Identification of a Transferable Sorting Domain for the Regulated Pathway in the Prohormone Convertase PC2

Creemers, John W.M.; Usac, E F; Bright, Nicholas A.; Loo, Jan-Willem Van de; Jansen, Erik; Ven, Wim J.M. Van de; Hutton, John C.

doi:10.1074/jbc.271.41.25284

Cited by 69 publications

(54 citation statements)

References 53 publications

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“…Regulated secretion experiments were performed essentially as described (8), except that cells were incubated overnight in nonsupplemented serum-free DMEM. Sample preparation and separation by SDS/PAGE have been described previously (32).…”

Section: Cell Lines and Transfections See Si Materials And Methods Fmentioning

confidence: 99%

“…Intracellularly furin is known to be concentrated in the trans-Golgi network (TGN), although ImmunoGold electron microscopy also revealed the presence of small amounts of furin on the periphery of immature secretory granules (ISG) (8). Furin is excluded from the mature secretory granules through phosphorylation of its cytoplasmic domain by casein kinase II and subsequent interaction with adaptor protein (AP)-1, a component of the TGN/ISG-localized clathrin sorting machinery (9).…”

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confidence: 99%

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Role of furin in granular acidification in the endocrine pancreas: Identification of the V-ATPase subunit Ac45 as a candidate substrate

Louagie

Taylor

Flamez

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Furin is a proprotein convertase which activates a variety of regulatory proteins in the constitutive exocytic and endocytic pathway. The effect of genetic ablation of fur was studied in the endocrine pancreas to define its physiological function in the regulated secretory pathway. Pdx1-Cre/loxP furin KO mice show decreased secretion of insulin and impaired processing of known PC2 substrates like proPC2 and proinsulin II. Both secretion and PC2 activity depend on granule acidification, which was demonstrated to be significantly decreased in furin-deficient ␤ cells by using the acidotrophic agent 3-(2,4-dinitroanilino)-3 amino-N-methyldipropylamine (DAMP). Ac45, an accessory subunit of the proton pump V-ATPase, was investigated as a candidate substrate. Ac45 is highly expressed in islets of Langerhans and furin was able to cleave Ac45 ex vivo. Furthermore, the exact cleavage site was determined. In addition, reduced regulated secretion and proinsulin II processing could be obtained in the insulinoma cell line ␤TC3 by downregulation of either furin or Ac45. Together, these data establish an important role for furin in regulated secretion, particularly in intragranular acidification most likely due to impaired processing of Ac45.endoprotease ͉ insulin ͉ islets of Langerhans ͉ proton pump ͉ proprotein convertase

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Section: Cell Lines and Transfections See Si Materials And Methods Fmentioning

confidence: 99%

mentioning

confidence: 99%

Role of furin in granular acidification in the endocrine pancreas: Identification of the V-ATPase subunit Ac45 as a candidate substrate

Louagie

Taylor

Flamez

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…An N-terminal sorting domain has been reported for proopiomelanocortin (POMC) (12,13), although this is controversial (14). A putative C-terminal sorting domain has also recently been proposed for the prohormone convertase PC2 (15). There is not as yet any direct experimental evidence for a sorting domain for proinsulin, although the comparison of structural features of many prohormones suggests that a region within the insulin B-chain may play such a role (16,17).…”

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confidence: 98%

Proinsulin Targeting to the Regulated Pathway Is Not Impaired in Carboxypeptidase E-deficientCpe /Cpe Mice

Irminger

Verchere

Meyer

et al. 1997

Journal of Biological Chemistry

103

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Sorting of proinsulin from the trans-Golgi network to secretory granules is critical for its conversion to insulin as well as for regulated insulin secretion. The proinsulin sorting mechanism is unknown. Recently, carboxypeptidase E (CPE) was proposed as a sorting receptor for prohormones. To know whether CPE is implicated in proinsulin sorting, pancreatic islets were isolated from CPE-deficient Cpe fat /Cpe fat mice and Cpe fat /؉ controls, pulse-labeled ([ 3 H]leucine), and then chased in basal medium (90 min) to examine constitutive secretion followed by medium with secretagogues (60 min) to stimulate regulated secretion. Secretion of labeled proinsulin via the constitutive pathway was <2% even in Cpe fat /Cpe fat islets. After a 150-min chase, only 13% of radioactivity remained as proinsulin in Cpe fat /؉ islets compared with 46% in Cpe fat /Cpe fat islets, reflecting slower conversion. Regulated secretion was stimulated to an equal extent from Cpe fat /؉ and Cpe fat /Cpe fat mice with 20% of the total content of labeled (pro)insulin released during the 60-min stimulatory period. It is concluded that in CPE-deficient Cpe fat / Cpe fat mice, proinsulin is efficiently routed to the regulated pathway and its release can be effectively stimulated by secretagogues. CPE is thus not essential for sorting proinsulin to granules.Proinsulin is directed to the regulated secretory pathway of the pancreatic ␤-cell and is converted to bioactive insulin in immature secretory granules (1). At least three enzymes are needed for generating native insulin, namely the conversion endoproteases PC1 (also known as PC3) and PC2 (2, 3) and carboxypeptidase E (also called carboxypeptidase H or CPE 1 ) for trimming residual basic C-terminal residues (4 -6). Correct targeting to secretory granules is essential for the cell to produce and store bioactive insulin, which can then be released upon stimulation of the ␤-cell by glucose or other secretagogues. One of the crucial steps in targeting takes place in the TGN (trans-Golgi network) (7). The proprotein must somehow be recognized selectively in the TGN and then be transferred to the immature secretory granules, instead of being released through the constitutive or default pathway (3).One hypothesis (reviewed in Ref.3) accounting for targeting within the TGN implicates a sorting receptor with a broad specificity in the TGN membrane, which recognizes a sorting domain and thereby binds proteins to be routed to the regulated pathway. Sorting domains have been reported for several prohormones including prosomatostatin (8, 9), chromogranin (10), and the basic proline-rich protein (11). An N-terminal sorting domain has been reported for proopiomelanocortin (POMC) (12, 13), although this is controversial (14). A putative C-terminal sorting domain has also recently been proposed for the prohormone convertase PC2 (15). There is not as yet any direct experimental evidence for a sorting domain for proinsulin, although the comparison of structural features of many prohormones suggests that a regio...

