [in this window] [in a new window]   Figure 2.. Structure of IgFLNa17 alone and in complex with the GPIb peptide. (A) A family of 20 NMR structures of the IgFLNa17 in solution. (B) Asymmetric unit of the crystal. Two molecules of IgFLNa17 are colored green and blue (chains A and B, respectively). The GPIb peptides are magenta (chain S) and gold (chain T). The N and C termini of all chains are shown. Note that the peptide T interacts with both FLN molecules. (C) Superimposition of the crystal structure (green) and the mean NMR structure (red). (D) Surface properties of the FLNa-GPIb556-577 interaction. The accessible surface of IgFLNa17 has been colored according to surface potential and the peptide is shown on the top (gold and atom color). This representation shows the extended conformation of the peptide in the complex and the hydrophopic character of the interaction. (E) Main chain hydrogen bonding between the peptide (green) and IgFLNa17 strand C. (F) Details of the peptide interaction. An electron density map calculated with the final model without the peptide at the level of 1.4 shows that the side chains of the peptide are well defined. The G1897 and C1912 residues of FLNa that were mutated in subsequent studies are shown in magenta. Residue numbers for the peptide are shown in black and for FLNa in green.   The crystal structure of FLNa domain 17 in complex with the GPIb peptide Well-diffracting crystals were obtained from cocrystallization experiments of IgFLNa17 with the GPIb556-577 peptide. The structure of the complex was solved by molecular replacement with a model from the IgFLNc24,29 and refined by using data to 2.3 Å resolution. The final model had good quality indicators and stereochemistry (Table 2). The asymmetric unit of the crystal contained 2 molecules of the IgFLNa17 and peptides binding to each of them (Figure 2B). The 2 molecules were refined independently and revealed structures and interactions very similar to the GPIb peptide. The difference was that the peptide T (IgFLNa17 chains A and B as well as peptide chains T and S are indicated in Figure 2B) binding to molecule A also interacted through its N-terminus with the molecule B. In the peptide S, the N-terminus was disordered in the crystal and not visible in the electron density. Our interpretation is that the interaction between the peptide that is the same in both molecules represents the physiologic interaction between the native proteins, and that the binding of the peptide T to molecule B is specific for the crystal arrangement. Interestingly, the IgFLNa17 does not undergo any major structural rearrangements during GPIb556-577 peptide binding, as the IgFLNa17 structures are essentially identical in both solution state (NMR) and in the complex form (x-ray) structures (Figure 2C). View this table: [in this window] [in a new window]   Table 2.. Crystallographic data collection statistics   In the crystal structure of the complex, residues 560 to 573 of the GPIb peptide (LPTFRSSLFLWVRP) interact with the strands C and D of IgFLNa17, and form a hydrogen-bonded strand next to strand C of IgFLNa17 (Figure 2D-E). The segment of the peptide adopting this conformation is flanked by 2 proline residues (Pro561 and Pro573). In addition to main-chain hydrogen bonding (Figure 2E), a side-chain hydroxyl group of Ser565 makes a hydrogen bond with the main-chain O atom of Ala1908 (Figure 2F). On the FLNa side, the interface contains 43% polar and 57% nonpolar atoms. The main hydrophobic contacts are between Leu567 (13.27% of the contact surface area) of the peptide and Ile1910 and Cys1912 at strand D of FLNa. Also Phe563 (11.21%), Leu569 (9.67%), Val571 (13.67%), Phe568 (5.44%), and Trp570 (6.52%) of the peptide make remarkable hydrophobic contacts with FLNa (Figure 2D). These findings are consistent with previous observations that mutations of Phe568 and Trp570 abolish the binding of GPIb to FLNa.28 Furthermore, the association between GPIb and FLNa resists chaotropic solvents over a wide pH range, although sodium dodecyl sulfate can separate the complex.30 This behavior is consistent with the many hydrophobic contacts in the FLNa-GPIb interface. Point mutations in domain 17 of full-length FLNa disrupt the GPIb/FLNa interaction in vivo To test if the GPIb complex interacts with FLNa through its IgFLNa17 in vivo, we engineered point mutations in IgFLNa17 residues (Gly1897, Ile1910, and Cys1912) that are located close to the peptide-FLNa interface seen in the