Secretory Granule Biogenesis in Sympathoadrenal Cells

Abstract

Chromogranin A (CgA) may be critical for secretory granule biogenesis in sympathoadrenal cells. We found that silencing the expression of CgA reduced the number of secretory granules in normal sympathoadrenal cells (PC12), and we therefore questioned whether a discrete domain of CgA might promote the formation of a regulated secretory pathway in variant sympathoadrenal cells (A35C) devoid of such a phenotype. The secretory granule-forming activity of a series of human CgA domains labeled with a hemagglutinin epitope, green fluorescent protein, or embryonic alkaline phosphatase was assessed in A35C cells by deconvolution and electron microscopy and by secretagoguestimulated release assays. Expression of CgA in A35C cells induced the formation of vesicular organelles throughout the cytoplasm, whereas two constitutive secretory pathway markers accumulated in the Golgi complex. The lysosome-associated membrane protein LGP110 did not co-localize with CgA, consistent with non-lysosomal targeting of the granin in A35C cells. Thus, CgA-expressing A35C cells showed electron-dense granules ϳ180 -220 nm in diameter, and secretagogue-stimulated exocytosis of CgA from A35C cells suggested that expression of the granin may be sufficient to restore a regulated secretory pathway and thereby rescue the sorting of other secretory proteins. We show that the formation of vesicular structures destined for regulated exocytosis may be mediated by a determinant located within the CgA N-terminal region (CgA-(1-115), with a necessary contribution of CgA-(40 -115)), but not the C-terminal region (CgA-(233-439)) of the protein. We propose that CgA promotes the biogenesis of secretory granules by a mechanism involving a granulogenic determinant located within CgA-(40 -115) of the mature protein.The regulated secretory pathway that exists in neurons and neuroendocrine cells is characterized by the concentration and sorting of a pool of secretory proteins into specialized intracellular organelles with a typical electron-dense appearance upon transmission electron microscopy, prompting the morphologic term dense-core granules. These granules may remain in the cell for an extended period of time after their formation, until prompted for exocytotic fusion with the plasma membrane by a secretagogue characteristic for a particular cell type.The mechanism underlying the initiation and regulation of dense-core secretory granule biogenesis is poorly understood. The formation of secretory granules is believed to be initiated at the trans-Golgi network (TGN) 2 apparatus, where aggregation and condensation of secretory proteins are typically viewed as the initial step prior to granulogenesis. Thus, formation of protein aggregates may occur in the mildly acidic environment that exists in the TGN and in the presence of millimolar concentrations of bivalent cations like Ca 2ϩ . Aggregation and condensation are the underpinning processes of two basic models of sorting of proteins within the regulated secretory pathway, viz. sorting for entry and sorting by ...

Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway

“…5C, the size of fluorescent SIG-SgII-GFP puncta was similar in either GalT-CFP-SgII-or GalT-CFP-expressing PC12 cells, with an average diameter of ϳ308 nm. Considering that the resolution of fluorescence puncta containing the photoprotein is diffraction-limited, this value is consistent with our earlier analyses by electron microscopy, reporting a DCG diameter of ϳ100 -130 nm in PC12 cells (21,25). The number of SIG-SgII-GFP puncta per xy optical section was significantly reduced in cells coexpressing GalT-CFP-SgII (22 Ϯ 4 puncta/xy plan, n ϭ 43; p Ͻ 0.05; Fig.…”

“…In addition, the subcellular distribution of SIG-GFP in PC12 and primary chromaffin cells ( Fig. 2A) was remarkably similar to that of the constitutive secretory pathway markers SgP-GFP (where SgP is the predicted 18-amino acid signal peptide of human CgA) (13) and SEAP (21,25) in the sympathoadrenal cell lineage, which further indicates that SIG-GFP is likely routed to the constitutive branch of the secretory pathway.…”

“…Trafficking of SgII Fusion Proteins into the Regulated Secretory Pathway-Taking our cue from the experimental paradigm developed in our earlier studies on the vesicular trafficking of CgA in sympathoadrenal cells (13,21,25), we fused full-length human SgII to GFP or EAP, for fluorescent or enzymatic tracing of the regulated secretory pathway. This study establishes that SgII flanked by a signal sequence is able to convert GFP or EAP to soluble (releasable) components of DCGs, and it thus reveals that SgII contains a dominant sorting signal for the regulated secretory pathway, whereas its signal peptide alone does not (Figs.…”

“…On the contrary, colocalization of SIG-GFP with endogenous CgB was poor ( Fig. 2B) with R p ϭ 0.13 Ϯ 0.05 and R o ϭ 0.17 Ϯ 0.04 (n ϭ 72; Table 1), as predicted for a fusion protein primarily destined to the constitutive secretory pathway (21,25). Partial rather than absolute overlap in the distribution of SIG-SgII-GFP with endogenous CgB can best be explained by the extended half-life of DCGs, which gives rise to pools of old granules (pre-transfection; lacking SIG-SgII-GFP) and young post-transfection granules (i.e.…”

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

“…A prominent role of chromogranin A (CgA) in the regulation of DCG formation in endocrine and neuroendocrine cells has been proposed. Thus, depletion of CgA in PC12 cells led to a dramatic decrease in the number of DCGs (12), and exogenously expressed CgA in these depleted PC12 cells, as in DCG-deficient endocrine A35C and 6T3 cells, restored DCG biogenesis (12,13). Besides, expression of granins in non-endocrine, constitutively secreting cells such as CV-1, NIH3T3, or COS-7 cells provoked the formation of DCG-like structures that release their content in response to Ca 2ϩ influx (12,14,15).…”