scholarly journals Molecular probing of the secretory pathway in peptide hormone-producing cells

1995 ◽  
Vol 108 (10) ◽  
pp. 3295-3305 ◽  
Author(s):  
J.C. Holthuis ◽  
E.J. Jansen ◽  
M.C. van Riel ◽  
G.J. Martens
Keyword(s):  
Secretory Pathway ◽  
Transmembrane Protein ◽  
Secretory Granules ◽  
Peptide Hormone ◽  
Mrna Levels ◽  
Black And White ◽  
Processing Enzymes ◽  
Black Background ◽  
Prohormone Processing ◽  
Associated Proteins

The biosynthetic machinery in the melanotrope cells of the Xenopus intermediate pituitary is primarily dedicated to the generation of proopiomelanocortin (POMC)-derived, melanophore-stimulating peptides. Transfer of the animal to a black background stimulates the production of these peptides and causes a dramatic increase in POMC mRNA levels. To identify genes involved in the biosynthesis and regulated release of peptide hormones, we differentially screened an intermediate pituitary cDNA library of toads adapted to a black background with cDNA probes derived from intermediate pituitary mRNA of black- and white-adapted animals. Here we report the identification of twelve distinct genes whose expression levels in the melanotropes are regulated in coordination with that of POMC. Four of these genes are novel while the others code for translocon-associated proteins, a lumenal cysteine protease of the endoplasmic reticulum, prohormone-processing enzymes, members of the granin family and a transmembrane protein presumably involved in the assembly and/or specific functioning of vacuolar H(+)-ATPase from secretory granules. Our results indicate that a wide variety of both soluble and membrane-associated components of the secretory pathway is recruited in physiologically activated, peptide hormone-producing cells.

scholarly journals Proinsulin Endoproteolysis Confers Enhanced Targeting of Processed Insulin to the Regulated Secretory Pathway

2000 ◽  
Vol 11 (6) ◽  
pp. 1959-1972 ◽  
Author(s):  
Regina Kuliawat ◽  
Daniel Prabakaran ◽  
Peter Arvan
Keyword(s):  
Secretory Pathway ◽  
Proteolytic Enzymes ◽  
Secretory Granules ◽  
Protein Solubility ◽  
Vitro Assay ◽  
Processing Enzymes ◽  
Prohormone Processing ◽  
Cargo Proteins ◽  
Regulated Secretory Pathway

Recently, two different prohormone-processing enzymes, prohormone convertase 1 (PC1) and carboxypeptidase E, have been implicated in enhancing the storage of peptide hormones in endocrine secretory granules. It is important to know the extent to which such molecules may act as “sorting receptors” to allow the selective trafficking of cargo proteins from the trans-Golgi network into forming granules, versus acting as enzymes that may indirectly facilitate intraluminal storage of processed hormones within maturing granules. GH4C1 cells primarily store prolactin in granules; they lack PC1 and are defective for intragranular storage of transfected proinsulin. However, proinsulin readily enters the immature granules of these cells. Interestingly, GH4C1 clones that stably express modest levels of PC1 store more proinsulin-derived protein in granules. Even in the presence of PC1, a sizable portion of the proinsulin that enters granules goes unprocessed, and this portion largely escapes granule storage. Indeed, all of the increased granule storage can be accounted for by the modest portion converted to insulin. These results are not unique to GH4C1 cells; similar results are obtained upon PC1 expression in PC12 cells as well as in AtT20 cells (in which PC1 is expressed endogenously at higher levels). An in vitro assay of protein solubility indicates a difference in the biophysical behavior of proinsulin and insulin in the PC1 transfectants. We conclude that processing to insulin, facilitated by the catalytic activities of granule proteolytic enzymes, assists in the targeting (storage) of the hormone.

scholarly journals Secretome and Tunneling Nanotubes: A Multilevel Network for Long Range Intercellular Communication between Endothelial Cells and Distant Cells

