From the Regulatory Biology Laboratory, Institute of Molecular and
Cell Biology, National University of Singapore, 30 Medical Dr.,
Singapore 117609, Republic of Singapore and the
Department of Immunology, The Scripps Research Institute,
La Jolla, California 92037
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ABSTRACT |
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The large gene family encoding the regulators of
G protein signaling (RGS) proteins has been implicated in the fine
tuning of a variety of cellular events in response to G protein-coupled receptor activation. Several studies have shown that the RGS proteins can attenuate G protein-activated extracellular signal-regulated kinase
(ERK) group of mitogen-activated protein kinases. We demonstrate herein
that the production of inositol trisphosphate and the activation of the
p38 group of mitogen-activated protein kinases by the G protein-coupled
platelet-activating factor (PAF) receptor was attenuated by RGS16 in
both CHO cells transiently and stably expressing RGS16. The inhibition
was not observed with RGS2, RGS5, and a functionally defective form of
RGS16, RGS16R169S/F170C. The PAF-induced p38 and ERK
pathways appeared to be preferentially regulated by RGS16 and RGS1,
respectively. Overexpression of a constitutively active form of
G A wide array of extracellular signals are transduced into cells by
membrane-bound receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G
proteins)1 (1-5). Various G
protein effectors have been identified (6-12), including the three
classes of highly conserved mitogen-activated protein kinases:
MAPK/ERK, c-Jun NH2-terminal kinase/stress-activated protein kinase, and p38 MAPK (3, 13-17). Understanding the functional linkage in the receptor-G protein-effector cascade is a formidable task. The recently appreciated gene family encoding regulators of G
protein signaling (RGS proteins) has provided new insights into the
perplexing G protein-mediated signaling pathways.
RGS proteins serve as GTPase-activating proteins of a variety of G
protein We have previously identified and characterized RGS16 (also known as
RGS-r; Ref. 29); and we have shown that RGS16 binds G Construction of Plasmids--
The cDNAs encoding RGS2, RGS5,
RGS16, and the mutant RGS16R169S/F170C were generated as
described previously (30). Plasmids of FLAG-tagged p38, FLAG-tagged
ERK2, and platelet-activating factor receptor (PAFR) were constructed
as before (32, 33). Expression vectors for G Transient Transfection of Cells--
Chinese hamster ovary (CHO)
cells were maintained in RPMI 1640 medium supplemented with 10% fetal
bovine serum, 100 IU penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. Cells plated in 60-mm dishes were transfected
with various plasmids using DOSPER according to the manufacturer's
instructions (Boehringer Mannheim). In each transfection mixture, the
total amount of transfected DNA was adjusted to 5 µg with the
empty vector pCMV5 where necessary. Two µg each of pCMV5-RGS
constructs was transfected together with 1 µg of pCDNA3-PAFR, and
2 µg of pCDNA3-FLAG-p38 MAPK or pCDNA3-FLAG-ERK2. For
cotransfection with G protein plasmids, 1.5 µg of each indicated G
protein subunit was cotransfected with 1.5 µg of pCMV5-RGS, 1.5 µg
of pCDNA3-FLAG-p38, and 0.5 µg of pCDNA3-PAFR. The
transfection medium was replaced with fresh growth medium after 24 h, and cells were harvested 40 h after transfection.
