We study the cell biology of endocrine and neuroendocrine cells. Our work focuses on the mechanisms of biosynthesis and intracellular trafficking of peptide hormones and neuropeptides and their processing enzymes. Our studies have led to the discovery of novel molecular mechanisms of protein trafficking to the regulated secretory pathway and of recycling of sorting receptors as well as to the understanding of diseases related to defects in hormone targeting. We have also identified a master on/off switch that regulates dense-core granule biogenesis in neuro-endocrine and endocrine cells.

Mechanism of Sorting Pro-Neuropeptides to the Regulated Secretory Pathway
Cawley, Zhang, Dhanvantari, Rodriguez-David, Arnaoutova, Loh
The intracellular sorting of pro-neuropeptides to the regulated secretory pathway (RSP) is essential for processing, storage, and release of active hormones in the neuroendocrine cell. We thus investigated the sorting of pro-opiomelanocortin (POMC, pro-ACTH/endor-phin) and pro-enkephalin (proEnk) to the RSP. We showed that, as a concentration step, these pro-proteins undergo homotypic oligomerization as they traverse the cell from the site of synthesis in the endoplasmic reticulum to the trans-Golgi network (TGN); there, 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 12–15Å apart and exposed on the surface of these molecules and two hydrophobic residues 5–7Å away from the acidic residues, which are necessary for sorting to the RSP. We identified an RSP sorting receptor that was specific for the sorting signal of POMC and proEnk as membrane carboxipeptidase E (CPE). The two acidic residues in the prohormone sorting motif specifically interact with two basic residues, R255 and K260, of the CPE sorting receptor to effect sorting to the RSP.

We showed that CPE is a transmembrane protein that is anchored in the TGN via its C-terminal amphipathic tail to unique cholesterol-glycosphingolipid–rich microdomains known as rafts. Removal of cholesterol from secretory granule membranes resulted in the inability of CPE to bind to cargo; by treating cells with lovastatin, cholesterol depletion 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. Transfection of CPE bearing R255A and K260A mutations into a CPE null clone of Neuro2a cells and transfection of a dominant negative CPE construct into AtT20 cells caused missorting of POMC to the constitutive pathway, indicating that the basic residues in the sorting domain of CPE interact with the acidic residues in the POMC sorting signal in vivo to effect sorting to the RSP. Using a mouse model that synthesizes a mutant CPE, which is degraded in the pituitary, we were able to show missorting of endogenous POMC in these neuroendocrine cells. Our studies provide evidence for a sorting signal/receptor-mediated mechanism for targeting prohormones to the RSP in neuro-endocrine cells.

Sorting of Proinsulin Mutants in Hyperpro-insulinemia Patients
Dhanvantari, Zhang, Loh; in collaboration with Mackin, Morris
Investigations into the sorting of proinsulin in pancreatic b-cells has identified an RSP sorting signal in proinsulin similar to POMC. In monomeric proinsulin, the sorting signal motif consists of residues E13 and L17 located on the B chain and L16 and E17 located on the A chain. In hexameric proinsulin, residue E13 on the B chain is buried, and two residues in the A chain from two adjacent proinsulin dimers in the hexamer contribute a motif. Depletion of CPE in these pancreatic cells with siRNA and the use of a dominant negative CPE construct demonstrated that CPE acts as a sorting receptor for proinsulin in b-cells. Furthermore, the binding of proinsulin and insulin to membrane CPE has pH optima of pH 6.5 and 5.5, respectively. We have proposed a “sorting by retention” model for proinsulin sorting in b-cells, which differs from the “sorting by entry” model for POMC sorting in neuroendocrine cells. In b-cells, proinsulin binds to CPE at the TGN pH of 6.5 and is sorted into immature secretory granules (ISG). However, other non–RSP proteins such as the lysosomal enzymes also enter the ISG in b-cells. Proinsulin is then released from CPE in the ISG and processed to insulin. Insulin then rebinds CPE at the more acidic pH of the ISG and is retained while other non-RSP proteins are removed from the ISG by a constitutive-like pathway (Fig. 22). To understand the molecular basis of hyperproinsu-linemia, we investigated the intracellular sorting of genetically mutated proinsulins found in such diabetes patients who, by definition, have abnormally high levels of plasma proinsulin. One form of mutant proinsulin found in the patients, HisB10Asp, which is unable to hexamerize but forms dimers, missorted to the constitutive pathway and was 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 between 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 secretogogue-dependent manner.

FIGURE 22

Other hyperproinsulinemia proinsulin mutants R65P and R65L were also secreted constitutively and were not stored. Binding studies showed that mutant R65P and R65L proinsulins bound poorly to CPE, accounting for the lack of sorting and retention in the ISG (Fig. 22). The high levels of secreted mutant proinsulins in the plasma of the hyperproinsulinemia patients are therefore attributable to defects in sorting of these molecules as a consequence of their genetic structural alterations.

Sorting and Recycling of the Sorting Receptor CPE
Zhang, Dhanvantari, Arnoutova, Loh; in collaboration with Donaldson, Jackson
Our studies showed that CPE, a transmembrane protein, is sorted into granules of the RSP in neuroendocrine cells by a mechanism involving insertion of its C-terminal domain into lipid rafts at the TGN. Upon stimulation and exocytosis, membrane CPE is localized on the plasma membrane and remains associated with rafts. It is subsequently recycled back to the TGN by a novel internalization mechanism. The inter-nalization of CPE from the plasma into early endosomes requires not only functional ARF6, a GTPase, but also physical interaction of ARF6 with the six-amino-acid cytoplasmic tail of CPE.

Regulation of Secretory Granule Biogenesis
Kim, Loh; in collaboration with Eiden, Cheng
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. Using antisense technology, we observed that depletion of CgA in rat PC12 cells resulted in the loss of LDCG and regulated secretion, and led to degradation of the granule proteins CgB and synapto-tagmin. Over-expression of bovine CgA in the same cells rescued the wild-type phenotype. In a mutant endocrine cell line, 6T3, which lacks CgA, LDCGs, and regulated hormone secretion, transfection of CgA restored the wild-type phenotype. Recently, we used microarrays to compare gene expression in 6T3 cells lacking LDCGs with 6T3 cells stably transfected with CgA. We found that the aquaporin 1 (AQP1, a water channel) and the granuphilin genes were highly up-regulated in 6T3 cells expressing CgA, indicating a new role of CgA at the transcriptional level. While it is known that granulphilin is present in LDCGs and is important in exocytosis, the subcellular localization and role of AQP1 in endocrine cells is unclear. We have now shown that 6T3 cells transfected with CgA and pituitary and chromaffin cell secretory granules contain high levels of AQP1 protein. Thus, AQP1 may be important in regulating secretory granule volume during granule maturation and exocytosis.