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J Biol Chem, Vol. 273, Issue 47, 31180-31185, November 20, 1998
From the Department of Cell Biology and Kaplan Cancer Center, New York University Medical Center, New York, New York 10016
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Carboxypeptidase E (CPE) is a
prohormone-processing enzyme and peripheral membrane protein of
endocrine/neuroendocrine secretory granules. CPE has been shown to bind
to an amino-terminal peptide of pro-opiomelanocortin (N-POMC) at pH 5.5 and hypothesized to be critically involved in the targeting of hormones
such as POMC to the regulated secretory pathway [Cool, D. R.,
Normant, E., Shen, F., Chen, H. C., Pannell, L., Zhang, Y., and
Loh, Y. P. (1997) Cell 88, 73-83]. To further
explore the possibility that CPE serves to mediate the association of
content proteins with the membrane during granule biogenesis, the
binding of CPE to granule content proteins was investigated using an
in vitro aggregation assay in which the selective
precipitation of granule content proteins is induced by titration of
the pH to <6.0. CPE was observed to co-aggregate efficiently with
pituitary and chromaffin granule content proteins at concentrations
well below those that promote its self-aggregation. In addition, CPE
co-precipitated at pH 5.8 with purified prolactin and with insulin,
which homophillically self-aggregate yet are structurally distinct from
N-POMC. N-POMC when added to the assays did not inhibit the aggregation
of CPE with prolactin or insulin, indicating that these interactions do
not involve a binding site for N-POMC. The data show that CPE interacts
at acidic pH with a variety of different content proteins, resembling
in this regard other granule membrane proteins. The results support the
idea that co-aggregation of abundant membrane proteins with content
proteins is an important general mechanism for the sorting and
retention of secretory granule proteins during granule maturation.
Aggregation of secretory granule proteins occurs as immature
secretory granules (ISGs)1
are formed from the trans-Golgi network (TGN) and continue to mature
and acidify (1, 2). Aggregation can also be achieved by lowering the pH
of isolated granule contents, including those derived from chromaffin
and anterior pituitary granules (3) as well as pancreatic zymogen
granules (4, 5). A number of purified granule content proteins,
including the chromogranins A and B (in the presence of calcium) (6,
7), prolactin (3), and insulin (8), aggregate in vitro as
the pH is lowered to <6.5 as well, reinforcing the view that changes
in the ionic milieu of the TGN and ISG are responsible for content
protein condensation (9, 10). Constitutively secreted proteins such as
albumin and IgG, when added to content proteins, do not precipitate,
suggesting that the formation of aggregates itself may directly
regulate the process of segregating constitutively secreted proteins
from granule content proteins (3).
We have previously demonstrated that the luminal domains of several
abundant granule membrane proteins can co-aggregate with content
proteins at pH 5.5-6 (3), which mimics the conditions existing in the
ISG, a site where sorting of proteins destined for storage from those
constitutively secreted is known to occur (11-13). This would be
consistent with the idea that membrane and content proteins use similar
routes of segregation during granule maturation and that the binding of
content proteins to the granule membranes is mediated by the major
membrane proteins themselves. Recently, Cool et al. (14)
showed that pro-opiomelanocortin (POMC) as well as a peptide consisting
of its amino-terminal 26 amino acids (N-POMC) bind to pituitary
secretory granule membranes with a pH optimum of 5.5-6. This peptide
was chosen because the amino-terminal portion of POMC can confer
granule targeting on heterologous proteins in transfected cells (15).
The binding of N-POMC to membranes was inhibited not only by POMC
itself but by proinsulin and proenkephalin, suggesting that common
sites of association are used by several different hormones. N-POMC could also be cross-linked at pH 5.5 to carboxypeptidase E (CPE), an
abundant granule membrane protein (14), suggesting that POMC binding to
membranes was mediated by CPE. Moreover, in cells depleted of CPE,
either from the pituitary of fat/fat mice, which bear a
mutation in CPE that prevents its exit from the endoplasmic reticulum
(16, 17), or in cells transfected with antisense DNA constructs, POMC
was observed to be secreted largely by the constitutive pathway (14).
