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J. Biol. Chem., Vol. 281, Issue 49, 38038-38051, December 8, 2006
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Secretory Granule Biogenesis in Sympathoadrenal Cells
IDENTIFICATION OF A GRANULOGENIC DETERMINANT IN THE SECRETORY PROHORMONE CHROMOGRANIN A*
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From the
Department of Medicine and the ||Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0838, the ¶Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom, and the Veterans Affairs San Diego Healthcare System, San Diego, California 92161
Chromogranin A (CgA) may be critical for secretory granule biogenesis in sympathoadrenal cells. We found that silencing the expression of CgA reduced the number of secretory granules in normal sympathoadrenal cells (PC12), and we therefore questioned whether a discrete domain of CgA might promote the formation of a regulated secretory pathway in variant sympathoadrenal cells (A35C) devoid of such a phenotype. The secretory granule-forming activity of a series of human CgA domains labeled with a hemagglutinin epitope, green fluorescent protein, or embryonic alkaline phosphatase was assessed in A35C cells by deconvolution and electron microscopy and by secretagogue-stimulated release assays. Expression of CgA in A35C cells induced the formation of vesicular organelles throughout the cytoplasm, whereas two constitutive secretory pathway markers accumulated in the Golgi complex. The lysosome-associated membrane protein LGP110 did not co-localize with CgA, consistent with non-lysosomal targeting of the granin in A35C cells. Thus, CgA-expressing A35C cells showed electron-dense granules 
180-220 nm in diameter, and secretagogue-stimulated exocytosis of CgA from A35C cells suggested that expression of the granin may be sufficient to restore a regulated secretory pathway and thereby rescue the sorting of other secretory proteins. We show that the formation of vesicular structures destined for regulated exocytosis may be mediated by a determinant located within the CgA N-terminal region (CgA-(1-115), with a necessary contribution of CgA-(40-115)), but not the C-terminal region (CgA-(233-439)) of the protein. We propose that CgA promotes the biogenesis of secretory granules by a mechanism involving a granulogenic determinant located within CgA-(40-115) of the mature protein.
Received for publication, April 27, 2006 , and in revised form, October 5, 2006.
* This work was supported by National Institutes of Health Grants DK59628 and HL58120 (to L. T.) and Grants DK60702 and HL58120 (to D. T. O.). The digital imaging and DNA sequencing performed at the Cancer Center Shared Resource Facilities of the University of California, San Diego, were supported in part by NCI, National Institutes of Health. 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 To whom correspondence should be addressed: Dept. of Medicine, University of California, San Diego, 9500 Gilman Dr., 0838, La Jolla, CA 92093-0838. Tel.: 858-534-0670; Fax: 858-534-0626; E-mail: ltaupenot{at}ucsd.edu.
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