From the Baker Medical Research Institute,
§ Department of Pathology and Immunology, Monash
University Medical School, Commercial Road, Prahran,
Victoria 3181, Australia
Received for publication, July 18, 2000, and in revised form, October 8, 2000
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ABSTRACT |
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The dynamin family of GTP-binding proteins has
been implicated as playing an important role in endocytosis. In
Drosophila shibire, mutations of the single dynamin gene
cause blockade of endocytosis and neurotransmitter release, manifest as
temperature-sensitive neuromuscular paralysis. Mammals express three
dynamin genes: the neural specific dynamin I, ubiquitous dynamin II,
and predominantly testicular dynamin III. Mutations of dynamin I result
in a blockade of synaptic vesicle recycling and receptor-mediated
endocytosis. Here, we show that dynamin II plays a key role in
controlling constitutive and regulated hormone secretion from mouse
pituitary corticotrope (AtT20) cells. Dynamin II is preferentially
localized to the Golgi apparatus where it interacts with G-protein
Dynamin is a polypeptide with a modular structure comprising a
GTP-binding domain in the N-terminal third, a middle domain of unknown
function, a pleckstrin homology
(PH)1 domain and a C-terminal
proline-rich or Src homology 3- (SH3-) binding domain (for reviews, see
Refs. 1-8). Mammals have at least three dynamin genes which code for
dynamin I, II, and III. Although the homology between dynamin I and II
proteins is 79% and between dynamin I and III proteins is 89%,
significant variation occurs at the C-terminal regions. In addition,
dynamin I is specifically expressed in neural tissues, whereas dynamin
II is ubiquitous and dynamin III is predominantly testicular. These
developmentally divergent dynamins may thus represent a large protein
family apparently performing a range of functions in association with
distinctive sites in mammals (1, 2).
While dynamin I has been implicated as playing an important role in
mediating synaptic vesicle recycling, little is known of the
physiological function of dynamin II. Since dynamin I complexes a large
ring-like structure surrounding the necks of clathrin-coated endocytic
pits on the cytoplasmic surface of presynaptic plasma membrane during
synaptic vesicle recycling (3, 6), it is thought that dynamin II plays
a similar role to that of dynamin I in receptor-mediated endocytosis in
non-neuronal cells (4-8). In support of this hypothesis are the
findings that the GTPase activities of both dynamin I and II, and of
their SH3-binding domain truncation mutants, are similarly stimulated
in vitro by phospholipids, grb2, and microtubules (9),
suggesting that the modes of interaction of the PH domains from dynamin
I and II with phosphatidylinositol (4,5)-bisphosphate and of the
SH3-binding domain with microtubules and grb2 are similar in
vitro (9). However, it is not yet established why dynamin I and II
are concomitantly present in neurons (4), and why rapid endocytosis in
adrenal chromaffin PC12 cells is significantly inhibited by the
microinjected PH domain of dynamin I but not by the PH domain of
dynamin II (10). Given the previous reports that dynamin II associates with the trans-Golgi network in human hepatic (HepG2) cells
(11), and that dynamin I and/or II immunoreactivity is present in the Golgi apparatus of human foreskin melanocytes and fibroblasts (12), it
is conceivable that dynamin II might have a different function in
membrane trafficking from the endocytic role of dynamin I in mammalian
cells (4). To establish a biological function of dynamin II, the
present study was undertaken to explore the activity of dynamin II in
mouse pituitary corticotropes (AtT20 cells). We found that dynamin II
plays a key role in controlling both constitutive and regulated hormone
secretions at the Golgi apparatus of these neuroendocrine cells.
Chemicals and Peptides--
ATP, GTP, creatine phosphate, and
creatine kinase were from Roche Molecular Biochemicals Australia Pty.
Ltd. (New South Wales, Australia). Na125I was from
PerkinElmer Life Sciences Australia Pty. Ltd. (NSW, Australia). The
peptide hormones rat CRH1-41, Tyr0-CRH, and
Preparation of Dynamin Expression Constructs and Expression of
Recombinant Proteins--
Expression vectors for wild-type,
GTP-binding mutation, SH3-binding mutation, and PH domain alone of
dynamin I and II were generated by subcloning PCR recombinant cDNA
fragments into pcDNA3HA plasmids with CMV and SV40 promoters
flanking the multiple cloning site to drive expression of the cDNA.