Establishment of RGS16-inducible Expression in CHO
Cells--
The ecdysone-inducible expression system, based on the
Drosophila molting induction system and modified for
mammalian cells, uses the steroid hormone ecdysone analog
muristerone A to activate expression of the gene of interest via a
heterodimeric nuclear receptor (Invitrogen). EcR-CHO Chinese hamster
ovary cells containing the ecdysone receptor (Invitrogen) were
transfected with pIND-RGS16 or pIND-RGS16R169S/F170C
constructs using DOSPER (Boehringer Mannheim), and 24 h after transfection, the cells from each dish were diluted into a 150-mm dish
and selected in medium containing 800 µg/ml of G418 (Life Technologies, Inc.). Clones resistant to G418 were isolated after 2-3
weeks and expanded to test for RGS16 expression in response to
muristerone A induction. Muristerone A was added to a final concentration of 1 µM; after 24 h, cell lysates
obtained before and after induction were analyzed by SDS-polyacrylamide
gel electrophoresis and immunoblotted with the RGS16 antibody (see
below). Multiple independent expression lines for both RGS16 and
mutant RGS16R169S/F170C were obtained, and at least
two clones with little leakiness for each construct were expanded and
tested for hormonal responses. These expression cell lines yielded
similar experimental results in both basal and stimulated
IP3 production as well as MAPK activities to their
respective lines expressing the wild type RGS16 (designated CHO-R16) or
the mutant RGS16R169S/F170C (CHO-M18).
Measurement of Inositol 1,4,5-Trisphosphate Production--
The
levels of IP3 in CHO cells were measured by a competitive
radioreceptor assay using the BiotrakTM
D-myo-inositol 1,4,5-trisphosphate assay system
(Amersham Pharmacia Biotech). Briefly, cells were separately treated
with the indicated ligands for 10 min; IP3 was extracted
with 15% (v/v) trichloroacetic acid and neutralized with
NaHCO3. The samples and working IP3 standards
were incubated with the binding protein in the presence of
[3H]IP3, and the amount of radioactivity
bound was measured by liquid scintillation counting. The amount of
IP3 in the samples was determined by interpolation from the
standard curve.
Immunoprecipitation of MAPK and Kinase Assays--
Cells were
serum-starved for 2 h and stimulated with the agonists indicated.
After a wash with PBS, the cells were lysed in ice-cold lysis buffer
(20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM
Production of RGS16 Antibody and Western Blot
Analysis--
Bacterially expressed glutathione
S-transferase fusion RGS16 proteins, generated as described
previously (30), were used to raise antibodies in rabbits
(Bioprocessing Technology Center, National University of Singapore).
The specific immunoglobulins were purified from serum samples by
affinity binding as before (35). For Western blotting, protein samples
were separated on 10% SDS-polyacrylamide gels and transferred onto
polyvinylidene difluoride membranes (Immobilon-P;
Millipore Corp.). The membranes were incubated for 2 h with rabbit polyclonal anti-RGS16 antibody (1:500), anti-FLAG,
anti-p38, or anti-ERK antibodies (1:1000, Santa Cruz Biotechnology),
and bound antibodies were visualized by enhanced chemiluminescence
(Amersham Pharmacia Biotech) using horseradish peroxidase-conjugated antibodies.
Inhibition of PAF-stimulated IP3 Production by
RGS16--
To study the biological function of RGS16 in G
protein-mediated signaling pathway, we employed the ecdysone-inducible
expression system to establish stable cell lines expressing RGS16
(CHO-R16) and its mutant RGS16R169S/F170C (CHO-M18). As
shown in Fig. 1, A and
B (insets), induction of these cells with 1 µM muristerone A significantly increased the expression
of the RGS16 proteins.
It has been shown that activation of the G protein-coupled PAF receptor
can induce IP3 production (27, 36, 37). We observed a
6-fold increase in IP3 production when CHO cells were
stimulated with PAF (Fig. 1A), confirming the presence of
endogenous PAF receptors in CHO
cells.2 However, this
PAF-induced increase in IP3 was almost entirely suppressed
by the induced expression of RGS16. On the other hand, induced
expression of the mutant RGS16R169S/F170C, which can no
longer bind any G PAF-stimulated p38 MAPK Activation Is Attenuated by RGS16--
The
G protein-coupled PAF receptor is known to activate other downstream
targets besides phospholipase C; these include the p38 group of MAPK
(15, 38). Since RGS16 expression attenuated the PAF-induced
IP3 production, we asked if the PAF-stimulated p38 activity
could be inhibited by RGS16 by measuring the activity of endogenous p38
using GST-ATF2 as substrate in stable cell lines before and after
induction with muristerone A. For both RGS16- and mutant
RGS16R169S/F170C-expressing cells, experiments were
conducted on at least two other clones for each construct, and the
results obtained were similar to CHO-R16 and CHO-M18 cells, respectively.
