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Secretory Protein
Trafficking and Granule Biogenesis in
Neuroendocrine Cells
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Y. Peng Loh, PhD, Head, Section on Cellular Neurobiology Niamh
X. Cawley, PhD, Staff Scientist Marjorie
Gondre-Lewis, PhD, Research Fellow Irina
Arnaoutova, PhD, Postdoctoral Fellow Masoumeh
Assadi, PhD, Postdoctoral Fellow Taeyoon
Kim, PhD, Postdoctoral Fellow Josh Park, PhD, Postdoctoral Fellow Tulin
Yanik, PhD, Postdoctoral Fellow Hong
Lou, MD, Senior Research Assistant Nimesh
Patel, BS, Predoctoral Fellow Chunfa Zhang, PhD, Guest Researcher |
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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. Such studies using cell lines, primary cell
cultures, and mouse models have provided a better understanding of diseases
related to defects in hormone and neuropeptide targeting, obesity, and
cholesterol deficiency. Mechanism
of sorting pro-neuropeptides and neurotrophins to the regulated secretory
pathway Cawley, Zhang, Lou, Loh; in collaboration
with Baum, Lu 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 RSP 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, which are necessary for sorting to the
RSP. While such a motif was found in BDNF, which is secreted in an
activity-dependent manner, nerve growth factor (NGF), which is primarily
secreted constitutively, was missing one amino acid residue of this motif.
Introduction of the missing residue by mutagenesis (Val20Glu) redirected NGF
to the RSP, further confirming the importance of the sorting motif in
targeting to the RSP. We identified an RSP sorting receptor, which is
specific for the sorting signal of POMC, pro-ENK, and BDNF, 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 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 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 achieve sorting
to the RSP. Using a CPE knockout mouse model, we were able to show missorting
of endogenous POMC in pituitary cells. Furthermore, we showed that BDNF is
not sorted to the regulated secretory pathway and is secreted constitutively
in cortical and hippocampal neurons of such mice. The studies provide
evidence for a sorting signal/receptor–mediated mechanism for targeting
prohormones, neuropeptides, and the neurotrophin BDNF to the regulated
secretory pathway in neuroendocrine cells and neurons. In collaboration with Bruce Baum, we 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. Arnaoutova I, Jackson CL, Al-Awar OS,
Donaldson JG, Loh YP. Recycling of raft-associated carboxypeptidase E
requires ARF6 interaction. Mol Biol Cell 2003;14:4448-4457. Cawley
NX, Rodriguez Y, Maldonado A, Loh YP. Trafficking of mutant
carboxypeptidase E to secretory granules in a beta-cell line derived from Cpefat/Cpefat
mice. Endocrinology
2003;144:292-298. Voutetakis A, Kok M, Zheng C, Bossis I, Wang
J, Cotrim A, Marracino N, Goldsmith C, Chiorini J, Loh YP, Nieman L, Baum B.
Reengineered salivary glands are stable endogenous bioreactors for systemic
gene therapeutics. Proc Natl Acad Sci
USA 2004;101:3053-3058. Aberrant
sorting of proinsulin mutants and mutant pro-CART in hyperproinsulinemia and
obese patients Dhanvantari, Zhang, Yanik, Loh; in
collaboration with Kuhar, Mackin, Morris Investigations into the sorting of proinsulin
in pancreatic beta cells has identified an RSP sorting signal in proinsulin
similar to that in POMC. In monomeric proinsulin, the sorting signal motif
consists of residues E13 and L17 located on the B chain and L16 and E17 on
the A chain. In hexameric proinsulin, residue E13 on the B chain is buried,
with the motif contributed by the two residues in the A chain from two
adjacent proinsulin dimers in the hexamer. Depletion of CPE in beta cells
using siRNA and the use of a dominant negative mutant of CPE demonstrated
that CPE acts as a sorting receptor for proinsulin in these cells. We investigated the intracellular sorting of
genetically mutated proinsulins found in hyperproinsulinemia patients with
abnormally high levels of plasma proinsulin in order 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, 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 the
proinsulin’s inability to be sorted to the RSP for processing to
insulin and secretion in a secretogog-dependent manner. We also found that
other hyperproinsulinemia proinsulin mutants, Arg65Pro and Arg65Leu, were
secreted constitutively rather than stored. Binding studies showed that
mutant Arg65Pro and Arg65Leu proinsulins bound poorly to CPE, accounting for
the lack of sorting and retention in the immature secretory granule. The high
levels of secreted mutant proinsulins in the plasma of such patients can
therefore be attributed to defects in sorting of these proninsulins that
result from their genetic structural alterations. We investigated the sorting and processing of
a mutant form of cocaine-amphetamine–regulated transcript (CART) found
in a family of obese patients. CART, found in brain, is an anorexigenic
peptide that has several physiological effects such as inhibiting feeding and
regulating energy expenditure. CART acts downstream of leptin in the
