| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, National Institute for Child and Human Development, National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Dr. Y. Peng Loh, Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, Building 49, Room 5A38, NIH/NICHD, Bethesda, Maryland 20892. E-mail: ypl{at}codon.nih.gov
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Two nonmutually exclusive hypotheses have been proposed to explain how proteins are selectively sorted at the level of the trans-Golgi network (TGN) to enter the RSP. The first is the aggregation passive sorting hypothesis, in which RSP proteins form an aggregate that condenses, thereby excluding other proteins from entering and being packaged into the nascent budding granule. Support for this hypothesis comes from showing that several RSP proteins such as chromogranin A (3, 4), chromogranin B (5), or chromogranin C (6), carboxypeptidase E (CPE), also known as CPH (7), prohormone convertase 2 (8), and other granule components (9) aggregate under acidic pH and high calcium concentration, conditions that exist within the TGN (10, 11, 12). However, there are studies suggesting that aggregation alone is not sufficient for sorting. For example, certain modifications on proteins such as chromogranin B (13) and insulin-like growth factor, IGF-1 (14), affected their correct sorting to the RSP, whereas their ability to aggregate was not hindered. The second hypothesis is the sorting signal-receptor binding mechanism which was proposed by Kelly in 1985 (2). This mechanism necessitates the binding of a specific signal domain of the RSP protein to a membrane-bound receptor, leading to segregation of the protein (or its aggregate) and subsequent packaging into secretory granules.
Various studies have provided evidence in support of the existence of sorting signal domains involved in the active sorting of proteins to the RSP. Studies with chimeric proteins (15, 16, 17, 18, 19, 20) have demonstrated that fusion of a constitutively secreted protein to a protein destined for the regulated secretory granules causes the redirection of the former to the RSP. Furthermore, RSP proteins such as POMC and chromogranin B, which have their sorting signal domain deleted or modified have been shown to be misrouted into the constitutive pathway (13, 21, 22). It has also been shown that an immunoglobulin (normally routed to the constitutive secretory pathway) against a regulated secretory granule component (chromogranin B) could be directed to the RSP if expressed in an endocrine cell line, where it can bind to its RSP protein antigen (23).
The RSP sorting domain has been identified for several proteins, i.e. POMC (21), proenkephalin (Bamberger et al., in preparation) chromogranin B (22), chromogranin A (20, 24), PC2 (16), PC5a (25), and PAM (26). More recently the specific sorting signal motif has been defined for POMC (21). It is a conformation-dependent motif consisting of a 13-amino acid amphipathic loop structure (N-POMC residues Cys8 to Cys20) with four residues exposed to the surface (D10, L11, E14, L18). The side chains of these residues may participate in binding to the sorting receptor. Recent studies have identified the membrane-bound form of CPE as a regulated secretory pathway sorting receptor for this POMC sorting signal (27). The functionality of CPE as a sorting receptor for POMC in vivo was demonstrated using stable Neuro-2a cell lines expressing CPE antisense RNA that have been transiently transfected with POMC complementary DNA (cDNA). Immunocytochemical and secretion studies showed lack of packaging in the regulated secretory granules and constitutive secretion of POMC in these CPE-depleted cells (27).
Because molecular modeling studies (C. R. Snell, Novartis Institute for Medical Research, London, UK, personal communication) have indicated that proenkephalin and proinsulin have similar putative sorting signal motifs as POMC, and both were capable of displacing the binding between POMC1–26 and CPE (27), we have now investigated the role of CPE in the sorting of these two prohormones. In addition, we have examined the role of CPE in sorting chromogranin A. Using the antisense strategy, we show that CPE is essential for the sorting of proinsulin and proenkephalin but not chromogranin A to the RSP. The experimental results presented emphazize that CPE is a common sorting receptor for several prohormones and proneuropeptides but that the sorting of other RSP proteins may use alternate membrane receptors.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Construction of insulin expression vector
The human preproinsulin cDNA insert was excised from the
pT7hPPI-1 vector kindly provided by Prof. Kevin Docherty (University of
Aberdeen, Fosterhill, UK) and inserted into the pcDNAIII vector. The
resulting vector was then transfected in CPE-depleted (clone
Neuro-2a-CPE-AS4) and wild-type Neuro-2a cells.
