Y.
Peng Loh, Ph.D., Principal Investigator
Niamh Cawley, Ph.D., Staff
Scientist
Chunfa Zhang, Ph.D., Senior
Fellow
Irina Arnaoutova, Ph.D., Postdoctoral
Fellow
Masoumeh Assadi, Ph.D., Postdoctoral
Fellow
Savita Dhanvantari, Ph.D., Postdoctoral Fellow,
Canadian Diabetes Association Fellowship
Taeyoon Kim, Ph.D., Postdoctoral
Fellow
Hong Lou, M.D., Senior
Research Assistant
Alex Maldonado, B.S., Predoctoral Fellow
Yazmin Rodriguez-David, B.S.,
Predoctoral Fellow
Tiffany Adams, B.S., NIH
Academy Predoctoral Fellow
For More
Information
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The
Section on Cellular Neurobiology, directed by Peng Loh, studies the cell
biology of endocrine and neuroendocrine cells. The goal of the group is
to understand the mechanisms of biosynthesis and intracellular trafficking
of peptide hormones and neuropeptides and the regulation of dense-core granule
and synaptic vesicle biogenesis. Our studies have led to the discovery of
the molecular mechanisms of protein trafficking in the regulated secretory
pathway, the understanding of diseases related to defects in hormone trafficking,
and the development of a novel procedure for hormone delivery through gene
therapy.
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](images/LOH_2001.jpg)
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-RaftAssociated 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-sphingolipidrich
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.
![](images/LOH_2002.jpg)
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.
![](images/LOH_2003.jpg)
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.
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PUBLICATIONS
- Cawley
NX, Normant E, Chen A, Loh YP. Oligomerization of pro-opiomelanocortin
is independent of pH, calcium and the sorting signal for the regulated
secretory pathway. FEBS Lett 2000;481:37-41.
- Dhanvantari
S, Arnaoutova I, Snell CR, Steinbach PJ, Hammond K, Caputo GA, London
E, Loh YP. Carboxypeptidase E, a prohormone sorting receptor, is
anchored to secretory granules via a C-terminal transmembrane insertion.
Biochemistry 2002, in press.
- Dhanvantari
S, Loh YP. Lipid raft association of carboxypeptidase E is necessary
for its function as a regulated secretory pathway sorting receptor.
J Biol Chem 2000;275:29887-29893.
- Kim
T, Tao-Cheng JH, Eiden LE, Loh YP. Chromogranin A, an on/off
switch controlling dense-core granule biogenesis. Cell 2001;106:477-509.
- 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 New York Acad Sci, in press.
- Loh YP, Maldonado A, Zhang C-F, Tam WH, Cawley NX. Mechanism of sorting
pro-opiomelanocortin and pro-enkephalin to the regulated secretory pathway
of neuroendocrine cells. Ann New York Acad Sci, in press.
a Bruce J. Baum, NIDCR.
b Lynette Nieman, NICHD.
c Kristine Miller, Goucher College,
Baltimore, MD, USA.
d Nigel Birch, University of Auckland,
New Zealand.
e Julie Donaldson, NHLBI.
f Erwin London, SUNY, Stoneybrook,
NY, USA.
g Lee Eiden, NIMH.
h Susan Cheng, NINDS.
i Robert Mackin, Creighton University
School of Medicine, Omaha, NE.
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