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Originally published In Press as doi:10.1074/jbc.M110210200 on April 4, 2002
J. Biol. Chem., Vol. 277, Issue 25, 22685-22691, June 21, 2002
Distinct Roles of Receptor Phosphorylation, G Protein Usage,
and Mitogen-activated Protein Kinase Activation on Platelet Activating
Factor-induced Leukotriene C4 Generation and Chemokine
Production*
Jasimuddin
Ahamed and
Hydar
Ali
From the Department of Pathology, University of Pennsylvania School
of Dental Medicine, Philadelphia, Pennsylvania 19104
Received for publication, October 24, 2001, and in revised form, March 6, 2002
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ABSTRACT |
Platelet activating factor (PAF) interacts
with cell surface G protein-coupled receptors on leukocytes to
induce degranulation, leukotriene C4
(LTC4) generation, and chemokine CCL2 production. Using a
basophilic leukemia RBL-2H3 cell line expressing wild-type PAF receptor
(PAFR) and a phosphorylation-deficient mutant (mPAFR), we have
previously demonstrated that receptor phosphorylation mediates
desensitization of PAF-induced degranulation. Here, we sought to
determine the role of receptor phosphorylation on PAF-induced LTC4 generation and CCL2 production. We found that PAF
caused a significantly enhanced LTC4 generation in cells
expressing mPAFR when compared with PAFR cells. In contrast,
PAF-induced CCL2 production was greatly reduced in mPAFR cells.
Pertussis toxin and U0126, which inhibit Gi and p44/42
mitogen-activated protein kinase (ERK) activation, respectively,
caused very little inhibition of PAF-induced CCL2 production (~20%
inhibition). In contrast, these inhibitors almost completely blocked
both PAF-induced ERK phosphorylation and LTC4 generation in
PAFR cells. However, in mPAFR cells pertussis toxin only partially
inhibited PAF-induced ERK phosphorylation. A
Ca2+/calmodulin inhibitor had no effect on PAF-induced ERK
phosphorylation in PAFR cells but completely blocked the response in
mPAFR cells. These data demonstrate that receptor phosphorylation,
which serves to desensitize PAF-induced LTC4 generation, is
required for chemokine CCL2 production. They also indicate a previously
unrecognized selectivity in G protein usage and ERK activation for
PAF-induced responses. Whereas PAF-induced CCL2 production is, in large
part, mediated independently of Gi activation or ERK
phosphorylation, LTC4 generation requires ERK
phosphorylation, which is mediated by different G proteins depending on
the phosphorylation status of the receptor.
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INTRODUCTION |
Platelet-activating factor
(1-O-alkyl-2-acetyl-sn-glycero-3 phosphocholine,
PAF)1 is an important
mediator of inflammation that is released from mast cells, platelets,
neutrophils, monocytes, and macrophages (1, 2). PAF activates cell
surface G protein-coupled receptors (GPCRs) to induce divergent
biological functions (3). PAF is a potent leukocyte chemoattractant (4)
that also induces degranulation (5, 6), leukotriene C4
(LTC4) generation (7, 8), and chemokine gene expression in
a wide variety of cells (9-13). Although much has been learned
regarding the signaling pathways involved in PAF-induced chemotaxis and
degranulation (4, 6, 14), very little information is available on the
mechanism by which PAF stimulates LTC4 generation and
chemokine production.
Receptor phosphorylation by G protein-coupled receptor kinase (GRK) and
the subsequent recruitment of -arrestin are essential for uncoupling
the receptor from G proteins (15). Thus, phosphorylation-deficient mutants of chemoattractant receptors expressed in basophilic leukemia RBL-2H3 cells respond to the ligand for enhanced G protein activation, a more sustained Ca2+ mobilization, and a greater extent of
degranulation when compared with cells expressing wild-type receptors
(14, 16, 17). Receptor phosphorylation and -arrestin recruitment
have recently been shown to mediate MAP kinase activation for many
GPCRs (18-21). However, the chemoattractants formylpeptide and the
complement component C3a stimulate ERK phosphorylation via pathways
that do not require receptor phosphorylation or -arrestin
recruitment (17, 22). We have recently shown that C3a receptor
phosphorylation by the G protein-coupled receptor kinase provides a
stimulatory signal that synergizes with ERK activation to induce
chemokine CCL2 (also known as monocytes chemoattractant protein-1 or
MCP-1) production (17).
