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PubMed2006

Lamina-specific abnormalities of AMPA receptor trafficking and signaling molecule transcripts in the prefrontal cortex in schizophrenia.

Ampakines, positive AMPA receptor modulators, can improve cognitive function in schizophrenia, and enhancement of AMPA receptor-mediated currents by them potentiates the activity of antipsychotics. In vitro studies have revealed that trafficking of AMPA receptors is mediated by specific interactions of a complex network of proteins that also target and anchor them at the postsynaptic density (PSD). The aim of this study was to determine whether there are abnormalities of the molecules associated with trafficking and localization of AMPA receptors at the PSD in the dorsolateral prefrontal cortex (DLPFC) in schizophrenia. We analyzed AMPA receptor expression in DLPFC in schizophrenia, major depression, bipolar disorder, and a control group, by examining transcript levels of all four AMPA receptor subunits by in situ hybridization. We found decreased GluR2 subunit expression in all three illnesses, decreased GluR3 in major depression, and decreased GluR4 in schizophrenia. However, autoradiography experiments showed no changes in AMPA receptor binding; thus, we hypothesized that these changes in receptor subunit stoichiometry do not alter binding to the assembled receptor, but rather intracellular processing. In situ hybridization for AMPA-trafficking molecules showed decreased expression of PICK1 and increased expression of stargazin in DLPFC in schizophrenia, both restricted to large cells of cortical layer III. These data suggest that AMPA-mediated glutamatergic neurotransmission is compromised in schizophrenia, particularly at the level of AMPA-related PSD proteins that mediate AMPA receptor trafficking, synaptic surface expression, and intracellular signaling.

Adult↗
PubMed1994

Tyrosine phosphorylation of phospholipase C-gamma 2 is involved in the activation of phosphoinositide hydrolysis by Fc receptors in human neutrophils.

The stimulation of phosphoinositide hydrolysis by a number of agonists (phosphoinositide response) is a ubiquitous transmembrane signalling process for the regulation of several cell functions. Two mechanisms of activation have been identified that involve different phospholipases C: one regulated by G-proteins and another regulated by receptors having an intrinsic tyrosine kinase domain or that stimulate intracellular tyrosine kinase activity. This last mechanism is activated in several immunological cells, including lymphocytes, mastocytes, NK cells and monocytes, in response to agonists that bind antigen receptors, and receptors for IgE and IgG. In the present study, we have investigated the role of tyrosine phosphorylation in the stimulation of phosphoinositide hydrolysis mediated by Fc gamma Rs in human neutrophils. The results demonstrated that: 1) the activation of Fc gamma Rs with insoluble immune complexes (IIC) induced a tyrosine phosphorylation of several proteins that was dose-dependently inhibited by the tyrosine kinase inhibitor, genistein; 2) the activation of Fc gamma Rs caused a stimulation of phosphoinositide hydrolysis measured as [3H]inositol phosphates formation; 3) genistein depressed the activation of phosphoinositide hydrolysis; 4) among the several proteins that became tyrosine phosphorylated upon Fc gamma Rs activation by IIC, one 145 kDa protein was identified as PLC-gamma 2, using a specific antiserum. The phosphorylation of PLC-gamma 2 was completely inhibited by genistein. These results demonstrate that the phosphoinositide response to activation of Fc gamma Rs involves the tyrosine phosphorylation of PLC-gamma 2.

Genistein↗
PubMed1995

Hydroxyl radical attack of low density lipoprotein decreases its cellular catabolism in the absence of significant lipid peroxidation.

Low density lipoprotein (LDL) has been submitted to oxidative modification induced by gamma radiolysis of water under conditions generating either hydroxyl radical (OH.) alone, or a mixture of superoxide anion and OH.. Treatment of LDL with hydroxyl radical alone did not lead to significant lipid peroxidation as assessed by thiobarbituric acid reactive substances (TBARS) and hydroperoxide measurement and induced only very small change in the electrophoretic mobility of the particle. In contrast, superoxide and hydroxyl radical mixture induced a dose-dependent increase in lipid peroxidation, with a marked elevation of the negative net charge of the LDL. However, in both cases, a similar reduction of the uptake and degradation of modified LDL by the apo B/E receptor pathway of human fibroblasts was observed. This suggests that factors other than lipid peroxidation could play a role in LDL modification and influence their cellular metabolism.