Cell culture, transfection, and selection of CPE antisense
clones
Neuro-2a cells were cultured in DMEM supplemented in 10% calf
serum and 20 mM glutamine, with streptomycin and penicillin
(100 U/ml) and amphotericin B (0.25 mg/ml). The CPE antisense cDNA was
transfected into the cells overnight using lipofectin in a 1:1 (wt:wt)
ratio. The clone selection was carried out with G418 (neomycin, Gibco
BRL, Gaithersburg, MD, 800 µg/ml). Several stably transfected clones
were selected, and their content of CPE checked, before each
experiment, by Western blotting, using a specific polyclonal antibody,
CPE 2–4, directed against the N-terminal part of the molecule (27).
The membrane and soluble forms were identified by the molecular weight
(28) and the membrane CPE was further verified by reprobing the Western
blot with a C-terminal CPE (CPE 7–4) antibody directed against the
last 15 residues of the enzyme. The human preproinsulin cDNA was
transfected as described above.
Secretion experiments
Confluent cells grown on 10-cm dishes were washed 3 times with
PBS, pH 7.4, at 37 C, and preincubated for 3 h at 37 C, in 3 ml of
medium 1 containing 25 mM HEPES, 125 mM NaCl,
4.8 mM KCl, 1.2 mM
KH2PO4, 5.6 mM glucose, 0.5
mM MgCl2, pH 7.4, and 7.7 kIU/ml aprotinin, 1
mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride
(AEBSF), 0.02% BSA. The preincubation buffer (medium 1) was collected,
and unstimulated cells were further incubated in the same medium for
another 3 h, whereas the cells to be stimulated were incubated in
3 ml medium 2, with high K+ (25 mM HEPES, 80
mM NaCl, 51 mM KCl, 1.2 mM
KH2PO4, 5.6 mM glucose, 0.5
mM MgCl2, 5.2 mM CaCl2,
and 7.7 KIU/ml aprotinin, 1 mM AEBSF, 0.02% BSA). The
second set of incubation buffers (medium 2) were collected and the
cells were then harvested by adding 3 ml of 10 mM Tris
buffer containing 1 mg/ml aprotinin, 1 mM AEBSF, and 1
mM EDTA. Cells were extracted by three freeze/thaw cycles
and centrifugation at 2000 x g for 2 min. The
supernatants of the cell extracts and incubation medium were then
passed through PD10 columns, and the eluate lyophylized overnight.
These samples were resuspended in PBS buffer containing 7.7 KIU/ml
aprotinin, 1 mM AEBSF, and an aliquot was loaded on either
12% acrylamide Tris-Glycine gels (for chromogranin and proenkephalin
analysis) or gradient 10–20% acrylamide Tricine gels. Proteins on the
gels were transfered onto a nitrocellulose membrane and analyzed by
quantitative Western blot. Secreted prohormones in secretion medium 2
was expressed as percentile of total immunoreactivity found in medium 1
+ 2 and the cell extract.
Western blot analysis
The primary antiserum against chromogranin A was a gift from Dr
S. Yoo (NIH, Bethesda, MD). To detect proinsulin and insulin on Western
blot, a mixture of three antibodies was used: two primary antisera
(from Incstar, Stillwater, MN and from Dako, Carpinteria, CA, used at
1/900 and 1/500 dilution respectively) detecting both proinsulin and
insulin and a monoclonal antibody prepared against insulin (from Sigma
Chemical Co., St. Louis, MO, used at a 1/500 dilution). The primary
antiserum against proenkephalin (RB13) was a gift from Dr S. Sabol
(NIH, Bethesda, MD). This antiserum detects Met-Enk and proenkephalin
(29). Detection of the immunoreactive proteins was by the enhanced
Chemiluminescence System (Pierce, Rockford, IL). The bands were scanned
on a Microtech Scanmaker II using the Adobe Photoshop 4.0 software,
followed by quantitation using the NIH Image 1.57 software. The values
were then corrected according to the percentage of sample loaded. The
RIA detection of Met-Enk was performed according to the instructions of
the Incstar kit.
