Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel
* Author for correspondence (e-mail: histol3{at}post.tau.ac.il )
Accepted 1 May 2002
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Summary |
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Key words: Protein kinase C, Synaptotagmin, TPA, Mast cells, Endosome
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Introduction |
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Conventional and novel PKCs serve as cellular targets for tumor-promoting
phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), which
induce tumors in initiated cells
(Nishizuka, 1986). Acute
exposure to TPA induces the translocation and activation of cytosolic
TPA-responsive PKCs to the plasma membrane, whereas prolonged incubation
results in the proteolytic degradation of the responsive PKCs and their
depletion from the cell (Nishizuka,
1986
; Mellor and Parker,
1998
). Both activation and downregulation of PKCs have appreciable
impacts on cellular processes and have been implicated in carcinogenesis
(Nishizuka, 1986
;
Mellor and Parker, 1998
).
Recent evidence indicates that degradation of PKC following
long-term exposure to TPA is traffic dependent and involves transport of the
activated kinase from the plasma membrane to caveolae and then to an endosomal
compartment (Mineo et al.,
1998
; Prevostel et al.,
2000
). Intracellular trafficking normally involves packaging of
cargo into transport vesicles, which subsequently fuse with their appropriate
target membranes. These processes are highly regulated, accommodating multiple
regulatory proteins, including the SNAREs, which are believed to constitute
the fusion machinery (Bennet, 1995; Lowe,
2000
; McNew et al.,
2000
), and the Rab GTPases
(Armstrong, 2000
). Another
family of proteins implicated in the control of protein traffic is the
synaptotagmin (Syt) family, which comprises thirteen evolutionarily conserved
and structurally related proteins (Sudhof
and Rizo, 1996
; Adolfsen and
Littleton, 2001
). Members of the Syt family display wide, yet
distinct, tissue distribution, suggesting that these proteins may regulate
discrete transport processes. Syt I and Syt II, the most characterized Syt
homologues, have been implicated as Ca2+ sensors in the control of
neurotransmission (Brose et al.,
1992
; Geppert et al.,
1994
). However, the role of these homologues in non-neural tissues
or the role of other members of this family remains obscure.
We have previously shown that rat basophilic leukemia cells (RBL-2H3), a
mucosal mast cell line, endogenously express the Syt homologues Syt II, III
and V (Baram et al., 1999;
Baram et al., 2001
). We have
further demonstrated that Syt II is located in the RBL-2H3 cells at a late
endosomal/lysosomal compartment, where it functions to negatively regulate
Ca2+- triggered lysosomal exocytosis
(Baram et al., 1999
). Because
RBL cells also express PKC
(Chang et
al., 1997
; Razin et al.,
1999
), we sought to employ the RBL cells to investigate whether
Syt II regulates the trafficking route leading to PKC
downregulation.
Here we show that in cells in which the level of Syt II expression is reduced
by >95% by antisense cDNA, the rate of TPA-induced PKC
degradation
is significantly reduced. We further show that this attenuation of PKC
downregulation results from the diversion of PKC
from a degradative
route towards the perinuclear recycling endocytic compartment. Our results
therefore identify Syt II as a novel and critical factor required for the
delivery of cargo from early endosomes to the degradative compartment as well
as a novel regulator of PKC
downregulation.
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Materials and Methods |
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Cell culture
Rat basophilic leukemia cells (RBL-2H3, hereafter termed RBL cells) were
maintained in adherent cultures in DMEM supplemented with 10% FCS in a
humidified atmosphere of 5% CO2 at 37°C.
Cell transfection
Syt II transfectants were generated as described previously
(Baram et al., 1999). Briefly,
full-length rat Syt II cDNA (generously provided by T. C. Sudhof, Howard
Hughes Medical Institute, University of Texas Southwestern Medical School,
Dallas, TX) was subcloned into the EcoRI site of the pcDNA3
expression vector (Invitrogen, San Diego, CA) both in the sense and antisense
orientations. RBL cells (8x106) were transfected with 20
µg DNA (recombinant or empty vector) by electroporation (0.25 V, 960 µF)
and immediately plated in tissue culture dishes containing growth medium
(supplemented DMEM). G418 (1 mg/ml) was added 24 hours after transfection, and
stable transfectants were selected within 14 days. The same procedure was
employed to generate stable Syt III transfectants, except that the cells were
transfected with a full-length rat Syt III cDNA (a generous gift from S.
