Isoproterenol increases sorting of parotid gland cargo proteins to the basolateral pathway

Exocrine cells have an essential function of sorting secreted proteins into the correct secretory pathway. A clear understanding of sorting in salivary glands would contribute to the correct targeting of therapeutic transgenes. The present work investigated whether there is a change in the relative proportions of basic proline-rich protein (PRP) and acidic PRPs in secretory granules in response to chronic isoproterenol treatment, and whether this alters the sorting of endogenous cargo proteins. Immunoblot analysis of secretory granules from rat parotids found a large increase of basic PRP over acidic PRPs in response to chronic isoproterenol treatment. Pulse chase experiments demonstrated that isoproterenol also decreased regulated secretion of newly synthesized secretory proteins, including PRPs, amylase and parotid secretory protein. This decreased efficiency of the apical regulated pathway may be mediated by alkalization of the secretory granules since it was reversed by treatment with mild acid. We also investigated changes in secretion through the basolateral (endocrine) pathways. A significant increase in parotid secretory protein and salivary amylase was detected in sera of isoproterenol-treated animals, suggesting increased routing of the regulated secretory proteins to the basolateral pathway. These studies demonstrate that shifts of endogenous proteins can modulate regulated secretion and sorting of cargo proteins. amylase; parotid secretory protein; polarized secretion     

Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells

In the beta-cells of pancreatic islets, insulin is stored as the predominant protein within storage granules that undergo regulated exocytosis in response to glucose. By pulse-chase analysis of radiolabeled protein condensation in beta-cells, the formation of insoluble aggregates of regulated secretory protein lags behind the conversion of proinsulin to insulin. Condensation occurs within immature granules (IGs), accounting for passive protein sorting as demonstrated by constitutive-like secretion of newly synthesized C- peptide in stoichiometric excess of insulin (Kuliawat, R., and P. Arvan. J. Cell Biol. 1992. 118:521-529). Experimental manipulation of condensation conditions in vivo reveals a direct relationship between sorting of regulated secretory protein and polymer assembly within IGs. By contrast, entry from the trans-Golgi network into IGs does not appear especially selective for regulated secretory proteins. Specifically, in normal islets, lysosomal enzyme precursors enter the stimulus-dependent secretory pathway with comparable efficiency to that of proinsulin. However, within 2 h after synthesis (the same period during which proinsulin processing occurs), newly synthesized hydrolases are fairly efficiently relocated out of the stimulus- dependent pathway. In tunicamycin-treated islets, while entry of new lysosomal enzymes into the regulated secretory pathway continues unperturbed, exit of nonglycosylated hydrolases from this pathway does not occur. Consequently, the ultimate targeting of nonglycosylated hydrolases in beta-cells is to storage granules rather than lysosomes. These results implicate a post-Golgi mechanism for the active removal of lysosomal hydrolases away from condensed granule contents during the storage process for regulated secretory proteins.     

Ammonium chloride alters secretory protein sorting within the maturing exocrine storage compartment

Addition of 20 mM ammonium chloride during in vitro chase incubation of [35S]methionine pulse-labeled parotid tissue does not perturb the magnitude or radiochemical composition of secretion stimulated by isoproterenol. An apparent inhibition of stimulated output of radiolabeled secretory proteins that was observed when ammonium chloride was added immediately postpulse (but not at later time points prior to stimulation) could be accounted for by slowdown in Golgi transit of exocrine secretory protein at a stage prior to completion of terminal glycosylation. Thus, ammonium chloride does not block entry of newly synthesized secretory proteins into the secretagogue-releasable storage granule compartment. By contrast, ammonium chloride increases the output and substantially alters the relative composition of newly synthesized protein in unstimulated secretion. The latter effects could be assigned to stages of intracellular transport that normally occur at chase times greater than 60 min postpulse and thus are focused within the maturing acinar storage granule. Notably, the compositional alterations cannot reflect the preferential exocytosis of immature granules. Taken together, these results suggest that the sorting of exocrine secretory proteins into the secretagogue-regulated pathway may not involve positive selection by a pH-based process initiated in a pregranule compartment. Rather, unstimulated secretion may arise by a negative sorting (or exclusion) process that occurs during compaction of proteins for storage within maturing granules and that is perturbed by weak base addition. Sorted (or excluded) proteins would appear to follow the vesicular (nongranular) secretory pathway that originates in maturing granules (von Zastrow, M., and Castle, J.D. (1987) J. Cell Biol. 105, 2675-2684).     