In the CHO-R16 cells, stimulation with PAF increased the p38 activity
4.5-fold (Fig. 2A,
top). Pretreatment of the same cells with muristerone A to
induce RGS16 expression abolished the PAF-stimulated p38 activation. By
contrast, in CHO-M18 cells, induced expression of the mutant
RGS16R169S/F170C, which is defective in G protein binding,
did not inhibit the p38 activation by PAF (Fig. 2A,
bottom). RGS16 and its mutant had negligible effect on p38
activation by sorbitol.
Since PAF has been demonstrated to activate ERK, another member of the
MAPK family (39), we examined the endogenous ERK activity of CHO-R16
cells using MBP as the substrate. As expected, in cells without RGS16
induction, PAF treatment markedly increased ERK activity (Fig.
2B). RGS16 expression diminished the PAF-stimulated ERK
activity slightly but did not impair ERK activation by the phorbol
ester PMA. Therefore, compared with the extent of p38 suppression, the
inhibitory effect of RGS16 on ERK was much less. We did not detect any
activation of c-Jun NH2-terminal kinase/stress-activated protein kinase by PAF, as measured by similar immunokinase assays (data
not shown), in agreement with previous results by Nick et al. (38).
Differential Regulation of PAF-activated p38 and ERK Pathways by
RGS1 and RGS16--
To determine whether the inhibition of
PAF-stimulated p38 activity was specific to RGS16, we separately
transfected RGS1, RGS2, RGS5, or RGS16, along with PAFR and FLAG-tagged
p38, into CHO cells. Assayed in the Saccharomyces cerevisiae
strain YDM400 (YPH499 sst2-
It has been shown that RGS1 markedly impaired ERK activation by PAF
(25). We asked whether ERK activation by PAF was affected by RGS1 and
RGS16 in a similar manner as p38 activation. In cells cotransfected
with FLAG-tagged ERK, pronounced activation of ERK was observed upon
treatment with the phorbol ester PMA, which was not affected by RGS1 or
RGS16 (Fig. 4A). The inability of RGS1 and RGS16 to inhibit
PMA-induced activation of ERK is consistent with the proposed role for
RGS proteins as direct regulators of G proteins. As previously reported
(25), RGS1 dramatically suppressed ERK activation by PAF (Fig.
4A). However, inhibition of PAF-stimulated ERK activity by
RGS16 was to a much lower extent (Fig. 4A), suggesting a
differential regulation by RGS proteins on PAF-activated MAPK pathways.
PAF-induced p38 Activation Is Pertussis Toxin-insensitive--
ERK
activation by the heptahelical PAF receptor has been shown to be
mediated by both pertussis toxin (PTX)-sensitive and -insensitive G
proteins (39). In the case of p38 activation by PAF, it is unclear
which G proteins are involved. We compared the effects of PTX on the
PAF-induced signals in transiently transfected CHO cells. Preincubation
with 100 ng/ml PTX for 24 h partially abolished PAF-stimulated ERK
activity (Fig. 5). However, p38
activation by PAF was insensitive to PTX treatment, indicating that p38
activation by PAF is independent of G G
In the absence of PAF stimulation, the basal levels of p38 activities
in cells transfected with G
In contrast, in the absence of PAF treatment, cells overexpressing the
GTPase-deficient mutant G RGS16 Does Not Affect G Our results demonstrate for the first time that activation of p38
MAPK by the G protein-coupled PAF receptor can be attenuated by an RGS
family member, RGS16. Such an inhibitory effect was not observed with
RGS2, RGS5, or the mutant RGS16R169S/F170C which is
defective in G protein binding. RGS1 and RGS16 showed preferential
regulation for ERK and p38 MAPK, respectively, in response to PAF
stimulation. The RGS16 attenuation of p38 can be inhibited by the
GTPase-deficient mutant G We and others have shown that RGS proteins are rather promiscuous with
respect to various G protein Several lines of evidence suggest that p38 activation by G
