scholarly journals Constitutive and basal secretion from the endocrine cell line, AtT-20.

1991 ◽  
Vol 112 (5) ◽  
pp. 843-852 ◽  
Author(s):  
L Matsuuchi ◽  
R B Kelly
Keyword(s):  
Cell Line ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Secretory Protein ◽  
Dense Core ◽  
Wild Type ◽  
Dna Encoding ◽  
Basal Secretion ◽  
Constitutive Secretion ◽  
Regulated Secretory Pathway

A variant of the ACTH-secreting pituitary cell line, AtT-20, has been isolated that does not make ACTH, sulfated proteins characteristic of the regulated secretory pathway, or dense-core secretory granules but retains constitutive secretion. Unlike wild type AtT-20 cells, the variant cannot store or release on stimulation, free glycosaminoglycan (GAG) chains. In addition, the variant cells cannot store trypsinogen or proinsulin, proteins that are targeted to dense core secretory granules in wild type cells. The regulated pathway could not be restored by transfecting with DNA encoding trypsinogen, a soluble regulated secretory protein targeted to secretory granules. A comparison of secretion from variant and wild type cells allows a distinction to be made between constitutive secretion and basal secretion, the spontaneous release of regulated proteins that occurs in the absence of stimulation.

scholarly journals Trafficking of Mutant Carboxypeptidase E to Secretory Granules in a β-Cell Line Derived from Cpefat/Cpefat Mice

Endocrinology ◽  
2003 ◽  
Vol 144 (1) ◽  
pp. 292-298 ◽  
Author(s):  
Niamh X. Cawley ◽  
Yazmin M. Rodriguez ◽  
Alex Maldonado ◽  
Y. Peng Loh
Keyword(s):  
Cell Line ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Prohormone Convertase ◽  
Β Cell ◽  
Carboxypeptidase E ◽  
Prohormone Convertase 2 ◽  
Glucagon Like Peptide 1 ◽  
Regulated Secretory Pathway ◽  
The Stability

Abstract We have reinvestigated the stability and intracellular routing of mutant carboxypeptidase E in NIT3 cells, a pancreatic β-cell line derived from the Cpefat/Cpefat mouse. Pulse-chase experiments demonstrated that this protein has a half-life of approximately 3 h in these cells and that up to 45% of the proCPE(202) can escape degradation by the proteosome. In double-label immunofluorescence microscopy, a portion of the mutant CPE did not colocalize with calnexin, an endoplasmic reticulum marker, but was found in prohormone convertase 2-containing secretory granules, demonstrating that it had escaped degradation and arrived at a post-Golgi compartment. The mutant CPE as well as prohormone convertase 2 were secreted into the medium in a stimulated manner by treatment with the physiological secretagogue, glucagon-like peptide-1, consistent with its presence in granules of the regulated secretory pathway. The presence of mutant carboxypeptidase E in granules supports a potential role for its involvement as a sorting/retention receptor in the trafficking of proinsulin to the regulated secretory pathway.

scholarly journals The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer.

1985 ◽  
Vol 101 (2) ◽  
pp. 639-645 ◽  
Author(s):  
T L Burgess ◽  
C S Craik ◽  
R B Kelly
Keyword(s):  
Gene Transfer ◽  
Cell Line ◽  
Anterior Pituitary ◽  
Endocrine Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Tumor Cell Line ◽  
Subcellular Fractionation ◽  
Regulated Secretory Pathway ◽  
Stimulation Of

The exocrine protein rat anionic trypsinogen has been expressed and is secreted from the murine anterior pituitary tumor cell line AtT-20. We examined which secretory pathway trypsinogen takes to the surface of this endocrine-derived cell line. The "constitutive" pathway externalizes proteins rapidly and in the absence of an external stimulus. In the alternate, "regulated" pathway, proteins are stored in secretory granules until the cells are stimulated to secrete with 8-Br-cAMP. On the basis of indirect immunofluorescence localization, stimulation of release, and subcellular fractionation, we find that trypsinogen is targeted into the regulated secretory pathway in AtT-20 cells. In contrast, laminin, an endogenous secretory glycoprotein, is shown to be secreted constitutively. Thus it appears that the transport apparatus for the regulated secretory pathway in endocrine cells can recognize not only endocrine prohormones, but also the exocrine protein trypsinogen, which suggests that a similar sorting mechanism is used by endocrine and exocrine cells.

