Previous studies have demonstrated roles for vesicle-associated membrane protein 2 (VAMP 2) and VAMP 8 in Ca 2؉ -regulated pancreatic acinar cell secretion, however, their coordinated function in the secretory pathway has not been addressed. Here we provide evidence using immunofluorescence microscopy, cell fractionation, and SNARE protein interaction studies that acinar cells contain two distinct populations of zymogen granules (ZGs) expressing either VAMP 2 or VAMP 8. Further, VAMP 8-positive granules also contain the synaptosome-associated protein 29, whereas VAMP 2-expressing granules do not. Analysis of acinar secretion by Texas red-dextran labeling indicated that VAMP 2-positive ZGs mediate the majority of exocytotic events during constitutive secretion and also participate in Ca 2؉ -regulated exocytosis, whereas VAMP 8-positive ZGs are more largely involved in Ca 2؉ -stimulated secretion. Previously undefined functional roles for VAMP and syntaxin isoforms in acinar secretion were established by introducing truncated constructs of these proteins into permeabilized acini. VAMP 2 and VAMP 8 constructs each attenuated Ca 2؉ -stimulated exocytosis by 50%, whereas the neuronal VAMP 1 had no effects. In comparison, the plasma membrane SNAREs syntaxin 2 and syntaxin 4 each inhibited basal exocytosis, but only syntaxin 4 significantly inhibited Ca 2؉ -stimulated secretion. Syntaxin 3, which is expressed on ZGs, had no effects. Collectively, these data demonstrate that individual acinar cells express VAMP 2-and VAMP 8-specific populations of ZGs that orchestrate the constitutive and Ca 2؉ -regulated secretory pathways.The exocrine pancreas is responsible for synthesizing and secreting a variety of digestive enzymes that are essential for assimilation of the diet. Secretion occurs by exocytosis of large dense core zymogen granules (ZGs) 3 localized in the apical cytoplasm of acinar cells. Similar to endocrine and neural cells, exocytosis is highly induced following acinar stimulation by secretagogues; however, acini are unique in that a significant proportion of exocytosis also occurs by a constitutive pathway under basal conditions. Secretagogue-stimulated exocytosis is mediated by G protein-coupled receptors, which signal through phospholipase C and/or adenylate cyclase, and The process of exocytosis and other membrane fusion events is widely held to be regulated by soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) interactions in cells (3-5). SNARE proteins are classified as either Q-SNARE or R-SNARE based on conserved glutamine (Q) and arginine (R) residues positioned within their characteristic coiled-coil motifs. When brought in close opposition, Q-and R-SNAREs on each membrane form a heterotrimeric complex that provides the driving force for membrane fusion. In neurons, one R-containing coil of the complex is derived from vesicle-associated membrane protein (VAMP/synaptobrevin) present on synaptic vesicles, and one Q-containing coil is contributed by syntaxin 1 located on the pre...

ISG maturation progresses by removal of lysosomal membrane and select content proteins, which enter endosomal intermediates prior to their apical exocytosis. Constitutive and stimulated secretion through this mechanism is termed the constitutive-like and minor-regulated pathways, respectively. However, the molecular components that control membrane trafficking within these endosomal compartments are largely unknown. We show that tumor protein D52 is highly expressed in endosomal compartments following pancreatic acinar cell stimulation and regulates apical exocytosis of an apically directed endolysosomal compartment. Secretion from the endolysosomal compartment was detected by cell-surface antigen labeling of lysosomeassociated membrane protein LAMP1, which is absent from ZGs, and had incomplete overlap with surface labeling of synaptotagmin 1, a marker of ZG exocytosis. Although culturing (16 -18 h) of isolated acinar cells is accompanied by a loss of secretory responsiveness, the levels of SNARE proteins necessary for ZG exocytosis were preserved. However, levels of endolysosomal proteins D52, EEA1, Rab5, and LAMP1 markedly decreased with culture. When D52 levels were restored by adenoviral delivery, the levels of these regulatory proteins and secretion of both LAMP1 (endolysosomal) and amylase was strongly enhanced. These secretory effects were absent in alanine and aspartate substitutions of serine 136, the major D52 phosphorylation site, and were inhibited by brefeldin A, which does not directly affect the ZG compartment. Our results indicate that D52 directly regulates apical endolysosomal secretion and are consistent with previous studies, suggesting that this pathway indirectly regulates ZG secretion of digestive enzymes.TPD52; constitutive-like pathway; minor-regulated pathway; endolysosomal secretion PANCREATIC ACINAR CELLS ARE a traditional model for studying membrane and protein trafficking within the secretory pathway of mammalian cells (53) because they mediate the constitutive and regulated secretion of digestive enzymes necessary for nutrient digestion in the intestine. Pancreatic acini are arranged in clusters of 8 -12 cells with their apical plasma membranes forming the terminal end of pancreatic ductules. Secretagogue stimulation at the basolateral membrane occurs through G protein-coupled receptors to activate PLC and/or adenylyl cyclase pathways that induce the exocytosis of large (ϳ1 m diameter) zymogen granules (ZGs). Acutely isolated acini are typically used to study secretion because they are highly sensitive to secretagogues and remain polarized. However, these features are lost in culture through unknown mechanisms, making use of mRNA silencing techniques to reduce protein expression difficult.In addition to the classic ZG secretory pathway, two unique parallel secretory pathways have been identified in acinar cells from the pancreas and parotid glands known as the constitutive-like (CLP) and minor regulated (MRP) pathways (1,10,11,33,35). Newly synthesized secretory proteins are selec...