Unlike the C3a receptor, which couples to Gi, the PAF
receptor interacts with the Gi and Gq family of
G proteins to induce distinct biological responses (4, 23). In the
present study, we sought to determine the roles of receptor
phosphorylation, G protein usage, and ERK phosphorylation on
PAF-induced LTC4 generation and CCL2 production. For this
purpose, RBL-2H3 cells expressing the wild-type PAF receptor (PAFR) and
a phosphorylation-deficient mutant of PAFR (mPAFR) were used (14).
Here, we demonstrate that receptor phosphorylation, which serves to
desensitize PAF-induced LTC4 generation, is required for
chemokine CCL2 production. Furthermore, CCL2 production is, in large
part, mediated independently of Gi activation or ERK
phosphorylation. In contrast, LTC4 generation is dependent
on ERK phosphorylation, which is mediated via different mechanisms
depending on the phosphorylation status of the receptor.
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EXPERIMENTAL PROCEDURES |
Materials--
PAF, fluphenazine, Ro-31-8220, and U0126 were
purchased from Calbiochem. [3H]PAF
(1-O-hexadecyl-[acetyl-3H(N)]) (499.5 GBq/mmol) was obtained from PerkinElmer Life Sciences. Rabbit
anti-p44/42 MAP kinase and anti-phospho-p44/42 MAP kinase antibodies
were obtained from New England Biolabs (Beverly, MA). 12CA5 and
anti-mouse IgG-R-phycoerythrin antibodies were obtained from
Roche Molecular Biochemicals and Southern Biotechnology Associates (Birmingham, AL), respectively. Pertussis toxin (PTX) and all tissue culture reagents were purchased from Invitrogen. Indo-1 acetoxymethyl and Pluronic F-127 were from Molecular Probes
(Eugene, OR). A cPLA2 assay kit was purchased from Cayman
Chemicals (Ann Arbor, MI). LTC4 sandwich EIA and ECL
Western blotting analysis kits were purchased from Amersham
Biosciences. A CCL2 sandwich ELISA kit was purchased from BioSource
International (Camarillo, CA). Texas Red-conjugated goat anti-mouse IgG
was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Cell Culture, Transfection, Ca2+ Mobilization, and
Degranulation--
RBL-2H3 cells stably expressing hemaglutinin-tagged
PAFR and mPAFR were used in this study (6, 14). The cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
15% fetal bovine serum, glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 µg/ml) (6, 24). To express
equivalent receptors, 7 µg of cDNA encoding PAFR and 25 µg
cDNA encoding mPAFR were used for transient transfection by
electroporation. For studies with -arrestin, 20 µg of total
cDNA in the ratio of 1:4 for PAFR/mPAFR and arr2-GFP (17) were
used. Cells were cultured in complete growth medium, and experiments
were performed 16-18 h after transfection. Cell surface receptor
expression was determined by incubating cells with 12CA5 or an
isotope-matched antibody followed by phycoerythrin-conjugated mouse IgG and analyzed on a FACStarPLUS flow cytometer (BD
Biosciences, Mountain View, CA). For Ca2+ mobilization,
cells (3 × 106) were loaded with 1 µM
Indo-1 acetoxymethyl in the presence of 1 µM Pluronic
F127 for 30 min at room temperature. Cells were washed, resuspended in
1.5 ml of HEPES-buffered saline, and intracellular Ca2+
mobilization was determined as described previously (6). For degranulation, cells (5 × 104 cells/well) were
cultured overnight in a 96-well tissue culture plate. Cells were washed
with HEPES-buffered saline, stimulated with PAF, and the extent of
degranulation was determined by measuring the release of
-hexosaminidase (6, 24).
