Chromogranin B (secretogranin I) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor.

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.

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Minireview: How Peptide Hormone Vesicles Are Transported to the Secretion Site for Exocytosis

Molecular Endocrinology ◽

10.1210/me.2008-0209 ◽

2008 ◽

Vol 22 (12) ◽

pp. 2583-2595 ◽

Cited By ~ 45

Author(s):

Joshua J. Park ◽

Y. Peng Loh

Keyword(s):

Plasma Membrane ◽

Secretory Pathway ◽

Hormone Secretion ◽

Release Site ◽

Peptide Hormone ◽

Adaptor Proteins ◽

Transport Systems ◽

Cytosolic Proteins ◽

Readily Releasable Pool ◽

Regulated Secretory Pathway

Abstract Post-Golgi transport of peptide hormone-containing vesicles from the site of genesis at the trans-Golgi network to the release site at the plasma membrane is essential for activity-dependent hormone secretion to mediate various endocrinological functions. It is known that these vesicles are transported on microtubules to the proximity of the release site, and they are then loaded onto an actin/myosin system for distal transport through the actin cortex to just below the plasma membrane. The vesicles are then tethered to the plasma membrane, and a subpopulation of them are docked and primed to become the readily releasable pool. Cytoplasmic tails of vesicular transmembrane proteins, as well as many cytosolic proteins including adaptor proteins, motor proteins, and guanosine triphosphatases, are involved in vesicle budding, the anchoring of the vesicles, and the facilitation of movement along the transport systems. In addition, a set of cytosolic proteins is also necessary for tethering/docking of the vesicles to the plasma membrane. Many of these proteins have been identified from different types of (neuro)endocrine cells. Here, we summarize the proteins known to be involved in the mechanisms of sorting various cargo proteins into regulated secretory pathway hormone-containing vesicles, movement of these vesicles along microtubules and actin filaments, and their eventual tethering/docking to the plasma membrane for hormone secretion.

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Retrovirus-mediated expression of preprosomatostatin: posttranslational processing, intracellular storage, and secretion in GH3 pituitary cells.

The Journal of Cell Biology ◽

10.1083/jcb.107.6.2087 ◽

1988 ◽

Vol 107 (6) ◽

pp. 2087-2095 ◽

Cited By ~ 20

Author(s):

T J Stoller ◽

D Shields

Keyword(s):

Amino Acids ◽

Growth Hormone ◽

Secretory Pathway ◽

Peptide Hormone ◽

Proteolytic Processing ◽

Gh3 Cells ◽

Single Pair ◽

Pituitary Cells ◽

Endoproteolytic Cleavage ◽

Regulated Secretory Pathway

Somatostatin (SRIF) is a 14-amino acid peptide hormone that is synthesized as part of a larger precursor, preproSRIF, consisting of a signal peptide and a proregion of 80-90 amino acids. The mature hormone, which is located at the carboxyl terminus of the precursor, is preceded by a single pair of basic amino acids. We are studying preproSRIF to investigate intracellular sorting, proteolytic processing, and storage of peptide hormone precursors in the secretory pathway. We used a retroviral expression vector to achieve the high levels of precursor synthesis which are necessary for detailed characterization of processing intermediates and mature somatostatin. Recombinant retroviruses containing RNA transcripts encoding anglerfish preproSRIF I were used to infect rat pituitary GH3 cells which secrete growth hormone and prolactin, neither of which are substrates for endoproteolytic cleavage. In these cells preproSRIF was accurately processed to the mature hormone with an efficiency of approximately 75%. Of the newly synthesized mature SRIF, 55% was sorted into the regulated secretory pathway and released in response to the secretagogue 8-Br-cAMP. The remaining 45% of mature SRIF and residual unprocessed precursor was rapidly secreted. In contrast to SRIF, only 5% of newly synthesized endogenous growth hormone was stored intracellularly, whereas 95% was sorted to the constitutive pathway and secreted rapidly with kinetics identical to proSRIF. Our results show that proSRIF processing is not necessarily dependent on a specific protease found only in SRIF-producing cells and suggest that proteolytic cleavage is not restricted to cells that process endogenous hormones. Moreover, these results demonstrate that GH3 cells have the capacity to discriminate between endogenous and foreign hormones and target the foreign molecule significantly more efficiently to the regulated secretory pathway.

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A Pilot Screen of a Novel Peptide Hormone Library Identified Candidate GPR83 Ligands

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555220934807 ◽

2020 ◽

Vol 25 (9) ◽

pp. 1047-1063

Author(s):

Nathan A. Sallee ◽

Ernestine Lee ◽

Atossa Leffert ◽

Silvia Ramirez ◽

Arthur D. Brace ◽

...

