Regulated apical secretion of zymogens in rat pancreas. Involvement of the glycosylphosphatidylinositol-anchored glycoprotein GP-2, the lectin ZG16p, and cholesterol-glycosphingolipid-enriched microdomains.
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We examined the role of glycosphingolipid- and cholesterol-enriched microdomains, or rafts, in the sorting of digestive enzymes into zymogen granules destined for apical secretion and in granule formation. Isolated membranes of zymogen granules from pancreatic acinar cells showed an enrichment in cholesterol and sphingomyelin and formed detergent-insoluble glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose density gradients and contained the glycosylphosphatidylinositol (GPI)-anchored glycoprotein GP-2, the lectin ZG16p, and sulfated matrix proteoglycans. Morphological and pulse-chase studies with isolated pancreatic lobules revealed that after inhibition of GPI-anchor biosynthesis by mannosamine or the fungal metabolite YW 3548, granule formation was impaired leading to an accumulation of newly synthesized proteins in the Golgi apparatus and the rough endoplasmic reticulum. Furthermore, the membrane attachment of matrix proteoglycans was diminished. After cholesterol depletion or inhibition of glycosphingolipid synthesis by fumonisin B1, the formation of zymogen granules as well as the formation of detergent-insoluble complexes was reduced. In addition, cholesterol depletion led to constitutive secretion of newly synthesized proteins, e.g. amylase, indicating that zymogens were missorted. Together, these data provide first evidence that in polarized acinar cells of the exocrine pancreas GPI-anchored proteins, e.g. GP-2, and cholesterol-sphingolipid-enriched microdomains are required for granule formation as well as for regulated secretion of zymogens and may function as sorting platforms for secretory proteins destined for apical delivery. (+info)
Stomatin, flotillin-1, and flotillin-2 are major integral proteins of erythrocyte lipid rafts.
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Lipid rafts are sphingolipid- and cholesterol-rich membrane microdomains that are insoluble in nonionic detergents, have a low buoyant density, and preferentially contain lipid-modified proteins, like glycosyl phosphatidylinositol (GPI)-anchored proteins. The lipid rafts were isolated from human erythrocytes and major protein components were identified. Apart from the GPI-anchored proteins, the most abundant integral proteins were found to be the distantly related membrane proteins stomatin (band 7.2b), flotillin-1, and flotillin-2. Flotillins, already described as lipid raft components in neurons and caveolae-associated proteins in A498 kidney cells, have not been recognized as red cell components yet. In addition, it was shown that the major cytoskeletal proteins, spectrin, actin, band 4.1, and band 4.2, are partly associated with the lipid rafts. Stomatin and the flotillins are present as independently organized high-order oligomers, suggesting that these complexes act as separate scaffolding components at the cytoplasmic face of erythrocyte lipid rafts. (+info)
Membrane lipid rafts are necessary for the maintenance of the (alpha)7 nicotinic acetylcholine receptor in somatic spines of ciliary neurons.
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Calcium-permeable neurotransmitter receptors are concentrated into structurally and biochemically isolated cellular compartments to localize calcium-mediated events during neurotransmission. The cytoplasmic membrane contains lipid microdomains called lipid rafts, which can gather into microscopically visible clusters, and thus the association of a particular protein with lipid rafts can result in its redistribution on the cell surface. The present study asks whether lipid rafts participate in the formation and maintenance of the calcium-permeable alpha7-subunit nicotinic acetylcholine receptor (alpha7nAChR) clusters found in somatic spines of ciliary neurons. Lipid rafts and alpha7nAChR become progressively colocalized within somatic spines during synaptogenesis. To determine whether these rafts are required for the maintenance of alpha7nAChR aggregates, cholesterol was extracted from dissociated ciliary neurons by treatment with methyl-beta-cyclodextrin. This treatment caused the dispersion of lipid rafts and the redistribution of alpha7nAChR into small clusters over the cell surface, suggesting that the integrity of lipid rafts is required to maintain the receptor clustering. However, lipid raft dispersion also caused the depolymerization of the F-actin cytoskeleton, which can also tether the receptor at specific sites. To assess whether interaction between rafts and alpha7nAChR is independent of F-actin filaments, the lipid raft patches were stabilized with a combination of the cholera toxin B subunit (CTX), which specifically binds to the raft component ganglioside GM1, and an antibody against CTX. The stabilized rafts were then treated with latrunculin-A to depolymerize F-actin. Under these conditions, large patches of CTX persisted and were colocalized with alpha7nAChR, indicating that the aggregates of receptors can be maintained independently of the underlying F-actin cytoskeleton. Moreover, it was found that the alpha7nAChR is resistant to detergent extraction at 4 degrees C and floats with the caveolin-containing lipid-rich fraction during density gradient centrifugation, properties that are consistent with a direct association between the receptor and the membrane microdomains. (+info)
Epstein-Barr virus coopts lipid rafts to block the signaling and antigen transport functions of the BCR.
