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At least 811 records · Page 45Linked to original sources
PubMed2000

Essential role for the C-terminal noncatalytic region of SHIP in FcgammaRIIB1-mediated inhibitory signaling.

The inositol phosphatase SHIP binds to the FcgammaRIIB1 receptor and plays a critical role in FcgammaRIIB1-mediated inhibition of B-cell proliferation and immunoglobulin synthesis. The molecular details of SHIP function are not fully understood. While point mutations of the signature motifs in the inositol phosphatase domain abolish SHIP's ability to inhibit calcium flux in B cells, little is known about the function of the evolutionarily conserved, putative noncatalytic regions of SHIP in vivo. In this study, through a systematic mutagenesis approach, we identified the inositol phosphatase domain of SHIP between amino acids 400 and 866. Through reconstitution of a SHIP-deficient B-cell line with wild-type and mutant forms of SHIP, we demonstrate that the catalytic domain alone is not sufficient to mediate FcgammaRIIB1/SHIP-dependent inhibition of B-cell receptor signaling. Expression of a truncation mutant of SHIP that has intact phosphatase activity but lacks the last 190 amino acids showed that the noncatalytic region in the C terminus is essential for inhibitory signaling. Mutation of two tyrosines within this C-terminal region, previously identified as important in binding to Shc, showed a reduced inhibition of calcium flux. However, studies with an Shc-deficient B-cell line indicated that Shc-SHIP complex formation is not required and that other proteins that bind these tyrosines may be important in FcgammaRIIB1/SHIP-mediated calcium inhibition. Interestingly, membrane targeting of SHIP lacking the C terminus is able to restore this inhibition, suggesting a role for the C terminus in localization or stabilization of SHIP interaction at the membrane. Taken together, these data suggest that the noncatalytic carboxyl-terminal 190 amino acids of SHIP play a critical role in SHIP function in B cells and may play a similar role in several other receptor systems where SHIP functions as a negative regulator.

Antigens, CD↗
PubMed2000

The transmembrane mutation G380R in fibroblast growth factor receptor 3 uncouples ligand-mediated receptor activation from down-regulation.

A point mutation, Gly380Arg, in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) leads to achondroplasia, the most common form of genetic dwarfism in humans. This substitution was suggested to enhance mutant receptor dimerization, leading to constitutive, ligand-independent activation. We found that dimerization and activation of the G380R mutant receptor are predominantly ligand dependent. However, using both transient and stable transfections, we found significant overexpression only of the mutant receptor protein. Metabolic pulse-chase experiments, cell surface labeling, and kinetics of uptake of radiolabeled ligand demonstrated a selective delay in the down-regulation of the mutant receptor. Moreover, this receptor was now resistant to ligand-mediated internalization, even at saturating ligand concentrations. Finally, transgenic mice expressing the human G380R mutant receptor under the mouse receptor transcriptional control demonstrated a markedly expanded area of FGFR3 immunoreactivity within their epiphyseal growth plates, compatible with an in vivo defect in receptor down-regulation. We propose that the achondroplasia mutation G380R uncouples ligand-mediated receptor activation from down-regulation at a site where the levels and kinetics of FGFR3 signals are crucial for chondrocyte maturation and bone formation.

Amino Acid Substitution↗
PubMed2004

A single pulse of agrin triggers a pathway that acts to cluster acetylcholine receptors.

Agrin triggers signaling mechanisms of high temporal and spatial specificity to achieve phosphorylation, clustering, and stabilization of postsynaptic acetylcholine receptors (AChRs). Agrin transiently activates the kinase MuSK; MuSK activation has largely vanished when AChR clusters appear. Thus, a tyrosine kinase cascade acts downstream from MuSK, as illustrated by the agrin-evoked long-lasting activation of Src family kinases (SFKs) and their requirement for AChR cluster stabilization. We have investigated this cascade and report that pharmacological inhibition of SFKs reduces early but not later agrin-induced phosphorylation of MuSK and AChRs, while inhibition of Abl kinases reduces late phosphorylation. Interestingly, SFK inhibition applied selectively during agrin-induced AChR cluster formation caused rapid cluster dispersal later upon agrin withdrawal. We also report that a single 5-min agrin pulse, followed by extensive washing, triggered long-lasting MuSK and AChR phosphorylation and efficient AChR clustering. Following the pulse, MuSK phosphorylation increased and, beyond a certain level, caused maximal clustering. These data reveal novel temporal aspects of tyrosine kinase action in agrin signaling. First, during AChR cluster formation, SFKs initiate early phosphorylation and an AChR stabilization program that acts much later. Second, a kinase mechanism rapidly activated by agrin acts thereafter autonomously in agrin's absence to further increase MuSK phosphorylation and cluster AChRs.

