The long coiled-coil protein NECC2 is associated to caveolae and modulates NGF/TrkA signaling in PC12 cells [corrected].

TrkA-mediated NGF signaling in PC12 cells has been shown to be compartimentalized in specialized microdomains of the plasma membrane, the caveolae, which are organized by scaffold proteins including the member of the caveolin family of proteins, caveolin-1. Here, we characterize the intracellular distribution as well as the biochemical and functional properties of the neuroendocrine long coiled-coil protein 2 (NECC2), a novel long coiled-coil protein selectively expressed in neuroendocrine tissues that contains a predicted caveolin-binding domain and displays structural characteristics of a scaffolding factor. NECC2 distributes in caveolae, wherein it colocalizes with the TrkA receptor, and behaves as a caveolae-associated protein in neuroendocrine PC12 cells. In addition, stimulation of PC12 cells with nerve growth factor (NGF) increased the expression and regulated the distribution of NECC2. Interestingly, knockdown as well as overexpression of NECC2 resulted in a reduction of NGF-induced phosphorylation of the TrkA downstream effector extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) but not of Akt. Altogether, our results identify NECC2 as a novel component of caveolae in PC12 cells and support the contribution of this protein in the maintenance of TrkA-mediated NGF signaling.journal articleresearch support, non-u.s. gov't20132013 09 06importe

Contribution à l'étude du rôle de la Chromogranine A dans la sécrétion neuroendocrine : identification de ses partenaires moléculaires impliqués dans la biogenèse des granules de sécrétion

ROUEN-BU Sciences (764512102) / SudocSudocFranceF

Contribution à la caractérisation des mécanismes moléculaires impliquant la chromogranine A dans l'établissement de la voie de sécrétion régulée dans les cellules neuroendocrines

Des études récentes d'invalidation du gène codant la chromogranine A (CgA) ont suggéré que cette protéine jouerait un rôle clé dans la biogenèse des granules de sécrétion (GS) au sein des cellules neuroendocrines. Néanmoins, les mécanismes moléculaires impliqués au cours de ce processus demeurent mal connus. Récemment, nous avons montré que l'expression de la CgA dans les cellules non-endocrines COS-7 induit la biogenèse de structures granulaires apparentées aux GS, capables de stocker et de sécréter la CgA et des hormones co-exprimées (NPY, GH), de manière calcium-dépendante. Les analyses des cellules COS-7 exprimant la CgA en microscope confocale et en vidéomicroscopie, associées à des approches biochimiques, ont permis d'établir des interactions entre les granules contenant la CgA, les microtubules et les filaments d'actine. Ces deux éléments du cytosquelette exercent des fonctions régulatrices sur le trafic intracellulaire et l'exocytose calcium-dépendante des granules contenant la CgA. Ces résultats suggèrent que la CgA est suffisante pour induire la biogenèse de GS et instaurer ainsi une voie de sécrétion régulée dans des cellules non-endocrines. Le clonage de la CgA de grenouille ayant révélé la conservation des régions N- et C-terminales au cours de l'évolution des vertébrés, nous avons émis l'hypothèse de leur implication en tant que déterminants fonctionnels de la CgA. Ainsi, nos résultats démontrent que les peptides conservés confèrent à la CgA sa capacité à former des GS et dirigent l'adressage et la libération régulée des hormones exogènes co-exprimées avec la CgA dans les cellules COS-7, ainsi que la POMC endogène dans les cellules corticotropes AtT20.The nature of the sorting signals for entry of proteins into the dense-core secretory granules (SG) and the molecular machinery required to generate SG remain unclear. Recent studies revealed that chromogranin A (CgA) deficiency is associated with hormone storage impairment, suggesting that CgA plays a major role in the formation of SG in neuroendocrine cells. The cloning of frog CgA revealed high conservation though evolution of the global acidity and of the terminal regions of the protein, suggesting that these features are essential for the biological activity of CgA. Expression of CgA in the non-endocrine COS-7 cells induced the formation of dense-core vesicles containing CgA. As SG in neuroendocrine cells, trafficking and exocytosis of CgA-containing granules required interactions with microtubules and actin filaments. These SG-like organelles were able to store hormones that could be released in a calcium-dependent manner. Deletion of the terminal regions of CgA resulted in a reorientation of the proteins from the regulated to the constitutive secretory pathway, indicating that these domains were essential for the formation of functional SG-like structures in COS-7 cells. Expression of CgA in the corticotrope AtT20 cells increased POMC levels in SG, whereas the expression of terminal deletion-mutants provoked retention of the hormone in the Golgi area. Thus, CgA, but not its truncated forms, promoted POMC sorting to the regulated secretory pathway. Our results demonstrate that CgA, through its conserved terminal domains, directs the formation of SG and the sorting and release of hormones.ROUEN-BU Sciences (764512102) / SudocSudocFranceF