Expression of pro-Muclin in pancreatic AR42J cells induces functional regulated secretory granules

It is not clear how protein cargo is sorted to and retained in forming regulated secretory granules (RSG). Here, the sulfated mucin-type glycoprotein pro-Muclin was tested for its ability to induce RSG in the poorly differentiated rat pancreatic cell line AR42J. AR42J cells express RSG content proteins, but they fail to make granules. Adenovirus-pro-Muclin-infected AR42J cells store amylase, accumulate RSG, and respond to hormonal stimulation by secreting the stored protein. Expression of pro-Muclin combined with the inducing effect of dexamethasone resulted in a significant enhancement of the efficiency of regulated secretion. The effect of pro-Muclin was a strong decrease in constitutive secretion compared with dexamethasone-induction alone. A pro-Muclin construct missing the cytosolic tail domain was less effective at improving the efficiency of regulated secretion compared with the full-length construct. Increased expression of cargo (using adenovirus amylase) also modestly enhanced regulated secretion, indicating that part of pro-Muclin's effect may be due to increased expression of cargo protein. Overall, the data show that pro-Muclin acts as a sorting receptor that can induce RSG, and that its cytosolic tail is important in this process. regulated secretion; protein sorting     

Not all secretory granules are created equal: Partitioning of soluble content proteins

Secretory granules carrying fluorescent cargo proteins are widely used to study granule biogenesis, maturation, and regulated exocytosis. We fused the soluble secretory protein peptidylglycine alpha-hydroxylating monooxygenase (PHM) to green fluorescent protein (GFP) to study granule formation. When expressed in AtT-20 or GH3 cells, the PHM-GFP fusion protein partitioned from endogenous hormone (adrenocorticotropic hormone, growth hormone) into separate secretory granule pools. Both exogenous and endogenous granule proteins were stored and released in response to secretagogue. Importantly, we found that segregation of content proteins is not an artifact of overexpression nor peculiar to GFP-tagged proteins. Neither luminal acidification nor cholesterol-rich membrane microdomains play essential roles in soluble content protein segregation. Our data suggest that intrinsic biophysical properties of cargo proteins govern their differential sorting, with segregation occurring during the process of granule maturation. Proteins that can self-aggregate are likely to partition into separate granules, which can accommodate only a few thousand copies of any content protein; proteins that lack tertiary structure are more likely to distribute homogeneously into secretory granules. Therefore, a simple "self-aggregation default" theory may explain the little acknowledged, but commonly observed, tendency for both naturally occurring and exogenous content proteins to segregate from each other into distinct secretory granules.     

Biosynthesis and secretion of pituitary hormones: dynamics and regulation

Production and secretion of hormones by the pituitary involve highly orchestrated intracellular transport and sorting steps. Hormone precursors are routed through a series of compartments before being packaged in secretory granules. These highly dynamic carriers play crucial roles in both prohormone processing and peptide exocytosis. We have employed the ACTH-secreting AtT-20 cell line to study the membrane sorting events that confer functionality (prohormone activation and regulated exocytosis) to these secretory carriers. The unique ability of granules to promote prohormone processing is attributed to their acidic interior. Using a novel avidin-targeted fluorescence ratio imaging technique, we have found that the trans-Golgi of live AtT-20 cells maintains a mildly acidic (approximately pH 6.2) interior. Budding of secretory granules causes the lumen to acidify to 相似文献   

Mannose 6–Phosphate Receptors Are Sorted from Immature Secretory Granules via Adaptor Protein AP-1, Clathrin, and Syntaxin 6–positive Vesicles

The occurrence of clathrin-coated buds on immature granules (IGs) of the regulated secretory pathway suggests that specific transmembrane proteins are sorted into these buds through interaction with cytosolic adaptor proteins. By quantitative immunoelectron microscopy of rat endocrine pancreatic β cells and exocrine parotid and pancreatic cells, we show for the first time that the mannose 6–phosphate receptors (MPRs) for lysosomal enzyme sorting colocalize with the AP-1 adaptor in clathrin-coated buds on IGs. Furthermore, the concentrations of both MPR and AP-1 decline by ~90% as the granules mature. Concomitantly, in exocrine secretory cells lysosomal proenzymes enter and then are sorted out of IGs, just as was previously observed in β cells (Kuliawat, R., J. Klumperman, T. Ludwig, and P. Arvan. 1997. J. Cell Biol. 137:595–608). The exit of MPRs in AP-1/clathrin-coated buds is selective, indicated by the fact that the membrane protein phogrin is not removed from maturing granules. We have also made the first observation of a soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 6, which has been implicated in clathrin-coated vesicle trafficking from the TGN to endosomes (Bock, J.B., J. Klumperman, S. Davanger, and R.H. Scheller. 1997. Mol. Biol. Cell. 8:1261–1271) that enters and then exits the regulated secretory pathway during granule maturation. Thus, we hypothesize that during secretory granule maturation, MPR–ligand complexes and syntaxin 6 are removed from IGs by AP-1/clathrin-coated vesicles, and then delivered to endosomes.     

