From the Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
Received for publication, November 19, 2002, and in revised form, January 6, 2003
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ABSTRACT |
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Phosphoinositide (PI) 3-kinases are critical
regulators of mast cell degranulation. The Class IA PI 3-kinases
p85/p110 Mast cells are important cellular mediators of allergic responses
in humans (1). Moreover, increased levels of mast cells and mast
cell-derived inflammatory mediators are found in brochoalveolar lavage
fluid from asthmatics, suggesting a role for mast cells in the etiology
of clinical asthma (2-4). Cross-linking of cell surface Fc The initial signaling events during antigen-stimulated degranulation
have been well studied. Fc G-protein-coupled receptors
(GPCRs)1 also regulate mast
cell degranulation. In bone marrow-derived mast cells and in a cell culture model, the RBL-2H3 basoleukemic line, adenosine can
synergistically enhance degranulation in response to FceRI
crosslinking, although it is not sufficient to stimulate degranulation
(6, 9, 10). Adenosine signaling in RBL-2H3 cells is primarily mediated
by the A3 adenosine receptor, a G The phosphoinositide 3-kinase inhibitor wortmannin is a potent
inhibitor of mast cell degranulation (15), and deletion of the gene for
the phosphatidylinositol trisphosphate-phosphatase SHIP markedly
enhances mast cell degranulation (16). These data demonstrate that PI
3-kinases are important regulators of antigen-stimulated degranulation.
Microinjection of isoform-specific inhibitory antibodies to p110 Cell Culture--
RBL-2H3 and RBL-2H3 cells expressing the M1
muscarinic receptor (RBL-2H3-M1) were cultured in Iscove's modified
Dulbecco's medium containing 15% fetal bovine serum on Nunc tissue
culture dishes or fibronectin-coated coverslips.
Antibodies, cDNA Constructs, and
Inhibitors--
Isoform-specific inhibitory antibodies against
p110 Microinjections and Transfections--
Cells were microinjected
using an Eppendorf 5171/5242 semi-automatic
micromanipulator/microinjector as described previously (19). Cells were
allowed to recover for 2 h after injection prior to stimulation as
described below.
Degranulation Assays--
RBL-2H3 cells were incubated overnight
in 0.1 µg/ml anti-DNP IgG. The cells were washed in Hanks' basic
salt solution (HBSS) and stimulated for 45 min at 37 °C with HBSS, 1 mM calcium containing 10 ng/ml DNP-albumin. For adenosine
experiments, cells were stimulated with 0.5 ng/ml DNP-albumin in the
absence or presence of 10 µM adenosine or carrier.
Alternatively, RBL-2H3-M1 cells were stimulated for 45 min at 37 °C
with HBSS containing 100 µM carbachol or carrier. In each
case, the supernatant was removed and brought to 100 mM citrate, pH 4.5, 1 mM
4-methylumbelliferyl-N-acetyl glucosamine (Sigma).
After 15 min at 37 °C, the reaction was stopped with 1/10 volume of
200 mM Na2CO3, glycine, pH 10.7, and substrate hydrolysis measured using a fluorescence
spectrophotometer (360 excitation/465 emission).
For detection of degranulated cells using annexin V, cells grown on
fibronectin-coated coverslips were preloaded as described above, washed
in HBSS, and stimulated as described with the additional presence of a
1:10 dilution of Alexa 594 or Alexa 488 annexin V reagent (Molecular
Probes, Eugene, OR). The cells were washed, fixed in 3.7% formaldehyde
for 10 min at 22 °C, and mounted. Cells were scored for annexin V
staining using a Nikon Eclipse 400 upright microscope with a 60 × 1.4 N.A. plan-apo infinity-corrected objective. Each measurement
reflects ~100 injected cells per condition, and the data are the mean
from three to five separate experiments. When indicated, cells were
transfected with LipofectAMINE Plus according to manufacturer's
instructions (Invitrogen). Images were acquired using a Cohu
4910 B/W CCD camera with NIH Image 1.62 analysis software.
A Single-cell Assay for Mast Cell Degranulation--
We and others
(19-21) have previously characterized specific inhibitory antibodies
to Class I and Class III PI 3-kinases. To use these reagents to study
mast cell degranulation in single cells, we modified a flow cytometry
assay developed by Demo et al. (23), in which the binding of
fluorescently labeled annexin V is used to identify degranulated cells.