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CRHSP-28 is a Ca2؉ -regulated heat-stable phosphoprotein, abundant in the apical cytoplasm of epithelial cells that are specialized in exocrine protein secretion. To define a functional role for the protein in pancreatic secretion, recombinant CRHSP-28 (rCRHSP-28) was introduced into streptolysin-O-permeabilized acinar cells, and amylase secretion in response to elevated Ca 2؉ was determined. Secretion was enhanced markedly by rCRHSP-28 over a time course that closely corresponded with the loss of the native protein from the intracellular compartment. No effects of rCRHSP-28 were detected until ϳ50% of the native protein was lost from the cytosol. Secretion was enhanced by rCRHSP-28 over a physiological range of Ca 2؉ concentrations with 2-3-fold increases in amylase release occurring in response to low micromolar levels of free Ca 2؉ . Further, rCRHSP-28 augmented secretion in a concentration-dependent manner with minimal and maximal effects occurring at 1 and 25 g/ml, respectively. Covalent cross-linking experiments demonstrated that native CRHSP-28 was present in a 60-kDa complex in cytosolic fractions and in a high molecular mass complex in particulate fractions, consistent with the slow leak rate of the protein from streptolysin-O-permeabilized cells. Probing acinar lysates with rCRHSP-28 in a gel-overlay assay identified two CRHSP-28-binding proteins of 35 (pp35) and 70 kDa (pp70). Interestingly, preparation of lysates in the presence of 1 mM Ca 2؉ resulted in a marked redistribution of both proteins from a cytosolic to a Triton X-100-insoluble fraction, suggesting a Ca 2؉ -sensitive interaction of these proteins with the acinar cell cytoskeleton. In agreement with our previous study immunohistochemically localizing CRHSP-28 around secretory granules in acinar cells, gel-overlay analysis revealed pp70 copurified with acinar cell secretory granule membranes. These findings demonstrate an important cell physiological function for CRHSP-28 in the Ca 2؉ -regulated secretory pathway of acinar cells.Exocrine cells specializing in protein secretion release a variety of factors necessary for normal function of the digestive, urogenital, respiratory, and ocular systems. Activation of these epithelia by neural and humoral agents stimulates the exocytosis of secretory granules at the apical plasma membrane in a process that is largely controlled by cellular Ca 2ϩ . In pancreatic acinar cells, Ca 2ϩ release is initiated in the apical pole and then propagates through the cell periphery to the basal cytoplasm. The cyclic reuptake and release of Ca 2ϩ from intracellular stores create an oscillatory mode of signaling with spatial and temporal characteristics that are unique to the specific type and concentration of physiologic stimulus (for review, see Refs. 1-3). Although the high concentrations of Ca 2ϩ generated in the apical cytoplasm are necessary for secretory granule trafficking and exocytosis to occur, a comprehensive understanding of the molecular events elicited by this ion is lacking.The secretory pathway in ac...

Tumor protein D52 (also known as CRHSP-28) is highly expressed in multiple cancers and tumor-derived cell lines; however, it is normally abundant in secretory epithelia throughout the digestive system, where it has been implicated in Ca(2+)-dependent digestive enzyme secretion (41). Here we demonstrate, using site-specific mutations, that Ca(2+)-sensitive phosphorylation at serine 136 modulates the accumulation of D52 at the plasma membrane within 2 min of cell stimulation. When expressed in Chinese hamster ovary CHO-K1 cells, D52 colocalized with adaptor protein AP-3, Rab27A, vesicle-associated membrane protein VAMP7, and lysosomal-associated membrane protein LAMP1, all of which are present in lysosome-like secretory organelles. Overexpression of D52 resulted in a marked accumulation of LAMP1 on the plasma membrane that was further enhanced following elevation of cellular Ca(2+). Strikingly, mutation of serine 136 to alanine abolished the Ca(2+)-stimulated accumulation of LAMP1 at the plasma membrane whereas phosphomimetic mutants constitutively induced LAMP1 plasma membrane accumulation independent of elevated Ca(2+). Identical results were obtained for endogenous D52 in normal rat kidney and HeLA cells, where both LAMP1 and D52 rapidly accumulated on the plasma membrane in response to elevated cellular Ca(2+). Finally, D52 induced the uptake of LAMP1 antibodies from the cell surface in accordance with both the level of D52 expression and phosphorylation at serine 136 demonstrating that D52 altered the plasma membrane recycling of LAMP1-associated secretory vesicles. These findings implicate both D52 expression and Ca(2+)-dependent phosphorylation at serine 136 in lysosomal membrane trafficking to and from the plasma membrane providing a novel Ca(2+)-sensitive pathway modulating the lysosome-like secretory pathway.