These data were interpreted to mean that CPE serves as the sorting
receptor for POMC and other related prohormones, although the
hypothesis remains controversial as proinsulin incorporation into
secretory granules was not greatly affected in either isolated islets
or an insulin-secreting cell line derived from fat/fat mice
(18, 19).
CPE is a processing enzyme found in all endocrine and neuroendocrine
cells (20). It associates with membranes at low pH by virtue of an
amphipathic helix in its carboxyl terminus (21), resembling in this
regard other processing enzymes such as prohormone convertase 2, which
has a membrane binding domain in its amino terminus (22). CPE undergoes
homophillic self-association at pH 5.5 when present at high
concentrations (23), another property shared by prohormone convertase 2 (22) and by the luminal segment of peptidyl glycine Antibodies and Purified Granule Content Proteins--
Purified
bovine CPE (mixture of membrane forms) and rabbit antibodies specific
for the amino- and carboxyl-terminal peptides were generously provided
by Dr. L. Fricker (Albert Einstein College of Medicine, Bronx, NY) and
were prepared as reported previously (23). Rabbit antibodies to
chromogranin B (CgB) were a gift from Dr. R. Angeletti (Albert Einstein
College of Medicine). Rabbit IgG, bovine insulin, and ovine prolactin
(Prl) were purchased from Sigma. Human growth hormone (GH) was obtained
from the National Institute of Diabetes and Digestive and Kidney
Diseases National Hormone and Pituitary Program, whereas rabbit
antibody was purchased from Accurate Chemical (Westbury, NY). N-POMC,
synthesized by Phoenix Pharmaceuticals (Mountain View, CA), was a gift
from Dr. Y.-P. Loh (National Institutes of Health, Bethesda, MD).
Preparation of Granule Content Proteins--
Bovine adrenal
chromaffin and pituitary granules were prepared as described previously
(3). Granules were lysed by freeze-thawing and sonication in 100 mM KCl, 25 mM HEPES, pH 7.5, in the presence of
protease inhibitors. The released content proteins were recovered from
the supernatant after centrifugation and desalted on Bio-Gel P-6 DG
columns (Bio-Rad).
In Vitro Aggregation Assay--
The assay was conducted as
described previously (3). In brief, 50-µl samples containing granule
content or the proteins of interest in 5 mM HEPES, 10 mM 4-morpholineethanesulfonic acid, pH 7.5, and 0.016%
Triton X-100, with KCl or CaCl2 where indicated, were
centrifuged for 30 min, and the pellet was discarded. The supernatants
were then titrated slowly to the indicated pH values by adding 0.125 N HCl while vortexing. The pH was measured with a
microelectrode (Microelectrodes Inc., Bedford, NH). CPE was generally
used at a concentration of 1.3 µg/ml, which is well below the
threshold necessary to induce self-aggregation at this pH (23). GEMSA
(Calbiochem) was added in some experiments after the prespin and 20 min
before pH titration. After incubation for 30 min at 23 °C., the
reaction mixture was centrifuged to separate the precipitates from the
supernatant fractions. Centrifugation in most cases was for 30 min at
15,000 × g in an Eppendorf centrifuge. In experiments
in which adrenal chromaffin extracts were incubated in the presence of
calcium, centrifugation was conducted at 56,000 × g in
a TL100 ultracentrifuge, which is required for efficient recovery of
CgA (3). CPE aggregation itself could be induced at concentrations of
40 µg/ml, with pellets recovered by centrifugation at 100,000 × g as described (23). A portion of the supernatant and the
entire pellet fractions were then subjected to immunoblotting (see
below). As a control, samples were subjected to pH titration but not
centrifuged. Under the conditions used, the amounts of protein
remaining in the tubes after removal of the assay mixture were
inconsequential (data not shown). Triton X-100 at 0.016% had no effect
on overall aggregation of pituitary or adrenal extracts but did prevent
binding of CPE itself to the tubes. To measure insulin aggregation,
pellets and supernatants from triplicate samples were subjected to a
Micro-BCA assay (Pierce), as described previously (3).