pcDNA3HA was derived from pcDNA3 (Invitrogen, NV Leek, The
Netherlands) by subcloning a double-stranded oligonucleotide sequence
containing a BamHI restriction site and a downstream
sequence coding for HA peptide (YPYDVPDYA). Dynamin II PCR fragments
were made with dynamin IIaa cDNA (13) as template and the following
oligonucleotides: gacccgggcaccatgggcaa (jp17c) and
gccttaagcagcctagtcgagcag (jp16) for dynamin wild-type, jp17c, and
acgaattctagctgctggtgttct (jp18) for the dynamin II SH3-binding
deletion, and ggagatcttggt (jp3) and cggaattcccagctcgcag (jp4) for the
PH domain only mutant. For dynamin GTP-binding mutation, the method of
PCR-ligation-PCR was used as described previously (14), using the
oligonucleotides jp17c, jp16, gccggcgctctgg (jp10b), and
gccagttcgtgtctcgagaac (jp9). For the dynamin I GTP-binding mutant, the
oligonucleotides used were: ggctctagatctaccatgggcaaccgcggcatg (jp19),
accgagcttgcgccggcgctctggcc (jp22), gctggagaatttcgtggg (jp21b), and
tcggaattcgatctggttagaggtcgaagg (jp20c). For the dynamin I PH domain
only mutant oligonucleotides were used: gatgagatcttggtcattcgaaaggggt (jp23) and acggaattcacgctcagggta (jp24). For antisense expression plasmids, two dynamin II cDNA fragments were cloned into
pcDNA3HA vectors in an antisense direction to generate dynamin II
antisense 1 (nucleotide
Cloned pcDNA3HA vectors with various dynamin cDNAs (except the
antisense constructs) were sequenced and used for in vitro translation with a commercial kit (Promega, Madison, WI) to verify the
sequences of the cDNA inserts and the molecular sizes of the expressed proteins. The expression vectors were then transfected into
monolayer cultures of AtT20 cells (D16-16, kindly provided by Karen
Sheppard) with LipofectAMINE (10 µl/2 µg of DNA/ml). After about 4 weeks of selection with G418 (400 µg/ml), cells were tested for
expression of recombinant dynamin cDNAs, protein localization,
receptor-mediated endocytosis, vesicular production, and hormone
release. In all cases, we used either normally cultured AtT20 cells or
stably transfected cells after G418 selection as indicated in the
individual experiments. For the glutathione S-transferase dynamin II PH domain fusion protein a DNA fragment, generated by
specific PCR using dynamin IIa cDNA as template and the synthetic oligonucleotides (ggagatcttggt (jp3) and (cggaattcccagctcgcag (jp4)) as
primers, was subcloned into the pGex5X-3 plasmid and after sequence
verification transformed into Escherichia coli BL21 to
produce the fusion proteins under
isopropyl-1-thio- Antibodies--
Dynamin II antibodies were raised in rabbits by
multiple immunization with the synthetic peptides against the PH domain
peptide (607CDSQEDVDSWKASFLRA) or the C-terminal peptide
(858PTIIRPAEPSLLD). The antibodies were affinity purified
against the PH domain peptide or the C-terminal peptide, respectively, as described elsewhere (15). Other antibodies used to detect proteins
include anti-HA epitope monoclonal antibody (12CA5 hybridoma), anti-rat
dynamin I antibody (DP4.1+) (16), anti-ACTH antibody (R72)
(17), anti-Rab6 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA), anti- Cell Culture, Receptor-mediated Endocytosis, Hormone Secretion,
MAP Kinase Activity, and DNA Synthesis--
Mouse pituitary
corticotrope (AtT20) cells or rat pheochromocytoma (PC12) cells were
maintained in exponential growth in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum, 2 mM
glutamine in an enclosed humidified atmosphere of 5% CO2 in air at 37 °C. For receptor-mediated endocytosis,
Tyr0-CRH and transferrin were iodinated by IODO-GEN
(Pierce). CRH (2.3 µg) or transferrin (40 µg) dissolved in
phosphate-buffered saline (PBS) was incubated with 1 mCi of
[125I]Na in an IODO-GEN-precoated tube for 30 min at room
temperature with intermittent mixing. Free iodine was removed from
[125I-Tyr0]CRH on a Sep-Pak C18
cartridge and from 125I-transferrin on a Sephadex
G-25 (PD10, Amersham Pharmacia Biotech). Cells (1 × 106) stably expressing wild-type or mutated dynamin II were
incubated in triplicate with 0.5 nM
[125I-Tyr0]CRH or
125I-transferrin in the presence or absence of 10 times
excess amount of relevant unlabeled ligand for different time periods
at 22 °C, and washed 10 times with binding buffer, twice with 50 mM glycine (pH 2.5) containing 150 mM NaCl, and
lyzed with 0.2 M NaOH containing 0.25% SDS. The rate and
degree of ligand binding and receptor-mediated endocytosis were
determined by counting surface-bound ligand in acid extracts and
internalized ligand in SDS lysates in a Subcellular Fractionation, Immunoblotting, and
Immunoprecipitation--
Sucrose gradient subcellular fractionation
was performed as described previously (20, 21). Briefly, cells (5 × 106) stably expressing wild-type or mutated dynamins
were lyzed in buffer containing 0.25 M sucrose and
centrifuged (500 × g for 5 min) to remove nuclei and
debris. Post-nuclear lysates were loaded on a stepwise sucrose gradient
(0.8-2 M) and centrifuged at 35,000 rpm for 15 h at
4 °C in a rotor (model SW4.1 Ti, Beckman). After fractionation,
proteins in each fraction were analyzed by immunoblotting and
immunoprecipitation. In some experiments, highly purified Golgi
fractions were used by diluting the fractions 3-5 with 1.8 M sucrose followed by sequential overlays with 1.18 and 0.96 M sucrose and centrifugation as described (11). For
immunoblotting, proteins in 15 µl of each fraction were separated by
electrophoresis on a 10% acrylamide minigel, transferred to 0.45-µm
nitrocellulose membranes, and detected by incubation with specific
primary antibodies followed by further incubation with a
peroxidase-conjugated anti-IgG and then with enhanced chemiluminescence
reagents (Amersham Pharmacia Biotech). All steps were performed at room
temperature. For immunoprecipitation, 100 µl of the Golgi
fraction was subjected to an incubation at 4 °C for 1 h with
specific primary antibodies in the presence or absence of glutathione
S-transferase-PH domain fusion protein (10 µg) followed by
another incubation with Gammabind plus Sepharose (Amersham Pharmacia
Biotech) and extensive washing. Immunoprecipitated proteins were
resolved in SDS-PAGE followed by immunoblotting with different antibodies.
Immunofluorescence Microscopy--
AtT20 cells or PC12 were
grown on glass coverslips and after washing in PBS subjected to
immunofluorescence staining as described previously (22). Briefly,
cells were prefixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 min, free aldehyde groups quenched in 50 mM
NH4Cl in PBS, and the cells permeabilized with 0.1% Triton X-100 for 5 min at room temperature. After washing and blocking for 30 min with 1% bovine serum albumin, cells were incubated for 1 h at
room temperature with the primary antibodies diluted in 1% bovine
serum albumin in PBS, followed by washing and another incubation with
fluorescein isothiocyanate-conjugated sheep anti-rabbit IgG (Silenus,
Australia) for 1 h at room temperature. After a further wash in
PBS, the coverslips were mounted onto glass slides with 2.6% DABCO
(1,4-diazabicyclo-(2,2,2)octane; Sigma) in 90% glycerol, 10% PBS (pH
8.6). Slides were then analyzed in a confocal laser scanning imaging
system (Bio-Rad MRC 1024 imaging system). The confocal images were
captured by a 60X/1.4 Nikon oil lens (Nikon ECLIPSE E600). For dual
labeling experiments, cells were incubated with affinity purified
rabbit anti-dynamin II antibody (1:2000 dilution in 1% bovine serum
albumin/PBS) for 1 h at room temperature, washed, and then
incubated with fluorescein isothiocyanate-conjugated sheep anti-rabbit
IgG, which was then followed by washing and further incubation with
human anti-p230 antibodies. After washing in PBS, cells were incubated
with tetramethylrhodamine isothiocyanate (TRITC)-conjugated sheep
anti-human IgG (Dako Corp., Botany, Australia). After a further wash,
the coverslips were mounted as above. Control incubations demonstrated
no cross-reactivity between the conjugated secondary antibodies or
between the primary antibodies and unrelated second antibodies.
