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 sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals

2013 ◽  
Vol 217 (2) ◽  
pp. 229-240 ◽  
Author(s):  
Rebecca McGirr ◽  
Leonardo Guizzetti ◽  
Savita Dhanvantari
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Alpha Helix ◽  
Intestinal Cell ◽  
Alpha Cells ◽  
Pancreatic Alpha Cells ◽  
Carboxypeptidase E ◽  
L Cells ◽  
Sorting Signals ◽  
Regulated Secretory Pathway

Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia. The sorting of proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced proglucagon sorting to granules. siRNA-mediated knockdown of CPE disrupted regulated secretion of glucagon from pancreatic-derived alphaTC1–6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals.

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.

scholarly journals Involvement of the membrane lipid bilayer in sorting prohormone convertase 2 into the regulated secretory pathway

2000 ◽  
Vol 349 (3) ◽  
pp. 843-852 ◽  
Author(s):  
Mercedes BLÁZQUEZ ◽  
Christoph THIELE ◽  
Wieland B. HUTTNER ◽  
Kevin DOCHERTY ◽  
Kathleen I. J. SHENNAN
Keyword(s):  
Membrane Proteins ◽  
Lipid Rafts ◽  
Lipid Bilayer ◽  
Secretory Pathway ◽  
Insoluble Fraction ◽  
Membrane Association ◽  
Prohormone Convertase ◽  
Binding Properties ◽  
Prohormone Convertase 2 ◽  
Regulated Secretory Pathway

Prohormone convertase 2 (PC2) is a neuroendocrine-specific protease involved in the intracellular maturation of prohormones and proneuropeptides. PC2 is synthesised as a proprotein (proPC2) that undergoes proteolysis, aggregation and membrane association during its transit through the regulated secretory pathway. We have previously shown that the pro region of proPC2 plays a key role in its aggregation and membrane association. To investigate this further, we determined the binding properties of a peptide containing amino acids 45–84 of proPC2 (proPC245–84) to trans-Golgi network/granule-enriched membranes from the AtT20 cell line. Removal of peripheral membrane proteins or hydrolysis of integral membrane proteins did not affect the binding properties of proPC245–84. Rather, proPC245–84 was shown to bind to protein-free liposomes in a pH- and Ca2+-dependent manner. To identify the component of the lipid bilayer involved in this membrane association, we used chromaffin-granule membranes and studied the binding properties of the endogenous PC2. Treatment of the membranes with saponin, a cholesterol-depleting detergent, failed to extract PC2 from the membranes, whereas chromogranin A (CgA) was removed. Treatment of the membranes with Triton X-100 yielded a low-density detergent-insoluble fraction enriched in PC2, but not CgA. The detergent-insoluble fraction also contained glycoprotein III, known to be part of the lipid rafts (membrane microdomains rich in sphingolipids). Finally, sphingolipid depletion of AtT20 cells resulted in the mis-sorting of PC2, suggestive of a link between the association of PC2 with lipid rafts in the membrane and its sorting into the regulated secretory pathway.

The C-Terminus of Prohormone Convertase 2 Is Sufficient and Necessary for Raft Association and Sorting to the Regulated Secretory Pathway

Biochemistry ◽  
2004 ◽  
Vol 43 (24) ◽  
pp. 7798-7807 ◽  
Author(s):  
Masoumeh Assadi ◽  
Juanita C. Sharpe ◽  
Christopher Snell ◽  
Y. Peng Loh
Keyword(s):  
Secretory Pathway ◽  
Prohormone Convertase ◽  
C Terminus ◽  
Prohormone Convertase 2 ◽  
Regulated Secretory Pathway

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 Altered Proglucagon Processing in an α-Cell Line Derived from Prohormone Convertase 2 Null Mouse Islets

2004 ◽  
Vol 279 (30) ◽  
pp. 31068-31075 ◽  
Author(s):  
Gene C. Webb ◽  
Arunangsu Dey ◽  
Jie Wang ◽  
Jeffrey Stein ◽  
Margaret Milewski ◽  
...  
Keyword(s):  
Cell Line ◽  
Null Mouse ◽  
Prohormone Convertase ◽  
Prohormone Convertase 2 ◽  
Mouse Islets

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.

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