Sendai virus binds to a dispersed population of NBD-GD1a.
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Receptor aggregation is believed to be an important step in the attachment of membrane enveloped virus' to target cell membranes. A likely receptor for Sendai virus is the ganglioside GD1a. In this work we have studied the membrane diffusion of the fluorescent ganglioside NBD-GD1a on the surface of CV-1 cells with standard photobleaching techniques. Using confocal laser scanning microscopy (CLSM) and Image Correlation Spectroscopy (ICS) NBD-GD1a is shown to exist in at least two populations: dispersed and aggregated. By quantifying the distribution of NBD-GD1a pre- and post-incubation with Sendai virus it is shown that the virus induces a dose-dependent clustering of NBD-GD1a. Image cross-correlation spectroscopy (ICCS) is used to further quantitatively characterize this clustering by demonstrating that it occurs due to binding of virus to the dispersed population of NBD-GD1a. (+info)
A mutation in the gene for the neurotransmitter receptor-clustering protein gephyrin causes a novel form of molybdenum cofactor deficiency.
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Gephyrin was originally identified as a membrane-associated protein that is essential for the postsynaptic localization of receptors for the neurotransmitters glycine and GABA(A). A sequence comparison revealed homologies between gephyrin and proteins necessary for the biosynthesis of the universal molybdenum cofactor (MoCo). Because gephyrin expression can rescue a MoCo-deficient mutation in bacteria, plants, and a murine cell line, it became clear that gephyrin also plays a role in MoCo biosynthesis. Human MoCo deficiency is a fatal disease resulting in severe neurological damage and death in early childhood. Most patients harbor MOCS1 mutations, which prohibit formation of a precursor, or carry MOCS2 mutations, which abrogate precursor conversion to molybdopterin. The present report describes the identification of a gephyrin gene (GEPH) deletion in a patient with symptoms typical of MoCo deficiency. Biochemical studies of the patient's fibroblasts demonstrate that gephyrin catalyzes the insertion of molybdenum into molybdopterin and suggest that this novel form of MoCo deficiency might be curable by molybdate supplementation. (+info)
Apoptosis of haematopoietic cells upon thymidylate synthase inhibition is independent of p53 accumulation and CD95-CD95 ligand interaction.
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Treatment of haematopoietic BA/F3 cells with the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (FUdR) activated apoptosis through a mechanism that required continuous protein synthesis and was inhibited by Bcl-2 over-expression. Analysis of p53 levels in cells treated with FUdR indicated a marked accumulation of this protein. Accumulation of p53 was also observed in cells over-expressing Bcl-2. In BA/F3 cells transfected with a cDNA coding for the human papilloma virus protein E6, p53 accumulation after FUdR treatment was inhibited markedly. However, apoptosis was induced in both control and E6 cells to a similar extent. The role of the CD95/CD95 ligand (CD95L) system in FUdR-induced apoptosis was also assessed. As determined by reverse transcriptase PCR, BA/F3 expressed a low constitutive level of CD95L mRNA, which decreased following FUdR treatment. Moreover, blocking CD95-CD95L interactions with antagonistic CD95 monoclonal antibody did not prevent drug-induced apoptosis. Furthermore, analysis of caspase involvement showed important differences in apoptosis induced by CD95-triggering or FUdR treatment. In summary, these results suggest that apoptosis induced by thymineless stress in haematopoietic BA/F3 cells occurs by a mechanism that does not require accumulation of p53 and which is independent of CD95-CD95L interactions. (+info)
Costimulation via lymphocyte function-associated antigen 1 in the absence of CD28 ligation promotes anergy of naive CD4+ T cells.
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The mechanisms controlling induction of anergy at the level of naive CD4+ T cells are poorly understood but thought to reflect limited contact with costimulatory molecules during T cell antigen receptor (TCR) ligation. To clarify this question, naive TCR transgenic CD4+ cells were exposed to specific peptide presented by transfected antigen-presenting cells (APC) expressing MHC class II molecules with defined accessory molecules. Significantly, culturing CD4(+) cells with APC expressing MHC II plus peptide alone elicited early TCR signaling but failed to induce either proliferation or anergy. Culture with APC expressing MHC II plus B7 molecules led to strong proliferation and T cell priming but no anergy. In marked contrast, conspicuous induction of anergy occurred after T cell culture with APC expressing MHC class II and intercellular adhesion molecule-1 (ICAM-1). Thus, at the level of naive CD4(+) cells, anergy induction appears to reflect selective contact with APC expressing ICAM-1 in the absence of B7. (+info)
Differential roles for alpha(M)beta(2) integrin clustering or activation in the control of apoptosis via regulation of akt and ERK survival mechanisms.