Secretory Vesicle Production--
Nascent vesicular formation
from the Golgi was assessed according to published methods with minor
modifications (20, 21). Briefly, 10 × 106 cells
stably expressing wild-type or mutated dynamin II were treated with
lysis buffer (0.25 M sucrose, 10 mM HEPES-KOH,
pH 7.2, 1 mM EDTA) or swelling buffer (10 mM
Hepes-KOH, pH 7.2, 10 mM NaCl) at 4 °C for 5 min for
vesicle formation analysis in vitro and in intact cells,
respectively. Cells in the lysis buffer were further lyzed by passing
them through a 22-gauge needle 5 times, followed by centrifugation at
600 × g for 10 min to remove the nuclei, and of the
post-nuclear supernatant at 14,000 × g for 10 min at
4 °C to obtain the Golgi-rich membrane fraction. In contrast, cells
in swelling buffer were passed through the 22-gauge needle once to
generate semi-permeable cells (90% by trypan blue staining), followed
by washing in 5 volumes of breaking buffer (100 mM KCl, 10 mM HEPES-KOH, pH 7.2, 100 units/ml Trasylol). The
Golgi-rich membranes and semi-permeable cells were incubated in the
nascent vesicle buffer (10 mM Hepes-KOH, pH 7.3, 0.5 mM CaCl2, 2.5 mM MgCl2,
110 mM KCl, 35 mM KOAc, 10 mM
creatine phosphate, 80 µg/ml creatine phosphate kinase, 0.5 mM phenylmethylsulfonyl fluoride, and 5 µl/ml Trasylol)
for different times at 37 °C, with or without cytosolic proteins (1 mg/ml), ATP (1 mM), and GTP (0.05 mM). The
incubations were then terminated by centrifugation at 14,000 × g for 10 min at 4 °C to separate the supernatant
presumably containing nascent vesicles from the Golgi or
Golgi-containing semi-permeable cells, followed by dilution of the
supernatants and resuspension of the pellets with 5 mM Tris
(pH 7.4) containing 0.1% Triton X-100 and different protease
inhibitors. The cytosol and nucleotide hydrolysis dependent vesicular
formation activity was assessed by measuring the percentage of
Expression of Dynamin II Regulates
To further explore the physiological role of dynamin II in hormone
secretion, two dynamin II antisense expression plasmids were generated
and transfected into AtT20 cells. Transfection of the cells with either
antisense construct resulted in a decrease of endogenous dynamin II by
50-60% (Fig. 2A) and a
reduction of Dynamin II Resides in the Golgi Apparatus--
To further
characterize the action of dynamin II during hormone secretion, the
subcellular distribution of dynamin II was studied by fractionation of
cultured AtT20 cells followed by immunoblotting. As shown in Fig.
3, dynamin II immunoreactivity was
predominantly found in fractions 3-5 from the top of a continuous
sucrose gradient (0.8-2 M). These were confirmed as Golgi
containing fractions by the presence of the Golgi marker proteins Rab6
and
Confocal immunofluorescence microscopy was also employed to localize
dynamin II in cultured AtT20 cells, using the antibody against dynamin
II C-terminal tail sequence (858PTIIRPAEPSLLD). Significant
immunofluorescent staining was observed in the juxtanuclear regions of
cultured AtT20 (Fig. 4). When the staining was compared with that seen with an antibody against the TGN
protein p230 (18, 22), dynamin II was found to colocalize with p230 in
cultured AtT20 cells, either nontransfected or transfected with control
plasmids (Fig. 4). In contrast, in cells transfected with dynamin II
antisense plasmids, staining for p230 was clearly visible but little
staining for dynamin II was observed, suggesting that levels of
endogenous dynamin II were significantly lowered by dynamin II
antisense RNA. To corroborate the Golgi localization of dynamin II in
another type of neuroendocrine cells, subcellular localization of
dynamin II and p230 was determined in adrenal chromaffin PC12 cells. As
shown in Fig. 5, dynamin II and p230 colocalized in the perinuclear regions of PC12 cells with or without treatment by nerve growth factor (NGF-7S, 1 µg/ml) for 24 h.
These data suggest that dynamin II is preferentially associated with the Golgi apparatus in both AtT20 and PC12 cells. Given that the antibody used to localize dynamin II is against the C-terminal tail
region that does not differentiate dynamin II alternative splicing
product (13), it is formally possible that the dynamin II associated
with the Golgi may be a dynamin II splice variant that is a predominant
form in the neuroendocrine cells.
Overexpression of Dynamin II PH Domain, or a Dynamin II Mutant
Lacking the SH3-binding Domain, Induces Translocation of Endogenous
Dynamin II from the Golgi Apparatus to the Plasma Membrane--
To
explore roles for the C-terminal PH- and SH3-binding domains of dynamin
II in subcellular localization, constructs coding for wild-type dynamin
II, dynamin II GTP-binding-domain mutant, dynamin II PH domain only
mutant, and the dynamin II mutant lacking the SH3-binding domain were
prepared and stably transfected into AtT20 cells. On confocal
microscopy these transfected cells showed a striking alteration of the
subcellular localization of endogenous dynamin II visualized with
antibody against the C-terminal tail region of dynamin II. As shown in
Fig. 6, in cells transfected with
wild-type dynamin II (panel A series) or dynamin II
GTP-binding domain mutant (panel B series) dynamin II
colocalized with the TGN protein p230 at the Golgi apparatus. In cells
transfected with the dynamin II SH3-binding domain deletion mutant,
however, staining for p230 remained unchanged, with the majority of
staining for dynamin II found on the plasma membrane, and the Golgi
apparatus almost completely devoid of dynamin II staining (Fig. 6,
panel C series). An identical pattern of staining for
dynamin II was also seen in cells transfected with the dynamin II PH
domain only construct (Fig. 6, panel D series). These
findings suggest that overproduction of either the dynamin II mutant
missing the C-terminal SH3-binding domain or the dynamin II PH domain
only mutant induces the translocation of endogenous dynamin II from the
Golgi apparatus to the plasmalemma. The predominant staining of dynamin
II at the plasma membrane showed a discrete punctate distribution to regions close to the Golgi apparatus in these mutant-transfected cells
(Fig. 6, panel C and D series).