Seino, Chiba University, School of Medicine, Japan) subcloned in an anti-sense
orientation into the HindIII/XbaI sites of the pcDNA3
expression vector.
Preparation of cell lysates
RBL cells (1x106) were washed in PBS and resuspended in 40
µl of lysis buffer [50 mM Hepes, pH 7.4, 150 mM NaCl, 10 mM EDTA, 2 mM
EGTA, 1% Triton-X 100, 0.1% SDS, 50 mM NaF, 10 mM NaPPi, 2 mM
Na3VO4, 1 mM PMSF, 10 µg/ml cocktail protease
inhibitors (Boehringer Mannheim, Germany)]. Cell lysates were left on ice for
10 minutes and then centrifuged at 12,000 g for 15 minutes at
4°C. The cleared supernatants were mixed with 5xLaemmli sample
buffer to a final concentration of 1x, boiled for 10 minutes and
subjected to SDS-PAGE and immunoblotting.
Isolation of cytosolic and membranal fraction
RBL cells (1x106) were washed twice with cold PBS. The
cytosolic fractions were extracted following 5 minutes of incubation on ice
with 200 µl of buffer (0.5 mg/ml digitonin, 20 mM Mops, pH 7.2, 10 mM EGTA,
5 mM EDTA), and the membranal fractions were collected following a further 7
minutes of incubation with 200 µl of the same buffer supplemented with 0.5%
Triton-X 100 (Pelech et al.,
1986).
Subcellular fractionation
Cells were fractionated as previously described
(Baram et al., 1999). Briefly,
RBL cells (8x107) were washed with PBS and suspended in
homogenization buffer [0.25 M sucrose, 2 mM MgCl2, 800 U/ml DNase I
(Sigma-Aldrich), 10 mM Hepes, pH 7.4, 1 mM PMSF and a cocktail of protease
inhibitors (Boehringer Mannheim, Germany)]. Cells were subsequently disrupted
by three cycles of freezing and thawing followed by 20 passages through a
21-gauge needle and 10 passages through a 25-gauge needle. Unbroken cells and
nuclei were removed by centrifugation for 10 minutes at 500 g,
and the supernatant was sequentially filtered through 5 and 2 µm filters
(Poretics Co.). The final filtrate was then loaded onto a continuous, 0.45-2.0
M sucrose gradient (10 ml), which was layered over a 0.3 ml cushion of 70%
(wt/wt) sucrose and centrifuged for 18 hours at 100,000 g.
SDS-PAGE and immunoblotting
Samples (normalized according to protein content or number of cells) were
separated by SDS-PAGE using 7.5 or 10% polyacrylamide gels. They were then
electrophoretically transferred to nitrocellulose filters. Blots were blocked
for 2 hours in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.05% Tween 20)
containing 5% skimmed milk followed by overnight incubation at 4°C with
the indicated primary antibody. Blots were washed three times and incubated
for 1 hour at room temperature with the secondary antibody
(horseradish-peroxidase-conjugated goat anti-rabbit or anti-mouse IgG; Jackson
Research Laboratories, West Grove PA). Immunoreactive bands were visualized by
the enhanced chemiluminescence method (ECL) according to standard
procedures.
Secretion from RBL cells
RBL cells were seeded in 24-well plates at 1x106 cells per
well and incubated overnight in a humidified incubator at 37°C. The cells
were then washed three times with Tyrode's buffer (20 mM Hepes, pH 7.4, 137 mM
NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 1.5 mM
CaCl2, 1 mM MgCl2, 5 mM glucose, and 0.1% BSA) and
stimulated in the same buffer with the indicated concentrations of the calcium
ionophore A23187 and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate
(TPA; Calbiochem). Secretion was allowed to proceed for 30 minutes at
37°C. Aliquots from the supernatants were taken for measurements of
released ß-hexosaminidase activity as described previously
(Baram et al., 1999).
