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

The mechanisms by which prohormone precursors are sorted to the regulated secretory pathway in neuroendocrine cells remain poorly understood. Here, we investigated the presence of sorting signal(s) in proneurotensin/neuromedin N. The precursor sequence starts with a long N-terminal domain followed by a Lys-Arg-(neuromedin N)-Lys-Arg-(neurotensin)-Lys-Argsequence and a short C-terminal tail. An additional ArgArg dibasic is contained within the neurotensin sequence. Mutated precursors were expressed in endocrine insulinoma cells and analyzed for their regulated secretion. Deletion mutants revealed that the N-terminal domain and the Lys-Arg-(C-terminal tail) sequence were not critical for precursor sorting to secretory granules. In contrast, the Lys-Arg-(neuromedin N)-Lys-Arg-(neurotensin) sequence contained essential sorting information. Point mutation of all three dibasic sites within this sequence abolished regulated secretion. However, keeping intact any one of the three dibasic sequences was sufficient to maintain regulated secretion. Finally, fusing the dibasic-containing C-terminal domain of the precursor to the C terminus of ␤-lactamase, a bacterial enzyme that is constitutively secreted when expressed in neuroendocrine cells, resulted in efficient sorting of the fusion protein to secretory granules in insulinoma cells. We conclude that dibasic motifs within the neuropeptide domain of proneurotensin/neuromedin N constitute a necessary and sufficient signal for sorting proteins to the regulated secretory pathway.Whereas all cells secrete proteins constitutively, neuroendocrine and exocrine cells are also able to release a number of proteins and peptides, including hormones and neuropeptides, prohormone convertases (PCs) 1 and digestive enzymes, through a regulated secretory pathway (RSP) (1-3). Furthermore, in neuroendocrine cells, neuropeptides and peptide hormones are generally synthesized as part of large precursors in which they are flanked by processing sites, usually pairs of basic residues. The precursors must be cleaved at processing sites by PCs (for review, see Ref. 4) to yield the biologically active peptides that will be secreted under stimulation. Sorting between the constitutive and the regulated secretory pathway is thought to occur in the trans-Golgi network (TGN) and/or in immature secretory granules (IG) that originate from the TGN (for review, see Ref. 5), ultimately leading to the packaging and storage of regulated proteins and peptides into mature secretory granules.Despite considerable advancement in our understanding of the regulated secretion machinery in specialized cells, the sorting mechanisms into the RSP remain unclear. Moore and Kelly (6) demonstrated that a constitutive protein can be re-routed to the RSP when fused with a regulated protein. This result led to the idea that the targeting of proteins into the RSP is an active process, whereas constitutive secretion occurs by default. Furthermore, it was recognized early that aggregation of secreted proteins occurs in a late Gol...

contrasting

confidence: 48%

The Role of Dibasic Residues in Prohormone Sorting to the Regulated Secretory Pathway

Féliciangéli

Kitabgi

Bidard³

2001

“…This proposal is based on studies examining the sorting of prohormones in the fat/fat mouse, and in cell lines that have a reduced level of CPE protein due to the expression of antisense RNA. However, proinsulin sorting is not dependent on CPE, based on studies of primary cultures of pancreatic beta cells (35) or the NIT3 beta cell line (21). The sorting of proinsulin and insulin into the regulated pathway of the beta cells proceeds in the complete absence of CPE from this pathway.…”

Section: Figmentioning

confidence: 99%

Glu300 of Rat Carboxypeptidase E Is Essential for Enzymatic Activity but Not Substrate Binding or Routing to the Regulated Secretory Pathway

Qian

Varlamov

Fricker

1999

Several recently discovered members of the carboxypeptidase E (CPE) gene family lack critical active site residues that are conserved in other family members. For example, three CPE-like proteins contain a Tyr in place of Glu 300 (equivalent to Glu 270 of carboxypeptidase A and B). To investigate the importance of this position, Glu 300 of rat CPE was converted into Gln, Lys, or Tyr, and the proteins expressed in Sf9 cells using the baculovirus system. All three mutants were secreted from the cells, but the media showed no enzyme activity above background levels. Wild-type CPE and the Gln 300 point mutant bound to a p-aminobenzoyl-Arg-Sepharose affinity resin, and this binding was competed by an active site-directed inhibitor, guanidinoethylmercaptosuccinic acid. The affinity purified mutant CPE protein showed no detectable enzyme activity (<0.004% of wildtype CPE) toward dansyl-Phe-Ala-Arg. Expression of the Gln 300 and Lys 300 mutant CPE proteins in the NIT3 mouse pancreatic beta-cell line showed that these mutants are routed into secretory vesicles and secreted via the regulated pathway. Taken together, these results indicate that Glu 300 of CPE is essential for enzyme activity, but not required for substrate binding or for routing into the regulated secretory pathway.

“…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%

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

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...