The intracellular sorting of pro-neuropeptides and neurotrophins to the regulated secretory pathway (RSP) is essential for processing, storage and release of active proteins and peptides in the neuroendocrine cell. The sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin), pro-enkephalin (pro-ENK), proinsulin and brain derived neurotrophic factor (BDNF) to the regulated secretory pathway (RSP) was investigated. We show that these pro-proteins undergo homotypic oligomerization, as a concentration step as they traverse the cell from the site of synthesis in the endoplasmic reticulum to the trans-Golgi network (TGN) where they are sorted into dense-core granules of the regulated secretory pathway for processing and secretion. Site-directed mutagenesis studies identified a consensus sorting motif consisting of two acidic residues, 12-15? apart from each other, exposed on the surface of these molecules, and two hydrophobic residues, 5-7? away from the acidic residues which are necessary for sorting to the RSP. A RSP sorting receptor that was specific for the sorting signal of POMC, pro-enkephalin proinsulin and BDNF was identified as membrane carboxypeptidase E (CPE). The two acidic residues in the prohormone/pro-BDNF sorting motif specifically interact with two basic residues, R255 and K260, of the sorting receptor, carboxypeptidase E (CPE), to effect sorting to the RSP. Transfection of a mutant CPE with R255 and K260 mutated to A in a CPE null clone of Neuro2a cells, and transfection of a dominant negative CPE mutant into INS cells, a pancreatic beta cell line, caused missorting of POMC and proinsulin to the constitutive pathway respectively, indicating that the basic residues in the sorting domain of CPE interacts with the acidic residues in the POMC and proinsulin sorting signal in vivo to effect sorting to the RSP. Using a mouse model which synthesizes a mutant CPE that is degraded in the pituitary, we were able to show missorting of endogenous POMC in these cells. These studies provide evidence for a sorting signal/receptor mediated mechanism for targeting prohormones to the regulated secretory pathway in neuro-endocrine cells.The intracellular sorting of genetically mutated proinsulins found in hyperproinsulinemia patients who have abnormally high levels of plasma proinsulin was investigated to understand the molecular basis of these forms of diabetes. One form of mutant proinsulin found in these patients, HisB10Asp, which is unable to hexamerize but forms dimers, was found to be missorted to the constitutive pathway and secreted in an unregulated manner when transfected into a cell line. Molecular modelling of the dimer of this mutant proinsulin predicted that the molecular distance of the two acidic residues of the RSP sorting signal motif would be too large to allow interaction with the basic residues in the binding site of the sorting receptor, CPE. Indeed in vitro binding studies showed that this mutant did not bind to CPE, thus resulting in its inability to be sorted to the RSP for processing to insulin and secretion in a secretogogue-dependant manner. Other hyperproinsulinemia proinsulin mutants, Arg65Pro and Arg65Leu were also found to be secreted constitutively and not stored. Binding studies showed that mutant Arg65Pro and Arg65Leu proinsulins bound poorly to CPE, accounting for the lack of sorting and retention in the immature secretory granule. The high levels of secreted mutant proinsulins in the plasma of these patients are therefore due to defects in sorting of these molecules, resulting from their genetic structural alterations.Recently we have also investigated the sorting of prohormone and neuropeptide processing enzymes, carboxypeptidase E (CPE) and prohormone convertases 1 and 2 (PC1 and PC2) to the regulated secretory pathway. We have shown that these enzymes are transmembrane proteins with an atypical membrane spanning domain at the C-terminus. They are sorted into granules of the RSP in neuroendocrine cells by a novel mechanism involving insertion of their C-terminal transmembrane domain into cholesterol-glycosphingolipid rich microdomains known as lipid rafts, at the TGN. Removal of cholesterol from secretory granule membranes resulted in the inability of CPE, the RSP sorting receptor to bind cargo; and cholesterol depletion by treatment of cells with lovastatin resulted in lack of sorting of CPE to the RSP. Thus membrane association with cholesterol-rich lipid rafts is essential for sorting of the prohormone processing enzymes to the TGN and secretory granules. We also showed that CPE is recycled back from the plasma membrane to the TGN after granule exocytosis, and the internalization of the enzyme is dependent on the interaction of its cytoplasmic domain with ARF 6. In another project we have studied the factors governing the formation of large dense-core granules (LDCG) at the TGN, which is essential for regulated secretion of hormones and neuropeptides from neuroendocrine cells. Our recent studies uncovered an on/off switch, chromogranin A (CgA), that controls the formation of LDCG in neuroendocrine cells. Depletion of CgA in rat PC12 cells using antisense technology resulted in the loss of LDCG, regulated secretion and degradation of granule proteins including CgB and synaptotagmin. Overexpression of bovine CgA in these cells rescued the wild type phenotype. In a mutant endocrine cell line, 6T3, lacking CgA, LDCGs and regulated hormone secretion, transfection of CgA restored the wild type phenotype in these cells. We have recently identified the Golgi as the site of degradation of the secretory granule proteins in the absence of granule biogenesis. Thus we propose that regulation of the stability of granule proteins at the Golgi by CgA may be a point of control of granule biogenesis in neuroendocrine cells. Recently, we compared gene expression in 6T3 cells lacking LDCGs and 6T3 cells stably transfected with CgA using microarrays. We found that aquaporin-1(AQP1, a water channel), a secretory granule protein was significantly up-regulated in 6T3 cells expressing CgA. These findings suggest that CgA may have a new regulatory role in secretory granule protein expression at the transcriptional level which in turn may regulate granule biogenesis.
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