protein-coupled receptor can be mediated by G The functional significance of RGS16 inhibition of PAF-stimulated p38
MAPK activity is as yet unclear. PAF exhibits a wide variety of
physiological and pathophysiological effects in various cells and
tissues, such as proto-oncogene expression in neuronal cells,
respiratory and cardiovascular functions, and inflammatory and immune
responses (50). It is conceivable that p38 mediates many aspects of the
PAF signaling. p38 MAPK is thought to play an important role in the
regulation of cellular responses during infection (51-53). Perhaps
RGS16 is recruited to fine tune the immune response through its effects
on the expression of proinflammatory molecules. Indeed, we have
observed changes in RGS16 expression when lymphoid cell lines are
challenged with PAF.5 As RGS1
and RGS16 display differential regulation of PAF-activated MAPK
pathways, it is possible that both RGS1 and RGS16 are necessary for
fine tuning the exquisitely orchestrated events elicited by PAF. Taken
together, our data show that RGS proteins display differential regulation of G protein-mediated p38 and ERK pathways, pointing to
distinct modulatory activities of different RGS proteins in G
protein-regulated signal transduction.
11 (G
11Q209L) prevented the
RGS16-mediated attenuation of p38 activity, suggesting that
G
q/11 is involved in PAF activation of p38. The
G
q/11 involvement is further supported by the
observation that p38 activation by PAF was pertussis toxin-insensitive. These results demonstrate for the first time that apart from ERK, p38
activation by a G protein-coupled receptor can be attenuated by an RGS
protein and provide further evidence for the specificity of RGS
function in G protein signaling pathways.
INTRODUCTION
Top
Abstract
Introduction
References
-subunits, terminating the signaling process by G
protein-coupled receptors (18-21). To date, about 20 mammalian RGS
proteins have been identified, all of which are defined by a highly
conserved domain of 120 amino acid residues in length. Underscoring the
significance of their sequence similarity within the RGS domain, RGS
proteins seem to have functional promiscuity as assayed by both their G
protein binding and functional resemblance to that of the Sst2 protein
in yeast. It is noteworthy that the RGS proteins vary in size, ranging
from 21 to 150 kDa, and contain divergent sequences flanking the
conserved RGS domain. The divergent sequences among their flanking
regions may be the specificity determinants for RGS function.
Functional specificity is best demonstrated by the finding that the RGS
domain-containing protein p115 RhoGEF specifically binds to
G
12 and G
13 and regulates Rho, mediating
cell morphology, adhesion, and cell proliferation (22, 23). In
addition, recent studies have shown that RGS4 and G
-interacting
protein block G
i-mediated inhibition of adenylyl cyclase
(24), whereas RGS1, RGS2, RGS3T, and RGS4 attenuate G
i-
or G
q-regulated activation of the ERK group of MAPK
(25-27). Furthermore, RGS3, RGS4, and G
-interacting protein
suppress G
q-mediated synthesis of inositol trisphosphate
(24, 26-28).
i2,
G
i3, and G
o subunits in the transition
state (30) and that RGS16 has GTPase-activating activity on these G
proteins (31). In this report, we show that RGS16 inhibits
platelet-activating factor (PAF)-stimulated p38 MAPK activation. The
RGS inhibition of p38 can be abolished by the mutant
G
11Q209L, which indicates an involvement of
G
q/11 in the PAF signaling. Moreover, we show that RGS
members have differential attenuating effects on the G protein-mediated
activation of ERK and p38. Our findings show that p38 activation by a G
protein-coupled receptor can be attenuated by an RGS protein and
provide further evidence that individual RGS members act as distinct
regulators for different G protein signaling pathways.