Cells, Cultured↗
PubMed1995

Nongenomic steroid action: independent targeting of a plasma membrane calcium channel and a tyrosine kinase.

It is known that steroids can produce nongenomic effects on cells, such as opening of ionic channels, membrane receptor aggregation or changes in protein phosphorylation status. However, it is not known whether these different effects, when occurring concomitantly, are linked by a causal relationship or whether they are triggered independently by the steroid ligand. Here we show that progesterone opens a plasma membrane Ca2+ channel and activates a protein tyrosine kinase of human spermatozoa independently of each other because, on the one hand, tyrosine kinase inhibition does not affect the progesterone-induced Ca2+ influx and, on the other hand, the absence of extracellular Ca2+ does not preclude tyrosine-kinase-mediated progesterone-induced acrosomal exocytosis. These data suggest that steroids interact with multireceptor systems on the surface of responsive cells.

Calcium Channels↗
PubMed1999

The proto-oncogene p120(Cbl) is a downstream substrate of the Hck protein-tyrosine kinase.

Hematopoietic cell kinase (Hck) is a member of the Src-family of protein tyrosine kinases. We have found that upon enzymatic activation of Hck by the heavy metal mercuric chloride, there was a rapid increase in the levels of tyrosine phosphorylation of several proteins including the proto-oncogene p120(Cbl). Fibroblasts that are transformed with an activated allele of Hck exhibit constitutive Cbl phosphorylation. Upon Fcgamma receptor activation, a more physiologically relevant extracellular signal, Cbl is tyrosine phosphorylated and the Src-family selective inhibitor, PP1, can prevent this phosphorylation on Cbl. Hck phosphorylates Cbl in vitro and the interaction between Cbl and Hck is direct, requiring Hck's unique, SH3 and SH2 domains for optimal binding. Using a novel estrogen-regulated chimera of Hck we have shown a hormone-dependent association between Hck and Cbl in murine fibroblasts. This work suggests that Cbl serves as a key mediator of Hck induced signalling in hematopoietic cells.

3T3 Cells↗
PubMed1999

Genetic and biochemical evidence for a critical role of Janus kinase (JAK)-3 in mast cell-mediated type I hypersensitivity reactions.

We investigated the role of JAK3 in IgE receptor/FcepsilonRI-mediated mast cell responses. IgE/antigen induced degranulation and mediator release were substantially reduced with Jak3-/- mast cells from JAK3-null mice that were generated by targeted disruption of Jak3 gene in embryonic stem cells. Further, treatment of mast cells with 3'bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154), a potent inhibitor of JAK3, inhibited degranulation and proinflammatory mediator release after IgE receptor/ FcepsilonRI crosslinking. Thus, JAK3 plays a pivotal role in IgE receptor/ FcepsilonRI-mediated mast cell responses and targeting JAK3 may provide the basis for new and effective treatment as well as prevention programs for mast cell-mediated allergic reactions.

Animals↗
PubMed2001

Enhanced infection of an X4 strain of HIV-1 due to capping and colocalization of CD4 and CXCR4 induced by capsianoside G, a diterpene glycoside.

We investigated whether capsianosides, diterpene glycosides, extracted from Capsicum plants could affect human immunodeficiency virus type 1 (HIV-1) infection. Significant effect on virus infection in MAGI/CCR5 cells was neither observed for the X4 virus by capsianosides II, XI, and A, nor for an R5 virus by capsianoside G. Apparent enhancement of X4 HIV-1 infection by capsianoside G was observed and exclusively related to the usage of the CXCR4 coreceptor. The capsianoside G-treated cells had no change in the expression level of CD4, CXCR4, and CCR5, however, colocalization and capping of CD4 and CXCR4, but not of CD4 and CCR5 was observed. Our results suggested that capsianoside G enhanced X4 virus infection at the level of viral penetration through the capping and colocalization of receptors needed for infection.

