Role of H+-ATPase-mediated acidification in sorting and release of the regulated secretory protein chromogranin A: evidence for a vesiculogenic function

The constitutive and regulated secretory pathways represent the classical routes for secretion of proteins from neuroendocrine cells. Selective aggregation of secretory granule constituents in an acidic, bivalent cation-rich environment is considered to be a prerequisite for sorting to the regulated secretory pathway. The effect of selective vacuolar H+-ATPase (V-ATPase) inhibitor bafilomycin A1 on the pH gradient along the secretory pathway was used here to study the role of acidification on the trafficking of the regulated secretory protein chromogranin A (CgA) in PC12 cells. Sorting of CgA was assessed by three-dimensional deconvolution microscopy, subcellular fractionation, and secretagogue-stimulated release, examining a series of full-length or truncated domains of human CgA (CgA-(1-115), CgA-(233-439)) fused to either green fluorescent protein or to a novel form of secreted embryonic alkaline phosphatase (EAP). We show that a full-length CgA/EAP chimera is sorted to chromaffin granules for exocytosis. Inhibition of V-ATPase by bafilomycin A1 markedly reduced the secretagogue-stimulated release of CgA-EAP by perturbing sorting of the chimera (at the trans-Golgi network or immature secretory granule) rather than the late steps of exocytosis. The effect of bafilomycin A1 on CgA secretion depends on a sorting determinant located within the amino terminus (CgA-(1-115)) but not the C-terminal region of the granin. Moreover, examination of chromaffin granule abundance in PC12 cells exposed to bafilomycin A1 reveals a substantial decrease in the number of dense-core vesicles. We propose that a V-ATPase-mediated pH gradient in the secretory pathway is an important factor for the formation of dense-core granules by regulating the ability of CgA to form aggregates, a crucial step that may underlie the granulogenic function of the protein.     

Secretory granule biogenesis in sympathoadrenal cells: identification of a granulogenic determinant in the secretory prohormone chromogranin A

Chromogranin A (CgA) may be critical for secretory granule biogenesis in sympathoadrenal cells. We found that silencing the expression of CgA reduced the number of secretory granules in normal sympathoadrenal cells (PC12), and we therefore questioned whether a discrete domain of CgA might promote the formation of a regulated secretory pathway in variant sympathoadrenal cells (A35C) devoid of such a phenotype. The secretory granule-forming activity of a series of human CgA domains labeled with a hemagglutinin epitope, green fluorescent protein, or embryonic alkaline phosphatase was assessed in A35C cells by deconvolution and electron microscopy and by secretagogue-stimulated release assays. Expression of CgA in A35C cells induced the formation of vesicular organelles throughout the cytoplasm, whereas two constitutive secretory pathway markers accumulated in the Golgi complex. The lysosome-associated membrane protein LGP110 did not co-localize with CgA, consistent with non-lysosomal targeting of the granin in A35C cells. Thus, CgA-expressing A35C cells showed electron-dense granules approximately 180-220 nm in diameter, and secretagogue-stimulated exocytosis of CgA from A35C cells suggested that expression of the granin may be sufficient to restore a regulated secretory pathway and thereby rescue the sorting of other secretory proteins. We show that the formation of vesicular structures destined for regulated exocytosis may be mediated by a determinant located within the CgA N-terminal region (CgA-(1-115), with a necessary contribution of CgA-(40-115)), but not the C-terminal region (CgA-(233-439)) of the protein. We propose that CgA promotes the biogenesis of secretory granules by a mechanism involving a granulogenic determinant located within CgA-(40-115) of the mature protein.     