Immunofluorescence microscopy
Wild-type Neuro-2a and Neuro-2a-CPE-AS4 cells,
either transiently transfected with pcDNAIII-insulin vector, or not
transfected, were grown on 8-well Nunc Permanox Lab-Tek tissue culture
chambers (Naperville, IL). The cells were fixed in 2%
paraformaldehyde and permeabilized with 0.1% Triton X100 for 30 min.
After 5 PBS washes, cells were preincubated with 10% goat serum and
then incubated with different primary antibodies overnight at 4 C.
After washing with PBS, the cells were incubated with biotinylated goat
antirabbit IgG (1/500 dilution) for 30 min and washed 5 x 5 min
with PBS. A solution of Texas Red conjugate (1/400 dilution, GIBCO-BRL)
was then added to the cells for 30 min, and the cells were washed
5 x 5 min with PBS. Images were captured using a TEC-470
charge-coupled device color camera on a Nikon (Tokyo, Japan) Optiphot
epifluorescent microscope coupled to a Macintosh (Cupertino, CA)
8100/100 PowerPC computer using Adobe Photoshop 4.0 software (San Jose,
CA).
Electron microscopy
Wild-type Neuro-2a and Neuro-2a-CPE-AS4 cells were
grown in 8-well Nunc Permanox Lab-Tek tissue culture chambers
(Naperville, IL). All the manipulations described below were at room
temperature. The cells were fixed for 1 h with 2% glutaraldehyde
in 0.1 M sodium cacodylate buffer (NCB), pH 7.4. The fixed
cells were then treated for 1 h with an OSO4 solution
in NCB and washed three times with NCB, three times with 0.1
N acetate buffer, incubated for 3 h in uranyl acetate
1%, and washed again with the acetate buffer. After dehydration in
ethanol, the cells were embedded in Epon followed by polymerization for
48 h at 60 C. The embedded cells were removed from the culture
slide, and thin sections were cut and counterstained with lead citrate
and micrographs taken on a JEOL100CX electron microscope at 80 kV.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). To determine whether the
transformation and/or selection had affected the neuroendocrine
phenotype of the Neuro-2a-CPE-AS4 cells, we examined these
cells for the presence of secretory granules by electron microscopy.
Figure 2 shows the presence of typical
dense core granules, a hallmark of neuroendocrine cells, in the
Neuro-2a-CPE-AS4 cells. Furthermore, the number of dense
core granules counted within randomly selected areas totaling 150
µm2 from different preparations, for wild-type and
CPE-depleted cells, did not show any significant difference between the
two cell lines (114 vs. 105 granules/150 µm2).
Immunocytochemical studies (see Fig. 9, B and D, below) indicated that
chromogranin A immunoreactivity was localized in punctate RSP granules.
In addition, the expression of endogenous proenkephalin and
chromogranin A, assayed by Western blot, was found to be similar in
both the wild-type and CPE-depleted cells (31 ± 5 arbitrary unit
(AU) vs. 27 ± 5 AU for enkephalin and 42 ± 7 AU
vs. 39 ± 9 AU for chromogranin A, see Fig. 3), suggesting no major difference in
neuroendocrine protein expression in these two cell types. These
results, together with secretion experiments showing stimulated release
of chromogranin A from CPE-depleted cells, confirmed that the phenotype
of Neuro-2a-CPE-AS4 cells is unchanged, and that
differences between the sorting of proenkephalin and proinsulin in
wild-type and CPE-depleted cells described below are not due to
phenotypic changes, such as the inability to form dense core secretory
granules in the transformed cells.