Keyword(s):

Secretory Pathway ◽

Cell Function ◽

Active Form ◽

Peptide Hormone ◽

Biologically Active ◽

Host Cells ◽

Functional Screening ◽

Posttranslational Processing ◽

Regulated Secretory Pathway ◽

G Protein Coupled

The identification of novel peptide hormones by functional screening is challenging because posttranslational processing is frequently required to generate biologically active hormones from inactive precursors. We developed an approach for functional screening of novel potential hormones by expressing them in endocrine host cells competent for posttranslational processing. Candidate preprohormones were selected by bioinformatics analysis, and stable endocrine host cell lines were engineered to express the preprohormones. The production of mature hormones was demonstrated by including the preprohormones insulin and glucagon, which require the regulated secretory pathway for production of the active forms. As proof of concept, we screened a set of G-protein-coupled receptors (GPCRs) and identified protein FAM237A as a specific activator of GPR83, a GPCR implicated in central nervous system and regulatory T-cell function. We identified the active form of FAM237A as a C-terminally cleaved, amidated 9 kDa secreted protein. The related protein FAM237B, which is 64% homologous to FAM237A, demonstrated similar posttranslational modification and activation of GPR83, albeit with reduced potency. These results demonstrate that our approach is capable of identifying and characterizing novel hormones that require processing for activity.

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

Biochemical Journal ◽

10.1042/bj20071382 ◽

2009 ◽

Vol 418 (1) ◽

pp. 81-91 ◽

Cited By ~ 26

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.

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

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2000.278.2.e285 ◽

2000 ◽

Vol 278 (2) ◽

pp. E285-E292 ◽

Cited By ~ 5

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.

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The Regulated Secretory Pathway in Neuroendocrine Cells

Frontiers in Endocrinology ◽

10.3389/fendo.2014.00048 ◽

2014 ◽

Vol 5 ◽

Cited By ~ 7

Author(s):

Rafael Vazquez-Martinez ◽

Keyword(s):

Secretory Pathway ◽

Neuroendocrine Cells ◽

Regulated Secretory Pathway

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Somatostatin is targeted to the regulated secretory pathway of gonadotrophs in transgenic mice expressing a metallothionein-somatostatin gene.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)66709-2 ◽

1986 ◽

Vol 261 (34) ◽

pp. 16260-16263

Author(s):

M J Low ◽

P J Stork ◽

R E Hammer ◽

R L Brinster ◽

M J Warhol ◽

...

Keyword(s):

Transgenic Mice ◽

Secretory Pathway ◽

Regulated Secretory Pathway

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amontillado, the Drosophila Homolog of the Prohormone Processing Protease PC2, Is Required During Embryogenesis and Early Larval Development

Genetics ◽

10.1093/genetics/163.1.227 ◽

2003 ◽

Vol 163 (1) ◽

pp. 227-237 ◽

Cited By ~ 1

Author(s):

Lowell Y M Rayburn ◽

Holly C Gooding ◽

Semil P Choksi ◽

Dhea Maloney ◽

Ambrose R Kidd ◽

...

Keyword(s):

Larval Development ◽

Secretory Pathway ◽

Larval Growth ◽

Peptide Hormones ◽

Complementation Group ◽

Prohormone Processing ◽

Complementation Groups ◽

Regulated Secretory Pathway ◽

Processing Protease ◽

Larval Molt

Abstract Biosynthesis of most peptide hormones and neuropeptides requires proteolytic excision of the active peptide from inactive proprotein precursors, an activity carried out by subtilisin-like proprotein convertases (SPCs) in constitutive or regulated secretory pathways. The Drosophila amontillado (amon) gene encodes a homolog of the mammalian PC2 protein, an SPC that functions in the regulated secretory pathway in neuroendocrine tissues. We have identified amon mutants by isolating ethylmethanesulfonate (EMS)-induced lethal and visible mutations that define two complementation groups in the amon interval at 97D1 of the third chromosome. DNA sequencing identified the amon complementation group and the DNA sequence change for each of the nine amon alleles isolated. amon mutants display partial embryonic lethality, are defective in larval growth, and arrest during the first to second instar larval molt. Mutant larvae can be rescued by heat-shock-induced expression of the amon protein. Rescued larvae arrest at the subsequent larval molt, suggesting that amon is also required for the second to third instar larval molt. Our data indicate that the amon proprotein convertase is required during embryogenesis and larval development in Drosophila and support the hypothesis that AMON acts to proteolytically process peptide hormones that regulate hatching, larval growth, and larval ecdysis.

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