Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor.
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Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defective lysosome-related organelles. Here, we report the identification of two HPS patients with mutations in the beta 3A subunit of the heterotetrameric AP-3 complex. The patients' fibroblasts exhibit drastically reduced levels of AP-3 due to enhanced degradation of mutant beta 3A. The AP-3 deficiency results in increased surface expression of the lysosomal membrane proteins CD63, lamp-1, and lamp-2, but not of nonlysosomal proteins. These differential effects are consistent with the preferential interaction of the AP-3 mu 3A subunit with tyrosine-based signals involved in lysosomal targeting. Our results suggest that AP-3 functions in protein sorting to lysosomes and provide an example of a human disease in which altered trafficking of integral membrane proteins is due to mutations in a component of the sorting machinery. (+info)
PETA-3/CD151, a member of the transmembrane 4 superfamily, is localised to the plasma membrane and endocytic system of endothelial cells, associates with multiple integrins and modulates cell function.
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The Transmembrane 4 Superfamily member, PETA-3/CD151, is ubiquitously expressed by endothelial cells in vivo. In cultured human umbilical vein endothelial cells PETA-3 is present on the plasma membrane and predominantly localises to regions of cell-cell contact. Additionally, this protein is abundant within an intracellular compartment which accounts for up to 66% of the total PETA-3 expressed. Intracellular PETA-3 showed colocalisation with transferrin receptor and CD63 suggesting an endosomal/lysosomal localisation which was supported by immuno-electronmicroscopy studies. Co-immunoprecipitation experiments investigating possible interactions of PETA-3 with other molecules demonstrated associations with several integrin chains including beta1, beta3, beta4, (alpha)2, (alpha)3, (alpha)5, (alpha)6 and provide the first report of Transmembrane 4 Superfamily association with the (alpha)6beta4 integrin. Using 2-colour confocal microscopy, we demonstrated similar localisation of PETA-3 and integrin chains within cytoplasmic vesicles and endothelial cell junctions. In order to assess the functional implications of PETA-3/integrin associations, the effect of anti-PETA-3 antibodies on endothelial function was examined. Anti-PETA-3 mAb inhibited endothelial cell migration and modulated in vitro angiogenesis, but had no detectable effect on neutrophil transendothelial migration. The broad range of integrin associations and the presence of PETA-3 with integrins both on the plasma membrane and within intracellular vesicles, suggests a primary role for PETA-3 in regulating integrin trafficking and/or function. (+info)
Role of autocrine stimulation on the effects of cyclic AMP on protein and lipid phosphorylation in collagen-activated and thrombin-activated platelets.
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We compared several responses in thrombin-stimulated and collagen (type I)-stimulated platelets with and without forskolin and inhibitors of autocrine stimulation (IAS: an ADP-removing system of creatine phosphate/creatine phosphokinase, Arg-Gly-Asp-Ser peptide to prevent fibrinogen/fibronectin binding to GPIIb/IIIa, SQ 29.548 as a thromboxane A2 receptor antagonist, cyproheptadine as a serotonin receptor antagonist, BN 52021 as a platelet-activating factor receptor antagonist). The pattern of tyrosine-phosphorylated proteins, the phosphorylation of lipids in the polyphosphoinositide cycle and phosphorylation of pleckstrin (P47) were studied as markers for signal-transducing responses, exposure of CD62 (P-selectin) and CD63 (Glycoprotein 53), as well as secretion of ADP + ATP and beta-N-acetyl-glycosaminidase were studied as final activation responses. Clear differences between thrombin-stimulated and collagen-stimulated platelets were observed. First, practically all protein-tyrosine phosphorylation induced by thrombin was inhibited by IAS, while a partial inhibition was observed for collagen; the phosphorylation due to collagen alone was apparently stimulated by elevation of cAMP. Secondly, the other responses to thrombin were inhibited by increased levels of cAMP, independent of autocrine stimulation. In contrast, only the autocrine part of the collagen-induced responses was inhibited by elevation of cAMP. Thus, the inhibition by elevated cAMP seen in collagen-stimulated platelets seems to be due to removal of the G-protein-mediated activation from secreted autocrine stimulators either by IAS or forskolin. The remaining activity is a pure collagen effect which is not affected by elevated levels of cAMP. (+info)
Detection of allergen-induced basophil activation by expression of CD63 antigen using a tricolour flow cytometric method.
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In the field of allergy diagnosis, most in vitro functional tests are focused on basophils. Nevertheless, the very small number of circulating basophils limits these experiments and their clinical benefit remains controversial. As flow cytometry is a valuable tool for identifying cell populations, even at low concentrations, we developed a tricolour flow cytometric method for the study of allergen-induced basophil activation. Identification of cells was based both on CD45 expression and on the presence of IgE on the cell surface, since basophils express high-affinity receptors for IgE (Fc epsilon RI). Cell activation upon allergen challenge was assessed by the expression of CD63 antigen on the plasma membrane. Basophil isolation and activation (with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine) were validated in 32 non-allergic patients. In 12 allergic patients, basophil stimulation by a relevant allergen was in most cases positive (10/12). Furthermore a concentration-dependent hook effect was observed. Of the allergic and non-allergic patients, none showed non-specific activation with an irrelevant allergen (specificity 100%). Overall, our preliminary results, even in a small population, suggest that this is a reliable and valuable method for the diagnosis of allergies complementing specific allergen IgE and skin test results. Obviously, additional clinical studies are needed to validate these first results. (+info)
Rapid down-regulation of CD63 transcription by progesterone in human endometrial stromal cells.