|
|
|
|
Wild-type Neuro-2a cells showed processing of proinsulin to insulin
(Fig. 4A), which was secreted into the
medium. This secretion was stimulated 4-fold under depolarizing
conditions (3.8% vs. 15.4%). In contrast,
Neuro-2a-CPE-AS4 cells secreted only unprocessed proinsulin
at a high basal level and was unresponsive to high
K+/Ca++ stimulation (Fig. 4B). These results
were correlated with immunocytochemical studies. In wild-type Neuro-2a
cells, immunoreactive proinsulin/insulin
(proinsulin/insulini) was detected in punctate granules
located in the cell body, along and at the tip of neurites, suggesting
localization in regulated secretory granules (Fig. 5, A and D). In contrast, when proinsulin
was expressed in Neuro-2a-CPE-AS4 cells, the
proinsulin/insulini were found in a perinuclear area (Fig. 5, B and E), overlapping with the wheat germ agglutinin (WGA) staining
of the Golgi (Fig. 5, C and F). Constitutive secretion and the lack of
proinsulin/insulini in punctate granules indicate that the
reduction of CPE in these cells caused a loss of sorting to the RSP
granules.
|
|
). The neuro-2a-CPE-AS4
cells, in contrast, showed no stimulated secretion. Instead, the amount
of proenkephalini released without stimulation was 9.5% of
the total. With stimulation, the amount released was about the same
(12% of the total). The release of high levels of
proenkephalini, i.e. 3.5 times the unstimulated
basal release, in a nonstimulatable manner indicates that the
Neuro-2a-CPE-AS4 cells released proenkephalin
constitutively. Similar results were obtained in one other
Neuro-2a-CPE-AS clone (Neuro-2a-CPEAS-7 (27), data not
shown). This observation is consistent with the lack of
proenkephalini in punctate granules in
Neuro-2a-CPE-AS4 cells (Fig 7, B and E), compared with wild-type
cells, which showed punctate immunostaining in the neurites and tips of
cells (Fig. 7, A and D).
|
|
).
The absence of met-enkephalin was confirmed by RIA (data not shown).
Nevertheless, wild-type Neuro-2a cells secreted proenkephalin in a
stimulatable manner (Fig. 6) and proenkephalini was
detected in punctate granules in these cells (Fig. 7). This indicates
that processing and maturation of this prohormone is not a prerequisite
for efficient sorting to and secretion via the RSP.
Chromogranin A is correctly sorted to the RSP in Neuro-2a cells
down-regulated in the expression of CPE
Chromogranin A, a matrix protein and major component of granules,
has been shown to weakly displace the binding of
N-POMC1–26 to CPE from the secretory granule membranes,
and N-POMC1–26 was able to weakly displace the binding of
chromogranin A to the same membranes (27). These in vitro
data raised the question whether chromogranin A required CPE to be
correctly sorted to the RSP in vivo. To address this issue,
the sorting of endogenous chromogranin A in wild-type Neuro-2a and
Neuro-2a-CPE-AS4 cells was investigated. The secretion
studies showed that both wild-type Neuro-2a and
Neuro-2a-CPE-AS4 cells released chromogranin A in a
stimulated manner with high K+ depolarization, yielding a
7-fold (3.5% vs. 24%) and 6-fold (3.7% vs.
22%) stimulation, respectively (Fig. 8).
Similar results were obtained in other Neuro-2a-CPE-AS clones
(Neuro-2a-CPEAS-7 (27), Neuro-2a-CPE-AS6 and
Neuro-2a-CPE-AS16, data not shown). This absence of effect
of depletion of CPE on the sorting of chromogranin A to the RSP was
supported by immunocytochemical studies. Chromogranin A
immunoreactivity (chromogranin Ai) was found localized in
punctate granules in cell bodies, along and at the tip of the neurites,
in both wild-type Neuro-2a and Neuro-2a-CPE-AS4 cells,
(Fig. 9). These results indicate that CPE
is not necessary for the sorting of chromogranin A to the RSP.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Based on these criteria, our data show that proinsulin sorting to the
RSP is dependent on the presence of CPE because CPE-depleted cells
(Neuro-2a-CPE-AS4 cells) secreted only proinsulin in a
nonstimulatable/constitutive manner (Fig. 4) and do not store this
prohormone in the secretory granules. In addition, previous binding
studies (27) showed that proinsulin displaced the binding of
N-POMC1–26 (which contain the sorting signal motif) to
secretory granule membranes, and vice-versa. Furthermore, molecular
modeling studies (C. R. Snell, personal communication) have
indicated that proinsulin shares a very similar conformation-dependent
sorting signal motif as POMC, and cross-linking studies (Cool, D. R.,
and Y. P. Loh, manuscript submitted) have shown that it binds to the
same sorting receptor, i.e. CPE. These in vitro
and in vivo data provide strong evidence that proinsulin is
sorted to the RSP, like POMC, via a receptor-mediated mechanism, by
binding to membrane-bound CPE.