To assess the effect of N-POMC on CPE aggregation with Prl or insulin,
a modification of the assay protocol was used. The final sample volume
was reduced to 25 µl, and a buffer of 5 mM HEPES, 20 mM 4-morpholineethanesulfonic acid, 130 mM KCl,
and 0.016% Triton X-100 was used. After the prespin, N-POMC (120 µg/ml final concentration) or an equivalent volume of 5 mM HEPES was added, and after a 15-min incubation the pH
was reduced to 5.8. After an additional 30 min at 37 °C,
centrifugation was conducted at 100,000 × g for 30 min. This protocol allowed titration of the amount of hormone necessary
to induce reproducible CPE co-aggregation to 40 µg/ml for insulin and
80 µg/ml for Prl. The aggregation of Prl and insulin was essentially
identical at 23 and 37 °C (data not shown).
Immunoblotting--
Immunoblotting was performed after SDS-PAGE,
transfer of the proteins to nitrocellulose paper, and staining with
Ponceau S to visualize the transferred proteins. In experiments in
which IgG was used, SDS-PAGE was performed without a reducing agent. Antibodies directed against the amino terminus of CPE were used in the
experiments shown, but carboxyl-terminal antibodies were used in
initial experiments with the identical results. All antibodies were
generally used at a 1:500-1:2000 dilution. The enhanced
chemiluminescence procedure was used with preflashed film to visualize
the reactive species (Amersham Pharmacia Biotech). The exposed
radiographs and the images of gels stained with Ponceau S were
digitized (LACIE Ltd., Beaverton, CA) and imported into Quark Express
(Denver, CO) for computerized labeling and printing of figures.
Quantitation of aggregation on the scanned images was conducted using
NIH Image (version 1.55) on exposures that were not oversaturated. All
of the data presented are representative experiments that were repeated two to four times with similar results.
Measurement of CPE Activity--
The assays of CPE activity were
performed using the method of Fricker and Devi (24). In brief, purified
CPE was diluted (1:500) in 0.1 M NaOAc, pH 5.5, 0.01%
Triton X-100, 10 µg/ml bovine serum albumin, and 1 mM
CoCl2 and incubated at 4 °C. for 20 min in the presence
or absence of 0.5 µM GEMSA. Dansyl-FAR (20 µM) was then added, and the reaction was allowed to
continue for 7 min at 37 °C and 5 min. at 4 °C before addition of
HCl (0.15 M final concentration) and chloroform. After
centrifugation, samples were read in a Perkin-Elmer 650-10S
fluorescence spectrophotometer using excitation and emission
wavelengths of 355 and 500 nm, respectively. All assays were conducted
in triplicate.
CPE Aggregates Together with Other Pituitary and Adrenal Chromaffin
Granule Proteins--
CPE is an endogenous protein of the pituitary
gland (20) and was readily detectable in granule content prepared from
bovine pituitary (Fig. 1). To assess
whether CPE could interact with pituitary content proteins, an in
vitro aggregation assay was used (3). This assay allows the
measurement of weak but specific protein-protein interactions at mildly
acidic pH that are difficult to detect by more direct procedures. The
pH of pituitary extracts was slowly acidified, and CPE was detected by
immunoblotting. As shown in Fig. 1, CPE aggregated effectively (>90%)
at pH 5.8 but remained almost entirely in the unaggregated supernatant
at pH 7.5. Similarly, as we have previously demonstrated (3), other
pituitary content proteins, including Prl and GH, aggregate as the pH
is reduced. This observation was not a consequence of the homophillic
self-aggregation of CPE, which is known to occur at pH 5.5 and high
concentrations of protein (23). CPE alone at levels (1.3 µg/ml)
comparable with those present endogenously (1.5 µg/ml estimated from
densitometric scanning) in the pituitary samples (Fig. 1, compare
lanes 2 and 10), did not aggregate either at pH
7.5 or pH 5.8 (
CPE was also detectable in the adrenal granule extracts but at much
lower levels (Fig. 2). Surprisingly, both
endogenous CPE and exogenously added CPE were recovered primarily
(>70% in this experiment) in the pellet fractions when the pH of the
chromaffin granule content was titrated to 5.8 even in the absence of
CaCl2, which is required for the aggregation of the major
content protein CgA (Fig. 2, lanes 3, 4, 9, and
10). Bands on the gels corresponding to CgB, however, were
detected in the pH 5.8 pellet fractions both by Ponceau staining (Fig.