Dynamin II Translocation from the Golgi Apparatus to Plasma
Membrane Is Associated with a Decrease of Hormone Release and an
Increase in Transferrin Receptor-mediated Endocytosis--
Given that
overexpression of the dynamin II mutant without the SH3-binding domain
or of the dynamin II PH domain only mutant induces translocation of
authentic dynamin II from the Golgi apparatus to plasma membrane, it is
clearly of interest to determine whether secretion of
What was unexpected was that transferrin receptor-mediated endocytosis
was significantly enhanced by either the dynamin II mutant lacking the
SH3-binding domain or the dynamin II PH domain only mutant (Fig.
7B). Overexpression of the SH3-binding domain deletion
mutant almost doubled the levels of receptor internalization in control
cells. Although less potent than the SH3-binding domain deleting
mutant, overexpression of the PH domain also stimulated receptor
internalization. Thus, in addition to the requirement of the
GTP-binding domain, the SH3-binding domain also plays an obligatory
role in dynamin II-regulated hormone release at the Golgi apparatus.
Once translocated by the overproduction of dynamin II mutants, the
endogenous dynamin II can then apparently functionally mimic dynamin I
in terms of receptor-mediated endocytosis at the plasma membrane.
Interaction between Dynamin II and G-protein Dynamin II Regulates The present study defines for the first time dynamin II as a Golgi
membrane trafficking protein required for both constitutive and
regulated hormone secretion from neuroendocrine cells. A striking consequence of loss of dynamin II function in neuroendocrine cells is
lowered hormone secretion with both basal and CRH-stimulated secretion
of In addition, structure-function analysis suggests a crucial role for
the PH domain in targeting dynamin II to the Golgi. The finding that
overexpressed dynamin II PH domain induces dissociation of endogenous
dynamin II from the Golgi and inhibition of hormone secretion strongly
suggests that the PH domain of dynamin II is involved in interacting
with Golgi membrane docking molecule(s) of dynamin II. Given that PH
domains from diverse proteins are capable of binding to the phosphate
groups of acidic phospholipids (such as phosphatidylinositol
(4,5)-bisphosphate) (30), it is possible that dynamin II interacts with
these lipid molecules but is specifically targeted at the Golgi
apparatus by protein-protein interactions involving its PH domain. In
an attempt to search for dynamin II interactive proteins, we detected
no interaction between dynamin II and In addition to the PH domain, our data also support a role for the
SH3-binding domain in dynamin II-mediated secretory vesicle production
at the Golgi apparatus. Overexpression of dynamin II lacking the
SH3-binding domain not only induces dislocation of endogenous dynamin
II from the Golgi but also produces inhibition of hormone secretion.
The translocation of endogenous dynamin II from the Golgi to the plasma
membrane suggests an involvement of the SH3-binding domain in dynamin
II association with secretory vesicle membranes (Fig.
10). Thus, uncoupling of the
SH3-binding domain renders dynamin II ineffective in mediating
secretory vesicle biogenesis, an effect paralleling previous finding
for dynamin I that disruption of the SH3-binding domain impairs
synaptic vesicle recycling (34).
subunit and regulates secretory vesicle release. The presence of
dynamin II at the Golgi apparatus and its interaction with the
subunit are mediated by the pleckstrin homology domain of the
GTPase. Overexpression of the pleckstrin homology domain, or a dynamin II mutant lacking the C-terminal SH3-binding domain, induces
translocation of endogenous dynamin II from the Golgi apparatus to the
plasma membrane and transformation of dynamin II from activity in the secretory pathway to receptor-mediated endocytosis. Thus, dynamin II
regulates secretory vesicle formation from the Golgi apparatus and
hormone release from mammalian neuroendocrine cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-endorphin were from Peninsula Laboratories, Inc. (San Carlos, CA).
The dynamin II peptides (607CDSQEDVDSWKASFLRA and
858PTIIRPAEPSLLD) were synthesized by Chiron Technologies
Pty. Ltd. (Victoria, Australia). Gel electrophoresis reagents were from Bio-Rad. Human transferrin (T2252), myelin basic protein,
protein A-Sepharose 4B, and all other chemicals were from Sigma.
8-129) and 2 (nucleotide
8-1617).
-D-galactopyranoside induction.
- and
-adaptin antibodies (Transduction
Laboratories, Lexington, KY), anti-G
antibody (T-20, Santa Cruz
Biotechnology, Inc.), anti-Golgi
-COP antibody (Sigma), anti-human
p230 antiserum (18), and anti-ERK (C-16) antibody (Santa Cruz
Biotechnology, Inc.).
-counter. For hormone
secretion, cells (1 × 105) stably expressing
wild-type or mutated dynamin II were incubated in triplicate with or
without CRH (10 nM) for different times, with
-endorphin
released into the incubation medium and retained within the cells
measured by radioimmunoassay using antibody (R56) that detects both
-endorphin and its precursor POMC peptide (17). For cellular
signaling pathways underlying cellular growth and differentiation,
mitogen-activated protein (MAP) kinase activity and DNA synthesis were
determined by the methods described elsewhere (19).
