Y. Peng Loh, PhD, Head, Section on Cellular Neurobiology

Niamh X. Cawley, PhD, Staff Scientist

Savita Dhanvantari, PhD, Postdoctoral Fellow

Irina Arnaoutova, PhD, Visiting Fellow

Masoumeh Assadi, PhD, Visiting Fellow

Taeyoon Kim, PhD, Visiting Fellow

Marjorie Gondre-Lewis, PhD, Research Fellow

Hong Lou, MD, Senior Research Assistant

Yazmin Rodriguez-David, BS, Postbaccalaureate Fellow

Chunfa Zhang, PhD, Guest Researcher

We study the cell biology of endocrine and neuroendocrine cells. Our focus is two-fold: to investigate the mechanisms of biosynthesis and intracellular trafficking of peptide hormones and neuropeptides and their processing enzymes and to uncover mechanisms involved in the regulation of dense-core secretory granule biogenesis. Our work has led to the discovery of novel molecular mechanisms of protein trafficking to the regulated secretory pathway and has identified players that control secretory granule biogenesis. Studies using cell lines, primary cell cultures, and mouse models have provided a better understanding of diseases related to defects in hormone targeting and cholesterol deficiency.

Mechanism of sorting pro-neuropeptides and neurotrophins to the regulated secretory pathway

Cawley, Zhang, Rodriguez-David, Lou, Loh

The intracellular sorting of pro-neuropeptides and neurotrophins to the regulated secretory pathway (RSP) is essential for the processing, storage, and release of active proteins and peptides in the neuroendocrine cell. We investigated the sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin), pro-enkephalin (pro-ENK), and brain-derived neurotrophic factor (BDNF) 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), where they are sorted into the 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 to 15Å apart and exposed on the surface of these molecules, and two hydrophobic residues, 5 to 7Å away from the acidic residues that are necessary for sorting to the RSP. We identified the RSP sorting receptor membrane carboxypeptidase E (CPE) that was specific for the sorting signals of POMC, pro-enkephalin, and BDNF. The two acidic residues in the prohormone/pro-BDNF sorting motif interact specifically 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 AtT-20 cells caused mis-sorting of POMC to the constitutive pathway, indicating that, in vivo, the basic residues in the sorting domain of CPE interact with the acidic residues in the POMC sorting signal to effect sorting to the RSP. Using a mouse model synthesizing a mutant CPE that is degraded in the pituitary, we were able to show mis-sorting of endogenous POMC in these cells. The studies provide evidence for a sorting signal/receptor-mediated mechanism for targeting prohormones to the regulated secretory pathway in neuroendocrine cells.

Loh YP, Maldonado A, Zhang CF, Tam WH, Cawley NX. Mechanism of sorting proopiomelanocortin

and proenkephalin to the regulated secretory pathway of neuroendocrine cells. Ann NY Acad Sci 2002;971:416-425.

Sorting of normal and proinsulin mutants in hyperproinsulinemia patients

Dhanvantari, Zhang, Loh; in collaboration with Mackin, Morris

Investigations into the sorting of proinsulin in pancreatic beta-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 the motif consists of the two residues in the A chain from two adjacent proinsulin dimers in the hexamer. Depletion of CPE in these cells using siRNA and the use of a dominant negative mutant of CPE demonstrated that CPE acts a sorting receptor for proinsulin in beta-cells.

To understand the molecular basis of hyperproinsulinemia, we investigated the intracellular sorting of genetically mutated proinsulins found in patients with abnormally high levels of plasma proinsulin. We discovered that one form of mutant proinsulin found in these patients, HisB10Asp, which is unable to hexamerize but forms dimers, was mis-sorted 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 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 secretagog-dependent manner. Arg65Pro and Arg65Leu hyperproinsulinemia proinsulin mutants were also found to be secreted constitutively and not stored. Binding studies showed that mutant Arg65Pro and Arg65Leu proinsulins bind poorly to CPE, accounting for the lack of sorting and retention in the immature secretory granules. The high levels of secreted mutant proinsulins in the plasma of hyperproinsulinemia patients are therefore attributable to defects in the sorting of these molecules as a consequence of their genetic structural alterations.

Dhanvantari S, Shen FS, Adams T, Snell CR, Zhang C, Mackin RB, Morris SJ, Loh YP. Disruption

of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsu-linemia.

Mol Endocrinology 2003;17:1856-1867.

Phenotypic characterization of the CPE knockout mouse

Cawley, Loh; in collaboration with Hill, Wetsel 

To test the function of CPE as a sorting receptor in vivo, we generated a CPE knock-out (KO) mouse by deleting exons 4 and 5 from the CPE gene and then characterized its phenotype. Behavioral analysis of the CPE KO mice carried out by Joanna Hill showed that the KO mice are docile, with diminished aggressiveness and sensitivity to pain. KO pups are runts for the first two weeks of age but become obese by 10 to 12 weeks of age. The obesity continues to develop until the mice reach weights of over 70g for both males and females at 40 weeks of age. In some cases, death occurs presumably as a result of obesity-related complications. To date, about 20 percent of the KO mice have died as compared with none of the control animals. Fasting levels of glucose and insulin were monitored regularly and found to be elevated in the KO mice. The onset of diabetes was evident at about 20 weeks of age and continues to be monitored in the older mice. Surprisingly, fasting levels of insulin-like immunoreactive (IR) material in the sera of the KO animals, which was subsequently identified as proinsulin, were more than 100 times higher those of normal animals, consistent with an elevated rate of basal secretion from the pancreas, as was the elevation of secreted IR from isolated pancreatic islets of KO animals. However, stimulated secretion of (pro)insulin from the KO islets was lower than from wild-type islets. The results indicate that the regulated secretory pathway in the beta-cell of KO animals is impaired, supporting a role for CPE as a sorting receptor for proinsulin in vivo. Furthermore, the CPE KO mouse may serve as an excellent model for obesity and neonatal nervous system development.

