Annexin VI

Journal of Cell Science, 1994

The Journal of Biological Chemistry, 1989

We have purified three 35-kDa calcium-and phospholipid-binding proteins from rat liver. These three calcimedins bind to phosphatidylserine in a calciumdependent manner and have been termed 35a, 358, and 357 based on their relative charge as determined by isoelectric focusing. Purification of the three 35-kDa calcimedins is achieved by phenyl-Sepharose, ion exchange, and gel filtration chromatography. Antibody was produced against the annexin consensus peptide, Lys-Ala-Met-Lys-Gly-Leu-Gly-Thr-Asp-Glu, which was derived from the sequence of several Ca2+/phospholipid-binding proteins including calpactin, lipocortin, endonexin 11, 67-kDa calelectrin, lymphocyte 68-kDa protein, and protein 11. Recognition of each 35-kDa calcimedin by anticonsensus sequence antibody places them in this protein family. Antibodies against each 35-kDa calcimedin were raised and purified by antigen-affinity chromatography. Each antibody is monospecific for the respective 35-kDa calcimedin. Immunological cross-reactivity defines 35a, 358, and 357 as lipocortins 111, IV, and V, respectively. Surveys by immunoblot analysis using these monospecific antibodies demonstrate a markedly different tissue expression pattern for each 35-kDa calcimedin. Furthermore, the levels of 35a, 358, and 357 are differentially regulated in maturing rat ovary and uterus. Each calcimedin has been localized by indirect immunofluorescence within specific cell types. These results support the concept that mediation of the intracellular calcium signal can occur via multiple pathways through several related yet independent mediator proteins.

Journal of Clinical Investigation, 1976

A B S T R A C T In dispersed acinar cells prepared from guinea pig pancreas, cellular uptake of 45Ca was moderately rapid and reached a steady state bv 60 min. At the steady state, 69% of total cellular 45Ca was membrane-bound. In acinar cells preloaded with 45Ca and then incubated with COOH-terminal octapeptide of cholecystokinin (CCK-OP) or carbanylcholine, total cellular 45Ca decreased by approximately 40% within 5-10 min and then steadily increased to control values by 60 min. Under identical conditions, membranebound 45Ca decreased by 40% within 5-10 min and remained constant for the duration of the incubation. Free cellular 45Ca did not change during the initial 30 min but then increased steadily to values three times those in control cells by 60 min. In cells preloaded with 45Ca and then incubated with EDTA, the loss of total cellular radioactivitv stimulated b)y CCK-OP could be accounted for by loss of membrane-bound 45Ca. CCK-OP failed to alter total cellular uptake of 45Ca when both tracer and peptide were added at the beginning of the incubation. Under identical conditions, membrane-bound 45Ca was not altered b1 CCK-OP during the first 30 min of incubation but was significantly below control values after this time. The effect of CCK-OP on free cellular 45Ca was the same as in cells preloaded with the tracer. These results suggest that CCK-OP catuses release of 45Ca from a membrane-botund compartment that equilibrates slowly with extracellular fluid and that the change in free cellular 45Ca is a secondary effect. NIETHODS Male Hartley strain albino guinea pigs (350-400 g) were obtained from the Small Animals Section, Veterinary Resources Branch, National Institutes of Health, Bethesda, md. 45Ca (12.5 mCi/mg) and ["sC]alpha-aminoisobutyric acid (AIB,' 5.1 mCi/mmol) were purchased from New England Nuclear (Boston, Mass.); 42K (2.6 mCi/mg) from ICN Corp., Irvine, Calif.); crude collagenase (Clostridium histolyticu mz, 'Abbreviations tised in this paper: AIB, alpha-aminoisobutyric acid; CCK-OP, COOH-terminial octapeptide of cholecvstokinin.

Bone and Mineral, 1992

Pfl�gers Archiv European Journal of Physiology, 1990

Regulation of intracellular free calcium ([C a2 +]i) in single epithelial duct cells of isolated rat and guinea pig pancreatic interlobular ducts by secretin, carbachol and cholecystokinin was studied by microspectrofluorometry using the Ca 2 +-sensitive, fluorescent probe Fura-2. Rat and guinea pig duct cells exhibited mean resting [Ca2+]i of 84 nM and 61 nM, respectively, which increased by 50%-100% in response to carbachol stimulation, thus demonstrating the presence of physiologically responsive cholinergic receptors in pancreatic ducts of both species. The carbachol-induced increase in [Ca2+]i involved both mobilization of Ca 2+ from intracellular stores and stimulation of influx of extracellular Ca z +. In contrast, neither cholecystokinin nor secretin showed reproducible or sizeable increses in [Ca z+]i. Both rat and guinea pig duct cells showed considerable resting Ca 2 + permeability. Lowering or raising the extracellular [Ca2+]i led, respectively, to a decrease or increase in the resting [Ca2+]i. Application of Mn 2+ resulted in a quenching of the fluorescence signal indicating its entry into the cell. The resting Ca 2+ and Mn 2+ permeability could be blocked by La 3 + suggesting that it is mediated by a Ca 2 + channel.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2001

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca 2-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-Lcyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-L-cyclodextrin complexes restored the Ca 2-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca 2 , annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca 2 concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca 2 concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.