Adsorption↗
PubMed1993

Inhibition of human T cell response to staphylococcal enterotoxin B by prior ligation of surface CD4 molecules.

The mechanisms whereby anti-CD4 monoclonal antibodies can block human peripheral blood lymphocyte response to staphylococcal enterotoxin B (SEB) were investigated. Preincubation of peripheral blood mononuclear cells (PBMC) with anti-CD4 mAbs resulted in a profound inhibition of SEB-induced DNA synthesis, while simultaneous addition of antibody and superantigen did not reproducibly decrease the proliferative response. Inhibition was achieved at a very low antibody concentration (0.1 microgram/ml). It was not increased by cross-linking of anti-CD4 mAb nor mediated by Fc-dependent signals as F(ab')2 antibody fragments were as effective as intact antibodies. Inhibition of proliferation was associated with a profound diminution of IL-2 and IFN-gamma secretion, CD25 (the alpha chain of IL-2 receptor) expression, and blast transformation. Stimulation by SEB after prior ligation of surface CD4 proteins by antibodies was associated with an increased percentage of lymphocytes with chromatin condensation and nuclear fragmentation. It was concluded that stimulation of mature peripheral T cells by SEB through T cell receptors induces an apoptotic signal providing that a small proportion of surface CD4 molecules has interacted with antibodies or F(ab')2 fragments before stimulation by SEB, while simultaneous addition of SEB and anti-CD4 mAb does not prevent the development of a complete activation program in this system. Possible implications of these observations regarding selective clonal deletion of autoreactive T cells by administration of anti-CD4 mAbs in patients with auto-immune diseases are discussed.

Antibodies, Monoclonal↗
PubMed1999

Antibody-induced CD3-CD4 coligation inhibits TCR/CD3 activation in the absence of costimulatory signals in normal mouse CD4(+) T lymphocytes.

The effect of CD3-CD4 coligation on CD3-mediated activation of normal mouse CD4(+) T lymphocytes has been analyzed in the absence of exogenous lymphokines. If anti-CD3 and anti-CD4 antibodies are adsorbed to culture wells by means of previously adsorbed anti-Ig antibodies (indirect binding), CD3-CD4 coligation inhibits activation measured as cell proliferation or as secretion of IL-2, IL-4, and IFN-gamma. Addition of IL-2, anti-CD28 antibodies, or phorbol esters, but not IL-1, IL-4, or ionomycin, blocked CD4-mediated inhibition and restored the response to levels equal or higher than those of cultures activated by anti-CD3 alone. In contrast, CD3-CD4 coligation by antibodies directly adsorbed to culture wells potentiated anti-CD3-induced activation, either in the absence or in the presence of exogenous costimuli. Similar results were observed when CD4(+) T cells of naive phenotype (CD44(low), CD45RB(high)) were used in the experiments. The analysis of early tyrosine phosphorylation in CD4(+) T cells shows that phosphorylation of many cell substrates is clearly enhanced upon CD3-CD4 coligation using indirectly or directly bound antibodies, yet certain substrates are mainly phosphorylated under inhibitory conditions. Although CD28 ligation does not produce any clear change in the tyrosine phosphorylation pattern in lysates from cells activated by indirectly bound anti-CD3 plus anti-CD4 antibodies, the analysis of active forms of the MAP kinase ERK suggests that downstream signaling pathways involved in IL-2 gene activation can be differentially activated depending on the direct or indirect CD3-CD4 adsorption and CD28 ligation.

Adsorption↗
PubMed1993

43K protein and acetylcholine receptors colocalize during the initial stages of neuromuscular synapse formation in vivo.