Role of cholesterol in prohormone processing enzyme sorting and granule biogenesis

Arnaoutova, Assadi, Lou, Gondre-Lewis, Loh; in collaboration with Birch, Porter, Sharpe, Snell

Our recent studies showed that the prohormone and neuropeptide processing enzymes CPE and prohormone convertases 1 and 2 (PC1 and PC2) are transmembrane proteins with an atypical membrane-spanning domain at

the C-terminus. In neuroendocrine cells, the enzymes are sorted at the TGN into granules of the RSP by a novel mechanism involving insertion of their C-terminal transmembrane domain into cholesterol-glycosphingolipid-rich microdomains known as lipid rafts. Removal of cholesterol from secretory granule membranes resulted in the inability of CPE, the RSP sorting receptor, to bind to cargo; cholesterol depletion by treatment of cells with lovastatin resulted in the 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.

To determine the role of cholesterol in dense-core secretory granule biogenesis and maturation in vivo, we used the Smith-Lemli-Opitz syndrome (SLOS) mouse model. SLOS is a developmental disorder caused by a defect of 7-dehydrocholesterol reductase (DHCR7), an enzyme necessary for the last step in cholesterol synthesis. Morphological analysis of neonatal exocrine pancreas zymogen granules by light and electron microscopy showed a marked decrease in the number of granules in SLOS versus control mice. Of the granules present in SLOS animals, most were of an immature phenotype as compared with control animals, appearing as partially formed spheres (see Figure 12.4). Thus, genetic inhibition of cholesterol synthesis in SLOS impairs RSP granule biogenesis and maturation, leading to deficits in the secretory function in the endocrine and nervous systems.

Arnaoutova I, Smith AM, Coates LC, Sharpe JC, Dhanvantari S, Snell CR, Birch NP, Loh YP. The

prohormone processing enzyme PC3 is a lipid raft-associated transmembrane protein. Biochemistry 2003;42:10445-10455.

Zhang CF, Dhanvantari S, Lou H, Loh YP. Sorting of carboxypeptidase E to the regulated secretory pathway requires interaction

of its transmembrane domain with lipid rafts. Biochem J 2003;369:453-460.

Regulation of secretory granule biogenesis by chromogranin A

Kim, Arnaoutova, Loh; in collaboration with Cheng, Eiden

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 chromogranin A (CgA), an on/off switch that controls the formation of LDCG in neuroendocrine cells. Depletion of CgA in rat PC12 cells by using antisense technology resulted in the loss of LDCG and regulated secretion, and degradation of granule proteins, including CgB and synaptotagmin. Overexpression of bovine CgA in these cells rescued the wild-type phenotype. In the mutant endocrine cell line 6T3, which lacks CgA, LDCGs, and regulated hormone secretion, transfection of CgA restored the wild-type phenotype. We have recently identified the Golgi apparatus 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. Supporting this hypothesis, we have recently found a protease inhibitor in the Golgi whose expression is coordinately regulated with CgA. 
 

Recently, we used microarrays to compare gene expression in 6T3 cells lacking LDCGs and 6T3 cells stably transfected with CgA. We found that the aquaporin-1(AQP1, a water channel) and granuphilin genes, both encoding secretory granule proteins, were significantly up-regulated in 6T3 cells expressing CgA. The findings suggest that CgA may play a previously unknown regulatory role in secretory granule protein expression at the transcriptional level. From these studies, we hypothesize that CgA, either directly or indirectly, regulates secretory granule biogenesis by adjusting the secretory granule protein levels at the transcriptional and post-translational level.

Kim T, Tao-Cheng JH, Eiden LE, Loh YP. Large dense-core secretory granule biogenesis is under

the control of chromogranin A in neuroendocrine cells. Ann NY Acad Sci 2002;971:323-331.

Kim T, Tao-Cheng JH, Eiden LE, Loh YP. The role of chromogranin A and the control of secretory

granule genesis and maturation. Trends Endocrinol Metab 2002;14:56-57.

COLLABORATORS

Nigel Birch, PhD, School of Biological Sciences, University of Auckland, New Zealand 

Susan Cheng, PhD, NINDS Electron Microscopy Facility, Bethesda MD

Lee Eiden, PhD, Laboratory of Cellular and Molecular Recognition, NIMH, Bethesda MD

Joanna Hill, PhD, Laboratory of Developmental Neurobiology, NICHD, Bethesda MD

Robert Mackin, PhD, Creighton University, Omaha NE

Stephen Morris, PhD, Aegera Therapeutics, Montreal, Canada

Forbes Porter, MD, PhD, Heritable Disorders Branch, NICHD, Bethesda MD

Juanita Sharpe, PhD, Surgical Neurology Branch, NINDS, Bethesda MD

Christopher Snell, PhD, Medivir UK Ltd., Cambridge, UK

William Wetsel, PhD, Duke University, Durham NC

For further information, contact ypl@codon.nih.gov