2021 ◽  
Vol 22 (15) ◽  
pp. 7971
Author(s):  
Béatrice Charreau
Keyword(s):  
Secretory Pathway ◽  
Transmembrane Protein ◽  
Secretory Granules ◽  
Immediate Release ◽  
Target Cells ◽  
Ectodomain Shedding ◽  
Tunneling Nanotubes ◽  
Long Distance ◽  
Transfer Of Information ◽  
Regulatory Functions

As a cellular interface between the blood and tissues, the endothelial cell (EC) monolayer is involved in the control of key functions including vascular tone, permeability and homeostasis, leucocyte trafficking and hemostasis. EC regulatory functions require long-distance communications between ECs, circulating hematopoietic cells and other vascular cells for efficient adjusting thrombosis, angiogenesis, inflammation, infection and immunity. This intercellular crosstalk operates through the extracellular space and is orchestrated in part by the secretory pathway and the exocytosis of Weibel Palade Bodies (WPBs), secretory granules and extracellular vesicles (EVs). WPBs and secretory granules allow both immediate release and regulated exocytosis of messengers such as cytokines, chemokines, extracellular membrane proteins, coagulation or growth factors. The ectodomain shedding of transmembrane protein further provide the release of both receptor and ligands with key regulatory activities on target cells. Thin tubular membranous channels termed tunneling nanotubes (TNTs) may also connect EC with distant cells. EVs, in particular exosomes, and TNTs may contain and transfer different biomolecules (e.g., signaling mediators, proteins, lipids, and microRNAs) or pathogens and have emerged as a major triggers of horizontal intercellular transfer of information.

Differential effects of temperature blockade on the proteolytic processing of three secretory granule-associated proteins

1994 ◽  
Vol 107 (3) ◽  
pp. 737-745 ◽  
Author(s):  
S.L. Milgram ◽  
R.E. Mains
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Proteolytic Processing ◽  
Prohormone Convertase ◽  
Trans Golgi Network ◽  
Processing Enzymes ◽  
Prohormone Convertase 1 ◽  
Associated Proteins ◽  
Mouse Pituitary ◽  
Golgi Network

Vesicular transport within the secretory pathway can be arrested by incubating cells at 15 degrees C or 20 degrees C to block exit from the endoplasmic reticulum or trans-Golgi network, respectively. Using this powerful tool we have compared the intracellular sites of endoproteolytic processing of proopiomelanocortin and two prohormone processing enzymes in AtT-20 mouse pituitary corticotrope tumor cells. For comparison, proopiomelanocortin processing was also evaluated in primary neurointermediate pituitary cultures. AtT-20 cells synthesize and store endogenous proopiomelanocortin and prohormone convertase 1; AtT-20 cells expressing high levels of integral membrane or soluble peptidylglycine alpha-amidating monooxygenase were generated by stable transfection. Cells were incubated with [35S]methionine and chased at 4 degrees C, 15 degrees C, 20 degrees C or 37 degrees C. The endoproteolytic processing of peptidylglycine alpha-amidating mono-oxygenase, prohormone convertase 1, and proopiomelanocortin was compared following immunoprecipitation. Endoproteolytic processing of integral membrane and soluble peptidylglycine alpha-amidating monooxygenase proteins was completely blocked by incubation of cells at 20 degrees C. In contrast, prohormone convertase 1 processing from the 87 kDa precursor to the 81 kDa intermediate proceeded to completion at both 15 degrees C and 20 degrees C, while cleavage to generate the 63 kDa prohormone convertase 1 protein was completely blocked at 20 degrees C. In AtT-20 cells and neurointermediate pituitary cultures, generation of beta-lipotropin from proopiomelanocortin continued at a slow but significant rate at 20 degrees C, while processing of beta-lipotropin to beta-endorphin was blocked. Thus prohormone convertase 1 processing begins in the endoplasmic reticulum and is not completed until after the trans-Golgi network, while peptidylglycine alpha-amidating monooxygenase processing begins after the trans-Golgi network. Selected proopiomelanocortin cleavages begin before entry into immature granules.