obesity-controlling signaling pathway. We found a mutant pro-CART (Leu34Phe)
in a 10-year-old Italian boy who has been obese since age two. This missense
Leu34Phe mutation co-segregates in three generations of his maternal
relatives along with the phenotype of severe obesity, but his father was not
obese. To investigate the trafficking of CART, we transiently transfected
AtT20 cells with wild-type (WT) or mutant (Leu34Phe) CART. While pro-CART was
substantially processed to active CART, mutant pro-CART was only minimally
processed to yield an intermediate form and an active form. Furthermore, WT
CART was secreted in a regulated manner with high potassium stimulation, but
mutant pro-CART/CART exhibited high basal release and no significant
stimulated secretion. Immunocytochemical studies revealed that 90 percent of
immunoreactive WT CART was co-localized in punctate granules with POMC, a
granule marker, in the processes of AtT20 cells. However, only 58 percent of
the cells showed punctate staining of immunoreactive mutant CART co-localized
with POMC in the cell processes. The results indicate that mutant pro-CART
was partially missorted and secreted via the constitutive pathway. Moreover,
the poor processing of mutant pro-CART may in part have resulted from the
missorting to the constitutive pathway. Thus, the missorting of mutant CART
(Leu34Phe) in neurons could provide a molecular basis for the obese phenotype
in such patients. 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
hyperproinsulinemia. Mol Endocrinol
2003;17:1856-1867. Endocrinological
and behavioral deficits of the CPE knockout mouse Cawley, Yanik, Loh; in collaboration with
Hill, Wetsel By deleting exons 4 and 5 from the CPE gene,
we generated a CPE knockout (CPE KO) mouse and characterized its phenotype.
KO mice became obese by 10 to 12 weeks of age and reached 60 to 80g by 40
weeks, more than double the weight of WT age-matched controls. The null
animals consumed more food overall, were less physically active during the
light phase of the light-dark cycle, and burned fewer calories as fat than WT
littermates. Fasting levels of glucose and insulin-like immunoreactivity (IR)
in plasma were elevated in both male and female KO mice at about 20 weeks;
males recovered from this state by 32 weeks, but females did not. Nevertheless,
at this time, insulin-like IR was 50 to 100 times higher than in the WT
animals. The plasma insulin-like IR material was identified primarily as
proinsulin. The KO mice showed impaired glucose clearance and were
insulin-resistant. High plasma levels of leptin
were present in the KO mice; however, they showed no circulating fully
processed CART, the peptide that is responsive to leptin-induced feedback
inhibition of feeding. In addition to obesity and diabetes phenotypes, the KO
mice were subfertile and showed deficits in GnRH processing in the
hypothalamus. Behavioral analyses revealed that KO animals had diminished
reactivity to stimuli and reduced muscle strength, coordination, visual
placing, and toe-pinch reflexes. Approximately 25 percent of the homozygous
mutants died prematurely, perhaps due to complications associated with
obesity. The CPE KO mice thus display a wide range of neural and endocrine
abnormalities, suggesting that CPE may have additional physiological roles
beyond those ascribed to peptide processing and sorting of prohormones in
cells. The CPE KO mouse will also be a useful model for studying type II
diabetes, obesity, and neural development. Cawley NX, Zhou J, Hill J, Abebe D, Romboz S,
Yanik T, Rodriguiz R, Wetsel W, Loh YP. The carboxypeptidase E knockout mouse
exhibits endocrinological and behavioral deficits. Endocrinology
2004;145:5807-5819. Role of cholesterol in prohormone processing enzyme sorting and
granule biogenesis Arnaoutova, Assadi, Lou, Gondre-Lewis,
Loh; in collaboration with Birch, Parsegian, 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, they are sorted into
granules of the RSP at the TGN by a novel mechanism involving transmembrane
association of their C-terminal 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 treating 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. To test the importance of cholesterol in
secretory granule biogenesis and in packaging of contents in vivo, we analyzed vesicles in the
pancreas of cholesterol-deficient mouse models of Smith-Lemli-Opitz syndrome
(SLOS) and lathosterolosis (Sc5d-/-). SLOS and lathosterolosis are
human disorders resulting from, respectively, defects in 7-dehydrocholesterol
reductase and lathosterol 5-desaturase, enzymes necessary for the final steps
of cholesterol synthesis. Morphological analysis by light and electron
microscopy of neonatal pancreas zymogen granules showed a marked decrease in
the number of granules in both SLOS and Sc5d-/- compared with
control mice. Of the granules present in SLOS and Sc5d-/-animals,
most were of a less mature phenotype than those of control animals, appearing
as partially formed spheres. The Sc5d-/- exocrine pancreas, which
lacks granules, was filled with rough ER ribbons and ribosomal structures,
indicating an inability to package materials into membrane-bound structures.
