From the Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut 06030-3401
Received for publication, September 4, 2000, and in revised form, October 30, 2000
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ABSTRACT |
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Unlike the neuroendocrine cell lines widely used
to study trafficking of soluble and membrane proteins to secretory
granules, the endocrine cells of the anterior pituitary are highly
specialized for the production of mature secretory granules. Therefore,
we investigated the trafficking of three membrane proteins in primary anterior pituitary endocrine cells. Peptidylglycine Peptidylglycine Soluble and membrane PAM are targeted to the regulated secretory
pathway in different ways (5, 6). By stably expressing wild type and
mutant PAM in AtT-20 corticotrope tumor cells, protein domains
containing trafficking information were identified (7-9). The two
catalytic domains of PAM can be expressed independently, and both are
efficiently targeted to secretory granules. In contrast, membrane PAM
is predominantly localized in the trans-Golgi network (TGN) at steady
state. The small amount of membrane PAM on the cell surface is rapidly
internalized and accumulates in the TGN region (7). The cytosolic
domain of PAM contains information necessary for the trafficking of
this membrane protein within the secretory pathway (7). Membrane
proteins lacking the cytosolic domain are less extensively cleaved by
secretory granule endoproteases, accumulate on the plasma membrane, and
fail to undergo internalization. A marker protein lacking both
catalytic domains of PAM, consisting only of the PAM signal sequence
followed by its transmembrane/cytosolic domain, is highly localized to
the TGN region of AtT-20 cells (10); appending the cytosolic domain of
PAM to Tac, a plasma membrane protein, rerouted Tac to the TGN (11).
The cytosolic tail of PAM interacts with proteins that affect
cytoskeletal organization and expression of membrane PAM alters the
organization of the actin cytoskeleton, perhaps explaining the ability
of membrane PAM to affect secretory granule biogenesis (12).
The endocrine cells of the anterior pituitary are highly specialized
for the production of peptide hormones, their storage, and regulated
release from mature secretory granules (4). Our initial studies of PAM
trafficking in anterior pituitary endocrine cells identified
similarities and clear differences in the trafficking of endogenous PAM
in primary cells and exogenous PAM in AtT-20 tumor cells. As in AtT-20
cells, the endogenous PAM in pituitary cells is subjected to
endoproteolytic cleavage, generating soluble PHM and PAL. In both
cases, PAM processing products are subjected to regulated exocytosis.
Unlike transfected AtT-20 cells, immunostaining and subcellular
fractionation identifies PAM mainly in pituitary secretory granules.
Anterior pituitary cells rapidly internalize membrane PAM from the cell
surface, but unlike AtT-20 cells, massive accumulation of retrieved PAM
in the TGN region is not observed in primary pituitary cells.
To elucidate the trafficking of membrane proteins in endocrine cells
and understand the distinctly different steady state localization of
endogenous PAM in primary pituitary cells and transfected PAM in AtT-20
cell lines, we investigated the trafficking of three membrane proteins
in primary anterior pituitary: membrane PAM (PAM-1); a truncated
version of PAM with only a small lumenal epitope (Myc-TMD/CD); and the
interleukin-2 receptor Primary Anterior Pituitary Cell Cultures--
Cultures were
prepared as described (4). Anterior pituitaries were dissected from the
neurointermediate lobes of 5-20 adult male Harlan Sprague-Dawley
rats (175-200 g; Charles River, Wilmington, MA) and dissociated with
collagenase (Worthington Biochemical Corporation, Lakewood, NJ),
hyaluronidase (Sigma), and benzonase (EM Science, Darmstadt,
Germany), followed by trypsin (Sigma). This procedure consistently
produced 1.5 × 106 cells/anterior pituitary. The
dissociated cells were plated on protamine-coated culture wells in
Dulbecco's modified Eagle's medium with Ham's F-12 medium
supplemented with 10% fetal clone III bovine serum (Hyclone, Logan,
UT) and 10% Nu-Serum IV (Collaborative Research, Bedford, MA) for the
first 24 h and then maintained in the same medium containing 10 µM cytosine arabinoside (Sigma).
Recombinant Adenovirus Constructs--
Where indicated, anterior
pituitary cells were infected with 2 × 103
plaque-forming units/ml of recombinant adenoviruses (V) 72 h after
plating. Recombinant adenoviruses have been described previously (15):
PAM-1V encodes full length rat PAM-1 (nucleotides 293-3245); POMCV encodes mouse POMC (nucleotides 1-920); Myc-TMD/CDV
encodes a chimeric protein described previously (10) in which the PAM signal (amino acids 1-26; nucleotides 298-375) and the pro-domain (amino acids 27-35; nucleotides 376-420) were fused in frame to Myc
epitope followed by the transmembrane domain (TMD) and cytoplasmic domain (CD) (amino acids 858-976; nucleotides 2871-3225) of PAM. For
recombinant adenovirus constructions, cDNAs were subcloned into the
virus shuttle vector (pAdLox) as described previously for PAM-1V and
POMCV (15). HEK293-CRE8 cells were then cotransfected with the shuttle
vector and purified Secretion Experiments and Analysis--
For each secretion
experiment, pituitary cell cultures were initially rinsed for three
30-min periods in complete serum free medium/BSA: Dulbecco's modified
Eagle's medium with Ham's F-12 medium with 1 µg/ml insulin, 0.1 µg/ml transferrin, and 0.1 mg/ml fatty acid-free BSA. Following this
equilibration period, medium was collected for two 1-h periods of basal
secretion (first basal and second basal) followed by a 1-h period of
stimulated secretion using either 1 mM BaCl2 or
1 µM phorbol 12-myristate 13-acetate (PMA) (Sigma)
diluted in complete serum free medium/BSA (4, 16). After the collected
media were centrifuged to remove cell debris, a mixture of protease
inhibitors was added (4), and the samples were stored at PHM and PAL Assays--
PHM and PAL activities were measured in
cell extracts or media as described previously using
ACTH Radioimmunoassay--
Anterior pituitary cells were
extracted in 5 N acetic acid containing protease
inhibitors; the soluble material was lyophilized, dissolved in
immunoassay buffer, centrifuged to remove insoluble material, and
stored frozen. Assays were performed on media and cell extracts using
antibody Kathy (1:20,000) and [125I]ACTH (1-39)
(PerkinElmer Life Sciences). Antiserum Kathy only recognizes POMC
products in which the COOH-terminal end of ACTH (1-39) is exposed
(18).