-endorphin produced in the supernatant over total cellular
-endorphin immunoreactivity after subtraction of the control values
obtained from the incubation in the absence of cytosolic proteins, ATP
and GTP, and this percentage then compared between different groups of
transfected cells. The levels of
-endorphin were measured by radioimmunoassay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Endorphin Secretion from
AtT20 Cells--
To explore the cellular function of dynamin II,
dynamin II mutants were generated and stably expressed in AtT20 cells.
While transfection of cultured AtT20 cells with wild-type or mutated dynamin II showed no effect on clathrin-dependent and
-independent receptor internalization (data not shown), both basal and
CRH-stimulated hormone secretion were modulated over 2-48 h of
observation by overexpression of either wild-type or mutated dynamin II
(Fig. 1), with the pattern of release of
-endorphin triggered by KCl (not shown) or CRH from the different
transformants remaining similar. Overexpression of wild-type dynamin II
significantly enhanced both basal and CRH-stimulated secretion of
-endorphin, whereas the single amino acid mutation in dynamin II
GTP-binding domain (K44A) lowered both basal and CRH-induced
-endorphin release (30-60% reduction relative to plasmid-only
control cells, and 50-75% to the wild-type). To determine whether
these effects of dynamin II on hormone secretion were due to
alterations in
-endorphin synthesis, cellular content of
-endorphin was examined. Cellular content of
-endorphin was not
significantly changed between the different groups of cells transfected
with control plasmids, wild-type, or GTP-binding domain-mutated dynamin
II over a period from 2 to 24 h (not shown). To determine whether
the effects of dynamin II on hormone secretion might have resulted from
nonspecific changes in cellular proliferation or signaling, basal DNA
synthesis and MAP kinase activity in response to epidermal growth
factor and the phorbol ester, phorbol 12-myristate 13-acetate were
measured in the different transformants. No significant difference was observed for either DNA synthesis or MAP kinase activity between control and dynamin II mutants (Fig. 1C). Given that changes
in hormone release induced by dynamin II variants are accompanied by no
change in cellular content and increased total synthesis of the
hormone, it is likely that dynamin II may be involved in regulating
secretory vesicle transport with the coupling between hormone secretion
and synthesis not being affected; enhanced effects on secretion may
lead to enhanced levels of synthesis to replenish the released stores
of
-endorphin.
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Fig. 1.
Overexpression of dynamin II and its
GTP-binding domain mutant regulates both basal and CRH-stimulated
secretion of -endorphin from neuroendocrine
AtT20 cells. A, cells stably transfected with empty
vectors (open bars), wild-type dynamin II (hatched
bars), or dynamin II GTP-binding domain mutant
(cross-bars) were incubated with (A) or without
(B) 10 nM CRH for different times as indicated
and
-endorphin release measured by radioimmunoassay using a specific
antibody (R56). Both basal and stimulated secretion of
-endorphin
were enhanced by wild-type dynamin II and suppressed by the dynamin II
GTP-binding domain mutant. Data are mean ± S.E. from two to three
similar experiments determined in duplicate. C,
overexpression of dynamin II GTP-binding domain mutant does not affect
MAP kinase activity of AtT20 cells. MAP kinase (ERK1/2) was
immunoprecipitated from the cultured cells transfected with empty
vector (control) or the dynamin II GTP-binding domain mutant (K44A)
following treatments of the cells for 20 min with or without the
phorbol 12-myristate 13-acetate (0.1 µM) or epidermal
growth factor (EGF) (0.1 µM) as indicated.
Immunoprecipitates were incubated with myelin basic protein
(MBP) at 32 °C for 15 min in appropriate protein
phosphorylation buffer (19), and phosphorylation of MBP was examined
through SDS-PAGE followed by autoradiography as shown in the
upper panel and immunoreactive MAP kinase (ERK1/2)
demonstrated by Western blotting shown in the lower panel.
The data were from a representative of two experiments.
-endorphin secretion at rest as well as after
stimulation by CRH at different concentrations (Fig. 2B). To
determine the specificity of the effect of dynamin II on
-endorphin
secretion, we examined whether the dynamin I GTP-binding domain mutant
might have such an effect. As shown in Fig. 2B, the dynamin
I GTP-binding mutant had no significant effect on
-endorphin
secretion. On the other hand, transfection with the dynamin II
antisense constructs showed no effect on transferrin receptor-mediated
endocytosis, whereas transfection with the dynamin I GTP-binding domain
mutant showed about 40% inhibition compared with the control (Fig.
2C). These findings, that decreasing or increasing cellular
expression of dynamin II inhibits or stimulates hormone release from
neuroendocrine cells, suggest that the dynamin II levels regulate the
level of hormone secretion in vivo.
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Fig. 2.
Expression of endogenous dynamin II plays an
important role in hormone secretion. A, expression of
two dynamin II antisense constructs inhibits endogenous dynamin II
synthesis in AtT20 cells. Cells were stably transfected with empty
vectors (lanes 1-3), dynamin II antisense construct 1 (lanes 4-6), or dynamin II antisense construct 2 (lanes 7-9), and 5, 10 and 20 µg of cellular extract
analyzed for each transformant by SDS-PAGE followed by immunoblotting
with anti-dynamin II antibody. B, underexpression of dynamin
II inhibits hormone secretion. Cells transfected with empty vectors
( ), dynamin II antisense 1 (
), dynamin II antisense 2 (
), or
dynamin I GTP-binding mutant (
) were incubated with or without
various concentrations of CRH as indicated for 8 h and
-endorphin release was determined. While the dynamin I mutant does
not significantly affect hormone secretion, underexpression of dynamin
II inhibits
-endorphin release which is similar to that observed
with the dynamin II GTP-binding mutant seen in Fig. 1. The
letter B denotes basal condition without CRH. Data are
mean ± S.D. from one of two similar experi ments. C, underexpression of dynamin II does not
inhibit transferrin receptor-mediated endocytosis. Cells transfected
with empty vectors (
), dynamin II antisense 1 (
), dynamin II
antisense 2 (
), or dynamin I GTP-binding mutant (
) were incubated
with 125I-transferrin for indicated times and after
extensive washing, the amount of the internalized
125I-transferrin receptor complex in the cells was
determined as described above. Nonspecific binding was determined in
the incubation of cells transfected with empty vectors with
125I-transferrin in the presence of 10 times excess cold
ligand (127). The extent of endocytosis is presented as percentage of
receptor internalization in vector-only control cells. Values shown are
mean ± S.D. (n = 3).