Secretory granule biogenesis: rafting to the SNARE

Regulated secretion of hormones occurs when a cell receives an external stimulus, triggering the secretory granules to undergo fusion with the plasma membrane and release their content into the extracellular milieu. The formation of a mature secretory granule (MSG) involves a series of discrete and unique events such as protein sorting, formation of immature secretory granules (ISGs), prohormone processing and vesicle fusion. Regulated secretory proteins (RSPs), the proteins stored and secreted from MSGs, contain signals or domains to direct them into the regulated secretory pathway. Recent data on the role of specific domains in RSPs involved in sorting and aggregation suggest that the cell-type-specific composition of RSPs in the trans-Golgi network (TGN) has an important role in determining how the RSPs get into ISGs. The realization that lipid rafts are implicated in sorting RSPs in the TGN and the identification of SNARE molecules represent further major advances in our understanding of how MSGs are formed. At the heart of these findings is the elucidation of molecular mechanisms driving protein--lipid and protein--protein interactions specific for secretory granule biogenesis.     

Immature granules are not major sites for segregation of constitutively secreted granule content proteins in NIT-1 insulinoma cells.

Immature secretory granules (ISG's) are sites of segregation of proteins destined for secretion by unregulated pathways from those stored in mature secretory granules in endocrine cells. To determine whether significant soluble protein sorting occurs in ISG's, the secretion of soluble versions of the pancreatic protein GP2 (GP2-GPI(-)) and placental alkaline phosphatase (SEAP) was analyzed in NIT-1 cells. By immunofluorescence microscopy, neither protein localized to SG's in transfected cells. Their secretion was secretagogue-independent in pulse-chase radiolabeling experiments even at early times of chase, while a small increase in the secretion of amylase, which is known to enter ISG's, could be detected. Finally, in sucrose gradient fractionation experiments, SEAP was present in light density fractions. We conclude that while some proteins, such as amylase, have a limited intrinsic capacity to enter ISG's, the segregation of proteins secreted via the constitutive pathway from SG content proteins occurs primarily in the trans Golgi network.     

Protein tyrosine phosphatase stimulates Ca(2+)-dependent amylase secretion from pancreatic acini.

Eukaryotic cells respond to various stimuli by an increase or decrease in levels of phosphoproteins. Phosphotyrosine levels on eukaryotic cellular proteins are tightly regulated by the opposing actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPases, EC 3.1.3.48). Studies on permeabilized mast cells suggest that the enabling reaction for exocytosis might involve protein dephosphorylation. In the present studies, a recombinant form of rat brain PTPase (rrbPTP-1) has been used to examine the potential role of PTPases in Ca(2+)-dependent amylase secretion from permeabilized rat pancreatic acini. Additionally, the concentrations and subcellular distributions of endogenous PTPase activity in rat pancreas were determined. The results from these experiments indicate that addition of exogenous PTPase stimulated Ca(2+)-dependent amylase secretion from pancreatic acinar cells and that endogenous PTPase activity was associated with the postgranule supernatant, zymogen granules, and in particular zymogen granule membranes. Our data suggest that protein tyrosine dephosphorylation is potentially involved in regulated secretion at a site(s) distal to receptor-mediated elevation of intracellular second messengers.     

Signal-mediated sorting of neuropeptides and prohormones: secretory granule biogenesis revisited

Neuropeptides and hormones, in contrast to constitutive secretory proteins, are sorted to and stored in secretory granules and released upon a stimulus. During the last two decades, signals and mechanisms involved in their sorting to the regulated pathway of protein secretion have been addressed in numerous studies. Taken together these studies revealed three important features of regulated secretory proteins: aggregation, sorting signal motifs and membrane binding. Here we try to dissect the sorting process with regard to these features and discuss their relevance in the context of current sorting models. We especially address the question where in the secretory pathway sorting takes place and discuss a possible role of sorting receptors.     