Expression of regulated secretory proteins is sufficient to generate granule-like structures in constitutively secreting cells

The formation of secretory granules and regulated secretion are generally assumed to occur only in specialized endocrine, neuronal, or exocrine cells. We discovered that regulated secretory proteins such as the hormone precursors pro-vasopressin, pro-oxytocin, and pro-opiomelanocortin, as well as the granins secretogranin II and chromogranin B but not the constitutive secretory protein alpha(1)-protease inhibitor, accumulate in granular structures at the Golgi and in the cell periphery in transfected COS-1 fibroblast cells. The accumulations were observed in 30-70% of the transfected cells expressing the pro-hormones and for virtually all of the cells expressing the granins. Similar structures were also generated in other cell lines believed to be lacking a regulated secretory pathway. The accumulations resembled secretory granules morphologically in immunofluorescence and electron microscopy. They were devoid of markers of the endoplasmic reticulum, endosomes, and lysosomes but in part stained positive for the trans-Golgi network marker TGN46, consistent with their formation at the trans-Golgi network. When different regulated proteins were coexpressed, they were frequently found in the same granules, whereas alpha(1)-protease inhibitor could not be detected in accumulations formed by secretogranin II, demonstrating segregation of regulated from constitutive secretory proteins. In pulse-chase experiments, significant intracellular storage of secretogranin II and chromogranin B was observed and secretion of retained secretogranin II was stimulated with the calcium ionophore A23187. The results suggest that expression of regulated cargo proteins is sufficient to generate structures that resemble secretory granules in the background of constitutively secreting cells, supporting earlier proposals on the mechanism of granule formation.     

Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network

Regulated secretory proteins are thought to be sorted in the trans-Golgi network (TGN) via selective aggregation. The factors responsible for this aggregation are unknown. We show here that two widespread regulated secretory proteins, chromogranin B and secretogranin II (granins), remain in an aggregated state when TGN vesicles from neuroendocrine cells (PC12) are permeabilized at pH 6.4 in 1-10 mM calcium, conditions believed to exist in this compartment. Permeabilization of immature secretory granules under these conditions allowed the recovery of electron dense cores. The granin aggregates in the TGN largely excluded glycosaminoglycan chains which served as constitutively secreted bulk flow markers. The low pH, high calcium milieu was sufficient to induce granin aggregation in the RER. In the TGN of pituitary GH4C1 cells, the proportion of granins conserved as aggregates was higher upon hormonal treatment known to increase secretory granule formation. Our data suggest that a decrease in pH and an increase in calcium are sufficient to trigger the selective aggregation of the granins in the TGN, segregating them from constitutive secretory proteins.     

Identification of a chromogranin A domain that mediates binding to secretogranin III and targeting to secretory granules in pituitary cells and pancreatic beta-cells

Chromogranin A (CgA) is transported restrictedly to secretory granules in neuroendocrine cells. In addition to pH- and Ca(2+)-dependent aggregation, CgA is known to bind to a number of vesicle matrix proteins. Because the binding-prone property of CgA with secretory proteins may be essential for its targeting to secretory granules, we screened its binding partner proteins using a yeast two-hybrid system. We found that CgA bound to secretogranin III (SgIII) by specific interaction both in vitro and in endocrine cells. Localization analysis showed that CgA and SgIII were coexpressed in pituitary and pancreatic endocrine cell lines, whereas SgIII was not expressed in the adrenal glands and PC12 cells. Immunoelectron microscopy demonstrated that CgA and SgIII were specifically colocalized in large secretory granules in male rat gonadotropes, which possess large-type and small-type granules. An immunocytochemical analysis revealed that deletion of the binding domain (CgA 48-111) for SgIII missorted CgA to the constitutive pathway, whereas deletion of the binding domain (SgIII 214-373) for CgA did not affect the sorting of SgIII to the secretory granules in AtT-20 cells. These findings suggest that CgA localizes with SgIII by specific binding in secretory granules in SgIII-expressing pituitary and pancreatic endocrine cells, whereas other mechanisms are likely to be responsible for CgA localization in secretory granules of SgIII-lacking adrenal chromaffin cells and PC12 cells.     