-COP (Fig. 3), and the trans-Golgi network (TGN)
protein p230 (18, 22) (data not shown). In contrast, no immunoreactive
dynamin II was found in the plasma membrane fractions containing plasma
membrane receptors for transforming growth factor
R1 (Fig. 3).
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Fig. 3.
On subcellular fractionation dynamin II
colocates with the Golgi proteins Rab6 and
-COP. Total cell lysates were subjected to
continuous sucrose gradient (0.8-2 M) centrifugation
followed by fractionation from the top of the centrifugation column.
Fractions from 1 to 13 were used to immunoblot for different proteins
with specific antibodies. While receptors for transforming growth
factor
(TGF
R1) were found in fraction
9-11 (A), dynamin II (B), Rab6 (C),
and
-COP (D) were detected in the Golgi-enriched
fractions (fractions 3-5; sucrose 1.1-1.2 M). Results are
representative of four similar experiments.
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Fig. 4.
The perinuclear distribution of dynamin II
overlaps with that of the TGN protein p230 by double immunofluorescent
labeling. Nontransfected AtT20 cells (A) and the
cells stably transfected with empty vector (B) were
incubated with rabbit anti-dynamin II C-terminal tail antibody and
fluorescein isothiocyanate-conjugated sheep antibodies to rabbit IgG,
with specific immunofluorescent staining shown in green. The
same cells (A' and B') as in A and
B were further incubated with human anti-p230 antibodies and
TRITC-conjugated sheep anti-human IgG, with the specific staining shown
in red. Comparison of the immunofluorescent staining
(A" and B") of dynamin II and p230 by
superimposing image (A and B) on the image
(A' and B') shows dynamin II and p230
colocalization (A" and B", yellow). No
significant staining for dynamin II in AtT20 cells transfected with
dynamin II antisense plasmid 1 (C) and 2 (D) was
seen, while the staining for p230 in these AtT20 cells (C'
and D') remained the same. C" and D"
show staining for both dynamin II and p230 by superimposing image
(C and D) on the image (C' and
D'), evidence for the expression of dynamin II specifically
inhibited in comparison with p230.
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Fig. 5.
Immunofluorescence micrographs illustrating
colocalization of dynamin II and p230 in PC12 cells. PC12 cells
under normal culture conditions were treated with or without nerve
growth factor (NGF-7S, 1 µg/ml) for 24 h before being
fixed in paraformaldehyde. The cells were probed with anti-dynamin II
C-terminal tail antibody followed by fluorescein
isothiocyanate-conjugated sheep antibodies to rabbit IgG, after
extensive wash, the cells were probed again with anti-p230 antibody
followed by TRITC-conjugated sheep anti-human IgG, and the cells were
then stained with bisbenzimide for DNA and examined by confocal
microscopy.
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Fig. 6.
Overexpression of dynamin II PH domain or a
mutant lacking the SH3-binding domain causes translocation of
endogenous dynamin II from the Golgi to plasma membrane. A,
A', A", visualization by confocal microscopy of AtT20
cells stably transfected with wild-type dynamin II shows colocalization
of dynamin II (in green, A) with p230 (in red,
A') and the superimposition (in yellow, A") of
A and A'. B, B', and B",
colocalization of dynamin II (in green) with p230 (in
red) at the Golgi (arrows) in the cells stably
transfected with the dynamin II mutant carrying a GTP-binding domain
mutation. C, C', C", dissociation of the
distribution of endogenous dynamin II (in green) in plasma
membrane from that of p230 (red) at the Golgi in the cells
stably transfected with the dynamin II mutant lacking the C-terminal
tail SH3-binding domain. The arrows indicate dynamin II and
p230 staining. D, D', and D", dissociation of the
distribution of endogenous dynamin II (in green) in plasma
membrane from that of p230 (red) at the Golgi in the cells
stably transfected with the PH domain of dynamin II. Arrows
indicate dynamin II and p230 staining.
-endorphin and
transferrin receptor-mediated endocytosis are affected by the dynamin
II variants. Overexpression of the dynamin II mutant lacking the
SH3-binding domain significantly reduced
-endorphin release from the
cells, with 60-70% of reduction in both basal and CRH-stimulated
-endorphin release (Fig.
7A). Similarly, overexpression
of the PH domain also inhibited both basal and stimulated
-endorphin
secretion, although the extent of inhibition was less than that with
the SH3-binding domain deletion mutant (Fig. 7A).
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Fig. 7.
Overexpression of dynamin II PH domain only
mutant, or a mutant lacking SH3-binding domain, causes inhibition
of -endorphin release but a stimulation of
transferrin receptor-mediated endocytosis. A, dynamin
II PH domain or the SH3-binding domain deletion mutant inhibits both
basal and CRH-stimulated release of
-endorphin. Cells stably
transfected with empty vector (
), dynamin II PH domain (
), or the
SH3-binding domain deletion mutant (
) were incubated with or without
CRH at the concentrations indicated, and
-endorphin release
determined by radioimmunoassay. The letter B represents at
the basal condition without CRH. Data are mean ± S.D. of two
experiments determined in duplicate. B, dynamin II PH domain
or SH3-binding domain deletion mutants stimulate transferrin
receptor-mediated endocytosis. Cells stably transfected with empty
vector (
), dynamin II PH domain (
), or the SH3-binding domain
deletion mutant (
) were incubated with 125I-transferrin
for different periods of time as indicated and the internalization of
125I-transferrin receptor complex was determined. Data are
mean ± S.D. of two experiments.