scholarly journals Differential trafficking of soluble and integral membrane secretory granule-associated proteins

1994 ◽  
Vol 124 (1) ◽  
pp. 33-41 ◽  
Author(s):  
SL Milgram ◽  
BA Eipper ◽  
RE Mains
Keyword(s):  
Cell Surface ◽  
Secretory Granule ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Posttranslational Processing ◽  
Endoproteolytic Cleavage ◽  
Associated Proteins ◽  
Storage Granules ◽  
Regulated Secretory Pathway ◽  
And Storage

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.

Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells

2009 ◽  
Vol 418 (1) ◽  
pp. 81-91 ◽  
Author(s):  
Hansruedi Stettler ◽  
Nicole Beuret ◽  
Cristina Prescianotto-Baschong ◽  
Bérengère Fayard ◽  
Laurent Taupenot ◽  
...  
Keyword(s):  
Chromogranin A ◽  
Endocrine Cells ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Peptide Hormone ◽  
Secretory Proteins ◽  
Cell Periphery ◽  
Granule Formation ◽  
Regulated Secretory Pathway

In endocrine cells, prohormones and granins are segregated in the TGN (trans-Golgi network) from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner on specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures that resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of CgA (chromogranin A) is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP (green fluorescent protein) or a short epitope tag was expressed in COS-1 fibroblast cells and analysed by fluorescence and electron microscopy and pulse-chase labelling. Full-length CgA as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that co-localized with co-expressed SgII (secretogranin II). These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 (pheochromocytoma cells) cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway.

scholarly journals Misrouting of Glucagon and Stathmin-2 Towards Lysosomal System of α-Cells in Glucagon Hypersecretion of Diabetes

2021 ◽  
Author(s):  
Farzad Asadi ◽  
Savita Dhanvantari
Keyword(s):  
High Glucose ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Glucagon Secretion ◽  
Mrna Levels ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Lysosomal System ◽  
Regulated Secretory Pathway ◽  
Cells Cultured

Glucagon hypersecretion from the pancreatic α-cell is a characteristic sign of diabetes, which exacerbates fasting hyperglycemia. Thus, targeting glucagon secretion from α-cells may be a promising approach for combating hyperglucagonemia. We have recently identified stathmin-2 as a protein that resides in α-cell secretory granules, and showed that it regulates glucagon secretion by directing glucagon towards the endolysosomal system in αTC1-6 cells. Here, we hypothesized that disruption of Stmn2-mediated trafficking of glucagon to the endolysosomes contributes to hyperglucagonemia. In isolated islets from male mice treated with streptozotocin (STZ) to induce diabetes, Arg-stimulated secretion of glucagon and Stmn2 was augmented. However, cell glucagon content was significantly increased (p<0.001), but Stmn2 levels were reduced (p<0.01) in STZ-treated mice, as measured by both ELISA and immunofluorescence intensity. Expression of Gcg mRNA increased ~4.5 times, while Stmn2 mRNA levels did not change. Using confocal immunofluorescence microscopy, the colocalization of glucagon and Stmn2 in Lamp2A+ lysosomes was dramatically reduced (p<0.001) in islets from diabetic mice, and the colocalization of Stmn2, but not glucagon, with the late endosome marker, Rab7, significantly (p<0.01) increased. Further studies were conducted in αTC1-6 cells cultured in media containing high glucose (16.7 mM) for two weeks to mimic glucagon hypersecretion of diabetes. Surprisingly, treatment of αTC1-6 cells with the lysosomal inhibitor bafilomycin A1 reduced K+-induced glucagon secretion, suggesting that high glucose may induce glucagon secretion from another lysosomal compartment. Both glucagon and Stmn2 co-localized with Lamp1, which marks secretory lysosomes, in cells cultured in high glucose. We propose that, in addition to enhanced trafficking and secretion through the regulated secretory pathway, the hyperglucagonemia of diabetes may also be due to re-routing of glucagon from the degradative Lamp2A+ lysosome towards the secretory Lamp1+ lysosome.

