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J. Biol. Chem., Vol. 280, Issue 50, 41595-41608, December 16, 2005

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

Angelo L. Garcia12, Shan-Kuo Han1, William G. Janssen, Zin Z. Khaing, Timothy Ito, Marc J. Glucksman¶, Deanna L. Benson, and Stephen R. J. Salton||3

From the Fishberg Department of Neuroscience, Kastor Neurobiology of Aging Laboratories, and the ^||Department of Geriatrics, Mount Sinai School of Medicine, New York, New York 10029 and the ^¶Midwest Proteome Center and Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science/Chicago Medical School, North Chicago, Illinois 60064

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 ⁵⁶⁴RRR⁵⁶⁶ PC cleavage site; mutation of the ⁵⁶⁴RRR⁵⁶⁶ 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 ⁵⁶⁷HFHH⁵⁷⁰ 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.

Received for publication, August 18, 2005 , and in revised form, October 11, 2005.

^* This work was supported in part by National Science Foundation Grant IBN-9986657 (to S. R. J. S. and D. L. B.), career scientist awards from the Irma T. Hirschl and Monique Weill-Caulier Trusts (to S. R. J. S. and D. L. B.), National Institutes of Health Grants DK57702 (to S. R. J. S.), DK071308 (to S. R. J. S.), and NS45305 (to S. R. J. S. and D. L. B.), and American Heart Association Grant 0350085N (to S. R. J. S.) The confocal laser scanning microscopy experiments performed at the Mount Sinai School of Medicine Microscopy Center were supported by National Institutes of Health Shared Instrumentation Grant 1 S10 RR0 9145-01 and National Science Foundation Major Research Instrumentation Grant DBI-9724504. The bioinformatics experiments performed at the Midwest Proteome Center were supported by National Institutes of Health Shared Instrumentation Grant S10 RR19325 (to M. J. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Supported in part by an American Psychological Association Diversity Program in Neuroscience predoctoral fellowship.

³ To whom correspondence should be addressed: Fishberg Dept. of Neuroscience, Box 1065, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Tel.: 212-659-5901; Fax: 212-996-9785; E-mail: stephen.salton{at}mssm.edu.

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