EXPERIMENTAL PROCEDURES
11 and its
GTPase-deficient mutant G
11Q209L were gifts from Dr. H. Itoh (Tokyo Institute of Technology, Yokohama, Japan). G
1 was amplified from rat pituitary cDNA, while
G
2 and RGS1 (34) were amplified from human brain
cDNA. The oligonucleotide sequences were as follows:
5'GAAGCTATGAGTGAGCTTGACCAGTTG-3' and 5'-GGGTTAGTTCCAGATCTTGAGGAA-3'
(for G
1); 5'-CCATGGCCAGCAACAACACCGCCA-3' and
5'-TCAGAGACTTAAAGGATGGCGCAG-3' (for G
2);
5'-CATATGCCAGGAATGTTCTTCTC-3' and 5'-GTCACTTTAGGCTATTAGCCTG-3' (for
RGS1). The G
i2 cDNA in pBluescript (30) was
used to generate G
i2Q205L by in vitro site-directed mutagenesis using the TransformerTM
site-directed mutagenesis kit (CLONTECH); the
oligonucleotide sequence used to create the mutant was
5'-TTTGATGTGGGTGGTCTGCGGTCTGAGCGCAAG-3'. pIND-RGS16 constructs
were generated by ligating the RGS16 or its mutant
(RGS16R169S/F170C) cDNA into the Asp718 and
BamHI sites of the ecdysone-inducible mammalian expression
vector pIND (Invitrogen). The mammalian expression vector used was
pCMV5 unless otherwise stated.
-glycerolphosphate, 1 mM sodium orthovanadate, 1 µg/ml
leupeptin, 1 mM phenylmethylsulfonyl fluoride). Kinases
were immunoprecipitated using either the mouse monoclonal anti-FLAG M2
(Eastman Kodak Co.), rabbit polyclonal anti-p38, or anti-ERK2
antibodies bound to Protein A/G Plus-agarose beads (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA). Kinase assays were performed on
the washed immunoprecipitates in a 50-µl reaction mixture comprising
the kinase buffer (25 mM Tris-HCl, pH 7.4, 5 mM
-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate, 10 mM
MgCl2), 5 µCi of [
-32P]ATP (Amersham
Pharmacia Biotech), kinase substrate (1 µg of affinity-purified
GST-ATF2 (residues 1-109; Ref. 33) for p38, 5 µg of myelin basic
protein (MBP) (Sigma) for ERK), and unlabeled ATP (50 µM
for p38; 100 µM for ERK). The reactions were carried out
at 30 °C for 30 min (p38) or 20 min (ERK) and terminated by adding
50 µl of 2× SDS-polyacrylamide gel electrophoresis sample buffer.
The boiled samples were separated by SDS-polyacrylamide gel
electrophoresis, and the radioactivity incorporated into the substrate
proteins was measured by an imaging analyzer (Molecular Dynamics model
425E) and detected by autoradiography. The amount of the kinase in each
immunoprecipitate was quantified by immunoblotting.
RESULTS
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Fig. 1.
Effects of RGS16 on ligand-stimulated
production of inositol 1,4,5-trisphosphate. The effects of RGS16
and its mutant RGS16R169S/F170C on the accumulation of
IP3 are shown in A and B,
respectively. Cells before (gray columns) and
after (dark columns) induction of RGS expression
by muristerone A were separately exposed for 10 min to 100 nM PAF, 100 ng/ml interleukin-8 (IL-8), 1 µM N-formyl-methionyl-leucyl-phenylalanine
(FMLP), or 10 µM lysophosphatidic acid
(LPA). The accumulation of IP3 in these
differently treated cells was measured as described under
"Experimental Procedures." The insets show relative
levels, assayed by Western blotting, of RGS16 (A) and
RGS16R169S/F170C (B) in cells before ( ) and
after (+) induction by muristerone A. The values represent the
means ± S.E. from three separate experiments performed in
quadruplicate. *, p < 0.05 compared with response in
cells before muristerone A induction.
subunit in vitro and has lost the
ability to inhibit pheromone signaling in yeast (30), did not affect
PAF-induced IP3 production (Fig. 1B). Treatment
of CHO-R16 cells with other agonists of G protein-coupled receptors, lysophosphatidic acid,
N-formyl-methionyl-leucyl-phenylalanine, and interleukin-8,
all marginally increased IP3 levels; these increases were
unaffected by induced RGS16 expression (Fig. 1A). These
results were repeated with at least two other clones.