As shown in Fig. 1, quiescent anti-DNP
IgE-loaded RBL-2H3 cells show no staining with FITC-annexin V (Fig.
1B). In contrast, after stimulation of Fc Role of Class IA PI 3-Kinases during Fc
We also examined the role of these PI 3-kinases in signaling by the
adenosine receptor, a physiologically important contributor to airway
inflammation and mast cell activation in asthma (6). Adenosine is not
sufficient to induce degranulation but can enhance Fc Carbachol-stimulated Degranulation in RBL-2H3-M1 Cells Requires
hVPS34--
The RBL-2H3-M1 line, which expresses the M1 acetylcholine
receptor, was developed as a model system to study cholinergic
regulation of secretion and neurotransmitter release (11). Carbachol
elicits a robust degranulation response in these cells. To examine the role of PI 3-kinases in M1 muscarinic receptor signaling, we
microinjected RBL-2H3-M1 cells with inhibitory anti-PI 3-kinase
antibodies and stimulated the cells with 100 µM carbachol
for 45 min. Unlike antigen-stimulated degranulation,
carbachol-stimulated degranulation was unaffected by inhibition of any
of the Class I PI 3-kinases (Fig.
3A). Surprisingly,
carbachol-stimulated degranulation was markedly reduced by inhibition
of the Class III enzyme, hVPS34 (Fig. 3A). To confirm the
role of hVPS34 in carbachol-stimulated degranulation, we transiently
transfected RBL-2H3 or RBL-2H3-M1 cells with an eGFP-linked construct
containing two FYVE domains from hepatocyte growth factor-regulated
tyrosine kinase substrate (Hrs) (22). Although this construct is
useful as a marker for PI(3)P-containing membranes when expressed at
low levels, high level overexpression of FYVE domain-containing
proteins has been shown to disrupt PI(3)P-mediated signaling,
presumably by titration of PI(3)P (24). We therefore used high level
expression of the construct to disrupt
PI(3)P-dependent signaling. We found that overexpression of
the eGFP-FYVE in mast cells had no effect on Fc Differential Utilization of PKC Isoforms in Fc In this report we have used well characterized isoform-specific
anti-PI 3-kinase antibodies (19-21), in conjunction with a single-cell
assay for degranulation, to identify the roles of distinct PI 3-kinases
during mast cell degranulation. The assay is based on a previously
published flow cytometry assay (23), which used fluorescently labeled
annexin V to quantitate the increase in exofacial phosphatidylserine
that occurs in the plasma membrane of degranulated cells. A similar
increase in exofacial phosphatidylserine occurs in apoptotic cells, and
fluorescent annexin V is a commonly used assay for apoptosis (25). In
the single cell assay presented here, degranulation is expressed as the
percentage of annexin V-positive cells per field. The dose-response
curve for antigen-stimulated degranulation using this assay correlates
well with biochemical assays for degranulation and shows similar
sensitivity to wortmannin. This assay provides a useful adjunct to
previous single-cell studies in RBL-2H3 cells, which focused on calcium
flux and membrane ruffling (17).
The importance of examining degranulation itself, as opposed to
degranulation-related events, is shown in the experiments on the role
of Class IA PI 3-kinases during antigen-stimulated degranulation.
Antigen-stimulated calcium flux has previously been shown to require
p85/p110 Our experiments also reveal a role for the p85/p110 We also studied degranulation in a heterologous system, the RBL-2H3-M1
cell line (11), which expresses the G How might hVPS34 modulate signaling from the M1 acetylcholine receptor?
hVPS34 signals via the production of PI(3)P and the recruitment and/or
activation of proteins containing FYVE or PX domains, which
specifically bind to PI(3)P (46-49). In both mammalian cells and
yeast, hVPS34 is targeting to membranes along with an associated
protein kinase (VPS15/p150) (40, 45, 50); in mammalian cells the Rab5
GTPase also regulates hVPS34 targeting to endosomes (38, 40).