Subcellular Fractionation--
Cultures prepared from 10 anterior pituitaries were harvested at 4 °C in 10 volumes of
homogenization buffer containing 0.32 M sucrose, 10 mM Tris-HCl, pH 7.4, and protease inhibitors, passed six
times through a 26-gauge needle, and then 12 times through a
ball-bearing homogenizer (H & Y Enterprises, Redwood City, CA) (8).
Cell debris (P1) was removed by centrifugation at 800 × g for 5 min. The resulting supernatant (S1) was separated
into a P2 pellet and soluble fraction (S2) by centrifugation at
20,000 × g for 30 min (2). The P2 pellet largely
enriched in secretory granules was resuspended in homogenization buffer
and fractionated further on a discontinuous sucrose gradient.
Resuspended P2 was layered onto a density gradient consisting of 200 µl each of 0.4, 0.6, 0.8, and 1.0 M sucrose, 350 µl
each of 1.2, 1.4, and 1.6 M sucrose, and 200 µl of 2.0 M sucrose; this gradient was designed to keep the densest
secretory granules from pelleting. Gradients were centrifuged for
2 h at 120,000 × g; 150-µl fractions were collected from the top of the gradient, and proteins in an equal fraction of each sample were analyzed. Finally, a P3 pellet and a
cytosolic fraction (S3) were obtained following centrifugation of S2 at
350,000 × g (2, 4).
Western Blot Analysis--
Samples were fractionated by 10 or
12% SDS-polyacrylamide gel electrophoresis and subjected to Western
blot analysis using rabbit polyclonal antisera to PHM (JH1761, 1:1000),
exon A (JH629, 1:1000), and PAL (JH471, 1:1000) or mouse monoclonal
antibodies to the cytosolic domain of PAM (6E6, 1:50; Refs. 4, 15, and 19), Myc epitope (9E10 hybridoma, 1:50; Ref. 10), and synaptobrevin-2 (VAMP-2, 1: 5000; Synaptic Systems; Ref. 20). Proteins were visualized
using the ECL kit (Amersham Pharmacia Biotech) (21).
Immunofluorescent Staining--
PAM, Myc-TMD/CD, POMC, TGN38,
and VAMP-2 were detected in anterior pituitary cells using indirect
immunofluorescence. Cells (200,000 cells/well of a 4-well slide) were
fixed with 4% paraformaldehyde in phosphate-buffered saline (50 mM sodium phosphate, 150 mM sodium chloride, pH
7.4) for 30 min, permeabilized with 0.075% Triton X-100, and blocked
with 2 mg/ml BSA in phosphate-buffered saline for 1 h at room
temperature. Cells were incubated overnight at 4 °C with rabbit
polyclonal antibodies against PAM exon A (JH629), PAM CD (JH571), or
TGN38 (JH1481) (19), and monoclonal antibodies to Myc (10), PAM-CD
(6E6), and ACTH (Novocastra Labs; Ref. 15), synaptobrevin-2 (VAMP-2;
Synaptic Systems; Ref. 20), Antibody Internalization Experiments--
Primary
pituitary cells were incubated in complete serum free medium/BSA
containing a 1:500 dilution of rabbit polyclonal antiserum to PAM exon
A (JH629) for 20 min at 37 °C and either prepared immediately for
immunofluorescence staining or chased in antibody-free medium for 1 or
2 h at 37 °C (4, 9). To determine whether the endocytosis of
PAM were affected by the binding of bivalent PAM antibody, control
experiments were performed using monovalent Fab fragments. We generated
Fab fragments by digestion of IgG prepared from PAM exon A antiserum
with immobilized papain as described by the manufacturer (Pierce); the
Fab fraction was separated from nondigested IgG and Fc fragments using
immobilized protein A-Sepharose (Sigma). The purity of the Fab
fragments was verified by SDS-polyacrylamide gel electrophoresis.
Primary pituitary cells were incubated with exon A antibody or Fab
fragments prepared from the same serum for 20 min at 37 °C and then
harvested or chased in the absence of antibody for additional time
periods, as described above.
Adenovirus-mediated Increase in PAM Expression in Anterior
Pituitary Cells--
To evaluate the level of protein expression
achieved following infection of primary cells with adenovirus, PHM and
PAL specific activities were compared in PAM-1V-infected pituitary
cells, uninfected pituitary cells (4), anterior pituitary tissue, and
stably transfected AtT-20 PAM-1 cells (Fig.
1, top panel). PHM and PAL specific activities increase 40-50-fold after PAM-1V infection, reaching levels substantially higher than in adult rat anterior pituitary or stably transfected AtT-20 PAM-1 cells (Fig. 1, top panel). Specific activities in PAM-1V-infected pituitary cells are
about 2-fold higher than in adult rat atrium, the richest source of PAM
in the body (24).
To assess the fraction of cells expressing virally encoded PAM, we used
indirect immunofluorescence (Fig. 1, bottom panels). Virally
encoded PAM-1 is expressed at varying levels in essentially all of the
primary pituitary cells (Fig. 1, A and B).
Consistent with the 50-fold increase in PHM and PAL specific
activities, endogenous PAM is not visualized under the same conditions
(Fig. 1, D and E). Because these cultures contain
very few nonendocrine cells (4), almost all of the virally encoded PAM
is expressed in cells proficient at producing secretory granules.
Punctate staining for virally encoded PAM is observed throughout the
cytosol of most of the endocrine cells (Fig. 1C). The
vesicular staining pattern observed for virally encoded PAM mimicks the
pattern observed previously for endogenous PAM in pituitary cells
(4).
Subcellular Localization of Virally Encoded PAM in Anterior
Pituitary Cell Culture--
To better evaluate the subcellular
localization of PAM, PAM-1V-infected pituitary cultures were
simultaneously visualized for PAM (Fig.
2A) and TGN38 (Fig.
