We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the ␤ 3 integrin and a P53L mutation in glycoprotein (GP) Ib␣. We show that GPIb␣-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The ␤ 3 -H723 causes constitutive, albeit partial, activation of the ␣ IIb ␤ 3 complex by disruption of the highly conserved cytoplasmic salt bridge with arginine 995 in the ␣ IIb integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO ␣ IIb ␤ 3 -H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, ␣ IIb ␤ 3 -H723, but not wild-type ␣ IIb ␤ 3 , generates a signal that leads to the formation of proplatelet-like protrusions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34 ؉ stem cell-derived megakaryocytes. We conclude that the constitutive activation of the ␣ IIb ␤ 3 -H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree. IntroductionInherited thrombocytopenias are a rare group of diseases with a wide spectrum of clinical phenotypes. Because of their rare occurrence, patients with an inherited low platelet count may be misdiagnosed with autoimmune thrombocytopenia (ITP) and receive inappropriate therapy. 1,2 Among the inherited thrombocytopenias, macrothrombocytopenia constitutes a subgroup in which Bernard-Soulier Syndrome (BSS), caused by mutations in the GP1BA, GP1BB, and GP9 genes, is the most common one, with large platelets and severe bleeding. 3 The molecular mechanisms of some of the rarer syndromes which are accompanied by macrothrombocytopenia have also been elucidated. Mutations in the myosin heavy-chain protein (MYH9) were identified in groups of patients with a spectrum of platelet disorders, such as the MayHegglin anomaly and the Epstein, Fechtner, and Sebastian syndromes. 4,5 The mode of inheritance of these disorders is generally autosomal recessive, but autosomal-dominant forms of a BSS-like disorder caused by nonsynonymous single-nucleotide polymorphisms (nsSNPs) in the GP1BA gene have also been reported. 6 More recently, mutations in the transcription factor GATA1 were defined as the cause of X-linked macrothrombocytopenia. 7 The most frequent autosomal-recessive platelet bleeding disorder is Glanzmann thrombasthenia (GT), which is caused by mutations in the ITGA2B or ITGB3 genes that encode for the integrin ␣ IIb ␤ 3 . This integrin, also named platelet glycoprotein (GP) IIbIIIa, is the the most abundantly expressed platelet membrane glycoprotein, 8 and its role is pivotal for platelet function. 9,10 Qualitative and quantitative defects...

We have investigated the structural requirements of the ␤ 3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during ␣ v ␤ 3 -mediated cell spreading. Using CHO cells transfected with various ␤ 3 mutants, we demonstrate a close correlation between ␣ v ␤ 3 -mediated cell spreading and tyrosine phosphorylation of FAK and paxillin, and highlight a distinct involvement of the NPLY 747 and NITY 759 motifs in these signaling processes. Deletion of the NITY 759 motif alone was sufficient to completely prevent ␣ v ␤ 3 -dependent focal contact formation, cell spreading, and FAK/paxillin phosphorylation. The single Y759A substitution induced a strong inhibitory phenotype, while the more conservative, but still phosphorylation-defective, Y759F mutation restored wild type receptor function. Alanine substitution of the highly conserved Tyr 747 completely abolished ␣ v ␤ 3 -dependent formation of focal adhesion plaques, cell spreading, and FAK/paxillin phosphorylation, whereas a Y747F substitution only partially restored these events. As none of these mutations affected receptorligand interaction, our results suggest that the structural integrity of the NITY 759 motif, rather than the phosphorylation status of Tyr 759 is important for ␤ 3 -mediated cytoskeleton reorganization and tyrosine phosphorylation of FAK and paxillin, while the presence of Tyr at residue 747 within the NPLY 747 motif is required for optimal ␤ 3 post-ligand binding events.Anchorage of cells to the extracellular matrix is mediated in part by integrins, a large family of heterodimeric cell surface receptors, that regulate numerous aspects of cell behavior, such as cell motility, proliferation, differentiation, and apoptosis (1). Cell engagement with extracellular matrix ligands induces integrin translocation to subcellular structures known as focal adhesion plaques that form at regions of close contact between the cell and its underlying substratum (2). Integrin clustering at focal contact sites in turn triggers major intracellular events, including cytoskeleton reorganization, intracellular ion transport, phosphoinositide turnover, kinase activation, and tyrosine phosphorylation of intracellular proteins (3). A large number of tyrosine-phosphorylated proteins have been identified within focal adhesion plaques. These include cytoskeletal proteins, kinases and adaptor proteins, growth factor receptors, and growth factor receptor-related signaling molecules, thus emphasizing the potential role of integrins as recruiting centers for molecules involved in various signaling pathways.Although the link of integrins with focal adhesions is well established, the precise mechanism by which integrins associate with cytoskeletal proteins, regulate focal adhesion plaque assembly, and participate in the activation of intracellular signaling cascades is still unclear. There is convincing evidence that integrin ␤ subunits are likely to play a major role in these processes: (i) truncation of the ␤ subuni...