Biology of the Cell, 1991

The results of immunoblot analysis performed with a specific monoclonal antibody showed that the intestinal mucosa, pancreas and liver are privileged tissues for the expression of annexin IV. Immunofluorescence labelling of thin frozen sections of these tissues showed a strong concentration of annexin IV along the basolateral domain of the plasma membrane of intestinal absorbing cells, hepatocytes and pancreatic acinar cells, whereas in intestinal mucous secreting cells and centro acinar pancreatic cells, annexin IV was found to be present throughout the cytoplasm. poladty I Iipocortin I immunofluorescence I eytoskeleton I plasma membrane

Journal of Cellular Biochemistry, 1991

Annexin VI is a member of a Ca2+-dependent, phospholipid-binding protein family. Although functions for this annexin have been proposed from in vitro studies, most remain controversial. Diaz-MuFioz et al. ( 1 Biol Chern 265:15894, 1990) demonstrated that annexin VI modified, in a Ca2+-dependent manner, the gating behavior of the sarcoplasmic reticulum Ca*+-release channel, reconstituted into artificial bilayers, by increasing both the open probability and the mean open time. This effect was specific to the trans chamber, which represents the luminal side of the sarcoplasmic reticulum. In agreement with those findings, we show herein that annexin VI produced no effect on Ca2+-uptake or -release by intact heavy sarcoplasmic reticulum vesicles (analogous to the cis chamber). We also used monospecific antibodies to evaluate the subcellular localization of annexin VI by immunofluorescent microscopy. Studies in rat skeletal muscle suggest that annexin VI is present surrounding individual myofibrils. Double immunolocalization studies with cultured muscle cells (chick myotubes) using anti-annexin V1 and anti-SR Ca2'-ATPase antibodies demonstrated superimposable staining patterns. In non-muscle tissue (normal rat kidney (NRK) cells), a punctate, perinuclear anti-annexin VI staining pattern was observed. Collectively, these data suggest that annexin VI may play a regulatory role in the Ca'+-release/uptake cycle in the sarcoplasmic reticulum as well as in non-muscle organelles, a key process in stimulus-response systems.

Journal of Biological Chemistry, 2002

Annexins are Ca 2؉-and phospholipid-binding proteins that are widely expressed in mammalian tissues and that bind to different cellular membranes. In recent years its role in membrane traffic has emerged as one of its predominant functions, but the regulation of its intracellular distribution still remains unclear. We demonstrated that annexin 6 translocates to the late endocytic compartment in low density lipoprotein-loaded CHO cells. This prompted us to investigate whether cholesterol, one of the major constituents of low density lipoprotein, could influence the membrane binding affinity and intracellular distribution of annexin 6. Treatment of crude membranes or early and late endosomal fractions with digitonin, a cholesterol-sequestering agent, displayed a strong reduction in the binding affinity of a novel EDTA-resistant and cholesterol-sensitive pool of annexin 6 proteins. In addition, U18666A-induced accumulation of cholesterol in the late endosomal compartment resulted in a significant increase of annexin 6 in these vesicles in vivo. This translocation/recruitment correlates with an increased membrane binding affinity of GST-annexin 6 to late endosomes of U18666A-treated cells in vitro. In conclusion, the present study shows that changes in the intracellular distribution and concentration of cholesterol in different subcellular compartments participate in the reorganization of intracellular pools of Ca 2؉-dependent and-independent annexin 6. * This work was supported by the Deutsche Forschungsgemeinschaft (DFG, Ja 421/3-1 and Be 829/5-1) and Grants from Ministerio de Ciencia y Tecnología (PM99-0166), Acciones Integradas (HA98-0007), and Generalitat de Catalunya (BE2000). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Recipient of a fellowship from the Institut d'Investigacions Biomèdiques August Pi i Sunyer.

Biochemical Journal, 2003

Store-mediated Ca# + entry (SMCE) is one of the main pathways for Ca# + influx in non-excitable cells. Recent studies favour a secretion-like coupling mechanism to explain SMCE, where Ca# + entry is mediated by an interaction of the endoplasmic reticulum (ER) with the plasma membrane (PM) and is modulated by the actin cytoskeleton. To explore this possibility further we have now investigated the role of the actin cytoskeleton in the activation and maintenance of SMCE in pancreatic acinar cells, a more specialized secretory cell type which might be an ideal cellular model to investigate further the properties of the secretion-like coupling model. In these cells, the cytoskeletal disrupters cytochalasin D and latrunculin A inhibited both the activation and maintenance of SMCE. In addition, stabilization of a cortical actin barrier by jasplakinolide prevented the