Insertion of Dibasic Residues Directs a Constitutive Protein to the Regulated Secretory Pathway

Féliciangéli, Sylvain; Kitabgi, Patrick

doi:10.1006/bbrc.2001.6137

Cited by 9 publications

(13 citation statements)

References 19 publications

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“…Proteins that are secreted without storage in a cellular compartment are said to transit through the constitutive or constitutive-like secretory pathway (4, 5). Scientific debate continues as to what targets prohormones and other proteins into the regulated secretory pathway with some favoring a receptor-mediated model (6 -8), self-aggregation of regulated secretory pathway cargo (9 -11), or binding of the prohormone to lipid rafts (12-14), but it is generally agreed that there are unique structural elements of prohormones, not necessarily universal, that result in their targeting to the regulated secretory pathway (15)(16)(17)(18)(19)(20)(21)(22). It has yet to be reported which elements of pro-TRH might be important in the targeting of this important prohormone to the regulated secretory pathway.…”

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confidence: 99%

“…Because the rat pro-TRH molecule contains several structural elements that have been implicated in the regulated sorting of other prohormone molecules such as dibasic residues (20,21), a disulfide bond (17,19,29), and two RGD sequences (30), we have hypothesized that the processing of pro-TRH before packaging into immature secretory granules is critical for its downstream sorting. The early and subsequent PC1 processing events may expose sorting signals, which then become accessible for targeting of the various pro-TRHderived peptides.…”

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Prohormone-Convertase 1 Processing Enhances Post-Golgi Sorting of Prothyrotropin-releasing Hormone-derived Peptides

Mulcahy

Vaslet

Nillni

2005

Journal of Biological Chemistry

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Rat prothyrotropin-releasing hormone (pro-TRH) is endoproteolyzed within the regulated secretory pathway of neuroendocrine cells yielding five TRH peptides and seven to nine other unique peptides. Endoproteolysis is performed by two prohormone convertases, PC1 and PC2. Proteolysis of pro-TRH begins in the transGolgi network and forms two intermediates that are then differentially processed as they exit the Golgi and are packaged into immature secretory granules. We hypothesized that this initial endoproteolysis may be necessary for downstream sorting of pro-TRH-derived peptides as it occurs before Golgi exit and thus entry into the regulated secretory pathway. We now report that when pro-TRH is transiently expressed in GH4C1 cells, a neuroendocrine cell line lacking PC1, under pulse-chase conditions release is constitutive and composed of more immature processing intermediates. This is also observed by radioimmunoassay under steady-state conditions. When a mutant form of pro-TRH, which has the dibasic sites of initial processing mutated to glycines, is expressed in AtT20 cells, a neuroendocrine cell line endogenously expressing PC1, both steady-state and pulse-chase experiments revealed that peptides derived from this mutant precursor are secreted in a constitutive fashion. A constitutively secreted form of PC1 does not target pro-TRH peptides to the constitutive secretory pathway but results in sorting to the regulated secretory pathway. These results indicated that initial processing action of PC1 on pro-TRH in the trans-Golgi network, and not a cargo-receptor relationship, is important for the downstream sorting events that result in storage of pro-TRH-derived peptides in mature secretory granules.All of the peptides derived from pro-TRH 2 are targeted to the regulated secretory pathway of neuroendocrine cells (1, 2), but it is not completely clear how pro-TRH and other prohormones are targeted to secretory granules from the trans-Golgi network (TGN). Proteins and peptides targeted to the regulated secretory pathway are ultimately stored in secretory granules until the cell receives a stimulus signaling for granule content release (3). Proteins that are secreted without storage in a cellular compartment are said to transit through the constitutive or constitutive-like secretory pathway (4, 5). Scientific debate continues as to what targets prohormones and other proteins into the regulated secretory pathway with some favoring a receptor-mediated model (6 -8), self-aggregation of regulated secretory pathway cargo (9 -11), or binding of the prohormone to lipid rafts (12-14), but it is generally agreed that there are unique structural elements of prohormones, not necessarily universal, that result in their targeting to the regulated secretory pathway (15)(16)(17)(18)(19)(20)(21)(22). It has yet to be reported which elements of pro-TRH might be important in the targeting of this important prohormone to the regulated secretory pathway.Rat pro-TRH is a prohormone composed of 255 amino acids, and when fully processed...