2B), a trans-Golgi network marker (25). PAM (in
red) is localized to vesicular structures distributed throughout the cell, whereas TGN38 (in green) is highly localized to a
compact, reticular structure in the perinuclear region of each cell
(Fig. 2B, arrows). Very little PAM is identified
in the TGN region (Fig. 2C). In contrast, double
immunofluorescent staining of PAM-1V-infected pituitary cells for PAM
(Fig. 2D) and VAMP-2 (Fig. 2E), a secretory
granule marker (20), reveals a largely overlapping distribution. As
with endogenous anterior pituitary PAM (4), overlaying the PAM and
VAMP-2 images (Fig. 2F, PAM in green and VAMP-2
in red) reveals a range of colors, with some structures
enriched in PAM and others in VAMP-2. Despite massive
adenovirus-mediated overexpression, membrane PAM is largely localized
to vesicular structures with no accumulation on the cell surface or in
the TGN area.
Localization of Myc-TMD/CD in Anterior Pituitary Cells--
The
routing of integral membrane PAM involves contributions from the two
lumenal, catalytic domains and from the COOH-terminal cytoplasmic
domain (11). To evaluate the importance of the transmembrane and
cytoplasmic domains of PAM in trafficking in anterior pituitary endocrine cells, we generated a recombinant adenovirus encoding Myc-TMD/CD (Fig. 3). In this chimeric
protein (Fig. 3A), the PAM signal and
NH2-terminal regions allow the Myc epitope tag, which precedes the transmembrane and cytoplasmic domains of PAM, to adopt a
lumenal location (10).
Primary anterior pituitary cells were infected with adenovirus encoding
Myc-TMD/CD, and the chimeric protein was visualized simultaneously
using antisera to PAM-CD and Myc (Fig. 3, B and C). With both antisera, vesicular staining distributes
throughout the cytosol of all of the pituitary cells (Fig. 3,
B and C). Antisera to the lumenal domain (Fig.
3C) and to the cytoplasmic domain (Fig. 3B)
exhibited identical patterns. Simultaneous visualization of Myc-TMD/CD
and TGN38 (Fig. 3, D and E) demonstrated
distinctly different patterns; Myc-TMD/CD does not accumulate in the
TGN area (Fig. 3F). In contrast, simultaneous visualization
of Myc-TMD/CD and VAMP-2 (Fig. 3, G and H)
suggests that Myc-TMD/CD, like intact PAM-1, is largely localized to
secretory granules (Fig. 3I).
Western blot analysis of extracts of cultures infected with the
Myc-TMD/CD virus identified a single 23-kDa protein that is recognized
by antibodies to Myc and to the cytosolic domain of PAM (Fig.
4A). To explore further the
trafficking of Myc-TMD/CD in anterior pituitary cells, we used
differential centrifugation followed by sucrose gradient fractionation
(Fig. 4, B and C). The majority of the Myc-TMD/CD
is recovered in the P2 pellet (Ref. 2 and Fig. 4B).
Visualization of VAMP-2 demonstrated the presence of secretory granules
in the P2 fraction (Fig. 4B). The P2 pellet was further
fractionated on a sucrose gradient (Fig. 4C). Fractions 9-11 are enriched in Myc-TMD/CD and in VAMP-2, confirming the localization of Myc-TMD/CD to secretory granules. A similar subcellular localization is observed for the majority of the endogenous anterior pituitary PAM (4) and for virally encoded PAM-1 expressed in anterior
pituitary cells (data not shown). Anterior pituitary endocrine cells
commit a significant percentage of their total protein synthesis to
production of secretory granules (26, 27). Therefore, we considered the
possibility that secretory granule localization might represent the
default trafficking pathway in these cells. To address this issue, we
transfected anterior pituitary cultures with Tac, the interleukin-2
receptor Processing of Overexpressed PAM-1 in Anterior Pituitary
Cells--
Despite the high levels of expression achieved using
adenovirus, anterior pituitary endocrine cells appear to be able to
accommodate the exogenous PAM in vesicular structures (4). To
investigate the ability of pituitary cells to cleave the increased
amounts of PAM, we used Western blot analysis (Fig.
5). Consistent with the dramatic increase
in enzyme activity, analysis of the same amount of protein from
infected versus uninfected cells fails to visualize
endogenous PAM when the exposure times are restricted to yield a
nonsaturated signal for the infected cells; therefore, longer exposures
are shown for uninfected cultures. In PAM-1V-infected pituitary cells,
intact PAM-1 (125 kDa) is processed into membrane PAL (70 kDa), soluble
PAL (50 kDa), and soluble PHM (45 kDa) (Fig. 5A). Production
of these proteins requires cleavage within exon A and between PAL and
the transmembrane domain. As described previously (4), PAM-1-derived
proteins of similar mass are identified in uninfected cell extracts
(Fig. 5A).
To clarify the identity of the cleavage products, antisera to PHM, PAL,
and CD were also used (Fig. 5B). Again, longer exposures of
blots from uninfected cells are shown to facilitate comparison of the
products of endogenous PAM processing (primarily PAM-2 and PAM-3, with
small amounts of PAM-1) and virally expressed PAM-1 processing. The PHM
antibody recognizes PAM-1 and PHM, and the PAL antibody
recognizes PAM-1, mPAL, and sPAL. The CD antibody identifies PAM-1
along with mPAL and a 27-kDa protein containing the transmembrane and
cytosolic domains of PAM (Fig. 5B). Despite the high level
of expression, substantial amounts of virally encoded PAM-1 are cleaved
into products resembling the endogenous products (Ref. 4 and Fig.
5B). The increased fraction of protein recovered as intact
PAM-1 or membrane PAL in virally infected cells suggests that the
cleavage capacity of the cells becomes limiting.
Basal and Stimulated Secretion of PAM Proteins from PAM-1V-infected
Pituitary Cells--
To evaluate the ability of pituitary endocrine
cells to store increased amounts of PAM in the regulated secretory
pathway, we compared the basal and stimulated secretion of PHM activity from PAM-1V-infected and uninfected cultures (Fig.
6A). PHM secretion was
examined during two sequential basal collection periods before stimulation with BaCl2 or PMA (4). Data are expressed in
units (Fig. 6A, left panel) and as percentages of
cell content of PHM activity (Fig. 6A, right
panel). Basal secretion of PHM activity increases about 10-fold
when cell content of enzyme rose 40-fold in response to the PAM-1V
infection. Overexpression of PAM-1 did not result in increased basal
secretion of PHM activity when expressed as a percentage of cell
content/h (0.55 ± 0.1% versus 1 ± 0.1% of cell
content/h, infected versus uninfected, respectively; Fig. 6A, right panel). Overexpression of membrane PAM
does not lead to increased basal secretion of enzyme.