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Fig. 2.
PAF-stimulated p38 activation is attenuated
by RGS16. A, suppression of PAF-stimulated p38 MAPK
activity by RGS16 but not RGS16R169S/F170C. Cells before
( ) and after (+) induction by muristerone A were separately exposed
for 90 s to 100 nM PAF or for 15 min to 0.4 M sorbitol. Endogenous p38 was immunoprecipitated and
assayed for its kinase activity using GST-ATF2 as the substrate. The
amount of the kinase in each immunoprecipitate was quantified by
immunoblotting. B, PAF-stimulated ERK activity is less
affected by RGS16. Cells were treated as above or with 160 nM PMA for 20 min. Endogenous ERK2 was immunoprecipitated
and assayed for its kinase activity using MBP as the substrate. Data
are expressed as -fold kinase activation compared with kinase activity
produced in unstimulated control cells. The values represent the
means ± S.E. from three separate experiments.
2 strain, provided by H. G. Dohlman and J. Thorner), RGS1, RGS2, and RGS5 exhibited similar
activity to that of RGS16 in the attenuation of pheromone
signaling.3 While activation
of p38 by PAF was significantly inhibited by RGS16 (Figs.
3 and
4B), it was only partially
inhibited by RGS1 (Fig. 4B) and was not inhibited by RGS2 or
RGS5 (Fig. 3).
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Fig. 3.
Comparison of inhibitory effects of RGS2,
RGS5, and RGS16 on PAF-stimulated p38 MAPK. CHO cells were
transiently transfected with 2 µg of FLAG-p38 and 1 µg of PAFR plus
2 µg each of RGS2, RGS5, or RGS16. Transfected cells were
unstimulated ( ) or stimulated (+) for 90 s with 100 nM PAF before immunoprecipitation of FLAG-p38; kinase
activity was assayed using GST-ATF2. The amount of the kinase in each
immunoprecipitate was quantified by immunoblotting. Data are expressed
as -fold p38 activation compared with p38 activity produced in
unstimulated, vector-transfected cells. The values represent the
means ± S.E. from three separate experiments.
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Fig. 4.
Differential attenuation of PAF-stimulated
MAPKs by RGS1 and RGS16. CHO cells were transiently transfected
with 2 µg of RGS1 or RGS16 plus 2 µg of FLAG-ERK2 (A) or
2 µg of FLAG-p38 (B) and 1 µg of PAFR. Transfected cells
were unstimulated ( ) or stimulated (+) for 90 s with 100 nM PAF or for 20 min with 160 nM PMA. Following
immunoprecipitation of FLAG-p38 and FLAG-ERK2, their kinase activities
were assayed using GST-ATF2 and MBP, respectively. The amount of the
kinase in each immunoprecipitate was quantified by immunoblotting. Data
are expressed as -fold kinase activation compared with kinase activity
produced in unstimulated, vector-transfected cells. The values
represent the means ± S.E. from three separate experiments.
i.
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Fig. 5.
Effects of pertussis toxin on PAF-stimulated
p38 and ERK activation. CHO cells were transiently transfected
with 2 µg of RGS16 or vector plus 2 µg of FLAG-p38 or FLAG-ERK2 and
1 µg of PAFR. Transfected cells were treated for 20 h with (+)
or without ( ) 100 ng/ml PTX before stimulation for 90 s with 100 nM PAF. Immunokinase assays were performed as described in
the legend to Fig. 4. Data are expressed as -fold kinase activation
compared with kinase activity produced in unstimulated,
vector-transfected cells. The values represent the means ± S.E.
from three separate experiments.