BackgroundInitially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion.Methodology/Principal FindingsTo study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-δ isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process.Conclusions/SignificanceAltogether these findings quantitatively demonstrate for the first time that L-plastin contributes to the fine-tuning of actin turn-over, an activity which is regulated by Ser5 phosphorylation promoting its high affinity binding to the cytoskeleton. In carcinoma cells, PKC-δ signaling pathways appear to link L-plastin phosphorylation to actin polymerization and invasion.

Talin establishes a major link between integrins and actin filaments and contains two distinct integrin binding sites: one, IBS1, located in the talin head domain and involved in integrin activation and a second, IBS2, that maps to helix 50 of the talin rod domain and is essential for linking integrin ␤ subunits to the cytoskeleton (Moes, M., Rodius, S., Coleman, S. J., Monkley, S. J., Goormaghtigh, E., Tremuth, L., Kox, C., van der Holst, P. P., Critchley, D. R., and Kieffer, N. (2007) J. Biol. Chem. 282, 17280 -17288). Through the combined approach of mutational analysis of the ␤3 integrin cytoplasmic tail and the talin rod IBS2 site, SPR binding studies, as well as site-specific antibody inhibition experiments, we provide evidence that the integrin ␤3-talin rod interaction relies on a helix-helix association between ␣-helix 50 of the talin rod domain and the membrane-proximal ␣-helix of the ␤3 integrin cytoplasmic tail. Moreover, charge complementarity between the highly conserved talin rod IBS2 lysine residues and integrin ␤3 glutamic acid residues is necessary for this interaction. Our results support a model in which talin IBS2 binds to the same face of the ␤3 subunit cytoplasmic helix as the integrin ␣IIb cytoplasmic tail helix, suggesting that IBS2 can only interact with the ␤3 subunit following integrin activation.Integrins are ␣␤ heterodimeric receptors that mediate attachment of cells to the extracellular matrix (ECM) and therefore play important roles in cell adhesion, migration, proliferation, and survival (2, 3). Integrin clustering at sites of cellular attachment to the ECM triggers the assembly of large multifunctional submembrane protein complexes called focal adhesions (FAs), 5 that orchestrate the two-directional processing of stimuli across the cell membrane (4, 5). Both the integrin ␣ and ␤ chains participate in regulating the integrin extracellular ligand binding capacity, while the ␤ chain alone appears to link integrins to the actin cytoskeleton. Among the cytoskeletal proteins that directly interact with the ␤ chain cytoplasmic domain, four proteins: talin, ␣-actinin, filamin, and tensin possess both integrin and actin binding affinities (6, 7). Talin and ␣-actinin also bind to vinculin, an additional major component of FAs with actin binding activity.Talin and ␣-actinin are of particular interest as they have two integrin binding sites as well as multiple vinculin binding sites and display a similar structural organization comprising an extended rod domain composed of ␣-helical bundles. The ␣-actinin central rod domain, which is composed of 4 spectrin repeats of 3 ␣-helices (8, 9), associates with integrin ␤ subunits as well as vinculin through distinct helix-helix interactions (10 -16). Also, the 3 major talin rod-vinculin head (Vh) contacts have a similar architecture based on hydrophobic helixhelix interactions (17)(18)(19). Other FA proteins, such as members of the paxillin supergene family comprising paxillin, Hic-5, leupaxin, and PaxB, rely on an ␣-helical LD motif for their inter...