PAF Binding--
Binding studies were performed to evaluate the
number of receptors present in PAFR and mPAFR cells as described by
Carlson et al. (25). Briefly, RBL-2H3 cells (2 × 105/well) stably expressing PAFR and mPAFR were plated in
24-well dishes. Cells were washed twice with ice-cold buffer and then resuspended in 0.5 ml of the same buffer containing 10 nM
[3H]PAF alone or with 10 µM unlabeled PAF.
Cells were then incubated for 4 h at 4 °C. Cells were washed
with same buffer three times and lysed with 0.5 ml of 0.1% Triton
X-100. Bound radioactivity was determined by scintillation counting of
the cell lysates. Bmax values were normalized on
the basis of cell number by counting the number of cells in three
individual wells.
cPLA2 Enzyme Activity--
RBL-2H3 cells (2 × 106/ml) were stimulated with 100 nM PAF for 2 min at 37 °C. The reaction was stopped by adding 3 volumes of
ice-cold buffer. Cell pellets were resuspended in 50 µl of phosphate-buffered saline containing protease inhibitors, homogenized briefly in a microhomogenizer (0.2 ml), and centrifuged at
12,000 × g at 4 °C for 15 min.
PLA2 activity of the cell free lysate was determined
using a cPLA2 assay kit as described in the
manufacturer's protocol (Cayman Chemicals).
Assay of Chemokine CCL2 Production and LTC4
Generation--
RBL-2H3 cells (0.4 × 106/well)
expressing PAFR or mPAFR were cultured in complete growth medium
overnight. Cells were stimulated with PAF for 6 h (CCL2) and 20 min (LTC4) unless otherwise stated. Supernatants were
collected and stored frozen at 80 °C until analysis. CCL2 (17) and
LTC4 levels were quantified using sandwich ELISA and EIA
kits, respectively, as described in the manufacturer's protocols.
Trafficking of GFP- Arrestin by Confocal
Microscopy--
Cells coexpressing hemagglutinin-tagged
receptors with arr2-GFP were plated on 35-mm glass bottom dishes
(Mat Tek, Ashland, MA). The cells were stimulated with 100 nM PAF for 1 min at 37 °C. The reaction was stopped by
adding 3 volumes of cold phosphate-buffered saline, and the cells were
then washed and fixed with 2% paraformaldehyde solution for 30 min at
room temperature. To visualize cell surface receptor expression, cells
were incubated with the 12CA5 antibody followed by the Texas
Red-conjugated secondary antibody (Jackson ImmunoResearch). Cells were
observed using a laser-scanning confocal microscope (Olympus FluoView,
Olympus, Melville, NY) with a 60× lens. The GFP was excited by using a
488-nm argon/krypton laser, and Texas Red was excited at 515-540- and
570-nm band pass filters, respectively (17).
Phosphorylation of ERK-1/ERK-2--
RBL-2H3 cells expressing
PAFR or mPAFR were stimulated with PAF (100 nM) in HEPES
buffered saline, and the reaction was stopped at different time periods
by the addition of a 3-fold excess ice-cold phosphate-buffered saline
containing 1 mM sodium orthovanadate. Cells were mixed with
an equal volume of 2× SDS sample buffer and heated to 90 °C for 10 min. Samples were electrophoresed in 10% SDS-polyacrylamide gels and
transferred onto a nitrocellulose filter. The filter was treated
with 3% nonfat milk in phosphate-buffered saline and incubated with an
antibody specific for phosphorylated p44/42 MAP kinase. The reaction
was detected by enhanced chemiluminescence. The membrane was stripped
and reprobed with an antibody that reacts with unphosphorylated p44/42
MAP kinase (17, 26).