11Q209L, but Not G
i2Q205L, Can
Overcome the Suppression Effect of RGS16 on PAF-stimulated p38
Activity--
Mutations in the catalytic domain of G
subunits that
inhibit their intrinsic GTPase activity are known to render these
proteins constitutively active (40). To identify the G
subunit(s)
involved in the RGS16-mediated inhibition, we tested the effect of PAF on p38 MAPK in CHO cells transiently transfected with GTPase-deficient mutants of G
11 (G
11Q209L) or
G
i2 (G
i2Q205L).
i2, G
i2Q205L,
or G
11 were similar (Fig.
6). Stimulation by PAF in the
G
i2-, G
i2Q205L-, or
G
11-transfected cells increased p38 activity by about
5-fold each. RGS16 expression significantly diminished p38 activity to levels close to basal, whereas RGS1 showed less effect, consistent with
results in Fig. 4B.
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Fig. 6.
G 11Q209L, but not
G
i2Q205L, can overcome the
suppression effect of RGS16 on PAF-induced p38 activation. CHO
cells were transiently transfected with 1.5 µg of FLAG-p38, 1.5 µg
of vector, RGS1 or RGS16, plus 1.5 µg of G
i2,
G
i2Q205L, G
q/11, or
G
q/11Q209L, and 0.5 µg of PAFR. Transfected cells were
unstimulated (
) or stimulated (+) for 90 s with 100 nM PAF. Following immunoprecipitation of FLAG-p38, the
kinase activities were assayed using GST-ATF2, and the amount of the
kinase in each immunoprecipitate was quantified by immunoblotting. Data
are expressed as -fold p38 activation compared with p38 activity
produced in unstimulated, vector-transfected cells. The values
represent the means ± S.E. from three separate experiments.
11Q209L showed a high basal level of p38 activity, which was not affected by the expression of RGS1
or RGS16 (Fig. 6). Stimulation by PAF in these
G
11Q209L-transfected cells did not further increase the
activity of p38, suggesting that PAF stimulation of p38 MAPK occurs via
G
q/11 and that RGS16 interacts with G
q/11
to attenuate p38 activation induced by PAF.
1
2-mediated
p38 Activation--
It has been reported that overexpression of
G
1
2 can stimulate p38 MAPK activity (16).
We asked whether accelerated G
inactivation by RGS16 could also lead
to suppression of the G
-stimulated p38 activity. We included
G
1 and G
2 in the cotransfections and assayed for p38 activity in the presence or absence of overexpressed RGS16. Cells cotransfected with G
1
2
showed approximately 3-fold higher p38 activity than the control (Fig.
7). Overexpression of either RGS1 or
RGS16 did not affect the G
1
2-stimulated
p38 activity. Furthermore, PAF treatment in the
G
1
2-overexpressing cells resulted in an
additive increase in p38 activity. Consistent with observations above
(Figs. 4B and 6), this additive increase was only partially
suppressed by RGS1 expression but was suppressed to uninduced levels by
the expression of RGS16. In other words, the PAF induction component
was entirely suppressed by RGS16. These results also suggest that the
p38 activation pathway mediated by G
1
2 is
different from that activated by PAF.
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Fig. 7.
RGS16 does not affect
G 1
2-mediated
p38 activation. CHO cells were transiently transfected with 1.5 µg of FLAG-p38, 1.5 µg of RGS1 or RGS16, plus vector alone or with
0.75 µg each of G
1 and G
2 and 0.5 µg
of PAFR. Transfected cells were unstimulated (
) or stimulated (+) for
90 s with 100 nM PAF. Immunokinase assays were
performed as described in the legend to Fig. 6. Data are expressed as
-fold p38 activation compared with p38 activity produced in
unstimulated, vector-transfected cells. The values represent the
means ± S.E. from three separate experiments.
DISCUSSION
11Q209L, but not G
i2Q205L, indicating that G
q/11 mediates
signaling between the G protein-coupled PAF receptor and p38 MAPK.
-subunits in manifesting their
GTPase-activating activity. RGS4, for instance, binds to and serves as
a GTPase-activating protein for, G
i1,
G
i2, G
i3, G
o,
G
t, G
z, and G
q (41-44).