Secretion was stimulated with Ba2+, which mimics the effect
of Ca2+ on regulated exocytosis (28), or with PMA, which
acts via protein kinase C to stimulate a cascade of protein
phosphorylations and induce Ca2+-independent release (29).
BaCl2 stimulates secretion from mature granules, whereas
PMA stimulates Ca2+-independent exocytosis from both
immature and mature pools of vesicles (30). BaCl2 is more
effective at stimulating secretion of PHM activity from uninfected
cells (3.5 ± 0.5-fold; 4.5 ± 0.5% of cell content/h) than
from infected cells (2.5 ± 0.5-fold over basal; 1.3 ± 0.2%
of cell content/h). In contrast, PMA is at least as effective on
infected cells (6 ± 0.4% of cell content/h) as on uninfected
cells (5 ± 0.5% of cell content/h). PMA stimulates PHM secretion
from infected cells 10-fold, whereas secretion from uninfected cells is
only stimulated 4-fold. The ability to stimulate secretion demonstrates
the presence of a significant amount of virally encoded PAM in the
regulated secretory pathway.
The PAM proteins secreted during basal and challenge periods were
subjected to Western blot analysis using antisera to PHM (Fig.
6B). Under basal conditions, soluble, bifunctional PAM
proteins of 110 and 105 kDa are secreted along with 45-kDa PHM (Fig.
6B). Addition of BaCl2 or PMA stimulated
secretion of all three PAM proteins. As expected from the enzyme
assays, PMA was a more potent secretagogue than BaCl2.
Secretion of the 105- and 110-kDa PAM proteins was more responsive to
PMA than to BaCl2. This result suggests that both bi- and
mono-functional PAM proteins derived from PAM-1 processing are stored
in the secretory granules of PAM-1V-infected pituitary cells.
Effect of Exogenous PAM-1 Expression on Pituitary Hormone
Secretion--
Overexpression of PAM-1 in AtT-20 cells impairs the
regulated secretion of the endogenous peptide hormone, ACTH (16). Given the extremely high levels of PAM expression achieved by infection of
primary pituitary cells, we wanted to determine whether secretion of
endogenous hormone were affected. Secretion of GH from
somatotropes and ACTH from corticotropes was evaluated under basal and
stimulated conditions (Fig. 7,
A and B). Based on Western blot analysis, BaCl2 is as effective at stimulating infected and
uninfected somatotropes (Fig. 7A). PMA is a more effective
GH secretagogue than BaCl2, with no differences observed
between infected and uninfected cells. PMA is more effective than
BaCl2 (10-fold versus 2-fold) at stimulating ACTH secretion, with no differences observed between infected and
uninfected cells (Fig. 7B). Unlike the situation in AtT-20 corticotrope tumor cells, overexpression of PAM-1 does not impair regulated secretion from primary somatotropes or corticotropes.
Internalization of Exogenous PAM-1 in Anterior Pituitary
Cells--
The steady state localization of PAM reflects a balance of
fluxes between different subcellular compartments. Internalization of
membrane PAM from the cell surface is an important component of PAM
trafficking and can be monitored by incubating live cells with
antiserum to a lumenal domain of PAM. In PAM-1 transfected AtT-20
cells, PAM internalized from the plasma membrane accumulates in the TGN
region (7). In contrast, endogenous PAM internalized from the surface
of primary pituitary cells is distributed to vesicular structures
dispersed throughout the cell (4). To determine whether overexpression
of PAM in anterior pituitary cells overloads the sorting machinery and
alters trafficking of membrane PAM in the endocytic pathway, we studied
PAM internalization in PAM-1V-infected pituitary cells.
Pituitary cells expressing virally encoded PAM were incubated with exon
A antiserum for 20 min, washed and either harvested or chased for 1 or
2 h. The exon A antiserum binds to PAM-1 on the cell surface, and
the PAM/PAM antibody complex can then undergo endocytosis. The
internalized PAM/PAM-antibody complex is visualized using a fluorescent
second antibody. Localization of the internalized PAM/PAM-antibody
complexes was then compared with the steady state distribution of
various organelle markers. Substantial amounts of PAM/PAM-antibody
complex are internalized from the surface of PAM-1V-infected pituitary
cells (Fig. 8). The signal is much more
robust than that observed from uninfected cells. The intensity of the
signal obtained from the internalized PAM/PAM-antibody complexes
remains constant for hours of chase, suggesting that the internalized
complexes are not degraded or rapidly resecreted. Identical
internalization of PAM-1 proteins was observed when pituitary cells
were incubated with monovalent Fab fragments prepared from the same
exon A antiserum (not shown). Thus, the internalization of PAM from the
surface of pituitary cells was not caused by the binding of bivalent
antibodies.
After the pulse incubation with the exon A antibody, PAM/PAM antibody
complexes are present in small vesicles of uniform size that are
distributed throughout the cytosol (Fig. 8A). These
PAM/PAM-antibody complexes are often colocalized with AP-2, an early
endosomal marker (Refs. 23 and 31 and Fig. 8B). After 1 h of chase, internalized PAM/PAM-antibody complexes are in a more
heterogeneous collection of vesicles predominantly in the TGN area
(Fig. 8E). These vesicles colocalize with Effect of Membrane Protein Overexpression on Filamentous Actin
Organization in Anterior Pituitary Cells--
The cytoskeleton, along
with many cytosolic proteins, governs secretory granule formation,
maturation, translocation, and exocytosis (33-35). PAM-1 interacts,
via its cytosolic domain, with proteins that play a powerful
role in cytoskeletal organization (12, 36). In AtT-20 cells, expression
of PAM-1 alters the organization of the actin cytoskeleton (12, 16).
Using FITC-phalloidin, we studied the distribution of filamentous actin
in pituitary endocrine cells expressing PAM-1 or Myc-TMD/CD (Fig.
9). For comparison, filamentous actin
staining was evaluated in uninfected cells and in POMCV-infected
pituitary cultures. Uninfected (Fig. 9, A and B)
or POMCV-infected (Fig. 9, C and D) cells show
organized patches of filamentous actin throughout the cytosol, with
more intense signal at the margins of the cell immediately beneath the
plasma membrane (Fig. 9, B and D).
