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

Abstract

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...

Semaphorin-3B Is an Angiogenesis Inhibitor That Is Inactivated by Furin-Like Pro-Protein Convertases

“…3B, site 1) generates an f60 kDa NH 2 -terminal fragment and an f20 kDa COOH-terminal fragment (23,24), suggesting that the f60 kDa cleavage product observed in the conditioned medium of sema3B-expressing HEK293 cells is also the product of PPC activity. When the activity of furin-like PPC of HEK293 cells expressing recombinant sema3B was inhibited with the furin inhibitor decanoyl-RVKR-CMK (24,28), the cleavage of sema3B was partially inhibited, leading to the Figure 1. Sema3B undergoes proteolytic cleavage by furin-like PPCs expressed in tumorigenic cells.…”

Functional Consequences of the Subdomain Organization of the Sulfs

“…To obtain further information about the furin-type proteinase, we employed a cell-permeable furin inhibitor (decanoyl-RVKR-chloromethyl ketone) and a polyarginine inhibitor (Furin inhibitor II) (31,32). MCF7 cells were exposed to the inhibitors at several concentrations for a total of 6 h (with replenishment at 3 h).…”

The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals

“…Additionally, the sequence of glucagon contains an internal pair of basic amino acids, Arg17Arg18, which does not serve as a PC1/3 or PC2 cleavage site (Bataille 2007). Sequences of basic residues within prorenin (Brechler et al 1996), proneurotensin (Feliciangeli et al 2001), proneuropeptide Y (Brakch et al 2002) progastrin (Bundgaard et al 2004) and VGF (Garcia et al 2005) have been identified as sorting signals for these prohormones. Therefore, proglucagon may contain a number of sorting signals to direct it into granules.…”