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The B cell antigen receptor (BCR) functions to initiate signaling and to internalize antigen for processing from within Lyn kinase-enriched membrane lipid rafts. The signaling function of the BCR is blocked by Epstein-Barr Virus (EBV) latent membrane protein 2A (LMP2A), which is constitutively phosphorylated by Lyn. Here, we show that LMP2A resides in lipid rafts and excludes the BCR from entering rafts by Lyndependent mechanisms, thus blocking both BCR signaling and antigen transport. Mutant LMP2A that permits BCR signaling and raft translocation still blocks antigen trafficking, indicating independent control of these BCR functions. Thus, EBV coopts the lipid rafts to disarm both the signaling and antigen-processing functions of the BCR by independent mechanisms. (+info)
Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins.
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The anaerobic bacterium Clostridium difficile is the etiologic agent of pseudomembranous colitis. C. difficile toxins TcdA and TcdB are UDP-glucosyltransferases that monoglucosylate and thereby inactivate the Rho family of GTPases (W. P. Ciesla, Jr., and D. A. Bobak, J. Biol. Chem. 273:16021-16026, 1998). We utilized purified reference toxins of C. difficile, TcdA-10463 (TcdA) and TcdB-10463 (TcdB), and a model intestinal epithelial cell line to characterize their influence on tight-junction (TJ) organization and hence to analyze the mechanisms by which they contribute to the enhanced paracellular permeability and disease pathophysiology of pseudomembranous colitis. The increase in paracellular permeability induced by TcdA and TcdB was associated with disorganization of apical and basal F-actin. F-actin restructuring was paralleled by dissociation of occludin, ZO-1, and ZO-2 from the lateral TJ membrane without influencing the subjacent adherens junction protein, E-cadherin. In addition, we observed decreased association of actin with the TJ cytoplasmic plaque protein ZO-1. Differential detergent extraction and fractionation in sucrose density gradients revealed TcdB-induced redistribution of occludin and ZO-1 from detergent-insoluble fractions constituting "raft-like" membrane microdomains, suggesting an important role of Rho proteins in maintaining the association of TJ proteins with such microdomains. These toxin-mediated effects on actin and TJ structure provide a mechanism for early events in the pathophysiology of pseudomembranous colitis. (+info)
Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor.
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In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor-CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 zeta chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on zeta chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation. (+info)
Released GFRalpha1 potentiates downstream signaling, neuronal survival, and differentiation via a novel mechanism of recruitment of c-Ret to lipid rafts.
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Although both c-Ret and GFRalpha1 are required for responsiveness to GDNF, GFRalpha1 is widely expressed in the absence of c-Ret, suggesting alternative roles for "ectopic" sites of GFRalpha1 expression. We show that GFRalpha1 is released by neuronal cells, Schwann cells, and injured sciatic nerve. c-Ret stimulation in trans by soluble or immobilized GFRalpha1 potentiates downstream signaling, neurite outgrowth, and neuronal survival, and elicits dramatic localized expansions of axons and growth cones. Soluble GFRalpha1 mediates robust recruitment of c-Ret to lipid rafts via a novel mechanism requiring the c-Ret tyrosine kinase. Activated c-Ret associates with different adaptor proteins inside and outside lipid rafts. These results provide an explanation for the tissue distribution of GFRalpha1, supporting the physiological importance of c-Ret activation in trans as a novel mechanism to potentiate and diversify the biological responses to GDNF. (+info)
c-Src is required for glial cell line-derived neurotrophic factor (GDNF) family ligand-mediated neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway.
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The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), consisting of GDNF, neurturin, persephin, and artemin, signal via a multicomponent complex composed of Ret tyrosine kinase and the glycosyl-phosphatidylinositol (GPI)-anchored coreceptors GFRalpha1-alpha4. In previous work we have demonstrated that the localization of Ret to membrane microdomains known as lipid rafts is essential for GDNF-induced downstream signaling, differentiation, and neuronal survival. Moreover, we have found that Ret interacts with members of the Src family kinases (SFK) only when it is localized to these microdomains. In the present work we show by pharmacological and genetic approaches that Src activity was necessary to elicit optimal GDNF-mediated signaling, neurite outgrowth, and survival. In particular, p60Src, but not the other ubiquitous SFKs, Fyn and Yes, was responsible for the observed effects. Moreover, Src appeared to promote neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway because the PI-3K inhibitor LY294002 prevented GFL-mediated neuronal survival and prevented activated Src-mediated neuronal survival. In contrast, the inhibition of Src activity had no effects on NGF-mediated survival, indicating that the requirement for Src was selective for GFL-mediated neuronal survival. These data confirm the importance of protein-protein interactions between Ret and raft-associated proteins in the signaling pathways elicited by GDNF, and the data implicate Src as one of the major signaling molecules involved in GDNF-mediated bioactivity. (+info)