2, bottom) and by immunoblotting (data not shown), as were
unidentified high molecular weight proteins (Fig. 2,
arrowheads). As expected, CPE itself did not undergo appreciable aggregation when incubated alone at pH 7.5 or pH 5.8 in the
presence or absence of CaCl2, and neither the endogenous nor the added CPE was recovered in the pellet fractions at pH 7.5. Similar results were obtained even when the centrifugation was
performed at 15,000 × g, which was used to generate
the quantitative assessment of CPE aggregation shown in Fig. 2
(bottom right). The half-maximal value for aggregation of
CPE was ~5.8 in the adrenal extracts under these conditions. Thus,
CPE is able to interact with aggregating chromaffin granule content
proteins, perhaps including CgB.
The interaction of CPE with granule content proteins was not dependent
on its enzymatic activity. GEMSA, an inhibitor of CPE (24), which at
this concentration (0.5 µM) decreased the activity of
purified CPE by >95% (data not shown), had no effect on the co-aggregation of CPE together with pituitary content proteins (Fig.
3A). Moreover, the interaction
of CPE with granule content proteins occurred just as readily at
physiological salt concentrations. Inclusion of 140 mM KCl
in the reaction mixture had no effect on the extent of CPE aggregation
(Fig. 3B). Similar results were obtained in two separate
experiments with >95% aggregation of CPE occurring under each set of
conditions at pH 5.8.
Similar results were obtained for CPE aggregation in adrenal extracts
(Fig. 4). GEMSA did not affect
aggregation of CPE significantly (mean, 65%; range, 61-68% for
control versus 63%; range, 56-69% for GEMSA;
n = 2). The addition of KCl did lower the efficiency of
aggregation somewhat, but the effects were small, with the amount of
CPE in the pellets decreasing from 74% without salt to 65% with KCl
(Fig. 4) or from 42 to 32% in another experiment (data not shown).
CPE Co-precipitates with Prolactin and Insulin--
Prolactin is
the major component of the pituitary granule extracts. We have
previously shown that Prl self-aggregates as the pH is titrated to
<6.5 (3). It was therefore of interest to determine whether CPE and
Prl could interact directly. CPE was incubated with Prl as the pH was
titrated to 5.8. As shown in Fig. 5, in
the absence of Prl, CPE did not aggregate appreciably (
The interaction of CPE with insulin, another hormone that undergoes
self-aggregation at acidic pH (8), was also assessed. Insulin
aggregates effectively as pH is titrated to <6.5. In fact, >99% of
insulin is recovered in the pellet fraction at pH 5.8 under our
standard assay conditions (data not shown). It proved necessary,
however, to include 140 mM KCl in the assay mixtures to
effectively prevent IgG, a marker of the constitutive secretory pathway, from co-aggregating to some extent. Fig.
6 shows the results of an experiment in
which CPE and IgG were mixed with insulin. In the absence of insulin,
CPE did not aggregate significantly at either pH (Fig. 6, lanes
1 and 3), whereas in the presence of insulin, CPE was
recovered exclusively in the supernatant at pH 7.5 (Fig. 6, lane
5) and at pH 5.8 (Fig. 6, lane 7), primarily in the
pellet (81 ± 6%; n = 3). IgG remained almost
entirely in the supernatant even in the presence of insulin (Fig. 6,
lanes 5 and 7, bottom panels). The amount of
insulin aggregation could not be directly quantitated using SDS-PAGE.