Essential Role of the Disulfide-bonded Loop of Chromogranin B for Sorting to Secretory Granules Is Revealed by Expression of a Deletion Mutant in the Absence of Endogenous Granin Synthesis

Sorting of regulated secretory proteins in the TGN to immature secretory granules (ISG) is thought to involve at least two steps: their selective aggregation and their interaction with membrane components destined to ISG. Here, we have investigated the sorting of chromogranin B (CgB), a member of the granin family present in the secretory granules of many endocrine cells and neurons. Specifically, we have studied the role of a candidate structural motif implicated in the sorting of CgB, the highly conserved NH₂-terminal disulfide– bonded loop. Sorting to ISG of full-length human CgB and a deletion mutant of human CgB (Δcys-hCgB) lacking the 22–amino acid residues comprising the disulfide-bonded loop was compared in the rat neuroendocrine cell line PC12. Upon transfection, i.e., with ongoing synthesis of endogenous granins, the sorting of the deletion mutant was only slightly impaired compared to full-length CgB. To investigate whether this sorting was due to coaggregation of the deletion mutant with endogenous granins, we expressed human CgB using recombinant vaccinia viruses, under conditions in which the synthesis of endogenous granins in the infected PC12 cells was shut off. In these conditions, Δcys-hCgB, in contrast to full-length hCgB, was no longer sorted to ISG, but exited from the TGN in constitutive secretory vesicles. Coexpression of full-length hCgB together with Δcys-hCgB by double infection, using the respective recombinant vaccinia viruses, rescued the sorting of the deletion mutant to ISG. In conclusion, our data show that (a) the disulfide-bonded loop is essential for sorting of CgB to ISG and (b) the lack of this structural motif can be compensated by coexpression of loop-bearing CgB. Furthermore, comparison of the two expression systems, transfection and vaccinia virus–mediated expression, reveals that analyses under conditions in which host cell secretory protein synthesis is blocked greatly facilitate the identification of sequence motifs required for sorting of regulated secretory proteins to secretory granules.     

Chromogranin A Promotes Peptide Hormone Sorting to Mobile Granules in Constitutively and Regulated Secreting Cells: ROLE OF CONSERVED N- AND C-TERMINAL PEPTIDES*S?

