Galectin-1 induces partial TCR zeta-chain phosphorylation and antagonizes processive TCR signal transduction.
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Galectin-1 is an endogenous lectin with known T cell immunoregulatory activity, though the molecular basis by which galectin-1 influences Ag specific T cell responses has not been elucidated. Here, we characterize the ability of galectin-1 to modulate TCR signals and responses by T cells with well defined hierarchies of threshold requirements for signaling distinct functional responses. We demonstrate that galectin-1 antagonizes TCR responses known to require costimulation and processive protein tyrosine phosphorylation, such as IL-2 production, but is permissive for TCR responses that only require partial TCR signals, such as IFN-gamma production, CD69 up-regulation, and apoptosis. Galectin-1 binding alone or together with Ag stimulation induces partial phosphorylation of TCR-zeta and the generation of inhibitory pp21zeta. Galectin-1 antagonizes Ag induced signals and TCR/costimulator dependent lipid raft clustering at the TCR contact site. We propose that galectin-1 functions as a T cell "counterstimulator" to limit required protein segregation and lipid raft reorganization at the TCR contact site and, thus, processive and sustained TCR signal transduction. These findings support the concept that TCR antagonism can arise from the generation of an inhibitory pp21zeta-based TCR signaling complex. Moreover, they demonstrate that TCR antagonism can result from T cell interactions with a ligand other than peptide/MHC. (+info)
Stimulatory function of gp49A, a murine Ig-like receptor, in rat basophilic leukemia cells.
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Murine gp49, a 49-kDa type I transmembrane glycoprotein, is a member of the Ig-like receptors expressed on the surface of cells involved in natural immunity such as mast cells, NK cells, and macrophages. The two major subtypes, gp49A and gp49B, are encoded by two different genes adjacent to each other. gp49B contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic region and is known to function as an inhibitory molecule. In contrast, gp49A does not harbor any specific motif for signal transduction, nor has its physiological role been determined. Here we report on the stimulatory nature of gp49A by analyzing biochemical characteristics of chimeric molecules consisting of an ectodomain of Fc receptor and a C-terminal half of gp49A, namely the pretransmembrane, transmembrane, and cytoplasmic portions, expressed on the rat basophilic leukemia mast cell line. Cross-linking of the chimeric receptors evoked cytoplasmic calcium mobilization, PGD(2) release, and transcription of IL-3 and IL-4 genes, but did not elicit degranulation of the cells. The chimeric molecule could be expressed as a singlet and a homodimeric form on the cell surface. A pretransmembrane cysteine residue of gp49A was necessary for dimer formation. Dimerization was be necessary for their incorporation into glycolipid-enriched membrane fraction (GEM) upon cross-linking stimuli. The calcium mobilization response was inhibited by treatment of cells with methyl-beta-cyclodextrin, an inhibitor of GEM formation. Together with these results, it was strongly suggested that gp49A could be expressed as a homodimer and elicit activation signals that lead to calcium mobilization, eicosanoid production, and cytokine gene transcription through its incorporation into GEM. (+info)
Tuning chemotactic responses with synthetic multivalent ligands.
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BACKGROUND: Multivalent ligands have been used previously to investigate the role of ligand valency and receptor clustering in eliciting biological responses. Studies of multivalent ligand function, however, typically have employed divalent ligands or ligands of undefined valency. How cells respond to multivalent ligands of distinct valencies, which can cluster a signaling receptor to different extents, has never been examined. The chemoreceptors, which mediate chemotactic responses in bacteria, are localized, and clustering has been proposed to play a role in their function. Using multivalent ligands directed at the chemoreceptors, we hypothesized that we could exploit ligand valency to control receptor occupation and clustering and, ultimately, the cellular response. RESULTS: To investigate the effects of ligand valency on the bacterial chemotactic response, we generated a series of linear multivalent arrays with distinct valencies by ring-opening metathesis polymerization. We report that these synthetic ligands elicit bacterial chemotaxis in both Escherichia coli and Bacillus subtilis. The chemotactic response depended on the valency of the ligand; the response of the bacteria can be altered by varying chemoattractant ligand valency. Significantly, these differences in chemotactic responses were related to the ability of the multivalent ligands to cluster chemoreceptors at the plasma membrane. CONCLUSIONS: Our results demonstrate that ligand valency can be used to tune the chemotactic responses of bacteria. This mode of regulation may arise from changes in receptor occupation or changes in receptor clustering or both. Our data implicate changes in receptor clustering as one important mechanism for altering cellular responses. Given the diverse events modulated by changes in the spatial proximity of cell surface receptors, our results suggest a general strategy for tuning biological responses. (+info)
CD73 engagement promotes lymphocyte binding to endothelial cells via a lymphocyte function-associated antigen-1-dependent mechanism.