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confidence: 99%

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confidence: 99%

Prohormone-Convertase 1 Processing Enhances Post-Golgi Sorting of Prothyrotropin-releasing Hormone-derived Peptides

Mulcahy

Vaslet

Nillni

2005

Journal of Biological Chemistry

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“…In addition, dibasic sequences from pro-neurotensin can partially re-direct the constitutively secreted protein ␤-lactamase into the regulated pathway (62). The precise mechanism by which these positively charged residues mediate secretion is unclear, although they have been suggested to interact with the acidic polar heads of phospholipids (63,64), possibly in lipid raft microdomains, or with enzymes that cleave or modify dibasic sites, including PCs and CPE, that could therefore act as sorting receptors (43).…”

Section: Discussionmentioning

confidence: 99%

“…The precise mechanism by which these positively charged residues mediate secretion is unclear, although they have been suggested to interact with the acidic polar heads of phospholipids (63,64), possibly in lipid raft microdomains, or with enzymes that cleave or modify dibasic sites, including PCs and CPE, that could therefore act as sorting receptors (43). However, neither PC1, PC2, nor CPE seems to function as a general sorting receptor for all secreted hormones and neuropeptides; down-regulation of CPE in vitro or in vivo blocks regulated secretion of select hormones and growth factors (43,(65)(66)(67)(68), whereas distribution studies have identified cells that do not express either PC1 or PC2 yet secrete polypeptides via the regulated pathway (62).…”

Section: Discussionmentioning

confidence: 99%

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A Prohormone Convertase Cleavage Site within a Predicted α-Helix Mediates Sorting of the Neuronal and Endocrine Polypeptide VGF into the Regulated Secretory Pathway

Garcia

Han

Janssen

et al. 2005

Journal of Biological Chemistry

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Distinct intracellular pathways are involved in regulated and constitutive protein secretion from neuronal and endocrine cells, yet the peptide signals and molecular mechanisms responsible for targeting and retention of soluble proteins in secretory granules are incompletely understood. By using confocal microscopy and subcellular fractionation, we examined trafficking of the neuronal and endocrine peptide precursor VGF that is stored in large dense core vesicles and undergoes regulated secretion. VGF cofractionated with secretory vesicle membranes but was not detected in detergent-resistant lipid rafts. Deletional analysis using epitope-tagged VGF suggested that the C-terminal 73-amino acid fragment of VGF, containing two predicted ␣-helical loops and four potential prohormone convertase (PC) cleavage sites, was necessary and sufficient with an N-terminal signal peptide-containing domain, for large dense core vesicle sorting and regulated secretion from PC12 and INS-1 cells. Further transfection analysis identified the sorting sequence as a compact C-terminal ␣-helix and embedded 564 RRR 566PC cleavage site; mutation of the 564 RRR 566 PC site in VGF-(1-65): GFP:VGF-(545-617) blocked regulated secretion, whereas disruption of the ␣-helix had no effect. Mutation of the adjacent 567 HFHH 570 motif, a charged region that might enhance PC cleavage in acidic environments, also blocked regulated release. Finally, inhibition of PC cleavage in PC12 cells using the membrane-permeable synthetic peptide chloromethyl ketone (decanoyl-RVKR-CMK) blocked regulated secretion of VGF. Our studies define a critical RRR-containing C-terminal domain that targets VGF into the regulated pathway in neuronal PC12 and endocrine INS-1 cells, providing additional support for the proposed role that PCs and their cleavage sites play in regulated peptide secretion.Regulated secretion of polypeptides from neuronal and endocrine cells, which relies on packaging of proteins into the appropriate vesicle pools within the cell, is a critical control point for regulating peptide levels and ultimately function. Polypeptide motifs that target cell surface proteins to specific vesicle populations or to discrete locations within the cell, such as the basolateral or apical surface of a polarized epithelial cell, have been relatively well characterized (1). Protein domains that are responsible for targeting soluble proteins into the regulated release pathway appear to be more heterogeneous, and thus generalized sorting domains and consensus motifs have been more difficult to identify. Perhaps as a consequence, several models that explain targeting into the regulated secretory pathway by selective sorting and/or selective retention have been proposed (2-5). Following signal sequence-directed translocation into the lumen of the endoplasmic reticulum, segregation of proteins into the constitutive or regulated pathways is suspected to occur in the trans-Golgi network (TGN). 4 Constitutive secretory vesicles transit directly from the TGN to the plasma membran...

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Neurosecretory Protein Trafficking and Dense-Core Granule Biogenesis in Neuroendocrine Cells

Kim¹,

Gondre²,

Cawley³

et al. 2007

Handbook of Neurochemistry and Molecular Neurobiology

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Insertion of Dibasic Residues Directs a Constitutive Protein to the Regulated Secretory Pathway

Cited by 9 publications

References 19 publications

Prohormone-Convertase 1 Processing Enhances Post-Golgi Sorting of Prothyrotropin-releasing Hormone-derived Peptides

Prohormone-Convertase 1 Processing Enhances Post-Golgi Sorting of Prothyrotropin-releasing Hormone-derived Peptides

A Prohormone Convertase Cleavage Site within a Predicted α-Helix Mediates Sorting of the Neuronal and Endocrine Polypeptide VGF into the Regulated Secretory Pathway

Neurosecretory Protein Trafficking and Dense-Core Granule Biogenesis in Neuroendocrine Cells

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