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RESULTS |
Characterization of PAF-induced LTC4 Generation and
Chemokine CCL2 Production in Transfected RBL-2H3 Cells--
We have
previously shown that PAF stimulates degranulation in RBL-2H3 cells
stably expressing PAFR with an EC50 value of 3 nM (6). In the present study, we stimulated these cells
with different concentrations of PAF and determined LTC4
generation and chemokine CCL2 production. As for degranulation, PAF
stimulated both LTC4 generation and CCL2 production with an
EC50 of ~3 nM (Fig.
1, A and B).
However, there were remarkable differences in the time course of these
responses. For example, LTC4 generation was essentially
complete within 1 min after stimulation (Fig. 1C). In
contrast, CCL2 production was not evident until 2 h, reached a
peak at ~6 h, and remained elevated for up to 18 h after
stimulation (Fig. 1D).

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Fig. 1.
Dose and time dependence of PAF-induced
LTC4 generation and CCL2 production. RBL-2H3 cells
stably expressing wild-type PAFR were stimulated with different
concentrations of PAF for 20 min or 6 h (A and
B) or with a fixed concentration of PAF (100 nM)
for the indicated time periods (C and D).
LTC4 generation and CCL2 production were quantified by EIA
and a sandwich ELISA, respectively. Basal values of 20.3 ± 1.2 (CCL2) and 16.6 ± 0.9 (LTC4) were subtracted from the
values shown. The data shown are from one of three similar
experiments.
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Roles of Receptor Phosphorylation and -Arrestin Recruitment on
PAF-induced LTC4 Generation and CCL2 Production--
We
have previously shown that receptor phosphorylation leads to the
desensitization of PAF-induced degranulation in leukocytes (14). To
determine the role of receptor phosphorylation on PAF-induced LTC4 generation and CCL2 production, RBL-2H3 cells
expressing wild-type PAFR and phosphorylation-deficient mutant mPAFR
were used (14). PAF stimulated an equivalent Ca2+
mobilization in PAFR and mPAFR cells (Fig.
2A). PAF-induced
LTC4 generation requires
Ca2+-dependent activation of cPLA2
(27). Therefore, the ability of PAF to stimulate cPLA2
activity in PAFR and mPAFR cells was determined. As shown in Fig.
2B, PAF caused equivalent cPLA2 activity in PAFR
and mPAFR cells. PAF also stimulated the generation of LTC4
in PAFR and mPAFR cells to similar levels (Fig. 2C). The incubation of PAFR cells with PAF for 6 h resulted in maximal CCL2
production (Figs. 1D and 2D). Under this
condition, PAF did not cause CCL2 production in mPAFR cells (Fig.
2D) despite the fact that this mutated receptor signals for
Ca2+ mobilization, PLA2 activity, and
LTC4 generation (Fig. 2, A-C). The
possibility that the lack of CCL2 production in mPAFR cells reflects a
slower rate of production is unlikely because incubation of these cells
with PAF for up to 18 h failed to induce any chemokine (Fig.
2D, inset).

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Fig. 2.
Effects of receptor phosphorylation on
PAF-induced Ca2+ mobilization, cPLA2 activity,
LTC4 generation, and CCL2 production in RBL-2H3 cells
stably expressing PAFR and mPAFR. A, RBL-2H3 cells
stably expressing wild-type PAFR or a phosphorylation-deficient mutant
(mPAFR) were loaded with Indo-1 acetoxymethyl and stimulated with PAF
(100 nM), and Ca2+ mobilization was determined.
B, cells were stimulated with PAF (100 nM) for 2 min, and cPLA2 activity in cell lysate was determined as
described in the "Experimental Procedures" section. C,
cells were stimulated with PAF (100 nM) for 20 min, and the
supernatants were removed and assayed for LTC4 generation
by EIA. D, cells were stimulated with PAF (100 nM) for 6 h, and the supernatants were removed and
assayed for CCL2 production by ELISA. Open bars,
PAF; filled bars, +PAF. The inset to
panel D shows the time course of CCL2 production in response
to 100 nM PAF in PAFR and mPAFR cells. The data shown are
from one of three similar experiments.