The 43K protein is a cytoplasmic peripheral membrane protein concentrated subsynaptically in skeletal muscle. Recombinant 43K has been shown to cause clustering of acetylcholine receptors (AChRs) in cultured cells. However, the role of 43K in vivo is disputed, because in some cases it appears only after AChRs have clustered. We therefore examined the expression and distribution of 43K and AChRs during synapse formation in embryonic mouse muscles. Messenger RNA for 43K was detected on Embryonic Day (E) 12, a day prior to the first AChR clusters. Immunofluorescence showed that both AChRs and 43K were colocalized in patches by E13, the stage at which intramuscular nerves were first detected. The AChR/43K patches were nerve associated, and more than 98% of AChR patches were accompanied by 43K. The precise colocalization of 43K and AChRs persisted through development. These results are consistent with 43K being involved in the nerve-induced clustering of AChRs during synapse formation.

Animals↗
PubMed1998

Modulation of cell-cell adherens junctions by surface clustering of the N-cadherin cytoplasmic tail.

Cadherins mediate the formation of cell-cell adherens junctions (AJ) by homophilic interactions through their extracellular domains as well as by interacting with the actin cytoskeleton via their cytoplasmic portions. Cadherin clustering initiates cytoplasmic signaling that results in the assembly of structural components into cell-cell AJ. To elucidate the function of the cytoplasmic tail of cadherins in initiating the assembly signal, we generated and characterized a chimeric cadherin tail fused to an inert transmembrane anchor. The chimera enabled us to cluster the cadherin cytoplasmic tail in the absence of extracellular portions of the molecule. The transfected cadherin tail chimera localized to cell-cell AJ of epithelial cells, indicating that the submembrane junctional plaque has the capacity to recruit additional cadherins, with no involvement of their extracellular domains. Expression of the chimera in cells of mesenchymal origin resulted in dominant negative effects on the formation of cell-cell AJ. Surface clustering of cadherin cytoplasmic tails induced the recruitment of components and structural assembly of cell-cell AJ, thereby reversing the initial dominant-negative effects. We conclude that the cadherin cytoplasmic tail contains information required to direct the molecule to cell-cell AJ. Its function as modulator of cell-cell AJ depends on cell type and on whether the tail is clustered.

3T3 Cells↗
PubMed1999

Annexin V staining due to loss of membrane asymmetry can be reversible and precede commitment to apoptotic death.

Signal-induced apoptosis is a normal phenomenon in which cells respond to changes in their environment through a cascade of intracellular biochemical changes culminating in cell death. However, it is not clear at what point in this process the cell becomes committed to die. An early biochemical change characteristic of cells undergoing apoptosis is the loss of plasma membrane asymmetry, such that high levels of phosphatidylserine become exposed on the outside cell surface. These cells can be recognized by staining with Annexin V, which binds to phosphatidylserine with high affinity. To investigate the mechanisms controlling signal-induced apoptosis we have examined the response of a B cell lymphoma to crosslinking of the membrane immunoglobulin (mIg) receptor. We have found that many of the cells that stain positive for Annexin V are viable and can resume growth and reestablish phospholipid asymmetry once the signal is removed. These results indicate that Annexin V staining, and thus loss of membrane asymmetry, precedes commitment to apoptotic death in this system.

Animals↗
PubMed1995

Interaction of Fc gamma receptor type IIIB with complement receptor type 3 in fibroblast transfectants: evidence from lateral diffusion and resonance energy transfer studies.

