Secretory Granule Biogenesis and Neuropeptide Sorting to the Regulated Secretory Pathway in Neuroendocrine Cells

Abstract

Neuropeptide precursors synthesized at the rough endoplasmic reticulum are transported and sorted at the trans-Golgi network (TGN) to the granules of the regulated secretory pathway (RSP) of neuroendocrine cells. They are then processed into active peptides and stored in large dense-core granules (LDCGs) until secreted upon stimulation. We have studied the regulation of biogenesis of the LDCGs and the mechanism by which neuropeptide precursors, such as pro-opiomelanocortin (POMC), are sorted into these LDCGs of the RSP in neuroendocrine and endocrine cells. We provide evidence that chromogranin A (CgA), one of the most abundant acidic glycoproteins ubiquitously present in neuroendocrine/endocrine cells, plays an important role in the regulation of LDCG biogenesis. Specific depletion of CgA expression by antisense RNAs in PC12 cells led to a profound loss of secretory granule formation. Exogenously expressed POMC was neither stored nor secreted in a regulated manner in these CgA-deficient PC12 cells. Overexpression of CgA in a CgA- and LDCG-deficient endocrine cell line, 6T3, restored regulated secretion of transfected POMC and the presence of immunoreactive CgA at the tips of the processes of these cells. Unlike CgA, CgB, another granin protein, could not substitute for the role of CgA in regulating LDCG biogenesis. Thus, we conclude that CgA is a key player in the regulation of the biogenesis of LDCGs in neuroendocrine cells. To examine the mechanism of sorting POMC to the LDCGs, we carried out site-directed mutagenesis, transfected the POMC mutants into PC12 cells, and assayed for regulated secretion. Our previous molecular modeling studies predicted a three-dimensional sorting motif in POMC that can bind to a sorting receptor, membrane carboxypeptidase E (CPE). The sorting signal consists of four conserved residues at the N-terminal loop structure of POMC: two acidic residues and two hydrophobic residues. The two acidic residues were predicted to bind to a domain on CPE (CPE254-273) containing two basic residues (R255 and K260) to effect sorting into immature secretory granules. Site-directed mutagenesis of the motif on POMC resulted in accumulation of the mutant in the Golgi, as well as high basal secretion, indicating that the mutant POMC was inefficiently sorted to the RSP. These results support the model that POMC is actively sorted to the RSP granules for processing and secretion by a sorting signal-mediated mechanism.

Transport of receptors, receptor signaling complexes and ion channels via neuropeptide-secretory vesicles

“…Double immunofluorescence staining showed that DORs were present in SP-and CGRP-positive LDCVs in small DRG neurons in mice ( Figure 2A and Supplementary information, Figure S2A). This immunostaining pattern, which was shown with antibodies against DOR1 [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] , was abolished by the deletion of Oprd1 exon 1 in mice (Supplementary information, Figure S2A). Furthermore, β2 AR, G αi2 , PLCβ2, VGCC α2δ1 and P2X purinoceptor 2 (P2X 2 ) were present in SP-positive LDCVs in small DRG neurons (Figure 2A).…”

“…Chromogranin B (CGB) [16,17] and DOR1 [9,12] were used as markers for the LDCV fractions ( Figure 1A). The specificity of the antibodies against DOR1 was confirmed by the absence of immunoblot signal in the spinal cord of Oprd1 exon 1-deleted mice (Supplementary information, Figure S1A).…”

“…After three PBS washes, cells were labeled with the primary antibodies. We used antibodies against DOR1 [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] (1:30 000; DiaSorin), G αi2 (1:100; Santa Cruz), G β1 (1:200; Santa Cruz), G γ5 (1:400; Santa Cruz), PLCβ2 (1:200; Santa Cruz), P2X 2 (1:1 000; Chemicon), Fzd8 (1:400; Everest Biotech), β1 AR (1:400; Santa Cruz), β2 AR (1:200; Santa Cruz), SP (1:500; Neuromics), CGRP (1:500; Biogenesis), Dvl1 (1:200; a gift from Dr. Zhen-Ge Luo), VGCC α2δ1 (1:100; Affinity BioReagents) and Myc (1:500; a gift from Dr Jin-Qiu Zhou).…”

Sorting of growth hormone–erythropoietin fusion proteins in rat salivary glands

“…Although no universal regulated pathway-sorting motifs or signals have been identified [7,8], studies of prohormone/proneuropeptide trafficking have provided evidence that sorting of these molecules to the RSP involves a receptor-mediated mechanism [29,30]. For example, in endocrine cells, sorting motifs of pro-opiomelanocortin (POMC), brain derived neurotrophic factor and proinsulin were shown to interact with membrane carboxypeptidase E, which acts as a sorting or retention receptor to target these prohormones to the RSP [31].…”

Secretogranin III Directs Secretory Vesicle Biogenesis in Mast Cells in a Manner Dependent upon Interaction with Chromogranin A

“…Moreover, emerging evidence suggests that these proteins play an active role in directing the biosynthesis of secretory granules. This biological role for granins has been proposed on the basis of work in neuroendocrine cells, where several granin proteins are expressed and have been shown to cooperatively contribute to secretory vesicle biogenesis (14,15). This cooperation is exemplified by the interaction between SgIII and CgA (15,16).…”