Immunofluoresence and confocal analysis
Treated or untreated RBL cells (1x105) were grown on 12 mm
round glass coverslips. For immunofluorescence experiments, cells were washed
twice with PBS and fixed for 30 minutes at room temperature in 3%
paraformaldehyde/PBS, washed three times with PBS and permeabilized for 30
minutes in 0.5% Triton X-100/5% FCS/2% BSA/PBS. Cells were subsequently
incubated for 1 hour at room temperature with the primary antibodies diluted
in the same permeabilization buffer, washed three times in PBS and incubated
for 1 hour in the dark with the secondary antibody (Rhodamine- or
FITC-conjugated donkey anti-rabbit or anti-mouse IgG, at 1:200 dilution).
Coverslips were then washed in PBS and mounted with Gel Mount mounting medium
(Biomedica corp. Foster city, CA). Samples were analyzed using a Zeiss laser
confocal microscope (Oberkochen, Germany).
For colocalization analyses of internalized FITC-conjugated transferrin, cells were incubated for the last 5 minutes of TPA treatment with 50 µg/ml FITC-conjugated human transferrin. Cells were subsequently processed for immunofluorescence as described above.
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Results |
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The effect of Syt II was isoform specific, as indicated by the fact that
suppression of Syt III, the second most abundant Syt homologue expressed in
the RBL cells (Baram et al.,
1999), by > 90% (Fig.
1B, lower panel), by transfection with Syt III antisense cDNA
(E.G., Z.P., I. Hammel and R.S-E., unpublished) failed to protect PKC
from TPA-induced downregulation. (Fig.
1B).
For the next step, we compared the kinetics of TPA-induced downregulation
of PKC in control cells, RBL-Syt II- cells and in RBL-Syt
II+ cells, in which the level of Syt II expression was increased by
two-fold by stable transfection with Syt II sense cDNA
(Baram et al., 1999
). Notably,
we have previously demonstrated that the overexpressed Syt II protein in the
RBL-Syt II+ cells was targeted to the same late endosomal/lysosomal
compartment that contains the endogenous Syt II protein
(Baram et al., 1999
). These
analyses revealed that reducing or increasing the cellular level of Syt II
indeed inhibited or facilitated TPA-induced downregulation, respectively.
Hence, whereas the half-life of the kinase in TPA-treated control RBL cells
was 2 hours, it was increased to 4 1/4 hours in the RBL-Syt II-
cells and decreased to 1 1/2 hours in the RBL-Syt II+ cells
(Fig. 2).
|
A proteasomal inhibitor prevents TPA-induced downregulation of
PKC
Previous studies have implicated the proteasome in TPA-induced degradation
of PKC (Lu et al.,
1998
). Thus, proteasome inhibitors prevented the depletion of PKC
(Lu et al., 1998
). Moreover, a
kinase-dead ATP-binding mutant of PKC
could not be depleted by TPA
(Lu et al., 1998
). Therefore,
to identify the proteolytic machinery involved in TPA-induced degradation of
PKC
in the RBL cells, we tested the effects of leupeptin, an inhibitor
of lysosomal proteases, and ALLN, an inhibitor of the proteasome, on
TPA-induced downregulation. In the absence of TPA, neither of these inhibitors
affected the levels of PKC
present in the cytosol or the plasma
membrane (Fig. 3A). Exposing
the cells for 12 hours to TPA resulted in the complete depletion of PKC
from the RBL cells and in the partial depletion of PKC
from the RBL-Syt
II- cells (Fig. 3A).
In both cell types, pretreatment with leupeptin had no effect
(Fig. 3A). Similarly,
inhibition of endosomal/lysosomal acidification by chloroquine (50 µM) or
NH4Cl (20 mM) failed to prevent the depletion of PKC
(data
not shown). By contrast, inclusion of the proteasome inhibitor ALLN prevented
TPA-induced downregulation of PKC
in both the parental RBL cells and in
the RBL-Syt II- cells (Fig.
3A).
|
Depletion of PKC was prevented when the activity of the enzyme was
inhibited by including the PKC inhibitor Go 6976, which specifically blocks
the activity of the PKC
and ß isoforms
(Fig. 3B).