To explore potential inter-receptor interactions between Fc gamma RIIIB, a GPI-linked protein, and the leukocyte integrin CR3, we have prepared transfected 3T3 fibroblast cell lines expressing Fc gamma RIIIB, CR3, or both Fc gamma RIIIB and CR3. We test the hypothesis that Fc gamma RIIIB and CR3 are physically associated in membranes using fluorescence recovery after photobleaching (FRAP) and resonance energy transfer (r.e.t.) microscopy. Cells expressing Fc gamma RIIIB alone displayed a diffusion coefficient (D) of 3.4 x 10(-9) (+/- 2.9 x 10(-9) cm2/second and a mobile fraction (m.f.) of 0.73 (+/- 0.10). In contrast, Fc gamma RIIIB exhibited D = 2.5 x 10(-9) (+/- 1.4 x 10(-9) cm2/second (n.s.) and a m.f. of 0.48 (+/- 0.08) (p < 0.01) on cells expressing both Fc gamma RIIB and CR3, thus indicating that co-expression of CR3 constrains the lateral diffusion of Fc gamma RIIIB. To further test for a direct physical interaction between these gene products, (r.e.t.) microscopy was performed. Donor-labeled anti-CR3 and acceptor-labeled anti-Fc gamma RIIIB on cells expressing both receptors yielded a r.e.t. photon count rate of 8.9(+/- 6.4) kilocounts/second (kC/s), whereas CR3-to-CR3 measurements gave 1.6(+/- 0.6) kC/s (p < 0.01). Moreover, the addition of exogenous agents such as N-acetyl-D-glucosamine, but not indomethacin, diminished the magnitude of these interactions in transfectant membranes. These data support the notion that a subpopulation of Fc gamma RIIIB is physically associated with CR3 and that this association can be affected by exogeneous compounds.

3T3 Cells↗
PubMed1995

In vitro binding of ciliary neurotrophic factor to its receptors: evidence for the formation of an IL-6-type hexameric complex.

Ciliary neurotrophic factor (CNTF) is a cytokine sharing structural and functional similarities with interleukin-6 (IL-6) and other helical cytokines that utilize the common signalling chain gp130. While IL-6 induces gp130 dimerization, CNTF, after the initial interaction with the specific, non-signalling receptor subunit, CNTFR, induces the formation of gp130/LIF-receptor heterodimers. Through immunoprecipitation experiments with tagged soluble receptor molecules, we recently demonstrated that IL-6 drives the formation of a hexameric receptor complex with a defined topology and composed of two IL-6, two IL-6R alpha and two gp130 molecules. Here, we apply the same strategy to study the assembly in vitro of the CNTF receptor complex. We present evidence that both the cytokine and the specific binding chain undergo dimerization in the presence of gp130. Furthermore, although gp130 and LIFR are able to bind independently to the CNTF/CNTFR sub-complex, they never form homodimers but only heterodimers. We propose that CNTF assembles a hexameric receptor complex composed of two CNTF, two CNTFR, one gp130 and one LIFR molecule, and present a model of the reciprocal interaction of these molecules based on similarities with the IL-6 hexameric complex.

Amino Acid Sequence↗
PubMed1998

Mapping the mAb 383C epitope to alpha 2(187-199) of the Torpedo acetylcholine receptor on the three-dimensional model.

Monoclonal antibody 383C is an anti-acetylcholine receptor antibody whose binding to the receptor is blocked by alpha-bungarotoxin and by carbamylcholine. Monoclonal antibody 383C binds to the alpha subunit of the Torpedo acetylcholine (ACh) receptor as well as to its V8-protease 20 kDa fragment that possesses the affinity alkylatable Cys192/193. In an epitope scanning experiment spanning the N-terminal 211 amino acid residues of the alpha subunit, 383C binds uniquely to three overlapping peptides; alpha(184-196), alpha(187-199) and alpha(190-202). These peptides span a cluster of amino acid residues implicated in the binding of acetylcholine, including Cys192/193. To map the location of these residues on the three-dimensional model of the ACh receptor, we have employed a combination of X-ray diffraction from oriented complexes of 383C with ACh receptor-enriched membrane vesicles and electron microscopy of negatively stained tubular arrays of 383C/receptor complexes. The X-ray diffraction study finds extra electron density in the presence of 383C centered 35 A above the synaptic side phosphate head groups. The electron micrographic images display extra stain exclusion from the antibody at a site adjacent to the alpha2 subunit on the periphery of the rosette clockwise to the alpha2 vertex. This mapping localizes several residues of the ACh receptor alpha subunit involved in the binding of acetylcholine. Despite these residues being present in both alpha subunits, only the alpha2 subunit is decorated with this monoclonal antibody.