Recently, a study by Irminger et al. (31) showed that proinsulin is sorted to the regulated secretory pathway in pancreatic cells of CPEfat/CPEfat mice, which expressed a mutated form of CPE that is degraded in the endoplasmic reticulum (ER) (27, 32), suggesting that CPE is not a sorting receptor for proinsulin. CPE levels were not measured in these mice. Recent analysis (Shen and Loh, unpublished data) have revealed that the current CPEfat/CPEfat mice from Jackson Laboratory have a wide range of CPE levels, unlike previously, where the levels were always less than 10% of the normal mice (27, 32). Thus, conclusions of Irminger et al. remain tentative. Also, Fricker and colleagues (33) have shown that NIT 3 cells (an immortalized cell line derived from pancreatic ß-islets of mutant CPEfat/CPEfat mice), which contain as much proCPE as CPE in NIT control cells, were able to sort some proinsulin/insulin to the regulated secretory pathway. Their data, however, do not rule out the possibility that some proCPE may have exited the ER, allowing it to function as a receptor to sort some proinsulin into the regulated secretory pathway. Thus, both these studies do not definitively preclude a role of CPE as sorting receptor for targetting prohormones to the regulated secretory pathway.
The stimulation and immunocytochemical data obtained with proenkephalin in neuro2a-CPE-AS4 cells were comparable with those of proinsulin, leading to the conclusion that this prohormone was not stored within the secretory granules of these cells, thus the sorting of proenkephalin to the RSP was also dependent on CPE. Previous studies showed that proenkephalin displaced N-POMC1–26 binding to secretory granule membranes with high relative potency (27), and cross-linking experiments confirmed the binding of proenkephalin to membrane-bound CPE (Cool, D. R., and Y. P. Loh, manuscript submitted). Furthermore, the molecular modeling and sorting studies using the chimeric protein proenk1–31-CAT (bacterial chloramphenicolacetyl transferase) have identified a conformation-dependent sorting signal motif similar to that of POMC and proinsulin, that was sufficient to target CAT to the RSP (Bamberger et al., in preparation). These results indicate that proenkephalin share the same RSP sorting receptor, i.e. CPE, with proinsulin and POMC, and that the sorting of this prohormone to the RSP is by a sorting signal-receptor mechanism.
Interestingly, analysis of cell extract and medium by Western blot
(Fig. 5) and RIA (data not shown) revealed that Neuro-2a cells process
proenkephalin very poorly or not at all. However, in previous studies,
a very low amount of met-enkephalin was detected in these cells (30).
The much lower number of cells used in the current experiments probably
precluded the detection of such a small amount, due to the limit of
sensitivity of the assay. The low amounts of the processing enzymes PC1
and PC2 in these cells (30, 34) may be sufficient for processing
proinsulin but not proenkephalin due to differences in catalytic
efficiency of the enzyme for different substrates. These results
indicate that nonprocessed prohormone can be correctly routed to the
RSP, i.e. processing is not a prerequisite for sorting to
the RSP, as was hypothesized for renin (35). A similar conclusion was
reached by Jung et al. (36), working on the ELH prohormone
(egg laying hormone), which is processed at a tetrabasic site, to yield
a N- and a C-terminal intermediate. The former was sorted
constitutively, whereas the latter was sorted to the RSP. The authors
showed that, after mutation of the tetrabasic site (and a tribasic
secondary cleavage site), the intact precursor was routed into the
regulated secretory pathway, indicating that preliminary processing in
the Golgi was not a prerequisite for the routing of the C-terminal
hormone to the RSP. Thus, the lack of processing of proinsulin and
proenkephalin observed in the Neuro-2a-CPE-AS4 cells is due
to their missorting to the constitutive secretory pathway, rather than
being incorrectly sorted because of absence of maturation.