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Receptor-ligand binding studies were performed to evaluate the number
of receptors present in the cells used in the experiments described
above. RBL-2H3 cells expressed 152,300 ± 2,906 (n = 3) PAFRs per cell. In contrast, mPAFR cells expressed 28,630 ± 753 (n = 3) receptors per cell. It is therefore quite
possible that the inability of PAF to stimulate CCL2 production in
mPAFR cells reflects the expression of lower receptor numbers than
PAFR. We were previously unsuccessful in generating stable
transfectants in RBL-2H3 cells expressing high levels of mPAFR. For
this reason, we optimized a transient transfection procedure to express
PAFR and mPAFR at similar levels. Flow cytometric analysis of receptor expression using the 12CA5 antibody is shown in Fig.
3A. Using this system, we
tested the effects of PAF on LTC4 generation, degranulation, and CCL2 production. PAF stimulated significantly enhanced LTC4 generation and degranulation in mPAFR cells
when compared with PAFR cells (Fig. 3, B and C).
In contrast, the ability of PAF to induce CCL2 production in mPAFR
cells was ~60% less than that observed in PAFR cells (Fig.
3D). These data suggest that receptor phosphorylation, which
desensitizes PAF-induced degranulation and LTC4 generation,
provides a stimulatory signal for CCL2 production.

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Fig. 3.
Effects of receptor phosphorylation on
PAF-induced LTC4 generation, degranulation, and CCL2
production in RBL-2H3 cells transiently expressing PAFR and mPAFR.
Transient transfectants were generated in RBL-2H3 cells expressing
hemagglutinin-tagged PAFR or mPAFR. Cell surface receptor expression
was determined by flow cytometry using the 12CA5 antibody
(A). Cells were stimulated with PAF (100 nM) for
20 min, and the supernatants were removed and assayed for
LTC4 generation (B) and -hexosaminidase
release (C). Cells were stimulated with PAF (100 nM) for 6 h; the supernatants were removed and assayed
for CCL2 production by ELISA (D). Con, control;
*, p < 0.05; **, <0.01; ***, <0.001
versus the response in PAFR cells.
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Ligand-induced receptor phosphorylation is associated with the
translocation of -arrestin from the cytosol to the plasma membrane
(28, 29). To determine whether -arrestin recruitment correlates with
PAF-induced responses, transient transfectants were generated in
RBL-2H3 cells coexpressing PAFR or mPAFR and the -arrestin 2/green
fluorescent protein conjugate ( arr2-GFP). As shown in Fig.
4A, PAF caused the
translocation of arr2-GFP from the cytosol to the plasma membrane in
PAFR cells. In contrast, PAF did not induce this response in mPAFR
cells (Fig. 4B).

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Fig. 4.
PAF causes translocation of
arr2-GFP from the cytoplasm to the plasma membrane
in PAFR but not in mPAFR cells. RBL-2H3 cells transiently
coexpressing PAFR and arr2-GFP (A) or mPAFR and
arr2-GFP (B) were stimulated with PAF (100 nM) for 1 min, and the translocation of arr2-GFP was
determined by confocal microscopy. The data shown are representative of
three similar experiments.
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Roles of G Protein Usage, Phospholipase C Activation, and ERK
Phosphorylation on PAF-induced CCL2 Production and LTC4
Generation--
PAFR couples to Gi in RBL-2H3 cells to
induce chemotaxis (4). PAF also stimulates ERK phosphorylation in
Chinese hamster ovary (CHO) cells via a PTX-sensitive G protein
(30). In contrast, PAF-induced degranulation requires both
Gi and Gq-mediated activation of phospholipase
C , resulting in the activation of protein kinase C (PKC) and the
mobilization of Ca2+ (4, 6). We first evaluated the role of
signaling through G proteins on PAF-induced CCL2 production and
LTC4 generation in PAFR cells. Cells were cultured
overnight with or without pertussis toxin (PTX, 100 ng/ml), and its
effect on PAF-induced responses was determined. As shown in Fig.