Chromogranin A (CgA) has been proposed to play a major role in the formation of dense-core secretory granules (DCGs) in neuroendocrine cells. Here, we took advantage of unique features of the frog CgA (fCgA) to assess the role of this granin and its potential functional determinants in hormone sorting during DCG biogenesis. Expression of fCgA in the constitutively secreting COS-7 cells induced the formation of mobile vesicular structures, which contained cotransfected peptide hormones. The fCgA and the hormones coexpressed in the newly formed vesicles could be released in a regulated manner. The N- and C-terminal regions of fCgA, which exhibit remarkable sequence conservation with their mammalian counterparts were found to be essential for the formation of the mobile DCG-like structures in COS-7 cells. Expression of fCgA in the corticotrope AtT20 cells increased pro-opiomelanocortin levels in DCGs, whereas the expression of N- and C-terminal deletion mutants provoked retention of the hormone in the Golgi area. Furthermore, fCgA, but not its truncated forms, promoted pro-opiomelanocortin sorting to the regulated secretory pathway. These data demonstrate that CgA has the intrinsic capacity to induce the formation of mobile secretory granules and to promote the sorting and release of peptide hormones. The conserved terminal peptides are instrumental for these activities of CgA.Eukaryotic cells share the capacity to rapidly secrete proteins through the constitutive secretory pathway. The fundamental feature of neuroendocrine and endocrine cells is the occurrence of dense-core secretory granules (DCGs),³ which are key cytoplasmic organelles responsible for secretion of hormones, neuropeptides, and neurotransmitters through the regulated secretory pathway (RSP). Storage at high concentrations of these secretory products is required for their finely tuned release in response to extracellular stimulation (1, 2). DCG biogenesis starts with the budding of immature secretory granules (ISGs) from the trans-Golgi network (TGN) through interactions between lipid rafts and protein components, in a similar manner to constitutive vesicle budding (2, 3). The ISG budding is followed by a multistep maturation process to form the mature secretory granules, including removal of the constitutive secretory proteins and lysosomal enzymes inadvertently packaged into ISGs (4).Despite increasing knowledge of the various steps of DCG formation, the nature of the sorting signals for entry of proteins into the DCGs and the molecular machinery required to generate secretory granules are not fully elucidated (5, 6). Several recent studies highlighted the role of members of the granin family, which may represent the driving force for granulogenesis in the TGN (2), although this notion has been a matter of debate (7). Granins are soluble acidic proteins widely distributed in endocrine and neuroendocrine cells, which are characterized by the ability to aggregate at acidic pH and a high Ca²⁺ environment (8, 9). These conditions are found in the lumen of the TGN allowing granins to aggregate in this compartment and to be segregated from constitutively secreted proteins (10, 11). The granin aggregates are believed to associate directly or indirectly with lipid rafts at the TGN to induce budding and formation of the ISGs. A prominent role of chromogranin A (CgA) in the regulation of DCG formation in endocrine and neuroendocrine cells has been proposed. Thus, depletion of CgA in PC12 cells led to a dramatic decrease in the number of DCGs (12), and exogenously expressed CgA in these depleted PC12 cells, as in DCG-deficient endocrine A35C and 6T3 cells, restored DCG biogenesis (12, 13). Besides, expression of granins in non-endocrine, constitutively secreting cells such as CV-1, NIH3T3, or COS-7 cells provoked the formation of DCG-like structures that release their content in response to Ca²⁺ influx (12, 14, 15). Further investigations performed in CgA null mice and transgenic mice expressing antisense RNA against CgA also revealed a reduction in the number of DCGs in chromaffin cells that was associated with an impairment of catecholamine storage, thus demonstrating the crucial role of CgA in normal DCG biogenesis (16, 17). In CgA knockout mice, the introduction of the gene expressing human CgA restored the regulated secretory phenotype (16). A different CgA null mice strain exhibited no discernable effect on DCG formation, but elevated catecholamine secretion (18), proving that CgA deficiency is associated with hormone storage impairment in neuroendocrine cells in vivo, a finding that was confirmed in vitro (19). The CgA^-/- mice strain generated by Hendy et al. (18) exhibited a compensatory overexpression of other granins, pointing to a possible overlap in granin function in secretory granule biogenesis.We reported previously that the frog CgA (fCgA) gene is coordinately regulated with the pro-opiomelanocortin (POMC) gene in the pituitary pars intermedia during the neuroendocrine reflex of skin color change, which allows amphibia to adapt to their environment through the release of POMC-derived melanotropic peptides (20, 21). Sequence comparison of fCgA with its mammalian orthologs revealed a high conservation of the N- and C-terminal domains, and far less conservation of the central part of the protein (Fig. 1A), suggesting that these domains may play a role in DCG formation and hormone release in various species (9, 20, 21). To assess the role of fCgA and its conserved N- and C-terminal regions in hormone sorting, storage, and secretion, we engineered different constructs that produce the native unmodified (no tag added) protein and truncated forms lacking the conserved N- and C-terminal domains, and we developed an antibody that specifically recognizes the central region of fCgA. Using the constitutively secreting COS-7 cells, which are devoid of DCGs, we could demonstrate for the first time that CgA is essential for targeting peptide hormones to newly formed mobile DCG-like structures. In the CgA-expressing AtT20 cells, which exhibit an only moderate capacity to sort secretory proteins to the regulated pathway (22), the granin plays a pivotal role in the sorting and release of POMC. The conserved terminal peptides of CgA are instrumental for these activities.Open in a separate windowFIGURE 1.Specificity of the antibody directed against frog CgA. A, scheme depicting the structure of fCgA and showing the high conservation of the terminal regions and the percentages of amino acid identity between frog and human CgA sequences. The highly conserved peptide WE14 and dibasic cleavage sites are also indicated. B, Western blot showing that the antibody developed against fCgA recognized the protein and several processing intermediates in frog but not rat pituitary extracts, whereas an antibody, directed against the WE14 conserved peptide, detected CgA and its processing products in both rat and frog pituitary extracts. C, immunofluorescence analysis of frog pituitary and adrenal glands, and rat adrenal gland using the antibodies against fCgA and WE14. cx, cortex; DL, distal lobe; IL, intermediate lobe; and m, medulla. Scale bars equal 10 μm.     