In contrast, PAM-1V-infected endocrine cells (Fig. 9, E and
F) exhibit a different pattern of filamentous actin. The
patches of filamentous actin are gone, and filamentous actin is no
longer concentrated under the plasma membrane (Fig. 9F). In
some PAM-1V-infected cells, filamentous actin localizes to the TGN
region of the cell, overlapping regions enriched in PAM protein (Fig.
9F). Filamentous actin staining in cells expressing
Myc-TMD/CD (Fig. 9, G and H) is indistinguishable
from staining in cells expressing PAM-1 and quite distinct from the
pattern in uninfected and POMCV-infected cells.
The single most striking difference between the behavior of
endogenous PAM in primary pituitary cells and exogenous PAM in AtT-20
cells is the steady state localization of the protein (4). In pituitary
cells, as in hypothalamic neurons and cultured atrial myocytes, PAM is
concentrated in secretory granules (3, 37). When integral membrane PAM
proteins are expressed in the neuroendocrine AtT-20 cell line, much of
the protein is found in the vicinity of the Golgi apparatus (4, 19). At
the immunoelectron microscopic level, a significant amount of overlap
is observed between transfected PAM and endogenous TGN38, with PAM
expression enriched in the more distal compartments of the TGN
(19).
To explore trafficking to secretory granules in cells equipped to
assemble a large number of secretory granules, we expressed three
membrane proteins in primary anterior pituitary cells. Using the
adenovirus system, PAM-1 is expressed at varying levels in essentially
all of the primary pituitary cells. Following infection, PAM enzyme
activity, both PHM and PAL, is 40-50-fold higher than in adult rat
anterior pituitary or stably transfected AtT-20 PAM-1 cells.
Adenovirally mediated PAM expression in pituitary is high, but only
double the physiological level of PAM in adult rat atrium (24). The
overexpressed membrane PAM is localized primarily to vesicular
structures, with very little PAM in the TGN region and no accumulation
of PAM on the cell surface. This distribution stands in sharp contrast
to the TGN accumulation observed in AtT-20 PAM-1 cells (19). The
largely overlapping PAM and VAMP-2 localization, with variable ratio of
PAM and VAMP-2, is consistent with current models of vesicle biogenesis
and with continuous removal of membrane proteins and lumenal content
via clathrin coated vesicles (38, 39).
Our earlier studies in AtT-20 cells clearly identified routing
determinants in both the lumenal and cytosolic domains of membrane PAM
(8, 9, 16, 19). Expressed independently, both lumenal domains are
efficiently stored in secretory granules (4, 40). Studies in AtT-20
cells indicated that the cytosolic domain of membrane PAM contains
endocytic trafficking information, but a role for cytosolic signals in
granule entry was not apparent. PAM mutants truncated so that they lack
most of the cytosolic domain are less extensively cleaved by secretory
granule endoproteases, are localized on the plasma membrane, and fail
to undergo internalization (19). Like intact PAM, Myc-TMD/CD, lacking
both catalytic (lumenal) domains, is highly localized to the TGN region
of AtT-20 cells (10), and appending the cytosolic domain of PAM to Tac
reroutes Tac to the TGN (11). In contrast, Myc-TMD/CD is enriched in the secretory granules of anterior pituitary endocrine cells. This
result suggests the presence of granule targeting signals in the
transmembrane and/or cytosolic domain of PAM.
AtT-20 cells contain far fewer secretory granules than pituitary
endocrine cells (18, 41). In many systems, lumenal proteins enter
vesicular traffic derived from the TGN by default (42). Sorting of
regulated secretory proteins is then accomplished passively by removal
from immature granules of a subset of protein components. Therefore, we
considered the possibility that secretory granules are a default
pathway for membrane proteins in pituitary cells. However, Tac, which
accumulates on the cell surface when expressed in a variety of
endocrine and nonendocrine cells (11, 13), also localizes to the plasma
membrane of anterior pituitary endocrine cells. Therefore, pituitary
secretory granules do not represent a default pathway, and the granule
localization of Myc-TMD/CD indicates that it contains a secretory
granule routing signal. P-selectin is a platelet and endothelial cell
granule membrane protein. In AtT-20 cells, exogenous P-selectin is
targeted to secretory granules by signals in its cytoplasmic tail and
transmembrane domain (43-45). Recently, Cutler and co-workers (46)
demonstrated that the same cytoplasmic signal is required for the
appearance of P-selectin in immature and mature dense core vesicles as
well as synaptic-like microvesicles in PC12 cells.
Anterior pituitary endocrine cells show an immense storage capacity for
exogenous PAM. Remarkably, overexpression of PAM-1 in pituitary cells
actually decreases the basal secretion of PHM activity as a percentage
of cell content. Although high levels of expression do not lead to PAM
accumulation on the cell surface, there is clearly some limitation to
PAM proteolytic processing, with intact membrane PAM accounting for a
higher percentage of the PAM protein in infected cells than in
uninfected cells. Secretion of PHM is stimulated by both
BaCl2 and PMA. Secretion of PHM from the BaCl2
responsive compartment is limited in pituitary cells overexpressing
PAM-1 (1.3% of cell content/h versus 4.5% from uninfected
cells). Assuming that BaCl2 is stimulating
Ca2+-dependent exocytosis from mature granules
(28, 47), these data suggest that access to mature granules is limited
upon overexpression of membrane PAM. BaCl2-stimulated
release of GH and ACTH occurs normally following overexpression of PAM,
indicating that mature granules form normally. Access of PAM to mature
granules seems to be impaired.
PMA stimulated release of a comparable percentage of enzyme content/h
from uninfected and PAM-1-infected pituitary cultures (5 ± 0.5%
and 6 ± 0.4% respectively). PMA stimulates protein kinase C,
inducing Ca2+-independent exocytosis via phospholipase D,
which is known to activate secretory vesicle budding from the TGN (29,
48). Thus PMA is able to affect secretion at earlier steps of the
secretory pathway, including immature as well as mature granules
(diagramed in Fig. 10). PAM-1
overexpression in pituitary endocrine cells results in a dramatic
expansion of the PAM content of the immature and mature pools of
granules (Fig. 10); basal secretion from infected cells is stimulated
10-fold by PMA, whereas basal secretion from uninfected cells is
stimulated only 4-fold. The limited endoproteolytic cleavage of virally
expressed PAM also suggests accumulation in immature granules. PMA
stimulated GH and ACTH secretion normally following overexpression of
PAM-1.