Agrin↗
PubMed2002

Clustering of very late antigen-4 integrins modulates K(+) currents to alter Ca(2+)-mediated monocyte function.

Endothelial cell vascular cell adhesion molecule-1 (VCAM-1) activates adherent monocytes by clustering their very late antigen-4 (VLA-4) receptors, resulting in the modulation of the inwardly rectifying (I(ir)) and delayed rectifying (I(dr)) K(+) currents, hyperpolarization of the cells, and enhanced Ca(2+) influx (Colden-Stanfield M and Gallin EK. Am J Physiol Cell Physiol 275: C267-C277, 1998; Colden-Stanfield M and Scanlon M. Am J Physiol Cell Physiol 279: C488-C494, 2000). The present study was undertaken to test the hypothesis that monoclonal antibodies (MAbs) against VLA-4 (MAbVLA-4) mimic VCAM-1 to cluster VLA-4 integrins, which play a key role in signaling an increase in the secretion of the proinflammatory cytokine interleukin-8 (IL-8). Whole cell ionic currents and IL-8 secretion from THP-1 monocytes that were incubated on polystyrene, VCAM-1-immobilized MAbVLA-4 or an isotype-matched MAb against CD45 (MAbCD45) were measured. Clustering of VLA-4 integrins with a cross-linked MAbVLA-4, but not a monovalent MAbVLA-4, modulated the K(+) currents in an identical manner to incubation of cells on VCAM-1. Similarly, cross-linked MAbVLA-4 or VCAM-1 augmented Ca(2+)-mediated IL-8 secretion from THP-1 monocytes and was completely abolished by exposure to CsCl, an I(ir) blocker. Thus VLA-4 integrin clustering by cross-linked MAbVLA-4 mimics VCAM-1/VLA-4 interactions sufficiently to be associated with events leading to monocyte differentiation, enhanced Ca(2+)-mediated macrophage function, and possibly atherosclerotic plaque formation.

Antibodies, Monoclonal↗
PubMed1988

Molecular events in synaptogenesis: nerve-muscle adhesion and postsynaptic differentiation.

The clustering of acetylcholine receptors (AChR) in the postsynaptic membrane of newly innervated muscle fibers is one of the earliest events in the development of the vertebrate neuromuscular junction. Here, we describe two hypotheses that can account for AChR clustering in response to innervation. The "trophic factor" hypothesis proposes that the neuron releases a soluble factor that interacts with the muscle cell in a specific manner and that this interaction results in the local accumulation of AChR. The "contact and adhesion" hypothesis proposes that the binding of the nerve to the muscle cell surface is itself sufficient to induce AChR clustering, without the participation of soluble factors. We present a model for the molecular assembly of AChR clusters based on the contact and adhesion hypothesis. The model involves the sequential assembly of three distinct membrane domains. The first domain to form serves to attach microfilaments to the cytoplasmic surface of the muscle cell membrane at sites of muscle-nerve adhesion. The second domain to form is clathrin-coated membrane; it serves as a site of insertion of additional membrane elements, including AChR. Upon insertion of AChR into the cell surface, a membrane skeleton assembles by anchoring itself to the AChR. The skeleton, composed in part of actin and spectrin, binds and immobilizes significant numbers of AChR, thereby forming the third membrane domain of the AChR cluster. We make several predictions that should distinguish this model of AChR clustering from one that invokes soluble, trophic factors.

Animals↗
PubMed1997

CD40 inhibits B cell apoptosis by upregulating bcl-xL expression and blocking oxidant accumulation.

Signaling through the CD40 receptor on human and murine B lymphocytes is necessary for germinal center formation and immunoglobulin class switching in vivo and rescues B cells from apoptosis triggered by cross-linking of surface immunoglobulin M in vitro. Ligation of CD40 on the immature mouse B cell line WEHI-231 with recombinant CD40 ligand (CD40L) was found to protect cells from apoptosis after gamma irradiation, as well as that following treatment with the sphingomyelin ceramide or compounds that deplete intracellular glutathione. CD40 signaling led to a rapid increase in the expression of the apoptosis inhibitory protein Bcl-xL. In addition, the apoptosis-induced accumulation of intracellular oxidants in WEHI-231 B cells was rapidly diminished by CD40 crosslinking. This antioxidant response was observed within 1 h and coincided with a preservation of intracellular thiols. These findings indicate that CD40 signaling induces a generalized cellular resistance to apoptosis characterized by an upregulation of Bcl-xL and changes in the intracellular redox potential.

