Modulation of Secretory Granule-targeting Efficiency by Cis and Trans Compounding of Sorting Signals

Lacombe, Marie-Josée; Mercure, Chantal; Dikeakos, Jimmy D.; Reudelhuber, Timothy L.

doi:10.1074/jbc.m408658200

Cited by 19 publications

(21 citation statements)

References 35 publications

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“…Our results indicate that both motifs may constitute proglucagon sorting signals, and that they may act independently; mutating the dibasic site does not affect alpha-helical content, and altering the helical content leaves the dibasic site intact (Guizzetti and Dhanvantari 2012, unpublished observations). It has been shown that multiple types of sorting signals increase the efficiency of sorting to granules (Lacombe et al 2005), which may then translate into a greater degree of physiological regulation of secretion. In this regard, the sorting efficiency of proglucagon may be similar to that of proinsulin, which also contains a number of different sorting signals (hexamerisation, CPE binding sites and dibasic sites).…”

Section: Discussionmentioning

confidence: 99%

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

McGirr

Guizzetti

Dhanvantari

2013

Journal of Endocrinology

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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.

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Section: Discussionmentioning

confidence: 99%

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

McGirr

Guizzetti

Dhanvantari

2013

Journal of Endocrinology

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“…Although no consensus secretory granule-sorting sequence has emerged from these studies, various structural motifs influencing targeting of proteins to secretory granules have been identified. These motifs include hydrophobic disulfide-bonded loop structures found in a variety of regulated secretory proteins such as pro-opiomelanocortin, CgA, and CgB (15,28,32,33); amphipathic ␣-helical domains that mediate interaction with membranes (15,34,35); and Ca 2ϩ -binding domains that promote protein aggregation prior to sorting (36,37). Several studies on the secretory prohormone CgA have suggested a direct correlation between the sorting process and the formation of secretory granules (9 -13).…”

Section: Discussionmentioning

confidence: 99%

Secretory Granule Biogenesis in Sympathoadrenal Cells

Courel

Rodemer

Nguyen

et al. 2006

Journal of Biological Chemistry

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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 secretagoguestimulated 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 ϳ180 -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.The regulated secretory pathway that exists in neurons and neuroendocrine cells is characterized by the concentration and sorting of a pool of secretory proteins into specialized intracellular organelles with a typical electron-dense appearance upon transmission electron microscopy, prompting the morphologic term dense-core granules. These granules may remain in the cell for an extended period of time after their formation, until prompted for exocytotic fusion with the plasma membrane by a secretagogue characteristic for a particular cell type.The mechanism underlying the initiation and regulation of dense-core secretory granule biogenesis is poorly understood. The formation of secretory granules is believed to be initiated at the trans-Golgi network (TGN) 2 apparatus, where aggregation and condensation of secretory proteins are typically viewed as the initial step prior to granulogenesis. Thus, formation of protein aggregates may occur in the mildly acidic environment that exists in the TGN and in the presence of millimolar concentrations of bivalent cations like Ca 2ϩ . Aggregation and condensation are the underpinning processes of two basic models of sorting of proteins within the regulated secretory pathway, viz. sorting for entry and sorting by ...

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“…11), but the search for sequence similarity with other proteins by BLAST (Basic Local Alignment Search Tool) and PSI-BLAST (Position-specific Iterative-BLAST) did not reveal homology below the threshold of significance E ϭ 0.01. ␣-Helical domains with amphipathic properties have been suggested to mediate targeting of several prohormones into the regulated pathway, for instance CgA (13), prosomatostatin (14), and PCs (15,16), possibly by virtue of interaction with membrane components (5,43). In support of this concept, we show that ␣-helix-containing domains SgII- (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41) and SgII-(333-348) function as transferable DCG-sorting signals; the hydrophobic "faces" of these two amphipathic helical segments in cis (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Sorting-for-entry proposes that a structural/conformational motif within an aggregate of secretory proteins mediates subsequent association to a protein receptor or a lipid component of the membrane of the trans-Golgi network (TGN). The quest for identifying a consensus sorting signal has proven elusive, and it now appears that such process may rather depend on a variety of motifs (5-7), including hydrophobic disulfide-bonded loop structures (8 -12) or amphipathic ␣-helices (13)(14)(15)(16). In the sorting-by-retention model, selective aggregation/condensation occurs in post-TGN immature secretory granules (ISGs), resulting in retention of regulated secretory proteins, whereas nonaggregated proteins are removed into constitutive-like secretory vesicles (3).…”

mentioning

confidence: 99%

Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway

Courel

Vasquez

Hook

et al. 2008

Journal of Biological Chemistry

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Modulation of Secretory Granule-targeting Efficiency by Cis and Trans Compounding of Sorting Signals

Cited by 19 publications

References 35 publications

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

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

Secretory Granule Biogenesis in Sympathoadrenal Cells

Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway

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