Subunit at the
Golgi Apparatus--
In an attempt to determine the proteins with
which dynamin II interacts at the Golgi apparatus, we
immunoprecipitated dynamin II from purified Golgi fractions and then
probed the immunoprecipitates with various antibodies. Since dynamin I
has been shown to interact with
-adaptin of AP-2 adaptor protein at
plasma membrane, we determined whether
-adaptin of AP-1 might
coimmunoprecipitate with dynamin II from the Golgi fraction, and found
no evidence for such an interaction (data not shown). Significant
levels of immunoreactive G-protein
subunit were, however, found in
the dynamin II immunoprecipitates by immunoblotting analysis, while no
G-protein
subunit was detected in the immunoprecipitates obtained
with antibodies against the Golgi proteins
-COP or Rab6 (Fig.
8A). In addition,
immunoreactive dynamin II was found in the immunoprecipitates obtained
with anti-G-protein
subunit antibody, but not in the
immunoprecipitates obtained with anti-
-COP antibody. Interestingly,
immunoprecipitated dynamin II was observed as a single major band under
denatured conditions (not shown), but as multiple oligomers when
partially denatured in 0.5 mM dithiothreitol in SDS-PAGE
(Fig. 8B). Furthermore, the coimmunoprecipitation of dynamin
II with G-protein
subunits was blocked by addition of a PH
domain glutathione S-transferase fusion protein to the lysates before immunoprecipitation (Fig. 8B). These data
suggest that dynamin II interacts with the
subunits of
G-proteins through its PH domain at the Golgi apparatus.
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Fig. 8.
Molecular interaction between dynamin II and
the subunit of G-proteins at
the Golgi. A, immunoblotting analysis of the
subunit of G-proteins in immunoprecipitates. Immunoprecipitates
obtained with mouse anti-
-COP (lane 1), rabbit
anti-dynamin II (lane 2), rabbit anti-Rab6 (lane
3) or rabbit anti-G
subunit antibodies were subjected to
SDS-PAGE followed by immunoblotting with anti-
subunit antibody. The
detected
subunit is indicated with an arrow. The less
anti-
-COP antibody signal was due to the low cross-reactivity by the
second antibodies against rabbit IgG. B, immunoblotting
analysis of dynamin II in immunoprecipitates. Immunoprecipitates
obtained with antibodies specific to
-COP (lane 1),
G-protein
subunit (lane 2), G-protein
subunit in the
presence of dynamin II PH domain-glutathione S-transferase
fusion protein (lane 3), dynamin II PH domain (lane
4), or dynamin II tail (lane 5) were subjected to
SDS-PAGE followed by immunoblotting with anti-dynamin II tail antibody.
Immunoreactive dynamin II as monomer, dimer, and other oligomers is
indicated by arrows.
-Endorphin Release from the Golgi Apparatus
in Vitro and in Semi-permeabilized AtT20 Cells--
To explore the
biological function(s) of dynamin II at the Golgi apparatus, we have
determined the effects of differential expression of dynamin II
constructs on the production of secretory vesicles from the Golgi
apparatus, by assessing the release of
-endorphin through nascent
vesicular budding from the Golgi apparatus in vitro and in
semi-intact cells. As shown in the bottom panel of Fig.
9, incubation of semi-permeabilized cells
in the presence of ATP, GTP, cytosolic proteins, Mg2+,
Ca2+, creatine phosphate, and creatine phosphate kinase led
to increases in
-endorphin immunoreactivity in the medium,
reflecting presumptive de novo biogenesis from the Golgi
apparatus of nascent vesicles containing
-endorphin. Compared with
control,
-endorphin immunoreactivity in the medium was reduced in
cells transfected with the dynamin II GTP-binding domain mutant, but
increased in cells transfected with wild-type dynamin II. Consistent
with these findings obtained with semi-permeabilized intact cells,
presumptive nascent vesicular release from the Golgi-rich membrane
fraction was also decreased in cells expressing the dynamin II
GTP-binding domain mutant, and increased in wild-type dynamin
II-transfected cells (Fig. 9). Since basal levels of
-endorphin
release were relatively high (>50%) in this particular experiment, it
is possible that nonspecific membrane degradation may contribute the
high background and some of
-endorphin release in control and wild
type cells, but the significant decrease in
-endorphin release from
the mutant provided clearer evidence for the involvement of dynamin II
in regulating Golgi biogenesis of secretory vesicles. Thus, dynamin II
may regulate secretory vesicle production from the Golgi apparatus, consistent with the recent report that dynamin II mediates coated vesicle formation from the Golgi apparatus in broken cell systems (23).
Additional studies are required to provide direct evidence for dynamin
II regulation of vesicular budding from the TGN of AtT20 cells at the
electron microscope level.
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Fig. 9.
Dynamin II regulates
-endorphin secretory vesicle formation from the
Golgi of neuroendocrine AtT20 cells. Golgi-enriched membrane
preparations (A) and permeabilized cells (B) were
incubated with or without cytosol (1 mg/ml) or both ATP (1 mM) and GTP (0.05 mM) in a buffer containing
Ca2+ (0.5 mM), Mg2+ (2.5 mM), K+ (110 mM), creatine
phosphate (10 mM), and creatine phosphate kinase (80 µg/ml) at 37 °C for different times as indicated. Samples were
subsequently separated into pellet and nascent vesicle-containing
supernatant fractions and
-endorphin immunoreactivity determined
after dilution with lysis buffer. Percentages of the cytosol- and
nucleotide hydrolysis-dependent
-endorphin release from
the Golgi are presented and compared between the cells transfected with
empty vector (open bars), wild-type dynamin II
(hatched bars), or dynamin II carrying a mutation in its
GTP-binding domain (cross bars). The results are mean ± S.D. of triplicate determinations from one of five similar
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-endorphin being affected. A similar phenotype is also found when
antisense mRNA is used to block the synthesis of endogenous dynamin
II. By contrast, an increase in
-endorphin secretion is observed
when wild-type dynamin II is overexpressed. Thus, the extent of both
constitutive and regulated hormone secretions from these neuroendocrine
cells may be limited by the expression levels of endogenous dynamin II
under physiological conditions. This finding is in line with previous
findings that dynamin II is associated with the Golgi vesicular budding
in vitro (23) and in cultured rat epithelial cells (24), and
that microinjection of dynamin II does not mimic that of dynamin I on
rapid endocytosis in adrenal chromaffin cells (10). Since dynamin II
has also been implicated in caveolae endocytosis in hepatocytes (25) and receptor endocytosis in adipocytes (26), HeLa cells and Madin-Darby
canine kidney cells without affecting exocytotic pathways (27, 28), it
is possible that the cellular functions of dynamin II are largely
determined by its specifically targeted subcellular localization in a
cell specific fashion. Subcellular localization studies in
neuroendocrine cells suggest the presence of dynamin II at the Golgi
apparatus, consistent with dynamin II-regulated hormone secretion.