Disruption of hVPS34 alters the post-endocytic trafficking of cell
surface receptors (19, 41) and therefore could affect the
post-endocytic sorting of carbachol-stimulated M1 receptors. This could
alter the signaling properties of the receptor, as has been suggested
in the In summary, we have used a single cell assay for mast cell
degranulation to study the role of different PI 3-kinases in this process. We find a general role for Class IA PI 3-kinases in response to antigen-stimulated degranulation and a specific requirement for
p85/p110 and p85/p110
but not p85/p110
are required for
antigen-mediated calcium flux in RBL-2H3 cells (Smith, A. J.,
Surviladze, Z., Gaudet, E. A., Backer, J. M., Mitchell,
C. A., and Wilson, B. S. et al., (2001)
J. Biol. Chem. 276, 17213-17220). We now examine the
role of Class IA PI 3-kinases isoforms in degranulation itself, using a
single-cell degranulation assay that measures the binding of fluorescently tagged annexin V to phosphatidylserine in the outer leaflet of the plasma membrane of degranulated mast cells. Consistent with previous data, antibodies against p110
and p110
blocked Fc
R1-mediated degranulation in response to Fc
RI ligation.
However, antigen-stimulated degranulation was also inhibited by
antibodies against p110
, despite the fact that these antibodies have
no effect on antigen-induced calcium flux. These data suggest that p110
mediates a calcium-independent signal during degranulation. In
contrast, only p110
was required for enhancement of
antigen-stimulated degranulation by adenosine, which augments mast
cell-mediated airway inflammation in asthma. Finally, we examined
carbachol-stimulated degranulation in RBL2H3 cells stably expressing
the M1 muscarinic receptor (RBL-2H3-M1 cells). Surprisingly,
carbachol-stimulated degranulation was blocked by antibody-mediated
inhibition of the Class III PI 3-kinase hVPS34 or by titration of its
product with FYVE domains. Antibodies against Class IA PI 3-kinases had
no effect. These data demonstrate: (a) a
calcium-independent role for p110
in antigen-stimulated
degranulation; (b) a requirement for p110
in adenosine
receptor signaling; and (c) a requirement for hVPS34 during
M1 muscarinic receptor signaling. Elucidation of the intersections
between these distinct pathways will lead to new insights into mast
cell degranulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI
receptors leads to the release of pre-formed mediators present in mast
cell granules, as well as the induction of cytokines and bioactive
lipids (5). Release of these inflammatory molecules in the lung is
likely to contribute to inflammation and vasoconstriction during
asthma. Antigen-mediated degranulation is enhanced by co-stimulation of
mast cells with adenosine, which is an important contributor to airway
inflammation in asthma (6).
RI cross-linking leads to recruitment and
activation of lyn and syk tyrosine kinases, with subsequent
phosphorylation of tyrosine residues in the Fc
RI
-chain (5, 7).
This leads to the recruitment, phosphorylation and activation of
phospholipase C
, and generation of inositol trisphosphate and
diacylglycerol from the hydrolysis of plasma membrane
phosphatidylinositol (4,5)-bisphosphate. Inositol
trisphosphate-mediated release of intracellular calcium stores and
activation of classical and novel isoforms of protein kinase C (8) are
required for the opening of plasma membrane calcium channels. The
increase in intracellular calcium levels is critical for degranulation,
as evidenced by the fact that thapsigargin or calcium ionophores can
induce mast cell degranulation in the absence of additional stimuli.
i-coupled GPCR (9). In
addition, Beaven and colleagues (11) demonstrated that stable
expression in RBL-2H3 cells of a heterologous GPCR, the M1 muscarinic
acetylcholine receptor, leads to carbachol-stimulated degranulation.
Carbachol versus antigen stimulation of RBL-2H3-M1 cells
lead to similar changes in calcium mobilization and activation of Erk
and phospholipase A2 (12-14), although carbachol-stimulated
degranulation used PLC
rather than PLC
to trigger PKC activation
and calcium flux (13).
