Mechanism of Sorting Prohormones to the Regulated Secretory Pathway
Cawley, Dhanvantari, Maldonado, Rodriguez-David, Arnaoutova, Loh in collaboration with Baum,a Nieman,b Millerc
The intracellular sorting of prohormones to the regulated secretory pathway (RSP) is essential for the processing, storage, and release of active hormones in the neuroendocrine cell. We investigated the sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin), pro-enkephalin (pro-ENK), and pro-insulin to the RSP. We showed that, as a concentration step, these prohormones undergo homotypic oligomerization 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 RSP for processing and secretion. Site-directed mutagenesis studies identified a consensus-sorting motif consisting of two acidic residues, 12 to 15Å apart, exposed on the surface of these molecules and two hydrophobic residues, 5 to 7Å away from the acidic residues, which are necessary for sorting to the RSP.

An RSP sorting receptor that was specific for the sorting signal of POMC, pro-insulin, and pro-enkephalin was identified as membrane carboxypeptidase E (CPE). The two acidic residues in the prohormone sorting motif specifically interact with two basic residues, R255 and K260, of the sorting receptor, CPE, to effect sorting to the RSP (Figure14).

Figure 14

Model of N-POMC1-26 and Carboxypeptidase E(CPE)254-273 showing complimentarity of Arg255 (R255), Lys260 (K260) in CPE and Asp10 (D10) and Glu14 (E14) in N-POMC1-26.

We also showed that CPE is a transmembrane protein, which is anchored via its C-terminal amphipathic tail to unique cholesterol-glycosphingolipid-rich microdomains, known as rafts, in the TGN. Removal of cholesterol from secretory granule membranes by methyl b-cyclodextrin resulted in the inability of CPE to bind to cargo. Cholesterol depletion by treatment of cells with lovastatin resulted in lack of sorting of CPE and POMC to the RSP. Thus, membrane association is essential for the prohormone sorting receptor function of CPE at the TGN. Depletion of CPE by antisense RNA in Neuro2a cells caused missorting of prohormones to the constitutive pathway, indicating that CPE functions as a sorting receptor in vivo. Using a mouse model, which synthesizes a mutant CPE that is differentially degraded in pituitary and pancreas tissues, we were able to show a correlation between lowered CPE levels and the degree of missorting of endogenous prohormones in the cells of these tissues. Our studies provide evidence for a sorting-signal/receptor-mediated mechanism for targeting prohormones to the regulated secretory pathway in neuroendocrine cells. We have now also identified a similar sorting signal for human growth hormone (HGH) and, using this information, are currently developing a novel procedure for delivery of a biologically active mutant form of HGH systemically via gene expression in the salivary gland.

Sorting and Recycling of Lipid-Raft–Associated Proteins
Zhang, Dhanvantari, Arnoutova, Loh in collaboration with Birch,d Donaldson,e Londonf
CPE and prohormone convertase 1/3 (PC1/3) are two prohormone processing enzymes that have been found to be associated with cholesterol-sphingolipid–rich lipid rafts. Both enzymes are sorted into granules of the RSP in neuroendocrine cells by a mechanism involving insertion into lipid rafts at the TGN via their C-terminal tail. Upon stimulation and exocytosis, membrane CPE is localized on the plasma membrane and is subsequently recycled back to the TGN by a unique clathrin-independent, ARF6-dependent mechanism.

Regulation of Secretory Granule Biogenesis
Kim and Loh in collaboration with Eiden,g Chengh
Formation of large dense-core granules (LDCG) at the TGN is essential for regulated secretion of hormones and neuropeptides from neuroendocrine cells. Our recent studies uncovered a master 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 (Figure 15A) resulted in the loss of LDCG (Figure15b), 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 (Figure 16). Thus, CgA serves an important higher-order physiological role in controlling hormone secretion through regulating secretory granule biogenesis in endocrine and neuroendocrine cells.

Figure 15

A. Western blot showing CGA expression in wild-type (WT), a vector-alone control clone (WTv), and CGA-antisense clone 5 (CGAAS-5) PC12 cells. B.Electron micrographs of WT and CGAAS-5 PC12 cells. Arrows indicates dense-core secretory granules.

Sorting of Proinsulin Mutants in Hyperproinsulinemia Patients
Dhanvantari and Loh in collaboration with Mackini
Hyperproinsulinemia patients have abnormally high levels of plasma proinsulin. We investigated the intracellular sorting of genetically mutated proinsulins to understand the molecular basis of these forms of diabetes. One form of mutant proinsulin found in hyperproinsulinemia patients, HisB10Asp, is unable to hexamerize but forms dimers. When transfected into a cell line, it was missorted to the constitutive pathway and secreted in an unregulated manner. 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 the mutant did not bind to CPE, resulting in its inability to be sorted to the RSP for processing to insulin and secretion in a secretogog-dependent manner. Other hyperproinsulinemia proinsulin mutants, Arg65Pro and Arg65Leu, were also found to be secreted constitutively and not stored. The high levels of secreted mutant proinsulins in the plasma of these patients are therefore due to defects in sorting, processing, storage, and secretion of these molecules, resulting from their genetic structural alterations.

Figure 16

A.Western blot of CGA secretion in the presence or absence of 50 mM KCl from 6T3 cells stably expressing bovine CGA. b. Western blot showing secretion of transfected POMC from wild-type (6T3-WT) or bCGA-expressing (6T3-bCGA) 6T3 cells.