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...

No abstract

Phagocytosis is a process of innate immunity that allows for the enclosure of pathogens within the phagosome and their subsequent destruction through the production of reactive oxygen species (ROS). Although these processes have been associated with increases of intracellular Ca2+ concentrations, the mechanisms by which Ca2+ could regulate the different phases of phagocytosis remain unknown. The aim of this study was to investigate the Ca2+ signaling pathways involved in the regulation of FcγRs-induced phagocytosis. Our work focuses on IgG-opsonized zymosan internalization and phagosomal ROS production in DMSO-differentiated HL-60 cells and neutrophils. We found that chelation of intracellular Ca2+ by BAPTA or emptying of the intracellular Ca2+ store by thapsigargin reduced the efficiency of zymosan internalization. Using an small interfering RNA strategy, our data establish that the observed Ca2+ release occurs through two isoforms of inositol 1,4,5-triphosphate receptors, ITPR1 and ITPR3. In addition, we provide evidence that phagosomal ROS production is dependent on extracellular Ca2+ entry. We demonstrate that the observed Ca2+ influx is supported by ORAI calcium release-activated calcium modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1). This result suggests that extracellular Ca2+ entry, which is required for ROS production, is mediated by a store-operated Ca2+ mechanism. Finally, our data identify the complex formed by S100A8 and S100A9 (S100 calcium-binding protein A8 and A9 complex), two Ca2+-binding proteins, as the site of interplay between extracellular Ca2+ entry and intraphagosomal ROS production. Thus, we demonstrate that FcγR-mediated phagocytosis requires intracellular Ca2+ store depletion for the internalization phase. Then phagosomal ROS production requires extracellular Ca2+ entry mediated by Orai1/STIM1 and relayed by S100A8–A9 as Ca2+ sensor.

No abstract