The sorting of chromogranin A to the RSP was not hindered by the depletion of CPE, as clearly shown by the localization of chromogranin Ai inside secretory granules of CPE-depleted cells and by the comparable increase of secretion of chromogranin A after depolarization of the wild-type and CPE-depleted cells. Binding studies showed that chromogranin A had a much lower efficiency in displacing N-POMC1–26 binding to secretory granule membranes than proinsulin or proenkephalin (27). Furthermore, recent experiments failed to show cross-linking of [125I] chromogranin A to CPE (Cool, D. R., and Y. P. Loh, manuscript submitted). In addition, the molecular modeling studies failed to identify a comparable sorting signal motif to POMC, proinsulin, and proenkephalin, in the Cys17-Cys38 sorting signal domain of chromogranin A (C. R. Snell, personal communication). Thus, the in vivo data presented here argue that the sorting of chromogranin A to the RSP is not dependent on CPE. This is consistent with the proposal of Huttner’s group that chromogranin A/B is sorted to the RSP by homotypic binding to membrane-associated chromogranin A/B at the trans Golgi network (37).
In conclusion, this study has provided in vivo evidence, to support the in vitro binding data, that proinsulin and proenkephalin are sorted to the RSP by a mechanism involving the binding of a sorting signal to membrane-bound CPE, which acts as a sorting receptor. Furthermore, we have recently established, in the CPEfat/CPEfat mutant mouse lacking CPE, that GH, which is actively sorted and can reroute a constitutively secreted protein to the RSP in AtT20 cells (17, 38), also requires CPE for its correct sorting to the RSP (39). Thus, the membrane-bound form of CPE acts as a common receptor for sorting prohormones including POMC, proinsulin, and proenkephalin to the RSP. Furthermore, these three prohormones share a similar conformation-dependent sorting signal motif, but chromogranin A does not, which could account for its lack of binding to CPE. Thus, the granins, which are major proteins in the regulated secretory pathway granules, appear to use an alternate sorting receptor, other than CPE, for targeting to the RSP. In addition, the present study also confirmed that prohormone processing is not a prerequisite for its sorting to the RSP. Finally, the presence of dense core granules in CPE-depleted Neuro-2a cells suggests that, whereas CPE acts as a sorting receptor for a number of prohormones, it is not essential for granulogenesis. Further work is necessary to determine the events occurring subsequent to binding of the prohormone complex to CPE and the involvement of the CPE/hormone complex in facilitating budding at the TGN.
|
Acknowledgments |
|---|
Received September 11, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  M. Courel, M. S. Vasquez, V. Y. Hook, S. K. Mahata, and L. Taupenot Sorting of the Neuroendocrine Secretory Protein Secretogranin II into the Regulated Secretory Pathway: ROLE OF N- AND C-TERMINAL {alpha}-HELICAL DOMAINS J. Biol. Chem., April 25, 2008; 283(17): 11807 - 11822. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Garcia, S.-K. Han, W. G. Janssen, Z. Z. Khaing, T. Ito, M. J. Glucksman, D. L. Benson, and S. R. J. Salton A Prohormone Convertase Cleavage Site within a Predicted {alpha}-Helix Mediates Sorting of the Neuronal and Endocrine Polypeptide VGF into the Regulated Secretory Pathway J. Biol. Chem., December 16, 2005; 280(50): 41595 - 41608. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. R. Mulcahy, C. A. Vaslet, and E. A. Nillni Prohormone-Convertase 1 Processing Enhances Post-Golgi Sorting of Prothyrotropin-releasing Hormone-derived Peptides J. Biol. Chem., December 2, 2005; 280(48): 39818 - 39826. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hosaka, T. Watanabe, Y. Sakai, T. Kato, and T. Takeuchi Interaction between secretogranin III and carboxypeptidase E facilitates prohormone sorting within secretory granules J. Cell Sci., October 15, 2005; 118(20): 4785 - 4795. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. X. Cawley, J. Zhou, J. M. Hill, D. Abebe, S. Romboz, T. Yanik, R. M. Rodriguiz, W. C. Wetsel, and Y. P. Loh The Carboxypeptidase E Knockout Mouse Exhibits Endocrinological and Behavioral Deficits Endocrinology, December 1, 2004; 145(12): 5807 - 5819. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hosaka, M. Suda, Y. Sakai, T. Izumi, T. Watanabe, and T. Takeuchi Secretogranin III Binds to Cholesterol in the Secretory Granule Membrane as an Adapter for Chromogranin A J. Biol. Chem., January 30, 2004; 279(5): 3627 - 3634. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Dhanvantari, F.-S. Shen, T. Adams, C. R. Snell, C. Zhang, R. B. Mackin, S. J. Morris, and Y. P. Loh Disruption of a Receptor-Mediated Mechanism for Intracellular Sorting of Proinsulin in Familial Hyperproinsulinemia Mol. Endocrinol., September 1, 2003; 17(9): 1856 - 1867. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Taupenot, K. L. Harper, N. R. Mahapatra, R. J. Parmer, S. K. Mahata, and D. T. O'Connor Identification of a novel sorting determinant for the regulated pathway in the secretory protein chromogranin A J. Cell Sci., March 14, 2003; 115(24): 4827 - 4841. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. X. Cawley, Y. M. Rodriguez, A. Maldonado, and Y. P. Loh Trafficking of Mutant Carboxypeptidase E to Secretory Granules in a {beta}-Cell Line Derived from Cpefat/Cpefat Mice Endocrinology, January 1, 2003; 144(1): 292 - 298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hosaka, T. Watanabe, Y. Sakai, Y. Uchiyama, and T. Takeuchi Identification of a Chromogranin A Domain That Mediates Binding to Secretogranin III and Targeting to Secretory Granules in Pituitary Cells and Pancreatic beta -Cells Mol. Biol. Cell, October 1, 2002; 13(10): 3388 - 3399. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Conn, A. Leanos-Miranda, and J. A. Janovick Protein Origami: Therapeutic Rescue of Misfolded Gene Products Mol. Interv., September 1, 2002; 2(5): 308 - 316. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.-y. Zhang, A. Chang, T. B. Kjeldsen, and P. Arvan Intracellular Retention of Newly Synthesized Insulin in Yeast Is Caused by Endoproteolytic Processing in the Golgi Complex J. Cell Biol., June 4, 2001; 153(6): 1187 - 1198. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Kuliawat, D. Prabakaran, and P. Arvan Proinsulin Endoproteolysis Confers Enhanced Targeting of Processed Insulin to the Regulated Secretory Pathway Mol. Biol. Cell, June 1, 2000; 11(6): 1959 - 1972. [Abstract] [Full Text]
|
|
|
|
|
|
D. J. Cowley, Y. R. Moore, D. S. Darling, P. B. M. Joyce, and S.-U. Gorr N- and C-terminal Domains Direct Cell Type-specific Sorting of Chromogranin A to Secretory Granules J. Biol. Chem., March 10, 2000; 275(11): 7743 - 7748. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. A. Nillni and K. A. Sevarino The Biology of pro-Thyrotropin-Releasing Hormone-Derived Peptides Endocr. Rev., October 1, 1999; 20(5): 599 - 648. [Abstract] [Full Text]
|
|
|
|
 |
|
 S.-U. Gorr, X. F. Huang, D. J. Cowley, R. Kuliawat, and P. Arvan Disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins Am J Physiol Cell Physiol, July 1, 1999; 277(1): C121 - C131. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Qian, O. Varlamov, and L. D. Fricker Glu300 of Rat Carboxypeptidase E Is Essential for Enzymatic Activity but Not Substrate Binding or Routing to the Regulated Secretory Pathway J. Biol. Chem., April 23, 1999; 274(17): 11582 - 11586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-F. Zhang, C. R. Snell, and Y. Peng Loh Identification of a Novel Prohormone Sorting Signal-Binding Site on Carboxypeptidase E, a Regulated Secretory Pathway-Sorting Receptor Mol. Endocrinol., April 1, 1999; 13(4): 527 - 536. [Abstract] [Full Text]
|
|
|
|
|
|
|