Animals↗
PubMed2005

Different mitogen-mediated Beta-adrenergic receptor modulation in murine T lymphocytes depending on the thyroid status.

OBJECTIVE: The aim of this work was to analyze beta-adrenergic receptor (betaAR) regulation of T-lymphocyte proliferation in mice according to different thyroid hormone statuses. METHODS: T cells from eu-, hypo- (by propylthiouracil treatment) and hyperthyroid (by thyroxine, T4 administration) mice were purified and specific radioligand binding assays were performed. The effects of the beta-agonist isoproterenol (ISO) on intracellular levels of cyclic AMP (cAMP) were determined. Mitogen-induced T-cell proliferation was measured by [(3)H]-thymidine incorporation. Finally, protein kinase C (PKC) activity in cytosol and membrane fractions were determined using radiolabelled enzymatic substrates. RESULTS: Adecrease or a non-significant increase in betaAR number was found on T lymphocytes from hypo- and hyperthyroid mice compared to euthyroid controls. ISO stimulation of cAMP levels was lower in hypothyroid and higher in hyperthyroid T lymphocytes compared to controls. T-selective mitogen-induced proliferation was increased in T4-treated animals, but decreased in hypothyroid mice. During the peak of proliferation, downregulation of betaAR was observed in all animals. However, a higher or a lower decrease was observed in hyper- and hypothyroid T cells, respectively. In parallel, a higher translocation of PKC activity was observed in hyperthyroid cells, and a lower one was found in hypothyroid lymphocytes with respect to controls. CONCLUSIONS: These results indicate that intracellular signals triggered by mitogen activation, namely PKC, would be related to differential betaAR downregulation in T lymphocytes depending on the thyroid hormone status, contributing to the distinct proliferative responses found in hypo- or hyperthyroidism compared to the euthyroid state.

Adrenergic beta-Agonists↗
PubMedDate not supplied

Human heart mast cells in anaphylaxis and cardiovascular disease.

All sections of human heart tissue demonstrate tryptase- and chymase-containing mast cells (HHMCs) which have for the first time been isolated, partially purified and studied in vitro. HHMCs contain similar histamine levels as lung and skin mast cells, but tryptase levels are lower than in skin and higher than in lung mast cells. Complement C5a causes rapid dose-dependent release of histamine from HHMCs, but they are refractory to substance P and fMLP. Cross-linking IgE receptors on HHMCs leads to arachidonic acid metabolism through both the cyclooxygenase and 5-lipoxygenase pathways. HHMCs and their vasoactive mediators may be involved in anaphylactic/anaphylactoid reactions in humans and in the pathogenesis of some cardiovascular diseases.

Anaphylaxis↗
PubMedDate not supplied

Expression and function of Fc gamma R in mouse mast cells.

Activation of mast cells for the release of secretory granule, membrane lipid, and cytokine mediators via Fc gamma R requires cell surface expression of Fc gamma RIII. A soluble factor(s) from fibroblasts up-regulates the surface expression of Fc gamma RIII in interleukin-3-dependent, bone-marrow-derived mast cells, which otherwise degrade Fc gamma RIII intracellularly. Using a receptor-specific rabbit polyclonal antibody made to a peptide from the cytoplasmic domain of Fc gamma RIII alpha chain, we show that steady-state levels of Fc gamma RIII alpha protein increase in bone-marrow-derived mast cells after coculture with fibroblasts. Thus, the posttranslational stability of this functionally relevant receptor in mast cells is probably regulated by a fibro-blast-derived cytokine(s).

3T3 Cells↗
PubMedDate not supplied

A new member of the C-type lectin family is a modulator of the mast cell secretory response.

A glycoprotein identified on RBL-2H3 cells as capable of inhibiting the secretory response induced by the type I Fc epsilon receptor was named mast-cell-function-associated antigen (MAFA). The amino acid sequence deduced from the cloned full-length cDNA has now shown that the MAFA has marked sequence homology with several members of the C-type (calcium-dependent) animal lectin family. The high conservation of cysteinyl residues suggests an important role for intrachain disulfide bonds in attaining its structure and biological activity. We further show that MAFA clustering by monoclonal antibody G63 also inhibits the de novo synthesis and secretion of interleukin-6 induced by the Fc epsilon RI stimulus. Though no ligand has yet been identified for the MAFA, experiments using antisense oligonucleotides suggest that this novel lectin may have a role in cell adhesion in addition to its immunomodulatory capacity.