Prohormone transport through the secretory pathway of neuroendocrine cells

2000 ◽  
Vol 78 (3) ◽  
pp. 289-298 ◽  
Author(s):  
Roland P Kuiper ◽  
Gerard JM Martens
Keyword(s):  
Secretory Pathway ◽  
Potential Role ◽  
Protein Transport ◽  
Secretory Granules ◽  
Secretory Protein ◽  
Neuroendocrine Cells ◽  
Secretory Proteins ◽  
Regulated Secretory Pathway ◽  
Made In

En route through the secretory pathway of neuroendocrine cells, prohormones pass a series of membrane-bounded compartments. During this transport, the prohormones are sorted to secretory granules and proteolytically cleaved to bioactive peptides. Recently, progress has been made in a number of aspects concerning secretory protein transport and sorting, particularly with respect to transport events in the early regions of the secretory pathway. In this review we will deal with some of these aspects, including: i) selective exit from the endoplasmic reticulum via COPII-coated vesicles and the potential role of p24 putative cargo receptors in this process, ii) cisternal maturation as an alternative model for protein transport through the Golgi complex, and iii) the mechanisms that may be involved in the sorting of regulated secretory proteins to secretory granules. Although much remains to be learned, interesting new insights into the functioning of the secretory pathway have been obtained.Key words: regulated secretory pathway, p24 family, vesicular transport, POMC, protein sorting, secretory granule, Xenopus laevis.

scholarly journals Mutant Proinsulin That Cannot Be Converted Is Secreted Efficiently from Primary Rat β-Cells via the Regulated Pathway

2003 ◽  
Vol 14 (3) ◽  
pp. 1195-1203 ◽  
Author(s):  
Philippe A. Halban ◽  
Jean-Claude Irminger
Keyword(s):  
High Performance ◽  
Secretory Pathway ◽  
Recombinant Adenovirus ◽  
Islet Cells ◽  
Secretory Granules ◽  
Wild Type ◽  
Β Cells ◽  
Human Proinsulin ◽  
Regulated Secretory Pathway ◽  
Golgi Network

Prohormones are directed from the trans-Golgi network to secretory granules of the regulated secretory pathway. It has further been proposed that prohormone conversion by endoproteolysis may be necessary for subsequent retention of peptides in granules and to prevent their release by the so-called “constitutive-like” pathway. To address this directly, mutant human proinsulin (Arg/Gly32:Lys/Thr64), which cannot be cleaved by conversion endoproteases, was expressed in primary rat islet cells by recombinant adenovirus. The handling of the mutant proinsulin was compared with that of wild-type human proinsulin. Infected islet cells were pulse labeled and both basal and stimulated secretion of radiolabeled products followed during a chase. Labeled products were quantified by high-performance liquid chromatography. As expected, the mutant proinsulin was not converted at any time. Basal (constitutive and constitutive-like) secretion was higher for the mutant proinsulin than for wild-type proinsulin/insulin, but amounted to <1% even during a prolonged (6-h) period of basal chase. There was no difference in stimulated (regulated) secretion of mutant and wild-type proinsulin/insulin at any time. Thus, in primary islet cells, unprocessed (mutant) proinsulin is sorted to the regulated pathway and then retained in secretory granules as efficiently as fully processed insulin.

scholarly journals Intracellular transport and sorting of mutant human proinsulins that fail to form hexamers.

1991 ◽  
Vol 113 (5) ◽  
pp. 987-996 ◽  
Author(s):  
D Quinn ◽  
L Orci ◽  
M Ravazzola ◽  
H P Moore
Keyword(s):  
Intracellular Transport ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Point Mutations ◽  
Wild Type ◽  
Sorting Process ◽  
Storage Granules ◽  
Self Association ◽  
Regulated Secretory Pathway ◽  
Mouse Pituitary

Human proinsulin and insulin oligomerize to form dimers and hexamers. It has been suggested that the ability of prohormones to self associate and form aggregates may be responsible for the sorting process at the trans-Golgi. To examine whether insulin oligomerization is required for proper sorting into regulated storage granules, we have constructed point mutations in human insulin B chain that have been previously shown to prevent formation of insulin hexamers (Brange, J., U. Ribel, J. F. Hansen, G. Dodson, M. T. Hansen, S. Havelund, S. G. Melberg, F. Norris, K. Norris, L. Snel, A. R. Sorensen, and H. O. Voight. 1988. Nature [Lond.]. 333:679-682). One mutant (B10His----Asp) allows formation of dimers but not hexamers and the other (B9Ser----Asp) prevents formation of both dimers and hexamers. The mutants were transfected into the mouse pituitary AtT-20 cells, and their ability to be sorted into regulated secretory granules was compared to wild-type insulin. We found that while B10His----Asp is sorted somewhat less efficiently than wild-type insulin as reported previously (Carroll, R. J., R. E. Hammer, S. J. Chan, H. H. Swift, A. H. Rubenstein, and D. F. Steiner. 1988. Proc. Natl. Acad. Sci. USA. 85:8943-8947; Gross, D. J., P. A. Halban, C. R. Kahn, G. C. Weir, and L. Villa-Kumaroff. 1989. Proc. Natl. Acad. Sci. USA. 86:4107-4111). B9Ser----Asp is targeted to granules as efficiently as wild-type insulin. These results indicate that self association of proinsulin into hexamers is not required for its targeting to the regulated secretory pathway.