Modulating zymogen granule formation in pancreatic AR42J cells

Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. To investigate ZG biogenesis, cargo sorting and packaging, suitable cellular model systems are required. Here, we demonstrate that granule formation in pancreatic AR42J cells, an acinar model system, can be modulated by altering the growth conditions in cell culture. We find that cultivation of AR42J cells in Panserin^? 401, a serum-free medium, enhances the induction of granule formation in the presence or absence of dexamethasone when compared to standard conditions including serum. Biochemical and morphological studies revealed an increase in ZG markers on the mRNA and protein level, as well as in granule size compared to standard conditions. Our data indicate that this effect is related to pronounced differentiation of AR42J cells. To address if enhanced expression of ZG proteins promotes granule formation, we expressed several zymogens and ZG membrane proteins in unstimulated AR42J cells and in constitutively secreting COS-7 cells. Neither single expression nor co-expression was sufficient to initiate granule formation in AR42J cells or the formation of granule-like structures in COS-7 cells as described for neuroendocrine cargo proteins. The importance of our findings for granule formation in exocrine cells is discussed.     

The granin (chromogranin/secretogranin) family

The chromogranins/secretogranins, referred to in abbreviated form as granins, are a family of acidic secretory proteins that are found in the secretory granules of a wide variety of endocrine cells and neurons, being stored together with many different peptide hormones and neuropeptides. The recent elucidation of their primary structure has provided insights into possible functions of these proteins. Moreover, the granins have been successfully used as markers for normal and neoplastic endocrine and neuronal cells, as well as model proteins to understand the sorting mechanism involved in the formation of secretory granules.     

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.     

Prohormone-convertase 1 processing enhances post-Golgi sorting of prothyrotropin-releasing hormone-derived peptides

Rat prothyrotropin-releasing hormone (pro-TRH) is endoproteolyzed within the regulated secretory pathway of neuroendocrine cells yielding five TRH peptides and seven to nine other unique peptides. Endoproteolysis is performed by two prohormone convertases, PC1 and PC2. Proteolysis of pro-TRH begins in the trans-Golgi network and forms two intermediates that are then differentially processed as they exit the Golgi and are packaged into immature secretory granules. We hypothesized that this initial endoproteolysis may be necessary for downstream sorting of pro-TRH-derived peptides as it occurs before Golgi exit and thus entry into the regulated secretory pathway. We now report that when pro-TRH is transiently expressed in GH4C1 cells, a neuroendocrine cell line lacking PC1, under pulse-chase conditions release is constitutive and composed of more immature processing intermediates. This is also observed by radioimmunoassay under steady-state conditions. When a mutant form of pro-TRH, which has the dibasic sites of initial processing mutated to glycines, is expressed in AtT20 cells, a neuroendocrine cell line endogenously expressing PC1, both steady-state and pulse-chase experiments revealed that peptides derived from this mutant precursor are secreted in a constitutive fashion. A constitutively secreted form of PC1 does not target pro-TRH peptides to the constitutive secretory pathway but results in sorting to the regulated secretory pathway. These results indicated that initial processing action of PC1 on pro-TRH in the trans-Golgi network, and not a cargo-receptor relationship, is important for the downstream sorting events that result in storage of pro-TRH-derived peptides in mature secretory granules.     