and
p110
reduce antigen-stimulated calcium flux and membrane ruffling in
RBL-2H3 cells (17). In addition, recent studies on a mouse knockout of
the Class IB PI 3-kinase, p110
, suggest that p110
is important in
both FceRI and adenosine-mediated degranulation (18). However, the role
of Class IA and Class III PI 3-kinases in degranulation itself has not
been directly examined. In this paper, we use specific inhibitory
antibodies against Class IA and Class III PI 3-kinases to test the
requirement for these enzymes during degranulation. Using a single-cell
degranulation assay in RBL-2H3 cells, we show that the three Class IA
PI 3-kinases are all required for optimal antigen-stimulated
degranulation. In contrast, synergistic enhancement of
antigen-stimulated degranulation by adenosine specifically requires
p85/p110
. Surprisingly, carbachol-stimulated degranulation in
RBL-2H3-M1 cells does not require Class I PI 3-kinases and instead
requires the Class III enzyme hVPS34. The utilization of PKC isozymes
in antigen versus carbachol-stimulated degranulation was
also different. These data suggest a novel role of hVPS34 in GPCR
signaling and highlight the regulatory complexity of ligand-stimulated
degranulation in mast cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, p110
, p110
, and hVPS34 have been described previously
(19-21). All antibodies were affinity-purified and concentrated to 4 mg/ml in phosphate-buffered saline. The eGFP-2X-FYVE construct
(22) was obtained from Dr Harald Stenmark, The Norwegian Radium
Hospital, Norway. Wortmannin was obtained from Sigma, and
rottlerin and Go6976 were obtained from Calbiochem.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI receptors
with DNP-albumin for 45 min, a clear increase in FITC-annexin V
staining is seen in most cells (Fig. 1D). To validate the
use of annexin V staining as a single-cell assay, we used the
percentage of FITC-annexin V-positive cells as a measure of
degranulation, and compared it witho degranulation as determined by a
biochemical assay of degranulation, the release of
-hexosaminidase
activity. Both assays showed a similar dose response for degranulation
in DNP-albumin-stimulated cells (Fig. 1E). In addition, both
assays showed similar inhibition by the PI 3-kinase inhibitor
wortmannin (Fig. 1F) and by the pan-PKC inhibitor
Bisindolmaleimide (data not shown). Thus, the annexin V-based assay
accurately reflects ligand-stimulated degranulation of RBL-2H3
cells.
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Fig. 1.
A single-cell assay for mast cell
degranulation. A-D, RBL-2H3 cells grown on
fibronectin-coated coverslips were preloaded with anti-DNP-IgE
overnight, then incubated for 45 min at 37 °C with a 1:10 dilution
of Alexa 488-annexin V reagent (Molecular Probes) in the absence
(A, B) or presence (C, D)
of 10 ng/ml DNP-albumin. The cells were then fixed and visualized by
phase contrast (A, C) or immunofluorescence
(B, D) microscopy. E, RBL-2H3 cells
were preloaded overnight with anti-DNP IgE, then stimulated with the
indicated concentration of DNP-albumin in the presence of Alexa
488-annexin V for 45 min at 37 °C. Medium from parallel
coverslips was assayed for release of -hexosaminidase
(
hex) activity or by scoring the percentage of cells that
were stained with annexin V. The data are expressed as percentage of
maximum and are representative of two separate experiments.
F, anti-DNP IgE-loaded cells were incubated in the absence
or presence of 100 nM wortmannin for 15 min, then
stimulated with DNP-albumin in the presence of Alexa 488-annexin V for
45 min. Degranulation was measured by
-hexosaminidase assay of the
medium or by annexin V staining as above. The data are
representative of three separate experiments. BSA, bovine
serum albumin.
RI-mediated
Degranulation--
It was previously shown that specific inhibition of
two isoforms of Class IA PI 3-kinase, p85/p110
and p85/p110
,
diminishes antigen-stimulated calcium flux and membrane ruffling in
RBL-2H3 cells (17). To directly examine their role in degranulation, anti-DNP IgE-loaded RBL-2H3 cells were microinjected with inhibitory antibodies to p110
, p110
, p110
, and the Class III PI 3-kinase hVPS34. After a recovery period, the cells were stimulated with 10 ng/ml DNP-albumin for 45 min. Whereas microinjection of rabbit IgG or
anti-hVPS34 antibodies had no effects on degranulation, microinjection
of antibodies against p110
, p110
, and p110
inhibited degranulation by 70%, 60%, and 45%, respectively (Fig.
2A).
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Fig. 2.
Degranulation in response to
Fc RI activation. A, RBL-3H3
cells were loaded with anti-DNP IgE overnight, then microinjected with
antibodies against p110
, p110
, p110
, or hVPS34 as indicated.
The cells were stimulated with 10 ng/ml albumin-DNP in the presence of
Alexa 488-annexin V for 45 min at 37 °C and fixed and stained with
Cy3-anti-rabbit antibodies (to detect injected cells). Degranulation
was expressed as annexin V-positive injected cells as a percentage of
annexin V-positive non-injected cells on the same coverslip. Data are
the mean ± S.E. from three experiments. B, RBL-3H3
cells were microinjected with antibodies against p110
, p110
,
p110
, or hVPS34 as indicated. The cells were then stimulated with
0.5 ng/ml DNP-albumin in the absence or presence of 10 µM
adenosine for 45 min at 37 °C. All incubations also included Alexa
488-annexin V. Degranulation was measured as described above. Data are
expressed as fold increase in the presence of adenosine and are the
mean ± S.E. from three experiments.