-amidating monooxygenase (PAM), an integral membrane protein essential to the production of many bioactive peptides, is cleaved and enters the
regulated secretory pathway even when expressed at levels 40-fold
higher than endogenous levels. Myc-TMD/CD, a membrane protein lacking
the lumenal, catalytic domains of PAM, is still stored in granules.
Secretory granules are not the default pathway for all membrane
proteins, because Tac accumulates on the surface of pituitary endocrine
cells. Overexpression of PAM is accompanied by a diminution in its
endoproteolytic cleavage and in its BaCl2-stimulated release from mature granules. Because internalized PAM/PAM-antibody complexes are returned to secretory granules, the endocytic machinery of the pituitary endocrine cells is not saturated. As in corticotrope tumor cells, expression of PAM or Myc-TMD/CD alters the organization of
the actin cytoskeleton. PAM-mediated alterations in the cytoskeleton may limit maturation of PAM and storage in mature granules.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amidating monooxygenase
(PAM)1 is a bifunctional
enzyme involved in the posttranslational processing of many prohormones
and neuropeptides. PAM catalyzes the formation of
-amidated peptides
from peptide precursor molecules with a COOH-terminal glycine. In
neurons and endocrine cells, biologically active peptides are stored in
secretory granules that undergo regulated release in response to
external stimuli. Localized in secretory granules of many neural and
endocrine tissues, PAM is one of a small number of
posttranslational processing enzyme occurring naturally in soluble
and membrane forms (1-4). For this reason, we have used PAM to
investigate the trafficking of soluble and membrane proteins into
secretory granules (5, 6).
chain (Tac), normally a T-lymphocyte plasma
membrane protein (13, 14). We used recombinant adenovirus to
overexpress membrane PAM or Myc-TMD/CD in anterior pituitary cells; Tac
was expressed in the same cells by transfection. Using
immunofluorescent staining and subcellular fractionation, we show that
the transmembrane and cytosolic domains of PAM are sufficient to target
the protein to the secretory granules. In parallel, the capacity of
pituitary endocrine cells to cleave and store greatly increased amounts of PAM has been assessed. Although overexpression of PAM alters the
organization of the actin cytoskeleton, neither the regulated secretion
or the endocytic machinery is affected in anterior pituitary cells.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5 adenoviral DNA to produce recombinant
adenovirus. The next day, the virus-containing cell culture medium was
replaced with serum-containing medium; for all experiments the cells
were used 48 h after infection, but cells were viable for at least
2 weeks after infection. pCDM·Tac expression vector, kindly provided
by Dr. Juan Bonifacino (Cell Biology and Metabolism Branch, National
Institutes of Health), was transiently transfected in anterior
pituitary cells using GenePorter (Gene Therapy Systems) following the
manufacturer's protocols. Mouse AtT-20 corticotrope tumor cells
expressing PAM-1 (AtT-20 PAM-1) were grown in Dulbecco's modified
Eagle's medium with Ham's F-12 medium supplemented with 10% fetal
clone III bovine serum, 10% Nu-Serum IV, and G418 as described
previously (6).
80 °C.
Cells were scraped into 20 mM NaTES, 10 mM
mannitol, 1% Triton X-100, pH 7.4, containing protease inhibitors and
used for PHM or PAL assays or for Western blot analysis. Rat anterior
pituitary tissue was homogenized in the same buffer. Before use,
samples were frozen and thawed three times and centrifuged for 5 min to
remove debris.
-N-acetyl-Tyr-Val-Gly and
-N-acetyl-Tyr-Val-OH-Gly substrates, respectively (17).
Samples were assayed in duplicate, and reactions were carried out for
1.5 h. PHM and PAL specific activity is expressed as picomoles of
product formed per hour (units) per microgram of protein or as a
percentage of the corresponding total enzyme activity in the cell extract.
-adaptin (AP-1; Transduction
Laboratories; Ref. 22), and
-adaptin (AP-2) (Transduction
Laboratories) (23). Permeabilized or nonpermeabilized anterior
pituitary cells expressing membrane Tac protein were stained using a
monoclonal antibody 7G7 (AMAC, Westbrook, ME) directed against the
lumenal domain of Tac. The antigen-antibody complexes were visualized
using FITC-conjugated goat anti-rabbit IgG (Caltag, San Francisco, CA)
or Cy3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West
Grove, PA). To detect filamentous actin, cells were stained with
FITC-phalloidin (0.1 µg/ml; Sigma). Cells were viewed with a Zeiss
Axioskop microscope (Carl Zeiss, Thornwood, NY) and photographed with a
Micromax CCD camera (Princeton Instruments, Princeton, NJ) or a Spot RT
camera (Diagnostic Instruments, Sterling Heights, MI).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PAM-1 overexpression in primary anterior
pituitary cells. After 3 days in culture, primary anterior
pituitary cells were infected with recombinant adenovirus encoding
PAM-1; infected and uninfected cultures were analyzed 2 days later.
Top panel, duplicate samples of pituitary cultures, adult
anterior pituitary (AP), or stably transfected AtT-20 PAM-1
cells were extracted for measurement of PHM and PAL activities. Data
are the means ± SD (n = 4). Bottom
panels, cultures were fixed and permeabilized, and PAM was
visualized using antibody to exon A and FITC-tagged goat anti-rabbit
IgG. PAM-1V-infected cells (A and B) and
uninfected cells (D and E) were visualized under
identical conditions; immunofluorescence (A and
D) and the corresponding phase-contrast image (B
and E) were used. At higher magnification (C),
the vesicular nature of the PAM staining (Ves) is apparent,
as is the unstained cell nucleus (N).
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Fig. 2.