5A, PTX inhibited PAF-induced CCL2 production by 27 ± 3.0%. In contrast, PTX blocked
LTC4 generation by 92.6 ± 4.6% (Fig. 5B).
To determine the role of phospholipase C -dependent
signaling, we tested the effects of the inhibitors of protein kinase C
(Ro-31-8220) and Ca2+/calmodulin (fluphenazine) on
PAF-induced responses. Both Ro-31-8220 and fluphenazine almost
completely blocked PAF-induced CCL2 production and LTC4
generation (>90% inhibition) (Fig. 5, A and B).
To test the role of p44/42 MAP kinase activation on PAF-induced
responses, the effect of U0126 was tested. This MAP kinase inhibitor
blocked PAF-stimulated CCL2 production by 24 ± 1.3% (Fig.
5A), but it inhibited LTC4 generation by 95 ± 1.5% in PAFR cells (Fig. 5B). The effects of these
inhibitors were also tested on PAF-induced LTC4 generation
in mPAFR cells. PTX blocked PAF-induced LTC4 generation in
mPAFR cells by 51.3 ± 3.7% (Fig 5C). This is in
contrast to the situation in PAFR cells where PTX inhibited PAF-induced
response by 92.6 ± 4.6% (Fig. 5B). However, as in
PAFR cells, Ro-31-8220, fluphenazine, or U0126 almost completely
blocked PAF-induced LTC4 generation in mPAFR cells (>90%
inhibition) (Fig. 5C).

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Fig. 5.
Effects of inhibitors on PAF-stimulated CCL2
production and LTC4 generation. RBL-2H3 cells stably
expressing PAFR (A and B) or mPAFR (C)
were preincubated with PTX (100 ng/ml, overnight), Ro-31-8220 (10 µM, 10 min), fluphenazine (FLU; 30 µM, 30 min) or U0126 (1 µM, 30 min), and
PAF (100 nM)-induced CCL2 production in PAFR (A)
and LTC4 generation in PAFR (B) and mPAFR
(C) cells were determined. CON, control; **,
p < 0.01 versus control.
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PAF Stimulates ERK Phosphorylation in RBL-2H3 Cells via Different
Mechanisms That Depend on the Phosphorylation Status of the
Receptor--
As shown above (Fig. 5, B and C),
PAF-induced LTC4 generation in PAFR and mPAFR cells appears
to be mediated by different G proteins. To test the role of different G
protein usage on PAF-induced ERK phosphorylation, the effects of PTX on
PAFR and mPAFR responses were determined. As shown in Fig.
6A, PTX caused substantial
inhibition of PAF-induced ERK phosphorylation in PAFR cells (91 ± 4.6% inhibition). In contrast, PTX was much less effective in
inhibiting this response in mPAFR cells (44 ± 3.0% inhibition)
(Fig. 6B). However, Ro-31-8220 caused almost complete
inhibition (>90%) of PAF-induced ERK phosphorylation in both cell
types (Fig. 6, A and B). Interestingly,
fluphenazine had no inhibitory effect on ERK phosphorylation mediated
by PAF in PAFR cells (Fig. 6A), but it inhibited the
response in mPAFR cells by 95.3 ± 2.3% (Fig 6B).
U0126 blocked ERK phosphorylation in response to PAF in both cell
types (Fig. 6, A and B).

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Fig. 6.