Furthermore, protein synthesis and regulated secretion were less efficient in
primary cultures of cholesterol-deficient secretory cells in the exocrine
pancreas than in control cells. We hypothesize that the defect in granule
biogenesis and maturation is attributable to different physical contributions
of sterols to membrane curvature. Indeed, preliminary biophysical studies
indicate that sterols such as lathosterol and cholesterol contribute
differently to membrane rigidity and bending modulus. Thus, genetic
inhibition of cholesterol synthesis in SLOS and Sc5d-/- impairs
granule biogenesis and maturation in the RSP, leading to deficits in the secretory
function in the exocrine pancreas, possibly also 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. Assadi M, Sharpe J, Snell C, Loh YP. The
C-terminus of prohormone convertase 2 is sufficient and necessary for raft
association and sorting to the regulated secretory pathway. Biochemistry 2004;43:7798-7807. Regulation
of secretory granule biogenesis by chromogranin A Kim, Arnaoutova, Zhang, Loh; in
collaboration with Pickel Formation of large dense-core granules (LDCGs)
at the TGN is essential for regulated secretion of hormones and neuropeptides
from neuroendocrine cells. Our recent studies uncovered an on/off switch,
chromogranin A (CgA), that controls the formation of LDCGs in neuroendocrine
cells. Depletion of CgA in rat PC12 cells using antisense technology resulted
in the loss of LDCGs, regulated secretion, and degradation of granule
proteins, including CgB and synaptotagmin. Overexpression of bovine CgA in
these cells rescued the WT phenotype. Transfection of CgB into 6T3, a mutant
endocrine cell line lacking CgA, LDCGs, and regulated hormone secretion,
resulted in CgB degradation, whereas transfection of CgA restored the WT
phenotype in the same cells. We 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 focal point for control of granule
biogenesis in neuroendocrine cells. Moreover, we recently found a protease
inhibitor in the Golgi that is transcriptionally activated by CgA. This
protease inhibitor is upregulated in cells actively forming LDCGs but
downregulated in cells that minimally express CgA and show low levels of LDCG
biogenesis. Transfection of the protease inhibitor into 6T3 cells lacking CgA
prevented LDCG protein degradation and rescued granule biogenesis. Thus, we
have uncovered a novel mechanism whereby CgA regulates LDCG biogenesis by
transcriptionally activating a protease inhibitor that stabilizes granule
proteins necessary for LDCG biogenesis. We recently demonstrated the
importance of CgA in large dense-core granule biogenesis in vivo in an antisense mRNA transgenic mouse model deficient in
CgA, which we generated in collaboration with James Pickel. The mice showed
severe aberrant granule formation in the adrenal medulla (see Figure 12.9).
Kim T, Tao-Cheng
J-H, Eiden LE, Loh YP. The role of chromogranin A and the control of
secretory granule genesis and maturation. Trends Endocrinol Metab 2003;14:56-57. Loh
YP, Kim T, Rodriguez Y, Cawley NX. Secretory granule biogenesis
and neuropeptide sorting to the regulated secretory pathway in neuroendocrine
cells. J Mol Neurosci
2003;22:63-71. COLLABORATORS Bruce Baum, DMD, PhD, Gene Therapy and Therapeutics Branch,
NIDCR, Nigel Birch, PhD, Julie Donaldson, PhD, Laboratory of Cell Biology, NHLBI, Joanna Hill, PhD, Laboratory of Developmental Neurobiology,
NICHD, Michael Kuhar, PhD, Bai Lu, PhD, Laboratory of Cellular and Synaptic
Neurophysiology, NICHD, Robert Mackin, PhD, Stephen Morris, PhD, Aegera Therapeutics, Adrian Parsegian, PhD, Laboratory of Physical and Structural
Biology, NICHD, James Pickel, PhD, Laboratory of Genetics, NIMH, Forbes Porter, MD, PhD, Heritable Disorders Branch, NICHD, Juanita Sharpe, PhD, Surgical Neurology Branch, NINDS, Christopher Snell, PhD, Medivir UK Ltd., William Wetsel, PhD, For further information, contact ypl@codon.nih.gov |
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