Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway
ROLE OF N- AND C-TERMINAL α-HELICAL DOMAINS*
- ‡Department of Medicine, §Department of Biology, and ¶Department of Pharmacology, University of California at San Diego, La Jolla, California 92093-0838 and ∥Veteran Affairs San Diego Healthcare System, San Diego, California 92161
- 1 To whom correspondence should be addressed: University of California at San Diego, Dept. of Medicine, 9500 Gilman Dr., #0838, La Jolla, CA 92093-0838. Tel.: 858-534-0670; Fax: 858-534-0626; E-mail: ltaupenot{at}ucsd.edu.
Abstract
Secretogranin II (SgII) belongs to the granin family of prohormones widely distributed in dense-core secretory granules (DCGs) of endocrine, neuroendocrine, and neuronal cells, including sympathoadrenal chromaffin cells. The mechanisms by which secretory proteins, and granins in particular, are sorted into the regulated secretory pathway are unsettled. We designed a strategy based on novel chimeric forms of human SgII fused to fluorescent (green fluorescent protein) or chemiluminescent (embryonic alkaline phosphatase) reporters to identify trafficking determinants mediating DCG targeting of SgII in sympathoadrenal cells. Three-dimensional deconvolution fluorescence microscopy and secretagogue-stimulated release studies demonstrate that SgII chimeras are correctly targeted to DCGs and released by exocytosis in PC12 and primary chromaffin cells. Results from a Golgi-retained mutant form of SgII suggest that sorting of SgII into DCGs depends on a saturable sorting machinery at the trans-Golgi/trans-Golgi network. Truncation analyses reveal the presence of DCG-targeting signals within both the N- and C-terminal regions of SgII, with the putative α-helix-containing SgII-(25-41) and SgII-(334-348) acting as sufficient, independent sorting domains. This study defines sequence features of SgII mediating vesicular targeting in sympathoadrenal cells and suggests a mechanism by which discrete domains of the molecule function in sorting, perhaps by virtue of a particular arrangement in tertiary structure and/or interaction with a specific component of the DCG membrane.
Footnotes
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↵2 The abbreviations used are: DCG, dense-core secretory granule; CgA, chromogranin A; CgB, chromogranin B; CPE, carboxypeptidase E; EAP, embryonic alkaline phosphatase; GalT, β1,4-galactosyltranferase; GFP, green fluorescent protein; IP3R, receptor for inositol 1,4,5-trisphosphate; ISG, immature secretory granule; TGN, trans-Golgi network; SEAP, secreted form of embryonic alkaline phosphatase; SgII, secretogranin II; SgIII, secretogranin III; SIG, predicted 30 amino acid signal peptide of human SgII; ANOVA, analysis of variance; PBS, phosphate-buffered saline; NGF, nerve growth factor; CFP, cyan fluorescent protein; EGFP, enhanced GFP.
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↵* This work was supported by postdoctoral research fellowships from the Fondation Pour la Recherche Médicale and The National Kidney Foundation (to M. C.) and by National Institutes of Health Grants DK59628 and HL58120 (to L. T.). 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.
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- Received December 3, 2007.
- Revision received February 5, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