crystal structure (Figures 2F and 3A). A fusion protein consisting of maltose-binding protein and IgFLNa17 or the I1910M mutant bound indistinguishably to GPIb556-577 (Figure 3B). However, mutations G1897D or C1912D reduced binding, and the double mutant did not bind at all (Figure 3B). NMR spectroscopy confirmed that the double mutant caused no major defects in folding of the domain (data not shown). We also expressed GFP-tagged full-length human FLNa in the CHO-GPIb/IX cells that stably express GPIb/IX complex,18 and were able to bring down FLNa with the GPIb/IX complex (Figure 3C). Double-mutant (G1879D and C1912D) GFP-FLNa constructs expressed at levels equivalent to endogenous FLNa in CHO cells did not associate at all with GPIb/IX (Figure 3C), confirming that IgFLNa17 is also the major GPIb-binding site in vivo. N-ethylmaleimide (NEM) treatment (thiol alkylation of cysteine residues) dissociates FLNa from the GPIb complex in detergent-extracted platelets.32 Figure 3D shows that NEM also dissociated endogenous FLN expressed in CHO-GPIb/IX cells. NEM also inhibited IgFLNa17 binding to the GPIb556-577 peptide (data not shown). This is consistent with the Cys1912 and Cys1920 residues locating at or near the binding interface as seen in the crystal structure (Figure 2F). Thus, NEM inhibition and the mutagenesis results confirm that the interface seen in the crystal is also functional in solution and in vivo and that IgFLNa17 is the major interaction site for GPIb. View larger version (35K): [in this window] [in a new window]   Figure 3.. Obliteration of the binding of full-length FLNa to the GPIb complex in CHO cells by the point mutations in IgFLNa17 and by thiol alkylation. (A) ClustalW alignment31 of domain 17 of FLN isoforms. Residues inferred from the atomic structure to be involved in interactions with GPIb and tested by mutagenesis are indicated by the gray bars. Potential targets of NEM are indicated by the black bars. (B) Double mutations at G1897D and C1912D of IgFLNa17 abolish IgFLNa17/GPIb binding. Increasing amounts of purified MBP-IgFLNa17 (WT, wild type) and its mutants (G1897D, I1910M, C1912D, and G1897D+C1912D) were incubated with GPIb556-577–peptide beads, and bound proteins were detected by SDS-PAGE followed by Coomassie blue staining. (C) GFP-FLNa and its mutants were expressed in CHO-GPIb/IX cells and coimmunoprecipitated with the GPIb-IX complex. GFP-FLNa is detected by immunoblot with anti-GFP mAb. The intensity of the band was analyzed by densitometry and the relative intensity graphed. Data are presented as mean ± SD of 3 independent experiments. (D) FLN is expressed in CHO-GPIb/IX cells and coimmunoprecipitated with the GPIb-IX complex. The linkage is uncoupled by treating the cell lysate with cystein-blocking reagent, NEM. Since only anti-FLNb, but neither anti-FLNa nor anti-FLNc antibodies available cross-react with hamster FLN, immunoblot was performed with anti-FLNb mAb (1-11c). IP indicates immunoprecipitation; IB, immunoblot.   Electron microscopy of the GPIb-FLNa complex prepared from human platelets To investigate its intact structure, the native GPIb-FLNa complex was purified from human platelets using affinity ligands, anti-FLNa mAbs and wheat germ agglutinin (WGA) immobilized on beads to ligate FLNa and GPIb, respectively. When the purified complex was examined by SDS-PAGE, Coomassie blue stained exclusively the FLNa subunits (Figure 4B, left blot). However, immunoblotting with anti-GPIb mAbs demonstrated the presence of GPIb in the purified complex (Figure 4B, center blot). Subsequent immunoblotting of the same membrane with anti-FLNa mAb showed a nearly equal ratio of GPIb to FLNa (Figure 4B, right blot). In rotary shadowed images, the purified GPIb-FLNa complexes were monodisperse (Figure 4C). As previously described for purified FLNa molecules, semiflexible filamentous strands connect at one end to assume a "V"-shape (Figure 4C, top row). The FLNa-GPIb-IX-V complexes appeared similar, except for their decoration by globules of 22.6 ± 3.7 nm in diameter (mean ± SD; n = 50) localized to a central position of the FLNa molecules (Figure 4C, bottom row). Given that FLNa molecules dimerize at the carboxy-terminus of each chain, and that the purified GPIb-IX complex has globular domains of comparable size (15.9 and 8.9 nm),33 the images are consistent with the localization of the GPIb-IX-V interaction site to C-terminus of FLNa (IgFLNa17-19),10 and with the general schematic depiction of how the complex holds together (Figure 4A). 