Amino Acid Sequence↗
PubMed1996

The synapse-associated protein rapsyn regulates tyrosine phosphorylation of proteins colocalized at nicotinic acetylcholine receptor clusters.

Protein tyrosine phosphorylation has been suggested to play an important role in the clustering of the nicotinic acetylcholine receptor (AChR) at the developing neuromuscular junction. Recent studies have shown that the 43-kDa synapse-associated protein rapsyn induces clustering of the AChR in heterologous expression systems. In this study we examined whether tyrosine phosphorylation is involved in this rapsyn-induced AChR clustering. Rapsyn-induced AChR clusters in fibroblasts contain phosphotyrosine, as detected using immunofluorescent labeling with anti-phosphotyrosine antibodies. No anti-phosphotyrosine staining of rapsyn clusters is seen in the absence of AChR expression, indicating that the AChR is required for the appearance of phosphotyrosine at clusters. In addition, coexpression of rapsyn with the AChR induces the tyrosine phosphorylation of the beta amd delta subunits of the AChR. Surprisingly, mutation of the tyrosine phosphorylation sites in the AChR did not inhibit rapsyn-induced clustering of the AChR and clusters of the mutant AChRs still contained high levels of phosphotyrosine. Experiments with single AChR subunits demonstrate that the alpha subunit of the AChR appears to be necessary and sufficient for codistribution of phosphotyrosine with rapsyn-induced clusters of AChR subunits. Finally, transfection of cells with rapsyn activates cellular protein tyrosine kinase activity, resulting in the tyrosine phosphorylation of several membrane-associated proteins. These results suggest that rapsyn may therefore regulate clustering at least in part by regulating the tyrosine phosphorylation of cellular proteins.

Animals↗
PubMed1996

The role of an agrin-growth factor interaction in ACh receptor clustering.

The clustering of acetylcholine receptors (AChRs) at the neuromuscular junction is mediated in part by the heparan-sulfate proteoglycan agrin. However, our previous studies have also suggested the role of heparin-binding growth-associated molecular (HB-GAM) in AChR clustering. Here the role of an agrin-HB-GAM interaction in this process was examined using cultured Xenopus muscle cells. Agrin-coated beads further treated with HB-GAM were highly effective in AChR cluster induction. Protein overlay assays showed specific binding of HB-GAM to agrin. In addition, agrin-enriched neuritic tracks bound HB-GAM in a manner that showed a high degree of colocalization between the neural agrin and the applied factor. Finally, the introduction of exogenous HB-GAM together with soluble agrin resulted in the appearance of AChR clusters on the dorsal surface of cells in an agrin isoform-dependent manner; a dramatic change from the characteristic ventral AChR clustering seen in response to agrin alone. These results suggest that agrin may mediate AChR clustering by interacting with muscle-bound heparin-binding growth factors such as HB-GAM.

Agrin↗
PubMed1997

Clustering of GABAA receptors by rapsyn/43kD protein in vitro.

Rapsyn, a 43-kDa protein on the cytoplasmic face of the postsynaptic membrane, is essential for clustering acetylcholine receptors (AChR) at the neuromuscular junction. When transfected into nonmuscle cells (QT-6), rapsyn forms discrete membrane domains and can cluster AChR into these same domains. Here we examined whether rapsyn can cluster other ion channels as well. When expressed in QT-6 cells, the GABAA receptor (human alpha 1, beta 1, and gamma 2 subunits) and the skeletal muscle sodium channel were each diffusely scattered across the cell surface. Rapsyn, when co-expressed, clustered the GABAA receptor as effectively as it clustered AChR in previous studies. Rapsyn did not cluster co-transfected sodium channel, confirming that it does not cluster ion channels indiscriminately. Rapsyn mRNA was detected at low levels in the brain by polymerase chain reaction amplification of reverse-transcribed RNA, raising the possibility of a broader role for rapsyn.

Animals↗