A Predicted α-Helix Mediates Targeting of the Proprotein Convertase PC1 to the Regulated Secretory Pathway

Jutras, Isabelle; Seidah, Nabil G.; Reudelhuber, Timothy L.

doi:10.1074/jbc.m004757200

Cited by 48 publications

(76 citation statements)

References 38 publications

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“…30 It has a N terminus leader peptide, and a di-leucine motif that may mediate intracellular transport of melanosomal proteins through binding to Adaptor protein 3 (AP-3), 31 and a helix motif in the C terminus (MGLLLKHMQDVRVLLGHLLMEL) that may mediate sorting signal to the secretary granules. 32 …”

Section: Gene Structure Of Fcrl/freb Expressed In Melanocytes and Melmentioning

confidence: 99%

Novel melanoma antigen, FCRL/FREB, identified by cDNA profile comparison using DNA chip are immunogenic in multiple melanoma patients

Inozume

Matsuzaki

Kurihara

et al. 2004

Intl Journal of Cancer

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Section: Gene Structure Of Fcrl/freb Expressed In Melanocytes and Melmentioning

confidence: 99%

Novel melanoma antigen, FCRL/FREB, identified by cDNA profile comparison using DNA chip are immunogenic in multiple melanoma patients

Inozume

Matsuzaki

Kurihara

et al. 2004

Intl Journal of Cancer

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“…A C-terminal ␣-helical domain in PC1 was shown previously to be critical for targeting PC1 fusion proteins to the regulated secretory pathway (59). We used a number of independent algorithms to confidently and reproducibly predict the secondary structure of the VGF Nand C-terminal domains, which suggested by consensus that the C-terminal region contained two ␣-helical loops (Fig.…”

Section: Discussionmentioning

confidence: 99%

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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“…The upper sequence corresponds to the sorting domain at the C terminus of the mouse prohormone convertase 1/3. The overlined sequence is predicted to form an ␣-helix, and its disruption by mutagenesis (insertion of 2 prolines as depicted below the sequence) abolishes secretory granule sorting (3). The lower sequence depicts the first 16 amino acids of the human prorenin prosegment.…”

Section: Figmentioning

confidence: 99%

“…Indeed, expression of a variety of granule cargo proteins including chromogranin A (11,12), provasopressin, prooxytocin, proopiomelanocortin, secretogranin II, and chromogranin B (12) is sufficient to induce aggregatecontaining cytoplasmic vesicles, although these vesicles do not have all of the characteristics of secretory granules (12). Proinsulin, proopiomelanocortin, and engineered proteins containing the C-terminal tail of PC1/3 also form multimers; however, this property alone is insufficient to direct granule sorting (3,13). A second group of granule-sorting domains may act by anchoring cargo proteins to other granuleresident proteins including carboxypeptidase E (2) and (14).…”

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

Lacombe¹,

Mercure²,

Dikeakos

et al. 2005

Journal of Biological Chemistry

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Several protein domains acting through seemingly different mechanisms have been reported to have the capacity to target proteins to dense core secretory granules. Because proteins enter secretory granules with different efficiencies and because some of these proteins contain more than one granule-targeting motif, we have investigated whether compounding sorting signals could alter the efficiency of protein entry into secretory granules. In the current study we demonstrate that a paired basic cleavage site from human prorenin and an ␣-helix-containing secretory granulesorting signal from the prohormone convertase PC1/3 can synergize to increase granule-sorting efficiency not only when located on the same protein, but also when located on distinct proteins that associate in the secretory pathway.

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A Predicted α-Helix Mediates Targeting of the Proprotein Convertase PC1 to the Regulated Secretory Pathway

Cited by 48 publications

References 38 publications

Novel melanoma antigen, FCRL/FREB, identified by cDNA profile comparison using DNA chip are immunogenic in multiple melanoma patients

Novel melanoma antigen, FCRL/FREB, identified by cDNA profile comparison using DNA chip are immunogenic in multiple melanoma patients

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

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

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