34 View larger version (96K): [in this window] [in a new window]   Figure 4.. Schematic drawing and electron micrograph of the GPIb-FLNa complex from human platelets. (A) The cytoplasmic domain of GPIb of the GPIb-IX-V complex binds to actin filaments through FLNa. (B) GPIb-FLNa complex from human platelets. Coomassie-blue-stained SDS-PAGE (10%) of a concentrated GPIb-FLNa complex (left). Immunoblot of the GPIb-FLN complex with anti-GPIb mAb (WM23) (center). The same blotting membrane was subsequently blotted with an anti-FLNa mAb 3-14 (right). (C) Electron micrographs of rotary-shadowed recombinant FLNa molecules (top) and GPIb-FLNa complexes (bottom).      Discussion Top Abstract Introduction Materials and methods Results Discussion References   The GPIb-IX-V receptor interacts with FLNa through IgFLNa17 The GPIb binding site has been previously mapped to a FLNa fragment consisting of IgFLNa17-19.10 Here we have further narrowed this region and showed that IgFLNa17 is the main interaction site, while IgFLNa19 has some affinity. Our competition experiments clearly showed that IgFLNa17 has a higher affinity for the GPIb556-577 peptide than IgFLNa19 and that IgFLNa17 can compete with full-length FLNa for binding to the peptide, whereas IgFLNa19 cannot. Furthermore, specific point mutations of IgFLNa17, designed according to the crystal structure, prevent full-length FLNa binding to GPIb in coimmunoprecipitation experiments. Our electron microscope images of the GPIb-FLNa complex were fully consistent with the interaction site residing close to the C-terminus of the FLNa subunit. The hydrophobic CD face of FLNa domains is a common binding interface for the GPIb and integrin The crystal structure of the IgFLNa17-GPIb556-577 peptide complex revealed that the peptide interacts with -strands C and D of the IgFLNa17. The peptide forms a -strand that is hydrogen-bonded to strand C and has several hydrophobic contacts with residues of both strands C and D. Apparently both the hydrophobic contacts and hydrogen bonding define the specificity of the interaction. It is interesting that the interaction of GPIb with IgFLNa17 is very similar to that of integrin 7 cytoplasmic tail peptide with IgFLNa21.45 Although the overall similarity is very low between the GPIb and integrin cytoplasmic domains, the FLNa interaction sites are in both cases flanked by Pro residues, contain conserved Phe/Tyr and Ser residues, and take a strand conformation in the complex (Figure 5). The strand is associated with the strand C of the IgFLN in both cases, and conserved, but not identical, hydrophopic residues point toward strand D. Interestingly, IgFLNc24 also self-associates via strands C and D, and we predicted that this is a common dimerization interface for all mammalian FLNs.29 Our present results allow us to generalize even further and suggest that the CD face of the immunoglobulin sandwich is a common interaction surface for IgFLN domains and their partners. For example, the primary structure of this interface of IgFLNa17 is highly conserved in the corresponding domains of other FLN isoforms (FLNb and FLNc; Figure 4A), and FLNb is known to interact with GPIb.35,36 Despite the finding of many FLN-binding partners, one of the major issues of FLN research is that critical information of the biological significance of their individual interaction is limited because in most cases the binding sites have not been narrowed down to a single IgFLN domain or to a short sequence on the partner. Although it is still too early to define the "FLN-binding motif" of FLN-binding partners with 1 GPIb and 4 -integrin sequences (Figure 5), our data suggest specific target amino acid for the mutagenesis experiments. For example, dopamine D2 and D3 receptors that bind IgFLNa19 have significant homology with the FLN-binding site of GPIb (Figure 5).37 Since both GPIb and integrins also associate with IgFLNa19 in vitro, it is highly possible that the D2 and D3 receptors share the same binding mode. Structural basis of the different regulatory mechanism for FLNa-binding to the GPIb and integrin The most striking specific feature of the GPIb-IgFLNa17 complex is the interaction of Phe568 and Trp570 side chains with the main chain of the IgFLNa17 strand C. These residues are not conserved in integrin tails. It has been recently shown that the mutations of these residues to alanines abrogate GPIb binding to FLNa.28 There might also be some