Phorbol-ester-sensitive PKCs remain active in TPA-treated RBL-Syt
II- cells
The Ca2+ ionophore and TPA interact synergistically to promote
exocytosis in RBL cells (Sagi-Eisenberg
and Pecht, 1984;
Sagi-Eisenberg et al., 1985
).
Long-term exposure to TPA inhibits this response by downregulating the
TPA-responsive PKCs (Gat-Yablonski and
Sagi-Eisenberg, 1990
). To examine whether protection from
degradation also retained PCK
active in the RBL-Syt II-
cells, we measured the release of ß-hexosaminidase, a marker for
exocytosis, in response to triggering with the combination of a
Ca2+ ionophore (A23187) and TPA before and after prolonged
treatment with TPA. In the absence of any stimulus, both RBL and RBL-Syt
II- cells spontaneously released less than 5% of their total
ß-hexosaminidase activity (Fig.
4). Upon exposure to A23187 (10 µM), RBL cells released 17% of
their total ß-hexosaminidase, whereas RBL-Syt II- cells
released 24% (Fig. 4). TPA
alone failed to stimulate exocytosis in either the control or the RBL-Syt
II- cells (data not shown). However, exposure to A23187 and TPA
resulted in the release of 54% and 67% of ß-hexosaminidase from the RBL
and RBL-Syt II- cells, respectively
(Fig. 4). By contrast, in cells
pretreated with TPA for 6 hours prior to triggering, subsequent exposure to
A23187 and TPA resulted in the release of less than 19% of
ß-hexosaminidase from RBL cells, whereas the RBL-Syt II- cells
retained their responsiveness and released 53% of ß-hexosaminidase
(Fig. 4). By contrast, 6 hours
of treatment of the RBL-Syt III- cells with TPA resulted in the
loss of their responsiveness to a subsequent TPA/ionophore trigger
(Fig. 4). These results
therefore support the notion that suppression of Syt III, unlike that of Syt
II, could not repress TPA-induced PKC
downregulation.
|
The route of PKC in TPA-treated cells
To investigate the step in PKC downregulation that was dependent on
Syt II, we fractionated TPA-treated cells on linear sucrose gradients to
identify the cellular organelles thar PKC
was associated with. This was
carried out in the control RBL cells and the RBL-Syt II- cells and
as a function of the TPA incubation period. In resting RBL cells, PKC
was distributed between three major peaks
(Fig. 5A). Approximately 65% of
the total amount of enzyme were present in fractions 3-10; a smaller amount of
the enzyme (
20% of total) was observed at fractions 13-18 and a minor
amount (13%) was present at fractions 19-24
(Fig. 5A). On the basis of our
previous analyses, fractions 3-10, which run at 0.33-0.70 M sucrose contain
the cytosol, as evidenced by lactate dehydrogenase activity
(Baram et al., 1999
), as well
as the early endosomes, as indicated by the migration of several endosomal
markers, including the early endosomal antigen 1 (EEA1), annexin II and
syntaxin 7. Fractions 13-18, that run at 0.88-1.09 M sucrose, contain the
plasma membrane (E.G., Z.P., I. Hammel and R.S-E., unpublished), whereas
fractions 19-24 at 1.23-1.45 M sucrose include the SG, on the basis that they
contain both histamine and ß-hexosaminidase activity
(Baram et al., 1999
). Thus, in
the absence of TPA, most (
65%) of PKC
is localized to the
fractions that include cytosol and early endosomes, whereas a smaller amount
is distributed between the plasma membrane and the cells SG
(Fig. 5C). Following 20 minutes
of incubation with TPA, the amount of PKC
present at the light
(cytosol/early endosome) fractions decreased to approximately 20% of total,
the amount of the plasma-membrane-associated enzyme increased to 40%, and the
SG fractions contained 15% of the total cellular amount of enzyme
(Fig. 5C). After 2 hours of TPA
treatment, the amount associated with the plasma membrane fractions decreased
with a concomitant increase in the amount of enzyme associated with the SG
fractions (Fig. 5). Finally,
after 6 hours of TPA incubation, the total amount of PKC
in the cell
was markedly reduced and most of the residual enzyme was present at the SG
fractions (Fig. 5).