PAF stimulates ERK phosphorylation in RBL-2H3
cells via different mechanisms that depend on the phosphorylation
status of the receptor. RBL-2H3 cells stably expressing PAFR
(A) or mPAFR (B) were incubated with medium
(CON, control), PTX (100 ng/ml, overnight), Ro-31-8220 (10 µM, 10 min), fluphenazine (FLU, 30 µM, 30 min), or U0126 (1 µM, 30 min) and
stimulated with PAF (100 nM) for 1 min, and ERK
phosphorylation was determined by Western blotting using a
phospho-ERK-specific antibody. The extent of ERK phosphorylation is
expressed as percent of PAF-stimulated responses. ***,
p < 0.001 versus the response in PAFR
cells.
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DISCUSSION |
PAF plays an important role in inflammatory and cardiovascular
diseases (31, 32). PAF stimulates chemotaxis and degranulation in
leukocytes (6, 33). It also causes LTC4 generation and chemokine production in a variety of cell types (7, 9-13, 27). We have
previously utilized RBL-2H3 cells stably expressing PAFR and a
cytoplasmic tail deletion mutant receptor (mPAFR) and demonstrated that
receptor phosphorylation plays an important role in the desensitization of PAF-induced degranulation (14). The goal of the present study was to
determine the role of receptor phosphorylation on PAF-induced LTC4 generation and chemokine CCL2 production. Here, we
demonstrate that receptor phosphorylation desensitizes PAF-induced
LTC4 generation but provides a stimulatory signal for
chemokine CCL2 production. We also show distinct differences in both G
protein usage and ERK phosphorylation on LTC4 generation
and CCL2 production.
We recently reported that complement component C3a stimulates CCL2
production via a pathway that requires receptor phosphorylation (17).
Furthermore, Schwarz and Murphy (34) showed that Kaposi's sarcoma-associated herpes virus stimulates chemokine gene expression via the activation of a GPCR. However, truncation of the final five
amino acids in the cytoplasmic tail of the receptor, which contains one
serine and two threonine residues, resulted in a significant decrease
in chemokine production. These findings suggest that receptor
phosphorylation likely provides a general mechanism for stimulating
GPCR-induced chemokine gene expression. The receptor phosphorylation-dependent signal that mediates chemokine
production is not known. A substantial and growing body of evidence
suggests that the interaction of phosphorylated receptors with the
adapter molecule -arrestin leads to the formation of a scaffold in
the cytoplasm of cells. This complex directly interacts with Src, raf-1, ERK, c-Jun amino terminal kinase 3 (JNK-3), and a small GTP-binding protein, ADP-ribosylation factor 6 (ARF-6), to induce their
activation (20, 21, 35). These findings suggest that -arrestin could
be involved in PAF-induced CCL2 production. The demonstration that PAF
caused the recruitment of -arrestin in cells expressing PAFR but not
mPAFR is consistent with this notion. It is, however, important to note
that the ligand-induced phosphorylation of PAFR and the interaction of
the phosphorylated receptor with -arrestin do not require G protein
activation (36). Furthermore, PTX, which had no effect on
ligand-induced PAFR phosphorylation (4), caused a substantial
inhibition of PAF-induced ERK phosphorylation but had very little
effect on CCL2 production. In addition, U0126, which completely blocked
PAF-induced ERK phosphorylation, did not cause a substantial inhibition
of CCL2 production. These findings suggest that if -arrestin
mediates PAF-induced CCL2 production, it does so via the
activation of a pathway that is mostly independent of G protein
activation and ERK phosphorylation.
