Chromogranin A Promotes Peptide Hormone Sorting to Mobile Granules in Constitutively and Regulated Secreting Cells

Abstract

Chromogranin A (CgA) has been proposed to play a major role in the formation of dense-core secretory granules (DCGs) in neuroendocrine cells. Here, we took advantage of unique features of the frog CgA (fCgA) to assess the role of this granin and its potential functional determinants in hormone sorting during DCG biogenesis. Expression of fCgA in the constitutively secreting COS-7 cells induced the formation of mobile vesicular structures, which contained cotransfected peptide hormones. The fCgA and the hormones coexpressed in the newly formed vesicles could be released in a regulated manner. The N-and C-terminal regions of fCgA, which exhibit remarkable sequence conservation with their mammalian counterparts were found to be essential for the formation of the mobile DCG-like structures in COS-7 cells. Expression of fCgA in the corticotrope AtT20 cells increased pro-opiomelanocortin levels in DCGs, whereas the expression of N-and C-terminal deletion mutants provoked retention of the hormone in the Golgi area. Furthermore, fCgA, but not its truncated forms, promoted pro-opiomelanocortin sorting to the regulated secretory pathway. These data demonstrate that CgA has the intrinsic capacity to induce the formation of mobile secretory granules and to promote the sorting and release of peptide hormones. The conserved terminal peptides are instrumental for these activities of CgA.Eukaryotic cells share the capacity to rapidly secrete proteins through the constitutive secretory pathway. The fundamental feature of neuroendocrine and endocrine cells is the occurrence of dense-core secretory granules (DCGs), 3 which are key cytoplasmic organelles responsible for secretion of hormones, neuropeptides, and neurotransmitters through the regulated secretory pathway (RSP). Storage at high concentrations of these secretory products is required for their finely tuned release in response to extracellular stimulation (1, 2). DCG biogenesis starts with the budding of immature secretory granules (ISGs) from the trans-Golgi network (TGN) through interactions between lipid rafts and protein components, in a similar manner to constitutive vesicle budding (2, 3). The ISG budding is followed by a multistep maturation process to form the mature secretory granules, including removal of the constitutive secretory proteins and lysosomal enzymes inadvertently packaged into ISGs (4).Despite increasing knowledge of the various steps of DCG formation, the nature of the sorting signals for entry of proteins into the DCGs and the molecular machinery required to generate secretory granules are not fully elucidated (5, 6). Several recent studies highlighted the role of members of the granin family, which may represent the driving force for granulogenesis in the TGN (2), although this notion has been a matter of debate (7). Granins are soluble acidic proteins widely distributed in endocrine and neuroendocrine cells, which are characterized by the ability to aggregate at acidic pH and a high Ca 2ϩ environment (8, 9). These conditions are found in the lum...

Pro-hormone Secretogranin II Regulates Dense Core Secretory Granule Biogenesis in Catecholaminergic Cells

“…Cell Culture-Pheochromocytoma PC12 cells, the variant sympathoadrenal cell line A35C (27), and the African green monkey kidney fibroblast-derived COS-7 cells (ATCC, CRL 1651) were routinely grown as described previously (10,20). Most experiments were performed on cells plated onto either poly-L-lysine (Sigma) plus collagen (Upstate)-coated 15-mm round glass coverslips (Fischer No.…”

“…The following excitation and emission wavelengths were used for imaging: GFP and fluorescein-conjugated antibodies (green), ex 490 Ϯ 10/ em 528 Ϯ 38 nm; RFP, MitoTracker, and Alexa Fluor 594 antibodies (red), ex 555 nm/ em 580 nm; CFP (blue), ex 436 Ϯ 10/ em 465 Ϯ 30 nm; Hoechst 33342 (nuclear DNA stain, blue), ex 350/ em 461 nm. COS-7 cells were observed on a Leica SP2 upright confocal laser scanning microscope (DMRAX-UV) equipped with the Acousto-Optical Beam Splitter system and with 25, 40, and 63ϫ oil immersion objectives (Leica, Microsystems, Reuil-Malmaison, France) as described previously (20).…”

“…Indeed, CgB depletion in PC12 cells may contribute to an 80% decreased number of DCGs (11), although CgB seems unable to rescue a regulated secretory pathway in secretory-deficient neuroendocrine cells (12,16). A granulogenic role for CgA and CgB is also documented across cell lineages, where their expression induces the formation of granule-like structures competent for exocytosis (11,12,(17)(18)(19)(20).…”

Chromogranins A and B as Regulators of Vesicle Cargo and Exocytosis

“…The presence of this water-soluble glycoprotein that co-secretes with catecholamines from adrenomedullary cells also provided the first direct evidence of exocytosis on the basis that these proteins could not be released in any other way (Banks and Helle 1965). This family has since grown to also include chromogranin B (CgB or SgI), secretogranin II (SgII or CgC), SgIII (or 1B1075), , SgV (or 7B2), SgVI (or NESP55), SgVII (or VGF), and pro-SAAS, which are also found in many other tissues such as the pituitary gland and islets of Langerhans, as well as in brain neurons (Taupenot et al 2003;Helle 2010;Zhao et al 2009;Montero-Hadjadje et al 2009). …”

Chromogranin A as a Crucial Factor in the Sorting of Peptide Hormones to Secretory Granules

“…More studies performed in non-endocrine cells as well as in PC12 cells devoid of regulated secretory pathway (A35C cells) revealed that CgA expression induced the biogenesis of dense-core granular structures, distinct from lysosomes, containing CgA and co-expressed hormones, and able to release their content upon Ca 2? influx (Huh et al 2003;Beuret et al 2004;Courel et al 2006;Montero-Hadjadje et al 2009;Stettler et al 2009). In contrast, other studies reported that expression of CgA in non-endocrine cells induced its targeting to the constitutive secretory pathway (Malosio et al 2004).…”