The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments

The proprotein convertase PC5 is encoded by multiple mRNAs, two of which give rise to the COOH-terminal variant isoforms PC5-A (915 amino acids [aa]) and PC5-B (1877 aa). To investigate the differences in biosynthesis and sorting between these two proteins, we generated stably transfected AtT-20 cell lines expressing each enzyme individually and examined their respective processing pattern and subcellular localization. Biosynthetic analyses coupled to immunofluorescence studies demonstrated that the shorter and soluble PC5-A is sorted to regulated secretory granules. In contrast, the COOH- terminally extended and membrane-bound PC5-B is located in the Golgi. The presence of a sorting signal in the COOH-terminal 38 amino acids unique to PC5-A was demonstrated by the inefficient entry into the regulated secretory pathway of a mutant lacking this segment. EM of pancreatic cells established the presence of immunoreactive PC5 in glucagon-containing granules, demonstrating the sorting of this protein to dense core secretory granules in endocrine cells. Thus, a single PC5 gene generates COOH-terminally modified isoforms with different sorting signals directing these proteins to distinct subcellular localization, thereby allowing them to process their appropriate substrates.     

In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.     

N- and C-terminal domains direct cell type-specific sorting of chromogranin A to secretory granules

Chromogranins are a family of regulated secretory proteins that are stored in secretory granules in endocrine and neuroendocrine cells and released in response to extracellular stimulation (regulated secretion). A conserved N-terminal disulfide bond is necessary for sorting of chromogranins in neuroendocrine PC12 cells. Surprisingly, this disulfide bond is not necessary for sorting of chromogranins in endocrine GH4C1 cells. To investigate the sorting mechanism in GH4C1 cells, we made several mutant forms removing highly conserved N- and C-terminal regions of bovine chromogranin A. Removing the conserved N-terminal disulfide bond and the conserved C-terminal dimerization and tetramerization domain did not affect the sorting of chromogranin A to the regulated secretory pathway. In contrast, removing the C-terminal 90 amino acids of chromogranin A caused rerouting to the constitutive secretory pathway and impaired aggregation properties as compared with wild-type chromogranin A. Since this mutant was sorted to the regulated secretory pathway in PC12 cells, these results demonstrate that chromogranins contain independent N- and C-terminal sorting domains that function in a cell type-specific manner. Moreover, this is the first evidence that low pH/calcium-induced aggregation is necessary for sorting of a chromogranin to the regulated secretory pathway of endocrine cells.     

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.     

A large form of secretogranin III functions as a sorting receptor for chromogranin A aggregates in PC12 cells

Granin-family proteins, including chromogranin A and secretogranin III, are sorted to the secretory granules in neuroendocrine cells. We previously demonstrated that secretogranin III binds chromogranin A and targets it to the secretory granules in pituitary corticotrope-derived AtT-20 cells. However, secretogranin III has not been identified in adrenal chromaffin and PC12 cells, where chromogranin A is correctly sorted to the secretory granules. In this study, low levels of a large and noncleaved secretogranin III have been identified in PC12 cells and rat adrenal glands. Although the secretogranin III expression was limited in PC12 cells, when the FLAG-tagged secretogranin III lacking the secretory granule membrane-binding domain was expressed excessively, hemagglutinin-tagged chromogranin A was unable to target to the secretory granules at the tips and shifted to the constitutive secretory pathway. Secretogranin III was able to bind the aggregated form of chromogranin A, suggesting that a small quantity of secretogranin III is enough to carry a large quantity of chromogranin A. Furthermore, secretogranin III bound adrenomedullin, a major peptide hormone in chromaffin cells. Indeed, small interfering RNA-directed secretogranin III depletion impaired intracellular retention of chromogranin A and adrenomedullin, suggesting that they are constitutively released to the medium. We suggest that the sorting function of secretogranin III for chromogranin A is common in PC12 and chromaffin cells as well as in other endocrine cells, and a small amount of secretogranin III is able to sort chromogranin A aggregates together with adrenomedullin to secretory granules.     

Exocrine granule specific packaging signals are present in the polypeptide moiety of the pancreatic granule membrane protein GP2 and in amylase: implications for protein targeting to secretory granules.

