Identification of a Chromogranin A Domain That Mediates Binding to Secretogranin III and Targeting to Secretory Granules in Pituitary Cells and Pancreatic β-Cells

Abstract

Chromogranin A (CgA) is transported restrictedly to secretory granules in neuroendocrine cells. In addition to pH- and Ca2+-dependent aggregation, CgA is known to bind to a number of vesicle matrix proteins. Because the binding-prone property of CgA with secretory proteins may be essential for its targeting to secretory granules, we screened its binding partner proteins using a yeast two-hybrid system. We found that CgA bound to secretogranin III (SgIII) by specific interaction both in vitro and in endocrine cells. Localization analysis showed that CgA and SgIII were coexpressed in pituitary and pancreatic endocrine cell lines, whereas SgIII was not expressed in the adrenal glands and PC12 cells. Immunoelectron microscopy demonstrated that CgA and SgIII were specifically colocalized in large secretory granules in male rat gonadotropes, which possess large-type and small-type granules. An immunocytochemical analysis revealed that deletion of the binding domain (CgA 48–111) for SgIII missorted CgA to the constitutive pathway, whereas deletion of the binding domain (SgIII 214–373) for CgA did not affect the sorting of SgIII to the secretory granules in AtT-20 cells. These findings suggest that CgA localizes with SgIII by specific binding in secretory granules in SgIII-expressing pituitary and pancreatic endocrine cells, whereas other mechanisms are likely to be responsible for CgA localization in secretory granules of SgIII-lacking adrenal chromaffin cells and PC12 cells.

Secretogranin III Binds to Cholesterol in the Secretory Granule Membrane as an Adapter for Chromogranin A

“…We previously demonstrated that SgIII binds strongly to CgA in an intragranular milieu at pH 5.5 and 10 mM Ca 2ϩ (8). In this study, we tested whether CgA requires SgIII mediation for the binding to the SGM-type liposome.…”

“…Disruption of the disulfide loop of CgB resulted in its missorting to a constitutive pathway in PC12 cells (13). However, removal of the N-terminal loop from CgA did not affect its targeting to SGs in GH 4 C 1 cells (14), AtT-20 cells (8), and PC12 cells (15). For recognizing the N-terminal loop of POMC, carboxypeptidase E (CPE) has been proposed for its sorting receptor (10) but the sorting of CgA was not disturbed in Neuro-2a cells depleting CPE (16), Thus, the Nterminal loop-carrying CgA, CgB, and POMC appear to depend on distinct mechanisms for their sorting to SGs.…”

“…Thus, the sorting of granins may be directed by other factors such as specific protein-to-protein interactions or specific sorting domain structures such as the N-terminal disulfide loop (7). We previously demonstrated that CgA binds to SgIII by the CgA sequence and that SgIII targets CgA to SGs in pituitary and pancreatic endocrine cells (8). Thus, CgA is sorted to SGs by the lead of SgIII but the sorting of SgIII to SGs remains to be investigated.…”

Rab27 Effector Granuphilin Promotes the Plasma Membrane Targeting of Insulin Granules via Interaction with Syntaxin 1a

“…The rabbit anti-secretogranin III antibodies (13) were gifts from Drs. M. Hosaka (Gunma University) and T. Watanabe (Asahikawa University School of Medicine).…”

“…The resultant supernatant then was centrifuged at high speed (12,000 ϫ g) to separate the heavy organelles including dense core granules (P2) and other components containing cytosol (S). In control cells expressing ␤-galactosidase protein, the plasma membrane-associated syntaxin 1a mainly was present in the P1 fraction, whereas peripherally granuleassociated Rab27a (10) and the granule content, secretogranin III (SgIII) (13), were distributed in the P2 fraction (Fig. 3, upper panels).…”