Subcellular localization of exogenous PAM-1
in primary pituitary cells. A-C, primary pituitary
cells infected with the PAM-1 virus were simultaneously visualized with
mouse monoclonal antibody to PAM-CD (A,
arrowheads) and rabbit polyclonal antibody to TGN38
(B, arrows). The primary antibodies were then
visualized with fluorescently tagged secondary antisera (C,
PAM, red; TGN38, green). D-F, Cells
were simultaneously visualized with antisera to PAM (exon A) and VAMP-2
(D and E, arrows). Primary antibodies
were visualized with fluorescently tagged secondary antisera (PAM,
green; VAMP-2, red). F, higher
magnification images were superimposed; areas visualized by both
antibodies appear yellow (arrows), vesicles
stained only by VAMP-2 antibody are indicated by lines, and those
stained only by PAM antibody are indicated by
arrowheads. The magnification was the same for
A-E.
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Fig. 3.
Myc-TMD/CD localization in anterior pituitary
cells. A, diagram showing Myc-TMD/CD relative to PAM-1.
B-I, primary pituitary cells infected with the Myc-TMD/CD
virus were simultaneously visualized with two antisera. B
and C, Myc-TMD/CD protein was localized using antisera to
PAM-CD (FITC) and to Myc (Cy3). Myc-TMD/CD (D,
arrowheads) was visualized simultaneously with TGN38
(E, arrows); the superimposed images are shown in
F. Myc-TMD/CD (G) was visualized simultaneously
with VAMP-2 (H). The superimposed images (I)
demonstrate PAM/VAMP colocalization (yellow,
arrows). The scale bar for B-H is
shown in B.
chain, a membrane protein that normally resides on the
surface of T-lymphocytes (13, 14). Tac was previously shown to
accumulate on the surface of AtT-20 cells (11). Immunofluorescent
staining of nonpermeabilized pituitary cells, using an antibody against
the lumenal domain of Tac, demonstrates that a significant amount of
the expressed protein is present on the cell surface (Fig.
4D). The localization of Tac is distinctly different from
that of PAM-1 or Myc-TMD/CD, which are not visualized without
permeabilization (11). Thus, the transmembrane/cytosolic domain of PAM
is sufficient to direct the protein to secretory granules in pituitary
endocrine cells. The ability of Myc-TMD/CD to direct trafficking to
secretory granules is not apparent in AtT-20 cells because both
Myc-TMD/CD and membrane PAM are largely localized to the TGN area of
AtT-20 cells at steady state.
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Fig. 4.
Biochemical analysis of pituitary cells
expressing Myc-TMD/CD. A, extracts of anterior
pituitary cultures infected with the Myc-TMD/CD virus were subjected to
Western blot analysis and visualized with antisera to Myc and PAM-CD.
Anterior pituitary cultures infected with the Myc-TMD/CD virus were
subjected to differential centrifugation (B), and the
secretory granule enriched 20,000 × g pellet (P2) was
further fractionated on discontinuous sucrose density gradients
(C) (2). Myc-TMD/CD was identified by Western blot analysis
using antibody (Ab) to Myc and secretory granules were
identified using a VAMP-2 antibody. D and E,
anterior pituitary cells were transfected with vector encoding Tac.
Transfected Tac was visualized by immunofluorescent staining of fixed,
nonpermeabilized (D), or permeabilized cells (E)
using a monoclonal antibody to Tac (11).
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Fig. 5.
Exogenous PAM-1 processing in anterior
pituitary cultures. The major cleavage products of PAM-1 are
diagrammed (A, left side; apparent molecular
masses in kDa are indicated in italics). Empty
lollipop, N-linked oligosaccharide; filled
lollipop, O-linked oligosaccharide. Equal amounts of
protein from PAM-1V-infected and uninfected cultures extracted with
TES-mannitol/Triton X-100 were subjected to Western blot analysis. PAM
proteins were visualized with antisera to exon A (A), PHM,
PAL, or PAM-CD (B). The molecular mass markers are shown.
Because the PAM proteins in uninfected cultures were barely visible
under conditions optimized for analysis of infected cultures, longer
exposures are shown for these samples (indicated by dashed
lines). Ab, antibody.
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Fig. 6.
Basal and stimulated secretion of PHM
activity and PAM protein from PAM-1V-infected pituitary cells.
Infected and uninfected cultures were incubated in basal medium for two
sequential 1-h periods and then exposed to either 1 mM
BaCl2 or 1 µM PMA for 1 h. Cultures were
extracted in 20 mM NaTES, 10 mM mannitol, 1%
Triton X-100, pH 7.4. A, PHM activity measured in duplicate
samples of medium was expressed as units of product formed per
microgram of protein (left) and as percentage of cell
content of enzyme activity (right). Data are the means ± S.D. for four cultures for each secretagogue; where error estimates
are small, error bars are not visible. Similar results were
obtained in three additional independent experiments. B,
equal amounts of basal and stimulated media from PAM-1V-infected cells
were analyzed by Western blot and probed with antiserum to PHM.
Ab, antibody.
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Fig. 7.
Overexpression of PAM-1 does not impair
regulated secretion. The secretion experiments described in the
legend to Fig. 6 were subjected to additional analysis. A,
GH secretion was evaluated by Western blot analysis of medium samples.
B, ACTH secretion was determined by radioimmunoassay.
Similar results were obtained in three additional complete experiments.
Ab, antibody.
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Fig. 8.
Internalization of PAM ectodomain antibody
under basal conditions. PAM-1V-infected anterior pituitary cells
were incubated with exon A rabbit antibody for 20 min and either
harvested (pulse, 20 min; A-D) or allowed to internalize
PAM-antibody complexes for 1 h or 2 h at 37 °C
(E-H). Fixed, permeabilized cells were immunostained
using monoclonal antibodies to various subcellular markers: AP-2 for
early endosome, AP-1 for immature granules, and VAMP-2 for secretory
granules. The internalized PAM-antibody complex (in green)
and the different markers (in red) were visualized as
described under "Materials and Methods." Arrows indicate
PAM containing vesicles; arrowheads show areas of yellow
staining when internalized PAM colocalizes with one of the subcellular
markers.
-adaptin AP-1,
part of the adaptor complex required for the assembly of
clathrin-coated buds from the Golgi (Refs. 31 and 32 and Fig.
8F). Although internalized PAM/PAM-antibody complexes
are observed in the TGN area, no accumulation is observed. After a 2-h
chase, the PAM/PAM-antibody containing vesicular structures are
coincident with secretory granules visualized by antiserum to VAMP-2
(20) (Fig. 8, G and H). Overexpression of PAM in
anterior pituitary cells does not appear to overload the endocytic
machinery; internalization of PAM-1 is similar in infected and
uninfected cells (4).
