Chromogranin A Promotes Peptide Hormone Sorting to Mobile Granules in Constitutively and Regulated Secreting Cells: ROLE OF CONSERVED N- AND C-TERMINAL PEPTIDES*S⃞

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

Overexpression of NECC2 inhibits NGF-mediated TrkA signaling pathway.

<p><i>A</i>. Representative confocal images of PC12 cells transfected with cMyc-<i>Necc2</i>ΔHR and double-stained with anti-cMyc and anti-TrkA antibodies. Immunofluorescent signals significantly overlap at the cell periphery and intracellularly as shown in the binary mask (right panels). Scale bars, 10 µm. <i>B</i>, <i>C</i>, <i>D</i> and <i>E</i>. PC12 cells transiently transfected with full-length cMyc-<i>Necc2</i>, cMyc-<i>Necc2</i>ΔHR, or the empty vector (mock) were grown to 90% confluence and exposed for 4 h to serum-low differentiation media before NGF stimulation for the indicated time points. Whole cell protein extracts were then subjected to immunoblot with Akt and phospho-Akt (pAkt) antibodies (<i>B</i> and <i>C</i>) or with ERK and phosphor-ERK (pERK) antibodies (<i>D</i> and <i>E</i>). Quantitative data were represented as ratio of pAkt <i>vs</i>. Akt or pERK vs. ERK, respectively. The data represent the means (± SEM) of three independent experiments. <i>P</i> < 0.05 <i>vs</i>. corresponding control (unpaired, 2-tailed t test).</p

In silico analysis of rat NECC2 sequence.

<p><i>A</i>. Schematic representation of the structural and functional motifs predicted in rat NECC2 amino acid sequence. <i>B</i>. Schematic representation of the genomic structure of rat <i>Necc2</i> coding for the <i>Necc2</i> isoform containing the HR domain and a newly identified transcript lacking this domain (<i>Necc2</i>α). Arrows indicate the location of the paired primers used to amplify both <i>Necc2</i> transcripts. <i>C</i>. Standard PCR amplification in PC12 cells shows two PCR products with the expected sizes for the two rat <i>Necc2</i> transcripts (left panel; primers a and b). Nested PCR amplification of <i>Necc2</i> transcript (central panel; primer c) or the <i>Necc2</i>α transcript (rightmost panel; primer d) using specific internal reverse primers. Non-DNA samples (C-) are shown as controls for exogenous contamination.</p

NECC2 associates to caveolae both as an integral membrane protein and a peripheral membrane protein.

<p><i>A</i>. Immunoblot analysis of whole cell lysates from PC12 cells with the anti-NECC2 antibody. As control, anti-NECC2 antibody was pre-incubated with an excess of antigen. <i>B</i>. Cytosolic (S) and crude membrane (P) fractions from PC12 cells were obtained by subcellular fractionation as described in Methods and subsequent analyzed by immunoblotting. As shown by anti-NECC2 antibody immunolabeling, NECC2 distributes in the cytosol and, to a lesser extent, it also associates with membrane fractions. In contrast, exogenous full-length cMyc-NECC2 distributed to both fractions, with a higher content in the membrane fraction. The distribution of EEA1, TrkA, GM130, actin and caveolin-1 was also analyzed. <i>C</i>. Caveolae-enriched membranes from PC12 cells were isolated by using a detergent-free method based in a discontinuous sucrose gradient (5-35-45%). Distribution of endogenous NECC2 and cMyc-tagged NECC2 variants were assayed by immunoblot. Neither NECC2 nor NECC2ΔHR co-migrated with caveolin-1 and TrkA to the buoyant fraction (fraction 2).</p