The mechanisms for segregation of secretory and membrane proteins incorporated into storage granules from those transported constitutively have been thought to be conserved in diverse cell types, including exocrine and endocrine cells. However, GP2, the major protein of pancreatic zymogen granule membranes, in its native glycosyl phosphatidylinositol (GPI)-linked form, is incorporated into secretory granules when expressed in exocrine pancreatic AR42J cells, but not in the endocrine cells such as pituitary AtT20. To determine whether the protein moiety of GP2 contains the cell-type specific information for packaging into granules, a secretory form of GP2 (GP2-GPI-), with the GPI attachment site deleted, was generated and introduced into AR42J and AtT20 cells. Like native GP2, GP2-GPI- localized to the zymogen-like granules of AR42J cells and underwent regulated secretion. In AtT20 cells expressing GP2-GPI-, however, the protein was secreted by the constitutive pathway. Thus, a granule packaging signal is present in the luminal portion of GP2 that is functional only in the exocrine cells. However, this cell-type dependent sorting process is not limited to GP2 or membrane proteins. Amylase, a major content protein of pancreatic acinar and serous salivary gland granules, was also secreted exclusively by the constitutive pathway when expressed in AtT20 cells. The cell-type specific targeting of GP2 to granules correlated with its behavior in an in vitro aggregation assay where it co-aggregated more effectively with content proteins from pancreatic zymogen granules than with those from pituitary granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Sorting of the neuroendocrine secretory protein Secretogranin II into the regulated secretory pathway: role of N- and C-terminal alpha-helical domains

Secretogranin II (SgII) belongs to the granin family of prohormones widely distributed in dense-core secretory granules (DCGs) of endocrine, neuroendocrine, and neuronal cells, including sympathoadrenal chromaffin cells. The mechanisms by which secretory proteins, and granins in particular, are sorted into the regulated secretory pathway are unsettled. We designed a strategy based on novel chimeric forms of human SgII fused to fluorescent (green fluorescent protein) or chemiluminescent (embryonic alkaline phosphatase) reporters to identify trafficking determinants mediating DCG targeting of SgII in sympathoadrenal cells. Three-dimensional deconvolution fluorescence microscopy and secretagogue-stimulated release studies demonstrate that SgII chimeras are correctly targeted to DCGs and released by exocytosis in PC12 and primary chromaffin cells. Results from a Golgi-retained mutant form of SgII suggest that sorting of SgII into DCGs depends on a saturable sorting machinery at the trans-Golgi/trans-Golgi network. Truncation analyses reveal the presence of DCG-targeting signals within both the N- and C-terminal regions of SgII, with the putative alpha-helix-containing SgII-(25-41) and SgII-(334-348) acting as sufficient, independent sorting domains. This study defines sequence features of SgII mediating vesicular targeting in sympathoadrenal cells and suggests a mechanism by which discrete domains of the molecule function in sorting, perhaps by virtue of a particular arrangement in tertiary structure and/or interaction with a specific component of the DCG membrane.     

Targeted ablation of the chromogranin a (Chga) gene: normal neuroendocrine dense-core secretory granules and increased expression of other granins

Chromogranin A (CgA), originally identified in adrenal chromaffin cells, is a member of the granin family of acidic secretory glycoproteins that are expressed in endocrine cells and neurons. CgA has been proposed to play multiple roles in the secretory process. Intracellularly, CgA may control secretory granule biogenesis and target neurotransmitters and peptide hormones to granules of the regulated pathway. Extracellularly, peptides formed as a result of proteolytic processing of CgA may regulate hormone secretion. To investigate the role of CgA in the whole animal, we created a mouse mutant null for the Chga gene. These mice are viable and fertile and have no obvious developmental abnormalities, and their neural and endocrine functions are not grossly impaired. Their adrenal glands were structurally unremarkable, and morphometric analyses of chromaffin cells showed vesicle size and number to be normal. However, the excretion of epinephrine, norepinephrine, and dopamine was significantly elevated in the Chga null mutants. Adrenal medullary mRNA and protein levels of other dense-core secretory granule proteins including chromogranin B, and secretogranins II to VI were up-regulated 2- to 3-fold in the Chga null mutant mice. Hence, the increased expression of the other granin family members is likely to compensate for the Chga deficiency.