However, in experiments conducted in the absence of added CPE and
measuring total protein recoveries, 64 ± 3% (n = 3) of insulin was found in the pellet fraction under these assay
conditions.
N-POMC Does Not Inhibit the Aggregation of CPE with Prolactin or
Insulin--
As noted in the Introduction, CPE has been shown to bind
to a peptide corresponding to the amino-terminal 26 amino acids of POMC
at pH 5.5 (14). The question therefore arises of whether CPE has a
single binding site capable of interacting with a number of different
hormones, including Prl and insulin. To test this hypothesis, N-POMC
was included in the assays together with CPE before pH titration. To
maximize the ratio of the peptide to the precipitating hormones, much
lower amounts of insulin (40 µg/ml) and Prl (80 µg/ml) were used,
and the assay protocol was modified to recover a higher proportion of
the aggregates (see "Experimental Procedures"). Fig.
7 shows that under these conditions CPE
when incubated alone remained in the supernatant fraction ( Acidification is thought to play a critical role in secretory
granule formation. Incubation of secretory cells with high
concentrations of lysosomotropic amines (2, 25-28) or with bafilomycin
A1 (29), an inhibitor of vacuolar ATPases, leads to inefficient
packaging of granule content proteins as well as inefficient prohormone processing and ISG maturation. The aggregation of CPE with granule proteins from pituitary and adrenal gland is a measure of its ability
to bind to content proteins and is consistent with a role for acidic pH
in the interaction of content proteins with the membrane during granule
formation. The pH dependence in vitro is consistent with
initiation of aggregation in the environment of the TGN (5.9-6.2) (30,
31), with the process continuing even more vigorously in ISGs (5.5-6)
(2). Given the fact that the concentrations of content proteins are
much higher (>100 mg/ml) in secretory granules than those used in our
assay,2 it is reasonable to
propose that stronger and more significant interactions between CPE and
granule content proteins would take place in vivo in the
TGN, where protein concentration would also be expected to be high.
An unexpected observation was that CPE aggregated well in the
chromaffin granule extracts in the absence of added calcium at pH 5.8. CgB and higher molecular weight components were also present in the
precipitates, and we have previously shown that dopamine
As noted in the Introduction, CPE has been postulated to be a
"sorting receptor" having a direct role in chaperoning certain hormones to secretory granules (14, 33). Evidence that N-POMC can be
directly cross-linked to CPE in granule membranes was interpreted as
reflecting a receptor-ligand interaction. Because N-POMC has a
disulfide loop that is also found in other prohormones such as
proenkephalin and proinsulin, and these prohormones inhibit the binding
of N-POMC to granule membranes, a common binding site in CPE for this
type of motif was postulated to mediate the sorting of these proteins
(14). The interaction of CPE with additional granule content proteins,
as shown in this study, would be consistent with a role for CPE as a
broad spectrum chaperone that could mediate the interaction of a
variety of content proteins with membranes during granule biogenesis,
particularly as aggregation was occurring. It is important to note,
however, that the binding of CPE to Prl, which is structurally
unrelated to POMC, and to insulin, which lacks the C peptide and does
not have a complete disulfide loop, was unaffected by N-POMC,
indicating that CPE has more than one binding site for content proteins.
Recent studies using pancreatic islets (18) or insulin-secreting cell
lines derived from fat/fat mice (19) contradict the view
that CPE is critical for peptide hormone storage. In both cases,
constitutive release of proinsulin was not enhanced in cells with
defective CPE, whereas regulated secretion of partially processed
insulin and proinsulin remained robust, and immature-looking secretory
granules were also present (16, 19). These results indicate that CPE is
not required for proinsulin targeting but is necessary for processing
and condensation.