Furthermore, overexpression of the wild type or dynamin II mutant
regulates de novo production of secretory vesicles from
purified Golgi apparatus and semi-permeabilized cells. Thus, dynamin II
may be a major form of the dynamin family playing an obligatory role in
secretory vesicle biogenesis at the Golgi apparatus during hormone
secretion from neuroendocrine cells, consistent with the most recent
findings that dynamin II regulates post-Golgi transport of a
plasma-membrane protein (29).
-adaptin by
immunoprecipitation, although previous studies have shown that dynamin
I interacts with
-adaptin (31) and dynamin II mediates
clathrin-coated vesicle budding from the TGN (23). In contrast, the
interaction between dynamin II and the
subunit of G-proteins via
the dynamin II PH domain is specific, since the small GTP-binding
protein Rab6 does not coimmunoprecipitate with G-protein
subunit,
nor does dynamin II with
-COP (a Golgi coatamer protein also
possessing a WD40 repeat). Previous studies have also shown that
purified
subunits bind to purified dynamin (32) and stimulate
secretory vesicle formation (33).
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Fig. 10.
A model of the mechanisms postulated to
underlie the actions of dynamin II and its variants in regulating
secretory vesicle budding from the TGN. Interactions between
dynamin II variants (colored), G-protein subunit
(
), and a putative SH3-containing protein (L shaped)
are illustrated with secretory vesicles on and off the TGN membrane.
A, decreased expression of dynamin II by specific antisense
expression is associated with a decreased dynamin II interaction with
G-protein
subunit and secretory vesicle formation from the TGN.
B, normally expressed dynamin II binds to the
subunit
at the TGN with its PH domain and to an SH3-containing protein on the
budding vesicle with its SH3-binding or proline-rich domain
(PR). Once the vesicle is pinched off, dynamin II is
retained at the Golgi and then directed to another vesicle undergoing
biogenesis, through an SH3-binding domain interaction. C,
overexpression of wild type dynamin II is associated with increased
dynamin II interactions with both
subunit and SH3-containing
protein(s) and release of the secretory vesicles from the TGN.
D, overexpression of dynamin II GTP-binding domain mutant
inhibits secretory vesicle release. By analogy with dynamin I, this may
reflect the inhibition of GTP hydrolysis-induced vesicle pinching off.
E, overexpression of the dynamin II mutant lacking the
SH3-binding domain displaces endogenous dynamin II at the Golgi but is
without activity in mediating vesicle budding. The dislocated
endogenous dynamin II is then carried on secretory vesicles to the
plasma membrane by an interaction involving the SH3-binding domain.
F, overexpression of dynamin II PH domain also causes a
re-distribution of endogenous dynamin II by competing for the binding
site of endogenous dynamin II at the Golgi and a decrease in secretory
vesicle biogenesis.
It is also noteworthy that the SH3-binding domain deletion mutant is
more potent than the PH domain mutant in causing inhibition of hormone
release (Fig. 7A) and stimulation of receptor-mediated endocytosis (Fig. 7B). This difference in potency may
reflect a difference between the mutants in competing with endogenous dynamin II at the Golgi. Since the region immediately C-terminal to the
PH domain of -adrenergic receptor kinase is also involved in binding
to the
subunit (35), it is possible that other regions outside
the PH domain of dynamin II are also involved in targeting the protein
to the Golgi, and thus that the SH3-binding domain deletion mutant is
more effective than the PH domain-only mutant in causing translocation.
Once on the plasma membrane, the misallocated dynamin II is capable of
mediating clathrin-dependent endocytosis, suggesting that
dynamin II is structurally competent to participate in both secretory
and endocytic vesicular trafficking, and that its physiological role in
the secretory pathway is determined by its localization to the Golgi.
Collectively, these data show that dynamin II GTPase is a dominant
regulator of both constitutive and regulated hormone secretions from
neuroendocrine cells, operating as a polymer at the Golgi controlling
secretory vesicle biogenesis. During this process, dynamin II interacts
with subunit of G-proteins via a region containing the PH
domain; disruption of the interaction causes translocation of dynamin
II from the Golgi to plasma membrane. The parallel increases and
decreases in dynamin II expression with constitutive and regulated
hormone secretion are consistent with expressed levels of dynamin II
playing an important physiological role in regulating the capacity of
cell secretion in response to environmental stimuli. Molecular
targeting to particular dynamin isoforms may therefore provide a
potential therapeutic means for controlling Golgi protein trafficking
and hormone secretion under particular circumstances (36).
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ACKNOWLEDGEMENT |
---|
We thank S. Kawashima, P. J. Robinson, and T. C. Sudhof for materials and advice, D. Autelitano and R. Dilley for methodological assistance, and S. L. Sabol and K. Sheppard for supplying the AtT20 cells.
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FOOTNOTES |
---|
* This work was supported by grants from the Australian Research Council, National Health and Medical Research Council of Australia, and National Heart Foundation of Australia.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: Molecular Signaling Laboratory, Baker Medical Research Institute, P. O. Box 6492, St. Kilda Road Central, Melbourne, Victoria 8008, Australia. Tel.: 61-3-95224333; Fax: 61-3-95211362; E-mail: jun-ping.liu@baker.edu.au.
Published, JBC Papers in Press, October 13, 2000, DOI 10.1074/jbc.M006371200
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ABBREVIATIONS |
---|
The abbreviations used are: PH, pleckstrin homology; SH3, Src homology domain 3; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; MAP, mitogen-activated protein kinase; PAGE, polyacrylamide gel electrophoresis; TRITC, tetramethylrhodamine isothiocyanate; TGN, trans-Golgi network..
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