specificity in the regulation of FLNa-integrin and FLNa-GPIb interactions. While the integrin cytoskeleton interaction may be regulated by phosphorylation of Tyr or Thr residues of integrin tails,38,39 such regulation cannot take place in the case of the GPIb-FLNa interactions. The phosphorylated Tyr residue in the integrin tail is replaced by Phe in GPIb, and the phosphorylated Thr residues are not conserved at all. On the other hand, our structure raises the possibility that phosphorylation of the GPIb Ser565 might negatively regulate the interaction with FLNa because this Ser makes a side-chain hydrogen bond with the FLNa. A phosphate group in this position would be expected to prevent close contact between the peptide and FLNa. The GPIb Ser566 is also a potential regulatory site, because dopamine D2 and D3 receptors have Ser residues at the same position (Figure 5) and the D2 S358D mutant receptor, which mimics its phosphorylation state, has lower affinity to FLNa than wild type.40 However, such phosphorylation of GPIb has not been detected. View larger version (46K): [in this window] [in a new window]   Figure 5.. ClustalW alignment of GPIb, integrin, and dopamine receptor cytoplasmic tail sequences. The FLNa-binding site of GPIb and integrin 7 from the crystal structures and the corresponding sites of other integrins and dopamine D2 and D3 receptors are boxed. Potential phosphorylation sites (Tyr, red; Thr and Ser, blue) of integrins are indicated. A putative PKC phosphorylation site in D2 and D3 receptors are marked in yellow.   On the other hand, in vitro, the GPIb-FLNa interaction can be dissociated by thiol alkylation with NEM.32 Since there are no Cys residues in the cytoplasmic domain of the GPIb, the GPIb-interacting portion of FLNa should contain Cys modifiable by NEM. Crystal structure confirms this prediction: Cys1912 is in direct hydrophobic contact with GPIb residue Leu567, while Cys1920 is located sufficiently close to the interface for its modifications to affect binding (Figure 2F). Both of these Cys residues are conserved in all 3 human isoforms of FLN (Figure 3A). Topologic structure of the GPIb-FLNa complex and its biological significance Isothermal titration calorimetry analysis showed that IgFLNa17 binds GPIb556-577 peptide with a Kd in the micromolar range (Figure S1), demonstrating that the tightness of the complex31 is not attributable to a single domain-domain interaction. Electron micrographs of the purified GPIb-FLNa complex and previous biochemical analyses support the general model of how FLNa interacts with the GPIb-IX-V complex (Figure 4A). To our knowledge, this is the first visualization of a native complex of a transmembrane receptor with a cytoskeletal protein. The micrographs support the general model derived indirectly of the overall structure of the complex, and suggest that the complex is stable as a monomeric unit in the resting platelet. Blood platelets maintain the GPIb-FLNa connection constitutively in both resting and activated states, including when subjected to high shear forces.26 Clustering of this complex is induced by platelet activation and by chilling, which are important phenomena in thrombosis41 and for transfusion medicine.42 Therefore, we propose that the higher-order (or topologic) structure determines the tight-binding avidity of FLNa to the GPIb-IX-V receptor. This immediately means that clustering of the GPIb-IX-V complex and anchoring of FLNa to actin filaments by activation could also increase avidity, which could withstand high shear stress.    Acknowledgements   Dr Helena Tossavainen is acknowledged for her assistance with AMBER and CYANA calculations, and Dr Tiila Kiema for help in crystallography. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities, and thank Dr Didier Nurizzio for assistance in using the beamline ID23-1.    Footnotes   Submitted October 4, 2005; accepted October 23, 2005. Prepublished online as Blood First Edition Paper, November 17, 2005; DOI 10.1182/blood-2005-10-3964. Supported by National Institutes of Health grants numbers 19429 (to T.P.S.) and 056252 (to J.H.H.) and the Academy of Finland grants numbers 51863, 202725, and 203675 (to J.Y.). F.N. and R.P. contributed equally to this work. The online version of this article contains a data supplement. An Inside Blood analysis of this article appears at the front of this issue. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. 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