Given the conflicting observations, what is the significance of the
specific interactions of CPE with granule proteins, and what role might
these interactions play, if any, in promoting content protein storage?
Noteworthy in this regard is the fact that a number of other membrane
proteins share with CPE the ability to bind content proteins at pH
5.5-6. These include dopamine
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-amidating
mono-oxygenase (3). In the current study, we have evaluated the
interaction of CPE with granule content proteins using low pH-induced
aggregation as an assay. The results indicate that CPE has the capacity
to interact with granule content proteins other than prohormones as pH
is titrated to the range of the TGN and ISG. Thus, in its ability to
interact with content proteins, CPE resembles other membrane-associated proteins including peptidyl glycine -amidating mono-oxygenase, dopamine 
-hydroxylase, and pancreatic glycoprotein 2 (3, 5), suggesting that these interactions are important in the segregation of
granule membrane and content proteins during granule formation and maturation.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1% in this experiment, never >6% in any experiment). However, addition of exogenous CPE (membrane form) to the
pituitary extract led to the precipitation (>90%) of both endogenous
and exogenous CPE (Fig. 1, lanes 7 and 8).
Quantitative assessment of CPE aggregation in the pH range 5.4-7.5 is
shown in Fig. 1 (right panel). CPE aggregation was more
efficient than that of Prl and GH with a half-maximal value ~6.0.
Although our gel system did not completely separate the different
forms, much of the endogenous CPE would be expected to be in a soluble
form lacking its amphipathic membrane anchor (23). Thus, the data indicate that both soluble and membrane forms have the ability to
co-aggregate with content proteins in this assay.
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Fig. 1.
CPE co-aggregates with pituitary granule
content proteins at pH 5.8. Left panels, pituitary granule
content (1 mg/ml) was incubated either alone (lanes 1-4) or
with purified CPE (1.3 µg/ml) added (Pit + CPE,
lanes 5-8) using the standard assay conditions (see
"Experimental Procedures"). CPE was incubated in the absence of
granule content as a control (lanes 9-12). The pH was
reduced as indicated. The entire pellet (P; odd
numbered lanes) and 20% of the remaining supernatant
(S) after centrifugation (even numbered lanes)
were then subjected to SDS-PAGE and transfer to nitrocellulose paper.
CPE (top panels) was identified by immunoblotting, whereas
the major pituitary content proteins Prl and GH were visualized by
staining with Ponceau S (bottom panels). Right
panel, the assay was performed as described above, but in this
case, equal aliquots (50%) of the supernatant and pellet samples were
used for SDS-PAGE and immunoblotting. CPE in the pituitary pellet ( 
)
or incubated alone (
) is depicted along with Prl and GH bands
quantitated after staining the bottom portion of the gels with
Coomassie Blue (
). Each data point represents the mean and S.E. of
three to six samples from three separate experiments.
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Fig. 2.
CPE aggregates with adrenal granule content
proteins at pH 5.8. Chromaffin granule content (1 mg/ml) was
incubated either alone (lanes 1-6) or with CPE added
(lanes 7-12). CPE was incubated in the absence of granule
content as a control (lanes 13-18). Samples were incubated
and analyzed as described in Fig. 1, except that the precipitate was
recovered by centrifugation at 56,000 × g for 30 min.
CaCl2 (25 mM) was included in one set of
samples to induce aggregation of CgA (lanes 5, 6, 11, 12, 17, and 18). Bands corresponding to CgB as well as
other unidentified high molecular weight proteins
(arrowheads) recovered in the pellet fractions at pH 5.8 are
also indicated. Bottom right panel, adrenal extracts with
CPE added were subjected to pH titration and analysis as described
above in the absence of added CaCl2 and with recovery of
the pellet fractions at 15,000 × g. CPE in the adrenal
pellet ( ) or incubated alone () is depicted. Total protein was
determined from computerized images of the nitrocellulose paper stained
with Ponceau S (). Each data point represents three samples from two
separate experiments. P, pellet; S,
supernatant.