scholarly journals Sorting and storage during secretory granule biogenesis: looking backward and looking forward

1998 ◽  
Vol 332 (3) ◽  
pp. 593-610 ◽  
Author(s):  
Peter ARVAN ◽  
David CASTLE
Keyword(s):  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Secretory Granules ◽  
Secretory Proteins ◽  
Membrane Composition ◽  
Granule Formation ◽  
Granule Membrane ◽  
Regulated Secretory Pathway ◽  
Secretory Products

Secretory granules are specialized intracellular organelles that serve as a storage pool for selected secretory products. The exocytosis of secretory granules is markedly amplified under physiologically stimulated conditions. While granules have been recognized as post-Golgi carriers for almost 40 years, the molecular mechanisms involved in their formation from the trans-Golgi network are only beginning to be defined. This review summarizes and evaluates current information about how secretory proteins are thought to be sorted for the regulated secretory pathway and how these activities are positioned with respect to other post-Golgi sorting events that must occur in parallel. In the first half of the review, the emerging role of immature secretory granules in protein sorting is highlighted. The second half of the review summarizes what is known about the composition of granule membranes. The numerous similarities and relatively limited differences identified between granule membranes and other vesicular carriers that convey products to and from the plasmalemma, serve as a basis for examining how granule membrane composition might be established and how its unique functions interface with general post-Golgi membrane traffic. Studies of granule formation in vitro offer additional new insights, but also important challenges for future efforts to understand how regulated secretory pathways are constructed and maintained.

scholarly journals PACS-1 and Adaptor Protein-1 Mediate ACTH Trafficking to the Regulated Secretory Pathway

2018 ◽  
Author(s):  
Brennan S. Dirk ◽  
Christopher End ◽  
Emily N. Pawlak ◽  
Logan R. Van Nynatten ◽  
Rajesh Abraham Jacob ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Adaptor Protein ◽  
Cell Types ◽  
Cellular Membrane ◽  
Extracellular Milieu ◽  
Specialized Form ◽  
Regulated Secretory Pathway ◽  
Clathrin Adaptor

ABSTRACTThe regulated secretory pathway is a specialized form of protein secretion found in endocrine and neuroendocrine cell types. Pro-opiomelanocortin (POMC) is a pro-hormone that utilizes this pathway to be trafficked to dense core secretory granules (DCSGs). Within this organelle, POMC is processed to multiple bioactive hormones that play key roles in cellular physiology. However, the complete set of cellular membrane trafficking proteins that mediate the correct sorting of POMC to DCSGs remain unknown. Here, we report the roles of the phosphofurin acidic cluster sorting protein – 1 (PACS-1) and the clathrin adaptor protein 1 (AP-1) in the targeting of POMC to DCSGs. Upon knockdown of PACS-1 and AP-1, POMC is readily secreted into the extracellular milieu and fails to be targeted to DCSGs.

scholarly journals An antibody against secretogranin I (chromogranin B) is packaged into secretory granules.

1989 ◽  
Vol 109 (1) ◽  
pp. 17-34 ◽  
Author(s):  
P Rosa ◽  
U Weiss ◽  
R Pepperkok ◽  
W Ansorge ◽  
C Niehrs ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Intracellular Localization ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Chromogranin B ◽  
Double Labeling ◽  
Protein Protein Interaction ◽  
And Function ◽  
Secretogranin I

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.

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

1987 ◽  
Vol 105 (2) ◽  
pp. 659-668 ◽  
Author(s):  
T L Burgess ◽  
C S Craik ◽  
L Matsuuchi ◽  
R B Kelly
Keyword(s):  
Signal Peptide ◽  
Secretory Pathway ◽  
Protein Targeting ◽  
Secretory Granules ◽  
Tumor Cell Line ◽  
Signal Peptidase ◽  
Secretory Proteins ◽  
In Vitro Mutagenesis ◽  
Regulated Secretory Pathway

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.

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