Amino Acid Sequence↗
PubMedDate not supplied

Multiple cytokine mRNA expression in human mast cells stimulated via Fc epsilon RI.

Cross-linkage of Fc epsilon RI on human lung mast cells purified by affinity magnetic selection with monoclonal antibody YB5.B8 against c-kit (purity > 90%) expressed mRNA for multiple cytokines. There was no constitutive expression of interleukin (IL)-4 mRNA. Mast cell stimulation with anti-IgE induced IL-4 mRNA expression which appeared maximal at 2 h and waned slowly over the next 24 h. IL-5, IL-6, IL-8 and tumour necrosis factor (TNF)-alpha mRNA were constitutively expressed. Mast cell activation with anti-IgE led to an increase of IL-5 and TNF-alpha mRNA signals within 2 h and which persisted for at least 24-48 h. On the other hand, IL-6 and IL-8 mRNA expression were not affected by anti-IgE challenge.

Antibodies, Anti-Idiotypic↗
PubMedDate not supplied

X-ray crystal structures of birch pollen profilin and Phl p 2.

BACKGROUND: Type 1 allergy affects 20% of industrialized populations and thus represents a major health care issue. The symptoms of type 1 allergy, which include rhinitis, conjunctivitis, dermatitis and asthma, are elicited by the cross-linking of IgE receptors through polyvalent allergens. A detailed understanding of the cell surface phenomena and the rational development of new therapies require high-resolution structural information. METHODS: The structures of two widespread allergens, birch pollen profilin (BPP) and Phl p 2 have been solved by multiple isomorphous replacement. Refinements are underway to 2.4 and 2.0 A, respectively. In addition, the IgE-reactive epitopes of BPP where identified by screening an epitope expression library with the serum IgE of an allergic individual. RESULTS: BPP exhibits an alpha/beta-fold which is similar to the mammalian and amoeba profilins. The structure of Phl p 2 is a compact eight-stranded beta-barrel. Screening an epitope library of BPP identified three major epitopic regions involved in IgE binding, including the amino and carboxy-terminal alpha-helices. These regions also interact with the physiologically relevant ligands of profilin, actin and proline-rich peptides. CONCLUSIONS: The distribution of IgE-binding sites on BPP allows for the productive interaction with IgE antibodies of different epitope specificities required for efficient signal transduction. These epitopes correspond to the most highly conserved regions of the profilin molecule and thus provide the molecular basis for allergen cross-sensitivity. Due to steric considerations, the involvement of these epitopic regions in the binding of physiologically relevant profilin ligands indicates that the native profilin is the species responsible for eliciting the allergic response. A comparison of the BPP and Phl p 2 structures shows that there is no preference for secondary structural elements in the allergic response. The detailed chemical and physical description of the major reactive epitopes provides a data base for the design of tight-binding monovalent ligands which can prevent receptor aggregation and thereby reduce the allergic response.

Allergens↗
PubMed1995

Sensitization of human atrial 5-HT4 receptors by chronic beta-blocker treatment.

BACKGROUND: Chronic treatment of patients with beta-blockers induces beta 2-adrenergic receptor hyperresponsiveness in atrium and sinoatrial node. To investigate whether other atrial Gs protein-coupled receptors also become hyperresponsive after chronic treatment with beta-blockers, we investigated 5-HT4 receptors in tissues and myocytes, which mediate serotonin-evoked increases of both contractile force and cAMP levels. METHODS AND RESULTS: Isolated right atrial strips from patients who had been chronically treated or not treated with a beta-blocker were set up to contract. In tissues from beta-blocker-treated patients (n = 27), the maximum inotropic response to serotonin was 56 +/- 3% (mean +/- SEM) of the effect elicited by (-)-isoproterenol (200 mumol/L) compared with a response of 19 +/- 6% in tissues from non-beta-blocker-treated patients (n = 13) (P < .001). The responsiveness of the tissues to Ca2+ was unchanged by chronic beta-blocker treatment. Serotonin (1 and 10 mumol/L) increased tissue cAMP levels, the increase with 10 mumol/L being significantly greater (P < .05) in tissues from beta-blocker-treated (n = 9) than in non-beta-blocker-treated (n = 7) patients. In paced atrial myocytes, serotonin caused concentration-dependent increases in contraction. Myocytes obtained from atria of beta-blocker-treated patients were more sensitive (P < .01) to the effects of serotonin (-log EC50, 7.9 +/- 0.2 mol/L; n = 12) than myocytes obtained from non-beta-blocker-treated patients (-log EC50, 7.3 +/- 0.2 mol/L, n = 12). Chronic beta-blocker treatment had no effect on forskolin-evoked myocyte responses. Carbachol (1 mumol/L) suppressed the effects of both serotonin (n = 6) and (-)-isoproterenol (n = 6) in the same atrial myocyte. CONCLUSIONS: Chronic treatment of patients with beta-blockers causes atrial 5-HT4 receptor inotropic hyperresponsiveness and enhanced serotonin-evoked increases in cAMP levels. This may be due to modified cross talk between 5-HT4 receptors, beta-adrenergic receptors, and muscarinic receptors.

