We have used recombinant or synthetic ␣ IIb and ␤ 3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. ␣⅐␤ heterodimerization occurred in a 1:1 stoichiometry with a weak K D in the micromolar range. Divalent cations were not required for this association but stabilized the ␣⅐␤ complex by decreasing the dissociation rate. ␣⅐␤ complexation was impaired by the R995A substitution or the KVGFFKR deletion in ␣ IIb but not by the ␤ 3 S752P mutation. Integrins are ␣␤ heterodimeric cell-surface receptors that promote not only adhesion to components present within the extracellular matrix or on the surface of opposite cells but also transfer information into and out of a cell (1). The adhesive functions of integrins can be regulated by intracellular processes referred to as "inside-out signaling." Conversely, ligand binding to the extracellular domain of integrins initiates a cascade of intracellular events termed "outside-in signaling" that generate a large spectrum of cellular responses, such as cell migration, proliferation, differentiation, and gene expression (2). Integrin cytoplasmic tails appear to be key elements in these bidirectional signaling pathways, despite their short size as compared with other signaling receptors and the absence of any demonstrable catalytic activity (3, 4). Integrin ␣ and ␤ cytoplasmic domains are thought to mediate signaling events through modifications of their own structural and spatial organization and/or through interactions with specific cytoplasmic components. Various proteins have been identified that bind, at least in vitro, to the cytoplasmic tail of ␣ and ␤ subunits and are likely to play a role in regulating integrin signaling functions. These include cytoskeletal components such as talin and ␣-actinin, as well as several signaling or regulatory proteins such as integrin-linked kinase p59 ILK , focal adhesion kinase pp125FAK , Grb2, ␤ 3 -endonexin, cytohesin-1, integrin cytoplasmic domain-associated protein ICAP-1, calreticulin and calcium-and integrin-binding protein CIB 1 (reviewed in Refs. 5 and 6).Recently used methods for studying protein-protein interactions, such as the two-hybrid system, have allowed the identification of integrin-specific intracellular ligands (7-12). These methods are based on the use of a unique linear amino acid sequence as a bait and consequently do not take into account the secondary and tertiary structural features of the interacting molecules. However, numerous studies tend to demonstrate that ␣ and ␤ cytoplasmic domains adopt a defined conformation and that the preservation of these structural constraints is crucial to maintain the functional properties of integrin receptors (13)(14)(15)(16)(17)(18)(19)(20).One of the best studied integrins is the platelet fibrinogen receptor, integrin ␣ IIb ␤ 3 , that undergoes conformational changes necessary for receptor function. In order to elucidate further the structural relationship of the cytoplasmic tails of ␣ IIb and ␤ 3 , we...

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...

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S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand Ca2+-binding proteins and are abundantly expressed in the cytosol of neutrophils. In addition to their intracellular roles, S100A8/A9 can be secreted in the extracellular environment and are considered as alarmins able to amplify the inflammatory response. The intracellular activity of S100A8/A9 was shown to be regulated by S100A9 phosphorylation, but the importance of this phosphorylation on the extracellular activity of S100A8/A9 has not yet been extensively studied. Our work focuses on the impact of the phosphorylation state of secreted S100A9 on the proinflammatory function of neutrophils. In a first step, we characterized the secretion of S100A8/A9 in different stimulatory conditions and investigated the phosphorylation state of secreted S100A9. Our results on neutrophil-like differentiated HL-60 (dHL-60) cells and purified human neutrophils showed a time-dependent secretion of S100A8/A9 when induced by phorbol 12-myristoyl 13-acetate and this secreted S100A9 was found in a phosphorylated form. Second, we evaluated the impact of this phosphorylation on proinflammatory cytokine expression and secretion in dHL-60 cells. Time course experiments with purified unphosphorylated or phosphorylated S100A8/A9 were performed and the expression and secretion levels of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor alpha, CCL2, CCL3, CCL4, and CXCL8 were measured by real-time PCR and cytometry bead array, respectively. Our results demonstrate that only the phosphorylated form of the complex induces proinflammatory cytokine expression and secretion. For the first time, we provide evidence that S100A8/PhosphoS100A9 is inducing cytokine secretion through toll-like receptor 4 signaling.