RI-mediated
degranulation in RBL-2H3 cells (9). Anti-DNP IgE-loaded RBL-2H3 cells
were microinjected as above and then stimulated with a suboptimal does
of DNP-albumin (0.5 ng/ml) in the absence or presence of 10 µM adenosine. In control cells, adenosine caused a
3-4-fold increase in the number of degranulated cells. This was
unaffected by microinjection of antibodies against p110
, p110
, or
hVPS34 (Fig. 2B). In contrast, the effects of adenosine were
markedly attenuated by antibodies against p110
. These data
demonstrate a general requirement for Class IA PI 3-kinase during
Fc
RI-mediated degranulation and a specific requirement for
p85/p110
during adenosine-stimulated degranulation.
RI-mediated
degranulation in RBL-2H3 cells, but it inhibited carbachol-stimulated
degranulation in the RBL-2H3-M1 cells (Fig. 3B). Thus, both
hVPS34 and its lipid product, PI(3)P, are required for
carbachol-stimulated degranulation.
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Fig. 3.
Degranulation in response to M1 muscarinic
receptor activation. A, RBL-3H3-M1 cells were
microinjected with antibodies against p110 , p110
, p110
, or
hVPS34 as indicated. The cells were then stimulated with 100 µM carbachol in the presence of Alexa 488-annexin V for
45 min at 37 °C, and degranulation was determined as described in
the legend to Fig. 2. Data are the mean ± S.E. from three
experiments. B, RBL-3H3-M1 cells were transfected with
expression plasmids for either eGFP or eGFP-FYVE as indicated. After
6 h, the cells were stimulated with 100 µM carbachol
in the presence of Alexa 594-annexin V for 45 min at 37 °C.
Degranulation in eGFP-labeled cells was determined as described above
and expressed as a percentage of degranulation in untransfected cells.
Data are the mean ± S.E. from three experiments.
RI Versus
M1-mediated Degranulation--
To further characterize differential
signaling during antigen versus carbachol-stimulated
degranulation, we examined the requirement for PKC
and PKC
. These
isoforms have previously been shown to be stimulatory for degranulation
in RBL-2H3 cells in response to antigen (8). We found that the PKC
inhibitor rottlerin had similar inhibitory effects on both
antigen and carbachol-stimulated degranulation, with IC50
values of ~3-10 µM in the two cell lines (Fig.
4B). In contrast, the
PKC
/
inhibitor Go6976 potently inhibited antigen-stimulated
degranulation in RBL-2H3 cells (IC50 10 nm), but had
no effect on carbachol-stimulated degranulation in the RBL-2H3-M1
cells (Fig. 4A). These data show that the utilization of
both PKC and PI 3-kinase isoforms is different in carbachol versus antigen-stimulated degranulation.
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Fig. 4.
Differential utilization of PKC isoforms in
antigen versus carbachol-stimulated cells.
A, anti-DNP-IgE-loaded RBL-2H3 cells (left panel)
and RBL-2H3-M1 cells (right panel) were treated with the
indicated concentrations of Go6976 for 1 h. The cells were then
stimulated with 10 ng/ml DNP-albumin (left panel) or 500 µM carbachol (right panel), and degranulation
was measured by assaying the media for -hexosaminidase release. Data
are the mean ± S.E. from three experiments. B,
anti-DNP-IgE-loaded RBL-2H3 cells (left panel) and
RBL-2H3-M1 cells (right panel) were treated with the
indicated concentrations of rottlerin for 1 h. The cells
were then stimulated with 10 ng/ml DNP-albumin (left panel)
or 500 µM carbachol (right panel), and
degranulation was measured by assaying the media for
-hexosaminidase
release. Data are the mean ± S.E. from three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and p85/p110
but not p85/p110
(17). The role of these
enzymes in calcium flux is likely to be mediated by the potentiation of
PLC
activation by phosphatidylinositol trisphosphate, either through
Tec-family tyrosine kinases (26), Vav1 (27), or via direct effects on
PLC
(28, 29). A direct effect of phosphatidylinositol trisphosphate
on calcium uptake has also been demonstrated (30). However, an
additional calcium-independent role of PI 3-kinases has been suggested
by the fact that thapsigargin or calcium ionophore-mediated
degranulation is still sensitive to PI 3-kinase inhibitors (15,
31-33). Our data show that p85/p110
is required for
degranulation despite the fact that it is not required for
antigen-stimulated calcium flux in RBL-2H3 cells (17). This suggests
that p85/p110
is a candidate for the wortmannin-sensitive, calcium-independent factor in antigen-stimulated degranulation.