Regulated apical secretion of zymogens in rat pancreas. Involvement of the glycosylphosphatidylinositol-anchored glycoprotein GP-2, the lectin ZG16p, and cholesterol-glycosphingolipid-enriched microdomains

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.     

Characterization of the TGN exit routes in AtT20 cells using pancreatic amylase and serum albumin

The AtT20 pituitary cell is the one that was originally used to define the pathways taken by secretory proteins in mammalian cells. It possesses two secretory pathways, the constitutive for immediate secretion and the regulated for accumulation and release under hormonal stimulation. It is in the regulated pathway, most precisely in the immature granule of the regulated pathway, that proteolytic maturation takes place. A pathway that stems from the regulated one, namely the constitutive-like pathway releases proteins present in immature granules that are not destined for accumulation in mature granules. In AtT20 cells proopiomelanocortin the endogenous precursor of the accumulated adrenocorticotropic hormone, is predominantly secreted in a constitutive manner without proteolytic maturation. In order to better understand by which secretory pathway intact proopiomelanocortin is secreted by a cell line possessing a regulated secretory pathway, it was transfected with rat serum albumin (a marker of constitutive secretory proteins), and pancreatic amylase (a marker of regulated proteins). COS cells were also transfected in order to serve as control of release by the constitutive pathway. It was observed that both the basal and stimulated secretions of albumin and proopiomelanocortin from AtT20 cells are identical. In addition, secretagogue stimulation when POMC is in transit in the trans-Golgi network decreases its constitutive secretion by 50%. It was also observed using cell fractionation and 20 degrees C secretion blocks that albumin and proopiomelanocortin are present in the regulated pathway, presumably in the immature granules, and are secreted by the constitutive-like secretory pathway. These observations show that stimulation can increase sorting into the regulated pathway, and confirm the importance of the constitutive-like secretory pathway in the model AtT20 cell line.     

A sulfated proteoglycan is necessary for storage of exocrine secretory proteins in the rat parotid gland

Sulfated proteoglycans have beenproposed to play a role in the sorting and storage of secretoryproteins in exocrine secretory granules. Rat parotid acinar cellsexpressed a 40- to 60-kDa proteoglycan that was stored in secretorygranules. Treatment of the tissue with the proteoglycan synthesisinhibitor paranitrophenyl xyloside resulted in the complete abrogationof the sulfated proteoglycan. Pulse-chase experiments in the presenceof the xyloside analog showed a significant reduction in the stimulatedsecretion and granule storage of the newly synthesized regulatedsecretory proteins amylase and parotid secretory protein. Inhibition ofproteoglycan sulfation by chlorate did not affect the sorting of theseproteins. The effect of proteoglycan synthesis inhibition on proteinsorting was completely reversed upon treatment with a weak acid. These results suggest that the sulfated proteoglycan is necessary for sortingand storage of regulated secretory proteins in the exocrine parotidgland. Preliminary evidence suggests that the mechanism involves themodulation of granule pH by the proteoglycan rather than a directinteraction with other granule components.

     

Resting (basal) secretion of proteins is provided by the minor regulated and constitutive-like pathways and not granule exocytosis in parotid acinar cells

Resting secretion of salivary proteins by the parotid gland is sustained in situ between periods of eating by parasympathetic stimulation and has been assumed to involve low level granule exocytosis. By using parotid lobules from ad libitum fed rats stimulated with low doses of carbachol as an in vitro analog of resting secretion, we deduce from the composition of discharged proteins that secretion does not involve granule exocytosis. Rather, it derives from two other acinar export routes, the constitutive-like (stimulus-independent) pathway and the minor regulated pathway, which responds to low doses of cholinergic or beta-adrenergic agonists (Castle, J. D., and Castle, A. M. (1996) J. Cell Sci. 109, 2591-2599). The protein composition collected in vitro mimics that collected from cannulated ducts of glands given low level stimulation in situ. Analysis of secretory trafficking along the two pathways of resting secretion has indicated that the constitutive-like pathway may pass through endosomes after diverging from the minor regulated pathway at a brefeldin A-sensitive branch point. The branch point is deduced to be distal to a common vesicular budding event by which both pathways originate from immature granules. Detectable perturbation of neither pathway in lobules was observed by wortmannin addition, and neither serves as a significant export route for lysosomal procathepsin B. These findings show that parotid acinar cells use low capacity, high sensitivity secretory pathways for resting secretion and reserve granule exocytosis, a high capacity, low sensitivity pathway, for massive salivary protein export during meals. An analogous strategy may be employed in other secretory cell types.     

Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary

The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place.     

The lumenal domain of the integral membrane protein phogrin mediates targeting to secretory granules

Phogrin, a transmembrane glycoprotein of neuroendocrine cells, is localized to dense-core secretory granules. We have investigated the subcellular targeting of phogrin by analyzing the sorting of a series of deletion mutants to the regulated pathway of secretion in AtT20 cells. The lumenal domain as a soluble protein was efficiently routed to granules, based on a combination of morphological analysis and secretion studies. Sorting was not dependent on a candidate targeting signal consisting of an N-terminal conserved cysteine-rich motif. Both the pro-region and the lumenal domain of mature, post-translationally processed phogrin independently reached the granule, although the pro-region was sorted more efficiently. Once within the regulated secretory pathway, all phogrin lumenal domain proteins were stored in functional granules for extended periods of time. Thus, phogrin possesses several domains contributing to its targeting to the secretory granule. Our findings support a model of granule biogenesis where proteins are sorted on the basis of their biochemical properties rather than via signal-dependent binding to a targeting receptor. Sorting of integral membrane proteins mediated by the lumenal domain may ensure that functionally important transmembrane molecules are included in the forming granule.     

Protein sorting and secretion granule formation in regulated secretory cells

Formation of secretion granules in regulated secretory cells involves packaging a subject of proteins undergoing intracellular transport into specific vesicular carriers that function in stimulus-dependent exocytosis. Recent findings suggest that immature granules are a site of passive sorting, involving condensation of regulated secretory proteins. Proteins that are not condensed are stored to a lesser degree and are enriched in unstimulated, constitutive-like secretion. While these observations have helped to distinguish possible mechanisms of secretory protein sorting, there are only recent hints about the sorting processes that may be required to create the regulated secretory carrier membranes.     

A hydrophobic patch in a charged alpha-helix is sufficient to target proteins to dense core secretory granules

Many endocrine and neuroendocrine cells contain specialized secretory organelles called dense core secretory granules. These organelles are the repository of proteins and peptides that are secreted in a regulated manner when the cell receives a physiological stimulus. The targeting of proteins to these secretory granules is crucial for the generation of certain peptide hormones, including insulin and ACTH. Although previous work has demonstrated that proteins destined to a variety of cellular locations, including secretory granules, contain targeting sequences, no single consensus sequence for secretory granule-sorting signals has emerged. We have shown previously that alpha-helical domains in the C-terminal tail of the prohormone convertase PC1/3 play an important role in the ability of this region of the protein to direct secretory granule targeting (Jutras, I. Seidah, N. G., and Reudelhuber, T. L. (2000) J. Biol. Chem. 275, 40337-40343). In this study, we show that a variety of alpha-helical domains are capable of directing a heterologous secretory protein to granules. By testing a series of synthetic alpha-helices, we also demonstrate that the presence of charged (either positive or negative) amino acids spatially segregated from a hydrophobic patch in the alpha-helices of secretory proteins likely plays a critical role in the ability of these structures to direct secretory granule sorting.     

Differential aggregation properties of secretory proteins that are stored in exocrine secretory granules of the pancreas and parotid glands

Low-pH- and calcium-induced aggregation of regulated secretory proteins has been proposed to play a role in their retention and storage in secretory granules. However, this has not been tested for secretory proteins that are stored in the exocrine parotid secretory granules. Parotid granule matrix proteins were analyzed for aggregation in the presence or absence of calcium and in the pH range of 5.5 to 7.5. Amylase did not aggregate under these conditions, although <10% of parotid secretory protein (PSP) aggregated below pH 6.0. To test aggregation directly in isolated granules, rat parotid secretory granules were permeabilized with 0.1% saponin in the presence or absence of calcium and in the pH range of 5.0 to 8.4. In contrast to the low-pH-dependent retention of amylase in exocrine pancreatic granules, amylase was quantitatively released and most PSP was released from parotid granules under all conditions. Both proteins were completely released upon granule membrane solubilization. Thus neither amylase nor PSP show low-pH- or calcium-induced aggregation under physiological conditions in the exocrine parotid secretory granules.