|
A similar pattern was detected when analyzing fractions derived from
non-treated RBL-Syt II- cells or cells subjected to short term (20
minutes or 2 hours) TPA treatment (data not shown). However, two major
differences were observed upon increasing the incubation period with TPA to 6
hours. First, the total amount of PKC present in the cells was
considerably higher than that present in the TPA-treated control cells
(Fig. 5B). Secondly, in the
RBL-Syt II- cells, >30% of the total PKC
was still
associated with plasma membrane fractions
(Fig. 5B,C). Hence, whereas in
both the RBL and RBL-Syt II- cells PKC
was distributed
between the cytosol/endosomes, plasma membrane and SG, with increasing
exposure time to TPA, the total amount of PKC
in the RBL-Syt
II- cells was larger, and a significant fraction of the enzyme
remained associated with the plasma membrane fractions.
To substantiate these results further, we also used immunofluorescence and
laser confocal microscopy to observe the TPA-induced route of PKC.
Moreover, because our fractionation studies could not distinguish between
cytosolic and endosomal fractions, we labeled the latter compartment by
allowing the cells to internalize Fluorescein-conjugated transferrin
(FITC-Tfn) for 5 minutes. Both non-treated RBL and RBL-Syt II-
cells showed a diffuse cytosolic pattern of staining for PKC
(Fig. 6A,A'). TPA
treatment for 20 minutes resulted in translocation of the enzyme from the
cytosol to the plasma membrane (Fig.
6B,B'). However, in RBL-Syt II- cells, but not in
the control RBL cells, PKC
was also present in a perinuclear location
(Fig. 6B'). Under these
conditions, Tfn localized to peripheral vesicles, which were scattered through
the cytosol in the RBL cells (Fig.
6C), whereas in the RBL-Syt II- cells, Tfn was
concentrated in a perinuclear structure
(Fig. 6C'), where it
colocalized with PKC
(Fig.
6D'). After 2 hours of TPA treatment, the amount of
PKC
in the RBL cells was markedly reduced, and most of the enzyme
showed a granular stain (Fig.
6E). In a few cells the enzyme was also associated with the
perinucleus (Fig. 6E). RBL-Syt
II- cells contained more PKC
, and the enzyme was still
distributed between the plasma membrane and the perinuclear structure
(Fig. 6E'). Notably,
under these conditions - namely TPA treatment for 2 hours following 5 minutes
of internalization - Tfn was targeted to peripheral early endosomes in RBL
cells (Fig. 6F) but to the
perinuclear location in the RBL-Syt II- cells
(Fig. 6F').
|
A longer exposure period to TPA (4 hours) almost completely depleted
PKC from the control RBL cells (Fig.
6H), leaving only a minute amount associated with peripheral
vesicles. However, these vesicles did not overlap with the Tfn-positive early
endosomes (Fig. 6I,J). In
contrast, a considerable amount of PKC
was still concentrated in the
perinuclear structure in the RBL-Syt II- cells
(Fig. 6H'), where it
colocalized with internalized Tfn (Fig.
6I',J'). The remaining enzyme localized to the plasma
membrane and to Tfn-negative peripheral vesicles
(Fig. 6J'). To
investigate whether the perinuclear compartment, which included both Tfn and
PKC
, corresponded to the recycling endocytic compartment (recycling
endosomes), we stained the cells with an antibody directed against the small
GTPase Rab 11, which resides at the recycling compartment
(Sheff et al., 1999
;
Trischler et al., 1999
).
Indeed, the Tfn-positive compartment present in the TPA-treated RBL-Syt
II cells, overlapped with the Rab 11 staining, confirming its
identification as the recycling endosomes
(Fig. 7A-C). Because our
biochemical fractionation data suggested that PKC
also comigrated with
SG containing fractions, we investigated whether the Tfn-negative vesicles
with which PKC
was associated corresponded to SG. To this end, we
labeled the latter with an antibody directed against the SG marker serotonin.