We have shown in the present study that when PAFR and mPAFR were
expressed at similar levels in RBL-2H3 cells, PAF was able to induce
CCL2 production in mPAFR cells but at lower level (Fig. 3). Schwarz and
Murphy (34) also made a similar observation for a wild-type and a
mutant GPCR for a Kaposi's sarcoma-associated herpes virus lacking
serine and threonine residues at its carboxyl terminus. These findings
suggest that receptor phosphorylation alone does not provide a full
signal for chemokine production. This contention is supported by the
finding that the inhibition of G q-mediated responses
such as Ca2+ mobilization and PKC activation leads to the
inhibition of PAF-induced CCL2 production in PAFR cells. We have
previously shown that C3a-induced chemokine production requires the
interaction of two signals, one receptor
phosphorylation-dependent and the other G
protein-dependent (17). In contrast to the situation with
PAFR, the G protein-dependent signal for the C3a receptor
(C3aR) involves Gi-mediated ERK phosphorylation (17). These
findings suggest that GPCR-induced chemokine production is mediated via
shared (receptor phosphorylation-dependent) and distinct
pathways that differ in the G protein usage of the receptor.
The demonstration in the present study that the treatment of cells with
U0126 or a Ca2+/calmodulin inhibitor leads to a substantial
inhibition of PAF-induced LTC4 generation is consistent
with the roles of ERK phosphorylation and Ca2+ mobilization
on LTC4 generation (7, 27, 37, 38). Although PAF-induced
ERK phosphorylation has been studied in some detail, the mechanism of
its activation has not been clearly defined (30, 39-41). The data
presented herein indicate that PAF-induced ERK phosphorylation in
RBL-2H3 cells is mediated by different mechanisms, depending on the
phosphorylation status of the receptor. For example, PAF-induced ERK
phosphorylation in PAFR cells requires activation of a PTX-sensitive G
protein. In contrast, this response in mPAFR cells involves both
PTX-sensitive as well as PTX-insensitive G proteins. We have previously
shown that PAF-induced Ca2+ mobilization depends on the
activation of G q (6). The demonstration that the
Ca2+/calmodulin inhibitor fluphenazine blocked ERK
phosphorylation in mPAFR but not PAFR cells raises the intriguing
possibility that receptor phosphorylation modulates the G
protein-coupling specificity of PAF-induced ERK phosphorylation.
In summary, we have previously shown that receptor phosphorylation
mediates the desensitization of PAF-induced degranulation (14). In the
present study, we demonstrate that receptor phosphorylation also serves
to desensitize PAF-induced LTC4 generation but provides a
stimulatory signal for chemokine CCL2 production. The activation of
many GPCRs leads to LTC4 generation and chemokine gene
expression in a variety of cell types (27, 34, 37, 42-46). Therefore, receptor phosphorylation is likely to have a greater impact on cellular
functions than previously recognized.
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ACKNOWLEDGEMENTS |
We thank Dr. Bruce Shenker and Ali Zekavat
(Fluorescence-activated Cell Sorter Core Facility, University of
Pennsylvania School of Dental Medicine) and Christopher S. Adams
(Confocal Core Facility, University of Pennsylvania School of Dental
Medicine), for assistance with fluorescence-activated cell sorter
analysis and confocal microscopy, respectively. We also thank Dr.
Marc Caron (Duke University) for providing the cDNA encoding
-arrestin 2-GFP.
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FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL-54166 and HL-63372.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pathology,
University of Pennsylvania School of Dental Medicine, 4010 Locust St.
(346 Levy Bldg.), Philadelphia, PA 19104-6002. Tel.: 215-573-1993; Fax:
215-573-2050; E-mail: ali@path.dental.upenn.edu.
Published, JBC Papers in Press, April 4, 2002, DOI 10.1074/jbc.M110210200
 |
ABBREVIATIONS |
The abbreviations used are:
PAF, platelet-activating factor;
GPCR, G protein-coupled receptor;
LTC4, leukotriene C4;
MAP, mitogen-activated
protein;
ERK, extracellular signal-regulated kinase;
CCL2, CC chemokine
receptor ligand 2 (formerly known as MCP-1);
PAFR, PAF receptor;
mPAFR, mutant PAFR;
PTX, pertussis toxin;
cPLA2, cytosolic
phospholipase A2;
EIA, enzyme immunoassay;
ELISA, enzyme-linked immunosorbent assay;
arr2, -arrestin 2;
GFP, green
fluorescent protein.
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