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Fig. 9.
Effect of PAM-1 overexpression on filamentous
actin in anterior pituitary cells. Uninfected pituitary cells
(A and B) and POMCV-infected (C and
D), PAM-1V-infected (E and F), and
Myc-TMD/CDV-infected (G and H) cells were stained
using the PAM exon A antiserum (A and E), an ACTH
(C), or PAM-CD (G) monoclonal antibody and
FITC-phalloidin. Cells were photographed under identical conditions.
Filamentous actin staining throughout the cytosol is indicated by
arrows; areas with an intense filamentous actin signal
around the cell edges (arrowheads) or in the TGN region
(asterisk) are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 10.
Trafficking of membrane PAM in primary
pituitary cells. A, directed by targeting signals
encoded by both the lumenal and the cytosolic domains, endogenous PAM
proteins exit immature secretory granules (ISG) and have
unlimited access to mature secretory granules (MSG), which
are stored for days; very little newly synthesized PAM enters endosomes
for recycling to the TGN or basal secretion. CLV,
constitutive-like vesicle. Very little PAM is on the cell surface at
steady state and PAM appearing on the plasma membrane (PM)
is rapidly endocytosed. B, when overexpressed in primary
pituitary cells, PAM basal secretion is not impaired. PAM proteins
accumulate in the immature secretory granules affecting the actin
cytoskeleton via PAM-CD interactors, which delay the maturation of
secretory granules and entry into the BaCl2 stimulatable
pool of vesicles. Therefore, PAM endoproteolytic cleavage is less
efficient. No PAM accumulation is observed on the cell surface, and PAM
proteins reaching the cell surface are retrieved via the endosomal
compartment and directed to the secretory pathway.
The cytosolic tail of PAM interacts with proteins involved in cytoskeletal organization and expression of membrane PAM or Myc-TMD/CD alters regulated secretion in AtT-20 cells, perhaps by affecting the actin cytoskeleton (12, 16). Similarly, virally overexpressed PAM and Myc-TMD/CD affect filamentous actin organization in anterior pituitary cells (Fig. 10). In contrast, overexpression of the soluble POMC precursor is without effect on the actin cytoskeleton, ruling out nonspecific effects of adenovirus-mediated expression. PAM-1 and Myc-TMD/CD-infected cells exhibit an absence of punctate actin aggregates, instead exhibiting diffuse staining for filamentous actin and some concentration of filamentous actin in the TGN region (Fig. 10). The accumulation of filamentous actin in the TGN area may be involved in the accumulation of membrane PAM in immature granules following overexpression.
As observed for endogenous anterior pituitary PAM and for PAM-1 transfected AtT-20 cells, robust internalization of PAM/PAM-antibody complexes from the plasma membrane occurs under basal conditions. In both cell systems, internalized PAM/PAM-antibody complexes are initially present in small uniform vesicles distributed all over the cell. Later a more heterogeneous collection of intracellular vesicular structures is observed. PAM retrieved from the surface of anterior pituitary cells passes through an early endosomal compartment recognized by antisera to the plasma membrane adaptor, AP-2 (23, 31) (Fig. 10). At later times, the recycled PAM/PAM-antibody complex is more localized with AP-1 (31, 32). By 2 h, internalized PAM largely colocalizes with VAMP-2 in secretory granules (20). As for endogenous PAM, the enzyme internalized from the surface of pituitary endocrine cells was never collected in the TGN region (4). Therefore, overexpression of membrane PAM in anterior pituitary endocrine cells does not overload the endocytic sorting machinery, and membrane PAM retrieved from the pituitary cell surface has rapid access to secretory granules. In the same context, it has been demonstrated that VAMP-2-stained synaptic vesicles in neuroendocrine PC12 cells form by budding from tubular extensions of sorting endosomes; granule proteins together with the transferrin receptor are delivered to the early endosomes, where they are sorted into synaptic-like microvesicles and recycling vesicles (49). In the current study we propose targeting of recycled membrane PAM from the pituitary cell surface to the secretory granules via the endosomal compartment.
In summary, we propose a model of PAM trafficking in primary pituitary
cells where the transmembrane and/or cytosolic domains of PAM is
sufficient to target the protein to secretory granules (Fig. 10). PAM
proteins leaving immature secretory granules largely go to mature
granules, with a smaller amount of PHM and the bifunctional enzyme
undergoing constitutive-like secretion (4). Little membrane PAM reaches
the cell surface, and the amount that does is rapidly internalized via
the endosomal compartment for recycling toward the TGN or toward basal
secretion (Fig. 10A). Upon overexpression, PAM proteins
accumulate in immature secretory granules (Fig. 10B). The
expression of membrane PAM affects cytoskeletal organization, probably
via interactor proteins that recognize the CD of PAM, resulting in a
delay of secretory granule maturation and an alteration of PAM
processing. Overexpression of membrane PAM does not lead to PAM
accumulation on the cell surface, nor does it overload the endocytic pathway.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Lixian Jin and Kate Deanehan for help with tissue culture and Marie Bell for general laboratory assistance. We thank Dr. Victor May for critically reading this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK-32949.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: Dept. of Neuroscience,
University of Connecticut Health Center, 263 Farmington Ave.,
Farmington, CT 06030-3401. Tel.: 860-679-8898; Fax:
860-679-1885; E-mail: eipper@uchc.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M008062200
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ABBREVIATIONS |
---|
The abbreviations used are:
PAM, peptidylglycine
-amidating monooxygenase;
PHM, peptidylglycine
-hydroxylating
monooxygenase;
PAL, peptidyl-
-hydroxyglycine
-amidating lyase;
TGN, trans-Golgi network;
TMD, transmembrane domain;
CD, cytoplasmic
domain;
BSA, bovine serum albumin;
PMA, phorbol 12-myristate
13-acetate;
NaTES, sodium
N-Tris[hydroxymethyl]methyl-2-aminoethansulfonic acid;
ACTH, adrenocorticotropic hormone;
FITC, fluorescein isothiocyanate;
GH, growth hormone;
POMC, proopiomelanocortin;
POMCV, POMC
adenovirus.
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