PI 3-kinase
during adenosine-stimulated degranulation in cells treated with
sub-optimal doses of antigen. The specific requirement for p85/p110
in signaling by this G
i-coupled receptor is consistent with reports that p110
is activated by
subunits from trimeric G-proteins (34, 35) and is required for lysophosphatidic
acid-mediated signaling (36). In addition to p110
, p110
is
also required for adenosine secretion by mast cells and
adenosine-mediated autocrine stimulation of degranulation in
antigen-stimulated mast cells (18). The coordination of these two
G
-regulated PI 3-kinases during degranulation, and their
potentially different downstream effectors, will be a subject of
interest for future studies.
q-coupled M1 muscarinic receptor. Previous studies have shown that
carbachol-stimulated degranulation in these cells is different in
several respects from FceRI-stimulated degranulation, for example with
regard to the role of Src-family tyrosine kinases (37). Similarly, we find that inhibitors of PKC
/
block degranulation in
antigen-stimulated RBL-2H3 cells but not carbachol-stimulated
RBL-2H3-M1 cells. Even given these differences, the requirement for
hVPS34 during carbachol-stimulated degranulation of RBL-2H3-M1 cells is
a surprising finding. The best documented roles for hVPS34 in mammalian
cells are in the regulation of traffic through the endocytic or
phagocytic systems (19, 38-43), and during the sorting of newly
synthesized membrane proteins in polarized cells (44). These reports
are consistent with the well characterized role of VPS34p in vesicular
trafficking in yeast (45). However, a role for hVPS34 during regulated
secretion has not been demonstrated. It seems unlikely that hVPS34 is
involved in degranulation at the level of granule fusion, since it
would then also be required for degranulation in response to Fc
RI
receptor activation. Instead, our data suggest a novel role for hVPS34 in G
q-coupled receptor signaling.
-adrenergic receptor system (51). Alternatively, a recently
described RGS protein, RGS-PX1, is a G
s-specific GAP
that contains a PX domain (52); such a protein could provide a
mechanism for the regulation of G
s-coupled receptors by hVPS34. It
is possible that PX- or FYVE-domain-containing RGS proteins might exist
for G
q-coupled receptors as well. Finally, it is
possible that hVPS34 is a direct target of activated M1 receptors.
However, we have so far been unable to detect changes in hVPS34
activity in immunoprecipitates from carbachol-stimulated RBL-2H3 cells
(data not shown).
during adenosine receptor-Fc
RI receptor synergy. We also
find an unexpected requirement for hVPS34 in carbachol-stimulated degranulation. These latter data suggest an unappreciated role for
hVPS34 in signaling by G
q-coupled receptors. It will be
important to determine whether hVPS34 also plays a role in signaling by other G-protein-coupled receptors.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Bart Vanhaesebroeck, Ludwig
Institute for Cancer Research, for anti-p110 antibodies.
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FOOTNOTES |
---|
* This work was supported by a grant from the Sandler Foundation for Asthma Research (to J. M. B.) and by National Institutes of Health Training Grant T32 DK07513 (to D. A. W.).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.
Current address: Integrated Dept. of Immunology, National Jewish
Center, Denver, CO 80206.
§ To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2153; Fax: 718-430-3749; E-mail: Backer@aecom.yu.edu.
Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M211787200
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ABBREVIATIONS |
---|
The abbreviations used are: GPCR, G-protein-coupled receptor; DNP, dinitrophenol; eGFP, enhanced green fluorescent protein; FITC, fluorescein isothyocyanate; FYVE domain, Fab1/YOTB/Vac1p/EEA1-homology domain; PI(3)P, phosphatidylinositol 3-phosphate; PLC, phospholipase C; PKC, protein kinase C; PX domain, Phox homology domain; RGS, regulator of G-protein signaling; PI, phosphoinositide; HBSS, Hanks' basic salt solution.
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REFERENCES |
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