Adrenergic beta-Antagonists↗
PubMed2001

Leukocyte integrin Mac-1 recruits toll/interleukin-1 receptor superfamily signaling intermediates to modulate NF-kappaB activity.

The leukocyte integrin Mac-1 (alphaMbeta2, CD11b/CD18) regulates important cell functions in inflammation, including adhesion, phagocytosis, and oxidative burst. Deficiency of Mac-1 reduces vessel wall inflammation and neointimal thickening after murine carotid artery injury. Although Mac-1 has been implicated in modulating AP-1 and NF-kappaB activity, the signal transduction pathways involved are undefined. cDNA array analysis of Mac-1-clustered compared with -nonclustered monocytic THP-1 cells showed increased expression of the signal transducer TRAF6 (TNF receptor-associated factor 6), leading us to consider the possibility that Mac-1 used a Toll/IL-1 receptor family-like signaling pathway. Mac-1-dependent activation of NF-kappaB was potentiated by wild-type, and attenuated by dominant negative, TRAF6- and TGF-beta-activated kinase (TAK1) constructs. IRAK1 (IL-1 receptor associated kinase), a kinase immediately upstream of TRAF6, coimmunoprecipitated with Mac-1. Taken together, these observations indicate that Mac-1 recruits a Toll/IL-1 receptor family-like cascade to modulate NF-kappaB activity. This represents a new pathway for integrin-dependent modulation of gene expression.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Fc gamma receptor IIIb enhances Fc gamma receptor IIa function in an oxidant-dependent and allele-sensitive manner.

Two classes of receptors for IgG, Fc gamma RIIa and Fc gamma RIIIb, both of which exist in two allelic forms, are expressed on human neutrophils. Neutrophils from normal donors, homozygous for the different allelic phenotypes of Fc gamma RIIIb, have significantly different levels of Fc gamma receptor-mediated phagocytosis of IgG-opsonized erythrocytes (EA). However, the observation that Fc gamma RIIIb mediates phagocytosis of specific mAb-targeted erythrocytes poorly suggests that this receptor may influence EA internalization by Fc gamma RIIa in an allele-sensitive fashion. Donors homozygous for the NA1 allele of Fc gamma RIIIb showed greater activation of Fc gamma RIIa after Fc gamma RIIIb cross-linking than donors homozygous for the NA2 allele of Fc gamma RIIIb. This increase in receptor-specific internalization reflects both an increase in ligand binding by Fc gamma RIIa and an increase in internalization efficiency of targets bound. Activation of Fc gamma RIIa by Fc gamma RIIIb is transferable by supernatants from activated cells and is blocked by inhibitors of reactive oxygen species and the H2O2-myeloperoxidase-chloride system and by serine protease inhibitors. Thus, cross-linking of Fc gamma RIIIb, which leads to neutrophil degranulation and the generation of reactive oxygen intermediates, in turn alters Fc gamma RIIa avidity and efficiency. These oxidant-mediated changes in Fc gamma RIIa function provide a novel mechanism for receptors to collaborate in both an autocrine and paracrine fashion. The allele sensitivity of these effects suggests that Fc gamma receptor polymorphisms may be inherited disease susceptibility factors in host defense against infection and in the development of autoimmunity.

Adult↗
PubMed2004

Eph receptors, ephrins, and synaptic function.

Compelling new findings have revealed that receptor tyrosine kinases of the Eph family, along with their ephrin ligands, play an essential role in regulating the properties of developing mature excitatory synapses in the central nervous system. The cell surface localization of both the Eph receptors and the ephrins enables these proteins to signal bidirectionally at sites of cell-to-cell contact, such as synapses. Eph receptors and ephrins have indeed been implicated in multiple aspects of synaptic function, including clustering and modulating N-methyl-D-aspartate receptors, modifying the geometry of postsynaptic terminals, and influencing long-term synaptic plasticity and memory. In this review, we discuss how Eph receptors and ephrins are integrated into the molecular machinery that supports synaptic function.

Animals↗