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Differentiation of endometrial stromal cells (decidualization) plays a crucial role in embryo implantation and maintenance of pregnancy. While progesterone is a key factor in regulating endometrial cell decidualization, the molecular mechanisms remain unclear. In the present study, we investigated the effect of gene transcription in human endometrial stromal cells (ESC) by progesterone, oestrogen or vehicle using the polymerase chain reaction-based differential display methodology. A transcript which is down-regulated by progesterone, but not by vehicle and oestrogen, was identified from a differential display band and the progesterone sensitivity of its expression was verified in Northern blot analysis. The level of the gene expression in progesterone-treated ESC was approximately 60% of that in the vehicle- and oestrogen-treated ESC. This cDNA was revealed to be human CD63 antigen, a recently identified member of the transmembrane 4 superfamily. The inhibitory effect of progesterone is observed within 30 min after hormone treatment. In human endometrium, CD63 mRNA levels were significantly decreased (P < 0.05) during the secretory phase compared with levels during the proliferative phase. This down-regulation of CD63 in vivo elevated levels of progesterone in the secretory phase. These results suggest that CD63 transcription is down-regulated by progesterone in human endometrium. (+info)
Monocyte activation in patients with Wegener's granulomatosis.
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OBJECTIVE: Wegener's granulomatosis (WG) is an inflammatory disorder characterised by granulomatous inflammation, vasculitis, and necrotising vasculitis and is strongly associated with anti-neutrophil cytoplasmic antibodies (ANCA). Activated monocytes/macrophages are present in renal biopsy specimens and participate in granuloma formation by synthesising and secreting a variety of chemoattractants, growth factors, and cytokines. In view of these findings, in vivo monocyte activation was evaluated in patients with WG and the findings related to parameters of clinical disease activity. METHODS: Monocyte activation was analysed by measuring plasma concentrations of soluble products of monocyte activation, that is neopterin and interleukin 6 (IL6), by ELISA, and by quantitating the surface expression of activation markers on circulating monocytes by flow cytometry. RESULTS: Twenty-four patients with active WG were included in this study. Ten of these patients were also analysed at the time of remission. Twelve patients with sepsis served as positive controls, and 10 healthy volunteers as negative controls for monocyte activation. Patients with active disease had increased monocyte activation compared with healthy controls as shown by increased concentrations of neopterin (p < 0.0001) and increased surface expression of CD11b (p < 0.05) and CD64 (p < 0.05). In those patients with increased concentrations of IL6 during active disease plasma concentrations of IL6 decreased during follow up when patients went into remission (p < 0.0001). In addition, neopterin (r = 0.37, r = 0.44), IL6 (r = 0.37, r = 0.60) and CD63 expression (r = 0.39, r = 0.45) correlated significantly with disease activity as measured by the Birmingham Vasculitis Activity Score and C reactive protein values, respectively. Compared with patients with sepsis, all markers of monocyte activation in patients with vasculitis were lower. CONCLUSION: It is concluded that disease activity in WG correlates with the extent of activation of monocytes, compatible with their role in the pathophysiology of this disease. (+info)
Y receptor-mediated induction of CD63 transcripts, a tetraspanin determined to be necessary for differentiation of the intestinal epithelial cell line, hBRIE 380i cells.
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Peptide YY (PYY) and neuropeptide Y (NPY) are peptides that coordinate intestinal activities in response to luminal and neuronal signals. In this study, using the rat hybrid small intestinal epithelial cell line, hBRIE 380i cells, we demonstrated that PYY- and NPY-induced rearrangement of actin filaments may be in part through a Y1alpha and/or a nonneuronal Y2 receptor, which were cloned from both the intestinal mucosa and the hBRIE 380i cells. A number of PYY/NPY-responsive genes were also identified by subtractive hybridization of the hBRIE 380i cells in the presence or absence of a 6-h treatment with PYY. Several of these genes coded for proteins associated with the cell cytoskeleton or extracellular matrix. One of these proteins was the transmembrane-4 superfamily protein CD63, previously shown to associate with beta(1)-integrin and implicated in cell adhesion. CD63 immunoreactivity, using antibody to the extracellular domain, was highest in the differentiated cell clusters of the hBRIE 380i cells. The hBRIE 380i cells transfected with antisense CD63 cDNA lost these differentiated clusters. These studies suggest a new role for NPY and PYY in modulating differentiation through cytoskeletal associated proteins. (+info)
CD63 associates with CD11/CD18 in large detergent-resistant complexes after translocation to the cell surface in human neutrophils.
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CD63 antibody binding to the neutrophil surface triggers a transient activation signal that regulates the adhesive activity and surface expression of CD11/CD18. Gel permeation chromatography demonstrated that all of the cell surface CD11/CD18 associated with CD63 eluted in the void volume, indicating that they were present in large detergent-resistant complexes. In contrast, the majority of the total cellular CD63, CD11 and CD18, which are largely intracellular, was not present in complexes. The data suggest that intracellular CD11, CD18 and CD63 are not in detergent-resistant complexes, but enter such complexes following translocation to the cell surface. (+info)