Identification of a novel sorting determinant for the regulated pathway in the secretory protein chromogranin A

Chromogranin A (CgA) is the index member of the chromogranin/secretogranin (or 'granin') family of regulated secretory proteins that are ubiquitously distributed in amine- and peptide-containing secretory granules of endocrine, neuroendocrine and neuronal cells. Because of their abundance and such widespread occurrence, granins have often been used as prototype proteins to elucidate mechanisms of protein targeting into dense-core secretory granules. In this study, we used a series of full-length, point mutant or truncated CgA-green fluorescent protein (GFP) chimeras to explore routing of CgA in neuroendocrine PC12 cells. Using sucrose gradient fractionation and 3D deconvolution microscopy to determine the subcellular localization of the GFP chimeras, as well as secretagogue-stimulated release, the present study establishes that a CgA-GFP fusion protein expressed in neuroendocrine PC12 cells is trafficked to the dense core secretory granule and thereby sorted to the regulated pathway for exocytosis. We show that information necessary for such trafficking is contained within the N-terminal but not the C-terminal region of CgA. We find that CgA's conserved N-terminal hydrophobic CYS17-CYS38 loop structure may not be sufficient for sorting of CgA into dense-core secretory granules, nor is its stabilization by a disulfide bond necessary for such sorting. Moreover, our data reveal for the first time that the CgA(77-115) domain of the mature protein may be necessary (though perhaps not sufficient) for trafficking CgA into the regulated pathway of secretion.