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“…PC2 is a constituent of the secretory granules of many neuroendocrine cells where it is involved in the post-translational processing of a number of prohormones and proneuropeptides [18]. The presence of a transferable sorting domain in the C-terminal 50 amino acids has previously been reported [19] but deletion of a similar region does not affect regulated secretion from transfected AtT20 cells (Taylor, N. A., Jan, G., Scougall, K. T., Docherty, K. and Shennan, K. I. J., unpublished results). The precursor form of PC2 undergoes a low-pH-dependent and calcium-dependent aggregation and membrane-association event [9] which was proposed to be important in its sorting to the regulated secretory pathway.…”

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The propeptide of prohormone convertase PC2 acts as a transferable aggregation and membrane‐association signal

Jan¹,

Taylor²,

Scougall³

et al. 1998

European Journal of Biochemistry

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Prohormone convertase 2 (PC2) is a subtilisin-like protease involved in the intracellular processing of prohormones and proneuropeptides. Like its substrates, it is synthesised as a prepropeptide which undergoes proteolysis during transit through the regulated secretory pathway. Previous studies have shown that aggregation and membrane association of proPC2 occurs in a calcium-dependent and pH-dependent manner and that the pro-region of PC2 may be involved in this process. These events may be involved in the sorting of proteins to the regulated secretory pathway. To investigate this further, we made a chimeric protein containing both the signal peptide and pro-region of PC2 and the N-terminal part of A1-antitrypsin, called pro2A1. PC2, A1-antitrypsin and pro2A1 were compared with regard to their membrane association and aggregation properties using, respectively, sucrose gradient centrifugation after expression in Xenopus oocytes, and an in vitro aggregation assay. The chimeric protein, pro2A1, underwent low-pHdependent aggregation and membrane association similar to wild-type PC2. Membrane association occurred at pH 5.5 in the absence of calcium and at pH 6.0 in the presence of 10 mM calcium but not at pH 6.5 or 7.0. A1-antitrypsin, as expected of a constitutively secreted protein, did not aggregate at low pH, nor associate with membranes. Pro2A1 thus exhibits the membrane association and aggregation properties of PC2, confirming the role of the pro-region in these processes.A series of deletions were performed within the 84-residue propeptide in order to define the sequences involved. Deletion of amino acids 52Ϫ77 reduced aggregation but large deletions in the pro-region had only a minimal effect on membrane association. These data suggest that several regions within the propeptide are important in these events.Keywords : prohormone convertase 2; protein targeting; membrane association.Secretion of proteins from neuroendocrine cells can occur through at least two different pathways: constitutive secretion, which occurs at a constant rate limited by the bioavailability of material, and regulated secretion, where the protein is stored in dense-core granules for some time prior to release being stimulated by a secretagogue [1]. The most distal point in the secretory pathway common to both types of secretion and hence the point of sorting of proteins into the appropriate pathway, has been considered to be either the trans-Golgi network (TGN), based on morphological studies and glycosylation events [2], or the immature secretory granule, due to the observation that secretion of lysosomal enzymes can be stimulated from pancreatic β-cells shortly after their synthesis [3]. Whether sorting takes place at the TGN or in immature secretory granules, the mechanism of sorting remains poorly understood. One hypothesis is that sorting involves the selective aggregation and membrane association of regulated secretory proteins [1].Aggregation is thought to be triggered by the low pH and calcium content of the TGN or immature g...

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Identification of a Transferable Sorting Domain for the Regulated Pathway in the Prohormone Convertase PC2

Cited by 69 publications

References 53 publications

Role of furin in granular acidification in the endocrine pancreas: Identification of the V-ATPase subunit Ac45 as a candidate substrate

Role of furin in granular acidification in the endocrine pancreas: Identification of the V-ATPase subunit Ac45 as a candidate substrate

Proinsulin Targeting to the Regulated Pathway Is Not Impaired in Carboxypeptidase E-deficientCpe /Cpe Mice

The propeptide of prohormone convertase PC2 acts as a transferable aggregation and membrane‐association signal

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