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CD73 is a GPI-anchored lymphocyte adhesion molecule possessing an ecto-5'-nucleotidase enzyme activity. In this work, we show that engagement of lymphocyte CD73 increases lymphocyte binding to cultured endothelial cells (EC) in an LFA-1-dependent fashion. Engagement of CD73 by an anti-CD73 mAb 4G4 increases the adhesion of lymphocytes to cultured EC by about 80% compared with that of lymphocytes treated with a negative control Ab, and the increased adhesion can be blocked by an anti-CD18 mAb. The CD73-regulated increase in lymphocyte adhesion is not due to a conformational change leading to high-affinity LFA-1 receptors as assayed using mAb 24 against an activation-induced epitope of the molecule. Instead, CD73 engagement induces clustering of LFA-1 that is inhibitable by calpeptin, indicating involvement of Ca(2+)-dependent activation of a calpain-like enzyme in this process. In conclusion, the results shown here demonstrate that CD73 regulates the avidity of LFA-1 by clustering. This indicates a previously undescribed role for CD73 in controlling the poorly characterized activation step in the multistep cascade of lymphocyte extravasation. Moreover, these results suggest that in physiological conditions the activation step may result in clustering of LFA-1 rather than in an affinity change of the molecule. (+info)
Mismatched appositions of presynaptic and postsynaptic components in isolated hippocampal neurons.
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To determine whether presynaptic input is necessary for postsynaptic differentiation, we isolated hippocampal neurons in microisland culture and thus deprived pyramidal cells of GABA input and GABAergic neurons of glutamate input. We find that glutamate input is necessary for clustering the AMPA-type glutamate receptor but not for clustering the NMDA receptor or the associated PSD-95 family scaffold in GABAergic cells; GABA input is not necessary for clustering the GABA(A) receptor or gephyrin in pyramidal cells. Isolated neurons showed a surprising mismatch of presynaptic and postsynaptic components. For example, in isolated pyramidal neurons, although GABA(A) receptor clusters covered <4% of the dendritic surface and presynaptic boutons covered <12%, a full two-thirds of the GABA(A) receptor clusters were localized inappropriately opposite the non-GABAergic, presumed glutamatergic, terminals. Furthermore, inhibitory and excitatory postsynaptic components were segregated into separate clusters in isolated cells and apposed to separate boutons of a single axon. Thus, GABA(A) receptors were clustered opposite some terminals, whereas NMDA receptors were clustered opposite other terminals of a single axon. These results suggest the involvement of a synaptogenic signal common to glutamate and GABA synapses that permits experimentally induced mismatching of presynaptic and postsynaptic components in isolated neurons, as well as a second specificity-conferring signal that mediates appropriate matching in mixed cultures. (+info)
Cbl-b is a negative regulator of receptor clustering and raft aggregation in T cells.
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Stimulation of T cells via the antigen and costimulatory receptors leads to the organization of a supramolecular activation cluster called the immune synapse. We report that loss of the molecular adaptor Cbl-b in T cells frees antigen receptor-triggered receptor clustering, lipid raft aggregation, and sustained tyrosine phosphorylation from the requirement for CD28 costimulation. Introduction of the cbl-b mutation into a vav1-/- background relieved the functional defects of vav1-/- T cells and caused spontaneous autoimmunity. Wiscott Aldrich Syndrome protein (WASP) was found to be essential for deregulated proliferation and membrane receptor reorganization of cbl-b mutant T cells. Antigen receptor-triggered Ca2+ mobilization, cytokine production, and receptor clustering can be genetically uncoupled in cbl-b mutant T cells. Thus, Cbl-b functions as a negative regulator of receptor clustering and raft aggregation in T cells. (+info)
Dystroglycan overexpression in vivo alters acetylcholine receptor aggregation at the neuromuscular junction.
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Dystroglycan is a member of the transmembrane dystrophin glycoprotein complex in muscle that binds to the synapse-organizing molecule agrin. Dystroglycan binding and AChR aggregation are mediated by two separate domains of agrin. To test whether dystroglycan plays a role in receptor aggregation at the neuromuscular junction, we overexpressed it by injecting rabbit dystroglycan RNA into one- or two-celled Xenopus embryos. We measured AChR aggregation in myotomes by labeling them with rhodamine-alpha-bungarotoxin followed by confocal microscopy and image analysis. Dystroglycan overexpression decreased AChR aggregation at the neuromuscular junction. This result is consistent with dystroglycan competition for agrin without signaling AChR aggregation. It also supports the hypothesis that dystroglycan is not the myotube-associated specificity component, (MASC) a putative coreceptor needed for agrin to activate muscle-specific kinase (MuSK) and signal AChR aggregation. Dystroglycan was distributed along the surface of muscle membranes, but was concentrated at the ends of myotomes, where AChRs normally aggregate at synapses. Overexpressed dystroglycan altered AChR aggregation in a rostral-caudal gradient, consistent with the sequential development of neuromuscular synapses along the embryo. Increasing concentrations of dystroglycan RNA did not further decrease AChR aggregation, but decreased embryo survival. Development often stopped during gastrulation, suggesting an essential, nonsynaptic role of dystroglycan during this early period of development. (+info)
PSD-95 involvement in maturation of excitatory synapses.
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PSD-95 is a neuronal PDZ protein that associates with receptors and cytoskeletal elements at synapses, but whose function is uncertain. We found that overexpression of PSD-95 in hippocampal neurons can drive maturation of glutamatergic synapses. PSD-95 expression enhanced postsynaptic clustering and activity of glutamate receptors. Postsynaptic expression of PSD-95 also enhanced maturation of the presynaptic terminal. These effects required synaptic clustering of PSD-95 but did not rely on its guanylate kinase domain. PSD-95 expression also increased the number and size of dendritic spines. These results demonstrate that PSD-95 can orchestrate synaptic development and are suggestive of roles for PSD-95 in synapse stabilization and plasticity. (+info)