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Fig. 3.
Low pH induced aggregation of CPE with the
pituitary granule content proteins is not dependent on its enzymatic
activity and occurs at physiological salt concentrations. A,
0.5 µM GEMSA, an inhibitor of CPE activity, was added to
sample mixtures 20 min before titration to pH 5.8 (lanes 3 and 4). B, the aggregation assay was performed at
pH 5.8 without added KCl (lanes 1 and 2) or in
the presence of 140 mM KCl (lanes 3 and
4). The assays were performed and the samples were analyzed
as described in Fig. 1. Top panels, CPE alone; middle
panels, aggregation of CPE; bottom panels, aggregation
of Prl and GH under the same conditions. P, pellet;
S, supernatant.
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Fig. 4.
Low pH induced aggregation of CPE with the
chromaffin granule content proteins is not dependent on its enzymatic
activity and occurs at physiological salt concentrations.
Aggregation assays in the presence of 0.5 µM GEMSA
(A) or 140 mM KCl (B) were also
performed using adrenal chromaffin granule content with exogenous CPE
(1.3 µg/ml) added (lanes 3 and 4). In each
case, parallel assays were conducted under the standard conditions
(lanes 1 and 2). The aggregation assays were
performed exactly as described in Fig. 3, i.e. in the
absence of added CaCl2 and with centrifugation at
15,000 × g. Top panels, purified CPE alone;
middle panels, CPE aggregation in the adrenal extract;
bottom panels, aggregation of the chromaffin granule content
proteins. P, pellet; S, supernatant.
4%) at either
pH (lanes 1 and 3), whereas in the presence of Prl, CPE was recovered almost exclusively in the supernatant at pH 7.5 (99%) and in the pellet at pH 5.8 (mean, 95%; range, 94-97%; n = 2) together with Prl (mean, 47%; range, 42-52;
n = 2). This interaction also was observed in the
presence of 140 mM KCl (data not shown), indicating that
the interaction is relatively strong.
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Fig. 5.
CPE co-precipitates with prolactin. CPE
was incubated in the aggregation assay in the presence (bottom
panels) or absence (top panels) of Prl, which
homophillically self-aggregates at mildly acidic pH. The pH was
titrated to 5.8 where indicated (lanes 3 and 4).
Pellet (P, odd numbered lanes) and supernatant (S,
even numbered lanes) samples were collected and analyzed as
described in Fig. 1, with CPE identified by immunoblotting and Prl by
Ponceau S staining (bottom).
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Fig. 6.
CPE co-precipitates with insulin. CPE
(top panels) was incubated in the aggregation assay (with
140 mM KCl added) in the absence (lanes 1-4) or
presence (lanes 5-8) of bovine insulin (1 mg/ml). As a
control for nonspecific binding, rabbit IgG (1.5 µg/ml), a
constitutively secreted protein that should not interact with insulin,
was incubated in the absence (lanes 1-4) or presence
(lanes 5-8) of insulin in another series of tubes
(bottom panels). The pH was titrated to 5.8 where indicated
(lanes 3, 4, 7, and 8), which induces of insulin
to precipitate. Pellet (P, odd numbered lanes) and
supernatant (S, even numbered lanes) samples were collected
and analyzed as described in Fig. 1, with CPE and IgG identified by
immunoblotting.
6%) in the presence or absence of N-POMC (top panel). In the
presence of insulin (Fig. 7, middle panel), CPE aggregated
well in the absence or presence of the peptide (88 versus
80%, respectively, in this experiment; 95 versus 97% in a
second experiment). Similarly, in the presence of Prl (Fig. 7,
bottom panel) the amount of CPE recovered in the pellet was
not affected by N-POMC (56 without versus 59% with peptide
in this experiment; 86 versus 93% in a second experiment).
