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

Chromogranins A and B and secretogranin II: evolutionary and functional aspects

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Expression de l'EM66, un nouveau peptide dérivé de la sécrétogranine II, dans l'hypothalamus de la gerboise et du rat: effet de la privation de nourriture

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Biochemical Characterisation and Immunohistochemical Localisation of the Secretogranin II-Derived Peptide EM66 in the Hypothalamus of the Jerboa (Jaculus orientalis): Modulation by Food Deprivation

International audienceThe neuroendocrine protein secretogranin II is the precursor of several neuropeptides, including secreto-neurin and a novel 66-amino acid peptide, EM66, the sequence of which has been highly conserved across the vertebrae phylum. The presence of EM66 has been detected in the adult and fetal human adrenal gland, as well as the rat pituitary and adrenal glands. The present study aimed to explore a possible neuroendocrine role of EM66 by analysing its occurrence and distribution within the jerboa hypothalamus, and its potential implication in the control of feeding behaviour. High-performance liquid chromatography analysis of jerboa hypothalamic extracts combined with a radioimmunoassay of EM66 revealed a single peak of immunoreactive material exhibiting the same retention time as recombinant EM66. Immuno-cytochemical labelling showed that EM66-producing neurones are widely distributed in several hypotha-lamic regions, including the preoptic area, the suprachiasmatic, supraoptic, parvocellular paraventricular and arcuate nuclei, and the lateral hypothalamus. Food deprivation for 5 days induced a significant increase in the number of EM66-containing neurones within the arcuate nucleus (105% increase) and the parvo-cellular aspect of the paraventricular nucleus (115% increase), suggesting that EM66 could be involved in the control of feeding behaviour and/or the response to stress associated with fasting. Altogether, these data reveal the physiological plasticity of the EM66 system in the hypothalamus and implicate this novel peptide in the regulation of neuroendocrine functions

Biochemical characterization and immunocytochemical localization of EM66, a novel peptide derived from secretogranin II, in the rat pituitary and adrenal glands

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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

Expression and processing of the neuroendocrine protein secretogranin II in benign and malignant pheochromocytomas.

International audienceThe aim of the present study was to compare the expression levels of secretogranin II (SgII), prohormone convertases (PC)1 and PC2, and the proteolytic processing of SgII in benign versus malignant pheochromocytomas. Quantitative (Q)-PCR experiments indicated that SgII, PC1, and PC2 mRNAs were overexpressed in pheochromocytoma compared to non-tumoral chromaffin cells (P<0.001) and in benign compared to malignant tumors (P<0.01). Western blot analysis using a human SgII antiserum revealed the occurrence of a 97-kDa band corresponding to the expected size of SgII, with significantly higher quantities in benign than in malignant tumors (P<0.05). Using antisera directed against sequential regions of SgII (N-terminal, secretoneurin [SN], EM66, internal, and C-terminal sequences), we observed distinct processing profiles between benign and malignant pheochromocytomas. In contrast, using PC1 and PC2 antisera no differences between the two types of tumors were found. RIA measurement showed that EM66 median values between benign and malignant chromaffin cell tumors were significantly different (128.5 vs. 6.3 ng/mg protein, respectively; P<0.001). Taken together, these results indicate that, in pheochromocytoma, malignancy is associated with reduced PC1, PC2, and SgII mRNA expression and decreased levels of processing products of SgII, in line with the low concentrations of EM66 that occur in malignant tumors. These data support the notion that SgII-processing products, such as EM66, could represent prognostic markers of pheochromocytomas