Indeed, a partial overlap between PKC
and serotonin was clearly
demonstrated in both RBL and RBL-Syt II- TPA-treated cells
(Fig. 7B,A-F). These results
have therefore confirmed that the Tfn-negative peripheral vesicles, with which
PKC
was associated in the TPA-treated cells, correspond to the SG.
|
![]() |
Discussion |
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The major difference in the trafficking of PKC in RBL versus RBL-Syt
II- cells is the amount of enzyme delivered to the perinuclear
recycling compartment. Whereas only a small amount of the enzyme seems to
reach this compartment in the control cells, a considerably larger amount
colocalizes with Tfn in this compartment in the RBL-Syt II- cells.
These results are compatible with a model whereby Syt II is required for the
delivery of internalized cargo from early/sorting endosomes to late endosomes
and degradation (Fig. 8).
Suppression of Syt II thus results in the deviation of endosomal cargo from
the degradative pathway to the recycling endosomes. Therefore, while in the
control RBL cells active PKC
is delivered from the early/sorting
endosomes to degradation, it is targeted to the recycling endosomes in the
RBL-Syt II- cells from where it cycles to the plasma membrane
maintaining its active state. Indeed, whereas TPA fails to potentiate
Ca2+-ionophore-induced secretion in control cells pretreated for 6
hours with TPA, it still increases by more than two-fold the ionophore-induced
secretion in RBL-Syt II- cells
(Fig. 4). Notably, previous
studies have implicated PKCs ß and
in mediating secretion
triggered by the Fc
RI (Ozawa et al.,
1993
; Chang et al.,
1997
). Our data suggest that PKC
may be involved in
Ca2+-ionophore/TPA-induced secretion, although the possibility that
additional isoforms of PKC may also be protected from TPA-induced
downregulation in the RBL-Syt II- cells can not be excluded.
|
Interestingly, inhibiting the exit from early endosomes towards the late
endosomal/lysosomal compartments increases the exit rate towards the recycling
endosomes. Thus, whereas in the control cells after 5 minutes of
internalization, the majority of Tfn is associated with peripheral vesicles
corresponding to the early endosomes, following 5 minutes of internalization
by the RBL-Syt II- cells, most of Tfn already resides in the Rab
11-positive perinuclear recycling compartment. Although the reason for this
facilitated delivery to the recycling endosomes is presently unknown, it is
possible that Syt II compete with a distinct Syt homologue that is involved in
exit from early endosomes towards the recycling compartment for shared
effector proteins [SNARE proteins, that constitute the fusion machinery
(Adolfsen and Littleton, 2001),
or clathrin adaptor proteins (Chapman et
al., 1998
)]. In the latter case, suppression of Syt II will
increase the pool of effectors available for binding to the other Syt
homologue and thereby facilitate the exit.
Several observations indicate that the effect of Syt II is isoform
specific. First, neither of the other endogenously expressed Syt homologues
can substitute for Syt II in the RBL-Syt II- cells. Second,
PKC is not protected from degradation in RBL-Syt III- cells
in which the expression level of Syt III is reduced by >90%. Finally, the
route taken by PKC
in TPA-treated RBL-Syt III- cells is
similar to that of control RBL cells (data not shown). Although not proven
here, the ability of Syt II to modulate PKC
downregulation suggests
that PKC
is delivered to late endosomes on its route to degradation.
Yet neither inhibition of endosomal acidification by chloroquine (50 µM) or
NH4Cl (20 mM) nor exposure to the lysosomal inhibitor leupeptin
prevent this degradation. By contrast, degradation is prevented by the
proteasome inhibitor ALLN. What the relationship is between the proteasome and
the endocytic compartments and how PKC
is delivered from the endosome
to the proteasome is presently unknown. Recent studies have demonstrated a
mutual requirement for the proteasome and late endosomes/lysosomes in the
downregulation of membrane receptors such as MET
(Hammond et al., 2001
), the GH
receptor (Van Kerhof and Strous, 2001) and the receptor for interleukin
2ß (Rocca et al., 2001
).
This may thus also be the case for PKC
downregulation.
In conclusion, our studies indicate that Syt II plays a an active regulatory and physiological role in membrane trafficking. Moreover, by targeting signaling molecules to lysosomes, Syt II may serve as an important regulator of cell signaling, whose level of expression and proper function may define the duration of a signal by controlling receptor as well as effector downregulation.
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Acknowledgments |
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References |
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