Novel salmon cardiac peptide hormone is released from the ventricle by regulated secretory pathway

2000 ◽  
Vol 278 (2) ◽  
pp. E285-E292 ◽  
Author(s):  
Kati Kokkonen ◽  
Heidi Vierimaa ◽  
Sari Bergström ◽  
Virpi Tervonen ◽  
Olli Arjamaa ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Mechanical Load ◽  
Secretory Granules ◽  
Fold Increase ◽  
Peptide Hormone ◽  
Biologically Active ◽  
The Novel ◽  
Endothelin 1 ◽  
Basal Expression ◽  
Regulated Secretory Pathway

We used the secretion of the novel salmon cardiac peptide (sCP) as a model to examine the mechanisms of ventricular hormone release. Mechanical load increased dose dependently the secretion of immunoreactive sCP from isolated perfused salmon ventricle, with 3.3-fold increase when a load of 13 cmH2O was applied. Endothelin-1 (5 nmol/l) was also able to rapidly increase the secretion of sCP. The released peptide corresponded to the biologically active sCP-29, whereas the large ventricular storage consisted of pro-sCP-sized material. With the use of immunoelectron microscopy, a large number of granules containing immunoreactive sCP could be detected in salmon ventricle. As judged by RNA blot analysis, there was very active basal expression of the sCP gene in the ventricle, which was not increased by mechanical load of up to 2-h duration. Our results show that the ventricle actively expresses the gene of sCP, stores the prohormone in secretory granules, and releases the peptide in response to mechanical load and endothelin-1. Thus the salmon ventricle uses the regulated pathway to produce and release a hormone structurally related to the mammalian natriuretic peptides.

scholarly journals Transport via the regulated secretory pathway in semi-intact PC12 cells: role of intra-cisternal calcium and pH in the transport and sorting of secretogranin II.

1994 ◽  
Vol 127 (3) ◽  
pp. 693-705 ◽  
Author(s):  
L Carnell ◽  
H P Moore
Keyword(s):  
Pc12 Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Ph Gradients ◽  
Secretogranin Ii ◽  
Prohormone Processing ◽  
Endoproteolytic Cleavage ◽  
Regulated Secretory Pathway ◽  
Gamma S

To gain insight into the mechanisms governing protein sorting, we have developed a system that reconstitutes both the formation of immature secretory granules and their fusion with the plasma membrane. Semi-intact PC12 cells were incubated with ATP and cytosol for 15 min to allow immature granules to form, and then in a buffer containing 30 microM [Ca2+]free to induce exocytosis. Transport via the regulated pathway, as assayed by the release of secretogranin II (SgII) labeled in the TGN, was inhibited by depletion of ATP, or by the inclusion of 100 microM GTP gamma S, 50 microM AlF3-5 or 5 micrograms/ml BFA. When added after immature granules had formed, GTP gamma S stimulated rather than inhibited exocytosis. Thus, exocytosis of immature granules in this system resembles the characteristics of fully matured granules. Transport of SgII via the regulated pathway occurred at a fourfold higher efficiency than glycosaminoglycan chains, indicating that SgII is sorted to some extent upon exit from the TGN. Addition of A23187 to release Ca2+ from the TGN had no significant effect on sorting of SgII into immature granules. In contrast, depletion of lumenal calcium inhibited the endoproteolytic cleavage of POMC and proinsulin. These results establish the importance of intra-cisternal Ca2+ in prohormone processing, but raise the question whether lumenal calcium is required for proper sorting of SgII into immature granules. Disruption of organelle pH gradients with an ionophore or a weak base resulted in the inhibition of transport via both the constitutive and the regulated pathways.

scholarly journals Ionic milieu controls the compartment-specific activation of pro-opiomelanocortin processing in AtT-20 cells.