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The role of integrins in leukocyte apoptosis is unclear, some studies suggest enhancement, others inhibition. We have found that beta(2)-integrin engagement on neutrophils can either inhibit or enhance apoptosis depending on the activation state of the integrin and the presence of proapoptotic stimuli. Both clustering and activation of alpha(M)beta(2) delays spontaneous, or unstimulated, apoptosis, maintains mitochondrial membrane potential, and prevents cytochrome c release. In contrast, in the presence of proapoptotic stimuli, such as Fas ligation, TNFalpha, or UV irradiation, ligation of active alpha(M)beta(2) resulted in enhanced mitochondrial changes and apoptosis. Clustering of inactive integrins did not show this proapoptotic effect and continued to inhibit apoptosis. This discrepancy was attributed to differential signaling in response to integrin clustering versus activation. Clustered, inactive alpha(M)beta(2) was capable of stimulating the kinases ERK and Akt. Activated alpha(M)beta(2) stimulated Akt, but not ERK. When proapoptotic stimuli were combined with either alpha(M)beta(2) clustering or activation, Akt activity was blocked, allowing integrin activation to enhance apoptosis. Clustered, inactive alpha(M)beta(2) continued to inhibit stimulated apoptosis due to maintained ERK activity. Therefore, beta(2)-integrin engagement can both delay and enhance apoptosis in the same cell, suggesting that integrins can play a dual role in the apoptotic progression of leukocytes. (+info)
Differential modulation of stimulatory and inhibitory Fc gamma receptors on human monocytes by Th1 and Th2 cytokines.
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Immune complex-mediated inflammatory responses are initiated by Fc gamma R on phagocytes. We report in this study that an inhibitory receptor, Fc gamma RIIb2, is expressed on circulating human monocytes, and when co-cross-linked with stimulatory Fc gamma R it down-regulates effector function. Fc gamma RIIb2 expression is increased by IL-4 and decreased by IFN-gamma, in contrast to the activating receptor, Fc gamma RIIa, which is increased by IFN-gamma and decreased by IL-4. Thus, Th1 and Th2 cytokines differentially regulate the opposing Fc gamma R systems, altering the balance of activating and inhibiting Fc gamma R. The detection and cytokine modulation of Fc gamma RIIb2 in human myeloid cells provide evidence of a negative regulator of immune complex-mediated responses in human phagocytes and offer a new approach to limit Ab-triggered inflammation in autoimmune disease. (+info)
Oligomerization of opioid receptors with beta 2-adrenergic receptors: a role in trafficking and mitogen-activated protein kinase activation.
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G-protein-coupled receptors (GPCRs) have recently joined the list of cell surface receptors that dimerize. Dimerization has been shown to alter the ligand-binding, signaling, and trafficking properties of these receptors. Recent studies have shown that GPCRs heterodimerize with closely related members, resulting in the modulation of their function. In this study, we have attempted to determine whether members of GPCR superfamilies that couple to different families of G-proteins can associate and form oligomers. We chose the beta2 adrenergic receptor that couples to stimulatory G-proteins and delta & kappa opioid receptors that couple to inhibitory G-proteins. beta2 and delta receptors undergo robust agonist-mediated endocytosis, whereas kappa receptors do not. We find that when coexpressed, beta2 receptors can form heteromeric complexes with both delta and kappa receptors. This heterooligomerization does not significantly alter the ligand binding or coupling properties of the receptors. However, it affects the trafficking properties of the receptors. For example, we find that delta receptors, when coexpressed with beta2 receptors, undergo isoproterenol-mediated endocytosis. Conversely, beta2 receptors in these cells undergo etorphine-mediated endocytosis. However, beta2 receptors, when coexpressed with kappa receptors, undergo neither opioid- nor isoproterenol-mediated endocytosis. Moreover, these cells exhibit a substantial decrease in the isoproterenol-induced phosphorylation of mitogen-activated protein kinases. Taken together, these results provide direct evidence of heteromerization of GPCRs that couple to different types of G-proteins, which results in the modulation of receptor trafficking and signal transduction. (+info)
Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site.
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Aggregation of neurotransmitter receptors at pre- and postsynaptic structures is crucial for efficient neuronal communication. In contrast to the wealth of information about postsynaptic specializations, little is known about the molecular organization of presynaptic membrane proteins. We show here that the metabotropic glutamate receptor mGluR7a, which localizes specifically to presynaptic active zones, interacts in vitro and in vivo with PICK1. Coexpression in heterologous systems induces coclustering dependent upon the extreme C terminus of mGluR7a and the PDZ domain of PICK1. mGluR7a and PICK1 localize to excitatory synapses in hippocampal neurons. Furthermore, whereas transfected mGluR7a clusters at presynaptic sites, mGluR7adelta3 lacking the PICK1 binding site targets to axons but does not cluster. These results suggest that PICK1 is a component of the presynaptic machinery involved in mGluR7a aggregation and in modulation of glutamate neurotransmission. (+info)