It should be pointed out that N-POMC was present in ~5-fold molar
excess in the case of insulin, ~11-fold in the case of Prl, and in
>1500-fold excess of CPE itself. Hence we conclude that the sites of
interaction of CPE with Prl and insulin are distinct from any involved
in its binding to N-POMC.
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Fig. 7.
N-POMC does not inhibit the interaction of
CPE with insulin or prolactin. CPE (1.3 µg/ml) was incubated
either alone (top panels) or with 40 µg/ml insulin
(middle panels) or 80 µg/ml Prl (bottom
panels). N-POMC (120 µg/ml) was included in one set of samples
(lanes 3 and 4), whereas in the other set an
equal volume of buffer was included (lanes 1 and
2). The pH was reduced in all samples to 5.8, and
precipitates were recovered by centrifugation at 100,000 × g for 30 min. The entire pellet (P) and in this
case 50% of the supernatant (S) fractions were then
subjected to SDS-PAGE and immunoblotting as described in Fig. 1.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-hydroxylase partially aggregates under these conditions (~50% of
the calcium induced precipitation) (3). The formation of aggregates
containing content and membrane components during acidification of the
secretory organelles, as occurs in vitro, is consistent with
a pH-dependent protein sorting mechanism involving condensation of granule components. Indeed, these findings are consistent with the observations made in streptolysin-permeabilized PC12 cells, in which neutralization of pH interfered with the sorting
of chromogranin C to secretory granules (32).
-hydroxylase, which co-precipitates
with chromaffin granule content proteins (3), peptidyl glycine
-amidating mono-oxygenase, which co-aggregates with chromaffin and
pituitary granule content (3), glycoprotein 2, which specifically
co-aggregates with pancreatic zymogens (5) and binds polymerized
amylase (34), and an inositol triphosphate receptor/calcium channel,
which has been reported to bind to CgA at acidic pH (35). Moreover,
several granule membrane proteins self-aggregate at acidic pH. One is
CPE (23), and another is prohormone convertase 2 (22), which also have
amphipathic helices that mediate their membrane binding. The integral
membrane proteins peptidyl glycine -amidating mono-oxygenase (3) and
glycoprotein 2 (36) can self-associate as well. The association of
membrane proteins with themselves and perhaps their neighbors in the
plane of the membrane could provide a scaffold for the binding and
envelopment of aggregating content proteins in the TGN and ISG. The
specific interaction of CPE with some granule content proteins and with other membrane-associated processing enzymes, as has been reported (37), is consistent with this model. Hence processing enzymes, which
represent major granule membrane components (38), could subserve roles
both in peptide hormone generation and, in concert with other major
membrane proteins, as the "receptors" for the selective membrane
recruitment, segregation, and retention of content proteins as their
aggregation is initiated by acidic pH. In pancreatic beta cells,
depletion of a single membrane component such as CPE may have little
effect, but in other cell types, such as neuro 2A, CPE may represent a
higher proportion of the membrane proteins and potentially could play a
more significant role.
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ACKNOWLEDGEMENTS |
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I thank G. Kicska, M. Jackson, and Y. Jin for expert technical assistance, Dr. D. Sabatini for continued encouragement and support, Dr. Y.-P. Loh for the gift of N-POMC, and Drs. L. Fricker and L. Devi for generously providing reagents and advice.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK44238 and by the American Heart Association, New York City affiliate.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 212-263-5812;
Fax: 212-263-8139; E-mail: rindler{at}mcrcr.med.nyu.edu.
The abbreviations used are: ISG, immature secretory granule; TGN, trans-Golgi network; CPE, carboxypeptidase E/H; GH, growth hormone; Prl, prolactin; CgA and CgB, chromogranins A and B; POMC, pro-opiomelanocortin; N-POMC, amino-terminal 26 amino acids of POMC; PAGE, polyacrylamide gel electrophoresis; GEMSA, 2-guanidinoethylthiosuccinic acid.
2 M. Rindler, unpublished observation.
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Abstract
Introduction
Procedures
Results
Discussion
References
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