Myosin 1b and F-actin are involved in the control of secretory granule biogenesis

Hormone secretion relies on secretory granules which store hormones in endocrine cells and release them upon cell stimulation. The molecular events leading to hormone sorting and secretory granule formation at the level of the TGN are still elusive. Our proteomic analysis of purified whole secretory granules or secretory granule membranes uncovered their association with the actomyosin components myosin 1b, actin and the actin nucleation complex Arp2/3. We found that myosin 1b controls the formation of secretory granules and the associated regulated secretion in both neuroendocrine cells and chromogranin A-expressing COS7 cells used as a simplified model of induced secretion. We show that F-actin is also involved in secretory granule biogenesis and that myosin 1b cooperates with Arp2/3 to recruit F-actin to the Golgi region where secretory granules bud. These results provide the first evidence that components of the actomyosin complex promote the biogenesis of secretory granules and thereby regulate hormone sorting and secretion.This work was supported by Institut National de la Santé et de la Recherche Médicale, the University of Rouen Normandy, the Conseil Régional de Normandie and the Ministère de l’Enseignement Supérieur et de la RecherchePeer Reviewe

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

Mono- and Poly-unsaturated Phosphatidic Acid Regulate Distinct Steps of Regulated Exocytosis in Neuroendocrine Cells

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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

NECC2 expression and distribution are regulated by NGF.

<p><i>A</i>. Representative confocal images of HA-<i>TrkA</i> transfected PC12 cells under basal conditions or treated with NGF at the indicated time points. After treatment, cells were subjected to double-immunofluorescence using anti-NECC2 and anti-HA antibodies. The colocalization channel was isolated using Imaris 6.4 (Bitplane) and shown alone in the images on the far bottom. Mander’s coefficient A (between NECC2 and TkA) was calculated to quantify the degree of colocalization and represented as the mean ±SEM of at least 5 cells per experimental group. a, <i>P</i> < 0.05 <i>vs</i>. control; b, <i>P</i> < 0.05 <i>vs</i>. 5 min; c, <i>P</i> < 0.05 <i>vs</i>. 30 min (unpaired, 2-tailed t test). <i>B</i>. Representative micrographs of PC12 cells immunolabeled with anti-NECC2 during long-term (1-4 days) stimulation with NGF. NECC2 staining localizes beneath the plasma membrane as well as in puncta/vesicular-like structures in growing neurites and tips. <i>C</i>. Double-immunostaining of NGF-differentiated PC12 cells with anti-NECC2 and anti-caveolin-1 sera. Scale bars, 10 µm. <i>D</i>. Protein extracts from NGF-stimulated PC12 cells during differentiation (1-4 days) were analyzed by immunoblotting using the anti-NECC2 antibody. Quantitative data were represented as ratio of NECC2 vs. Ponceau. The data represent the mean (± SEM) of three independent experiments. a, <i>P</i> < 0.05 <i>vs</i>. corresponding control; b, <i>P</i> < 0.05 <i>vs</i>. 1- or 2-days treated cells (one-way ANOVA followed by <i>Newman–Keuls</i> test).</p