The intracellular sorting of pro-neuropeptides, prohormones 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-insulin, pro- cocaine-amphetamine regulated transcript (CART) and brain derived neurotrophic factor (BDNF) to the RSP was investigated. Such studies have led to the better understanding of diseases related to defects in hormone and neuropeptide targeting, obesity, diabetes, memory and learning.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, 12-15Ang apart from each other, exposed on the surface of these molecules, and two hydrophobic residues, 5-7Ang away from the acidic residues which are necessary for sorting these pro-proteins to the RSP. A RSP sorting receptor that was specific for the sorting signal of POMC, pro-insulin 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, CPE, to effect sorting to the RSP. Using a CPE knockout (KO) mouse model, we showed missorting of endogenous POMC and proinsulin in pituitary and pancreatic islet cells, respectively, in these animals. Furthermore BDNF which modulates synaptic plasticity was missorted in cortical and hippocampal neurons, which could account for the memory deficits observed in CPE KO mice. These studies provide evidence for a CPE-dependent, sorting signal/receptor mediated mechanism for targeting prohormones, neuropeptides and the neurotrophin, BDNF, to the regulated secretory pathway in endocrine cells and neurons. 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 shown to be missorted to the constitutive pathway and secreted in an unregulated manner when transfected into a cell line. Molecular modeling 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. The high levels of secreted mutant proinsulin in the plasma of these patients are therefore due to defects in sorting of these molecules, resulting from their genetic structural alterations. In another study, the sorting and processing of a mutant form of CART (CART Leu34Phe), found in a family of obese patients was investigated. CART, found in brain, is an anorexigenic peptide that has several physiological effects such as inhibiting feeding, regulating energy expenditure, and stress. CART acts downstream of leptin in the obesity controlling signaling pathway. We show that while pro-CART was substantially processed to active CART, mutant pro-CART was only minimally processed to yield an intermediate form. Furthermore, mutant pro-CART was partially missorted and secreted via the constitutive pathway, providing a possible molecular basis for the obese phenotype in these patients. We have also used our knowledge of the sorting motif of hormones to engineer biologically active mutant hormones that are redirected to the constitutive pathway. Such mutant hormones are currently being expressed in salivary glands for systemic secretion, with the ultimate aim of applying such technology to gene therapeutics. Recently we have also investigated the sorting of prohormone processing enzymes, 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 novel physical interaction of its cytoplasmic domain with ARF 6, a GTPase ADP-ribosylation factor. 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 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. The importance of CgA in large dense-core granule biogenesis in vivo was recently demonstrated using an antisense mRNA transgenic mouse model deficient in CgA.These mice showed severe aberrant granule formation quantitatively and qualitatively in the adrenal medulla.
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