1995 ◽  
Vol 6 (10) ◽  
pp. 1271-1285 ◽  
Author(s):  
W K Schmidt ◽  
H P Moore
Keyword(s):  
Intracellular Transport ◽  
Secretory Pathway ◽  
Intact Cell ◽  
Secretory Granules ◽  
Cell System ◽  
Proteolytic Processing ◽  
Prolonged Incubation ◽  
Trans Golgi Network ◽  
Processing Enzymes ◽  
Golgi Network

Newly synthesized prohormones and their processing enzymes transit through the same compartments before being packaged into regulated secretory granules. Despite this coordinated intracellular transport, prohormone processing does not occur until late in the secretory pathway. In the mouse pituitary AtT-20 cell line, conversion of pro-opiomelanocortin (POMC) to mature adrenocorticotropic hormone involves the prohormone convertase PC1. The mechanism by which this proteolytic processing is restricted to late secretory compartments is unknown; PC1 activity could be regulated by compartment-specific activators/inhibitors, or through changes in the ionic milieu that influence its activity. By arresting transport in a semi-intact cell system, we have addressed whether metabolically labeled POMC trapped in early secretory compartments can be induced to undergo conversion if the ionic milieu in these compartments is experimentally manipulated. Prolonged incubation of labeled POMC trapped in the endoplasmic reticulum or Golgi/trans-Golgi network did not result in processing, thereby supporting the theory that processing is normally a post-Golgi/trans-Golgi network event. However, acidification of these compartments allowed effective processing of POMC to the intermediate and mature forms. The observed processing increased sharply at a pH below 6.0 and required millimolar calcium, regardless of the compartment in which labeled POMC resided. These conditions also resulted in the coordinate conversion of PC1 from the 84/87 kDa into the 74-kDa and 66-kDa forms. We propose that POMC processing is predominantly restricted to acidifying secretory granules, and that a change in pH within these granules is both necessary and sufficient to activate POMC processing.

scholarly journals Endoplasmic reticulum Ca2+ is important for the proteolytic processing and intracellular transport of proinsulin in the pancreatic β-cell

1997 ◽  
Vol 323 (2) ◽  
pp. 445-450 ◽  
Author(s):  
Paul C. GUEST ◽  
Elaine M. BAILYES ◽  
John C. HUTTON
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Proteolytic Processing ◽  
Pulse Labelling ◽  
Atpase Inhibitor ◽  
Prohormone Processing ◽  
Proinsulin Processing ◽  
Rat Islets Of Langerhans

The role of intracellular Ca2+ in the proteolytic processing and intracellular transport of secretory granule proproteins was investigated by pulse–chase radiolabelling of isolated rat islets of Langerhans. The conversion of proinsulin was inhibited by depletion of medium Ca2+ with EGTA and by blocking the transport of Ca2+ into cells with the Ca2+-channel antagonists verapamil, nifedipine and NiCl2. Proinsulin conversion was also reduced by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, indicating that the process requires transport of Ca2+ into the endoplasmic reticulum. This was supported by the finding that proinsulin processing was inhibited when Ca2+ was depleted before or during pulse-labelling, but not after transport of the protein to post-endoplasmic-reticulum compartments. Similarly, the inhibition of proinsulin processing was reversed by re-introduction of medium Ca2+ around the time of radiolabelling, but not after 15 min of chase incubation. Ca2+ depletion also decreased proteolytic maturation of the prohormone convertases PC1, PC2 and carboxypeptidase H. Secretion experiments suggested that the rate and extent of proinsulin transport into secretory granules were inhibited marginally by Ca2+ depletion, whereas those of the convertases were markedly impeded. Inhibition of proinsulin conversion by Ca2+ depletion was thus not simply related to the Ca2+-dependencies of mature PC1 and PC2, but also to a requirement for endoplasmic reticulum Ca2+ in proteolytic maturation of the convertases and in their transfer to secretory granules. The results also suggest that the Ca2+ required for prohormone processing in the granules enters the secretory pathway via the endoplasmic reticulum.

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