Differences in pH Optima and Calcium Requirements for Maturation of the Prohormone Convertases PC2 and PC3 Indicates Different Intracellular Locations for These Events

Shennan, K. I. J.; Taylor, Nick; Jermany, Joanne L.; Matthews, Glenn; Docherty, Kevin

doi:10.1074/jbc.270.3.1402

Cited by 97 publications

(101 citation statements)

References 31 publications

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“…The processing of many neuroen- docrine peptides begins in the TGN and continues in the immature secretory granules. The acidification of the TGN and secretory granules is thought to play an important role in the activation of the processing enzymes such as prohormone convertase 1 and 2 (28,45). Importantly, CPD has a broad pH optimum (14,23) and could function both in the environment of the TGN and within acidic secretory granules.…”

Section: Resultsmentioning

confidence: 99%

Localization of Metallocarboxypeptidase D in AtT-20 Cells

Varlamov

Eng

Novikova

et al. 1999

Journal of Biological Chemistry

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Carboxypeptidase D (CPD) is a recently discovered metallocarboxypeptidase that is predominantly located in the trans-Golgi network (TGN), and also cycles between the cell surface and the TGN. In the present study, the intracellular distribution of CPD was examined in AtT-20 cells, a mouse anterior pituitary-derived corticotroph. CPD-containing compartments were isolated using antibodies to the CPD cytosolic tail. The immunopurified vesicles contained TGN proteins (TGN38, furin, syntaxin 6) but not lysosomal or plasma membrane proteins. The CPD-containing vesicles also contained neuropeptide-processing enzymes and adrenocorticotropic hormone, a product of proopiomelanocortin proteolysis. Electron microscopic analysis revealed that CPD is present within the TGN and immature secretory granules but is virtually absent from mature granules, suggesting that CPD is actively removed from the regulated pathway during the process of granule maturation. A second major finding of the present study is that a soluble truncated form of CPD is secreted mainly via the constitutive pathway in AtT-20 cells, indicating that the lumenal domain does not contain signals for the sorting of CPD to mature secretory granules. Taken together, these data are consistent with the proposal that CPD participates in the processing of proteins within the TGN and immature secretory vesicles.

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Section: Resultsmentioning

confidence: 99%

Localization of Metallocarboxypeptidase D in AtT-20 Cells

Varlamov

Eng

Novikova

et al. 1999

Journal of Biological Chemistry

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“…PC2 is also synthesized as a zymogen of 75 kDa that undergoes proteolysis to yield 68 -71-kDa forms (33)(34)(35). The binding protein, 7B2, regulates the maturation of PC2.…”

Section: Discussionmentioning

confidence: 99%

Impaired Prohormone Convertases in Cpe fat/Cpe fat Mice

Berman¹,

Mzhavia²,

Polonskaia³

et al. 2001

Journal of Biological Chemistry

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. (1995) Nat. Genet. 10, 135-142). Examination of the level of neuropeptides in these mice showed a decrease in mature bioactive peptides as a result of a decrease in both carboxypeptidase and prohormone convertase activities. A defect in CPE is not expected to affect endoproteolytic processing. In this report we have addressed the mechanism of this unexpected finding by directly examining the expression of the major precursor processing endoproteases, prohormone convertases PC1 and PC2 in Cpe fat mice. We found that the levels of PC1 and PC2 are differentially altered in a number of brain regions and in the pituitary. Since these enzymes have been implicated in the generation of neuroendocrine peptides (dynorphin A-17, ␤-endorphin, and ␣-melanocyte-stimulating hormone) involved in the control of feeding behavior and body weight, we compared the levels of these peptides in Cpe fat and wild type animals. We found a marked increase in the level of dynorphin A-17, a decrease in the level of ␣-melanocyte-stimulating hormone, and an alteration in the level of C-terminally processed ␤-endorphin. These results suggest that the impairment in the level of these and other peptides involved in body weight regulation is mainly due to an alteration in carboxypeptidase and prohormone convertase activities and that this may lead to the development of obesity in these animals.

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“…In vitro translation was performed as described previously [22] in a Xenopus egg extract [23] containing 10% (by vol.) nuclease-treated rabbit reticulocyte lysate, 10 mM creatine phosphate, 0.2 mM spermidine and 1 mCi/ml [ 35 S]methionine.…”

Section: Methodsmentioning

confidence: 99%

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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Differences in pH Optima and Calcium Requirements for Maturation of the Prohormone Convertases PC2 and PC3 Indicates Different Intracellular Locations for These Events

Cited by 97 publications

References 31 publications

Localization of Metallocarboxypeptidase D in AtT-20 Cells

Localization of Metallocarboxypeptidase D in AtT-20 Cells

Impaired Prohormone Convertases in Cpe fat/Cpe fat Mice

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

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