Similarly, RGS16 binds to G
i2, G
i3,
G
o, G
t (29, 30), and
G
q.3 The promiscuity of RGS proteins is also
suggested by the fact that many if not all of the RGS proteins can
complement the function of Sst2 in yeast pheromone signaling when
assayed using a
sst2 strain3 (25, 30, 45, 46). However,
investigations of RGS function in mammalian cells indicate that
individual RGS family members seem to be selective in the regulation of
specific G protein-linked signaling pathways. RGS1, RGS2, RGS3, and
RGS4 differ in their ability to impair interleukin-8 receptor signaling
of ERK, with RGS4 showing the greatest inhibition followed by RGS3,
RGS1, and RGS2 (25). G protein-gated inward rectifier potassium
channels evoked by agonist activation of muscarinic m2 receptors are
dramatically accelerated by coexpression of RGS1, RGS3, or RGS4, but
not RGS2 (47). In a separate study, RGS3, but not RGS1, RGS2, or RGS4, can suppress the IP3 responses induced by the
gonadotropin-releasing hormone (28). The present findings that
PAF-stimulated p38 MAPK activity is substantially attenuated by RGS16
and partially attenuated by RGS1 (but not by RGS2, RGS5, or
RGS16R169S/F170C) and that RGS1 is more effective than
RGS16 in blocking PAF-stimulated ERK activity provide further evidence
of selectivity. Moreover, it should be noted that assays in yeast may
not completely reflect what actually occurs in mammalian cells, since
yeasts do not contain the same repertoire of components. Indeed, we
have identified a mammalian membrane protein that specifically binds to
the NH2-terminal portion of a small subset of RGS
proteins.4 It is conceivable
that this membrane protein may somehow determine RGS functional
specificity in G protein interaction or cross-talk with other signaling
pathways. In addition, it is likely that other factors such as tissue
and cell type distribution, temporal expression, and post-translational
modification act in concert to dictate the specificity of RGS function.
q/11 and
G
(16, 48, 49). G
q can directly stimulate the
nonreceptor Bruton's tyrosine kinase Btk, which is required for the
activation of p38, as demonstrated in cells deficient for Btk (48). The
G
q/11-coupled receptor agonist phenylephrine activates
p38 MAPK in perfused rat heart (49). Moreover, p38 MAPK activation by
m1 muscarinic acetylcholine receptor involves both G
q/11
and G
, while m2 muscarinic acetylcholine and
-adrenergic
receptors act through G
in human embryonic kidney 293 cells (16).
It is intriguing that RGS4 can inhibit G
-activated inwardly
rectifying potassium channels (47), whereas RGS16 appears not to play a
role in the modulation of G
1
2-stimulated
p38 MAPK activation. The difference may be explained by the possibility
that different RGS proteins may be linked to different receptors,
although it cannot be ruled out that RGS16 may interfere with signaling
pathways mediated by other G
dimers.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. H. Itoh (Tokyo Institute of
Technology, Yokohama, Japan) for G11 and
G
11Q209L and C. Chen and X. Wang for providing invaluable reagents used in this study. We also thank Drs. W. Hong and
B. L. Tang for critical comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by grants from the National Science and Technology Board of Singapore (to S. C. L.) and by National Institutes of Health Grants GM51417 and AI41637 (to J. H.).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. Tel.: 65-779-4560; Fax: 65-779-1117; E-mail: mcblinsc{at}imcb.nus.edu.sg.
The abbreviations used are: G protein, guanine nucleotide-binding regulatory protein; ATF2, activating transcription factor-2; ERK, extracellular signal-regulated kinase; IP3, inositol 1,4,5-trisphosphate; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; PAF, platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine); PAFR, PAF receptor; PMA, phorbol 12-myristate 13-acetate; PTX, pertussis toxin; RGS, regulator of G protein signaling; CHO, Chinese hamster ovary.
2 A. Schonbrunn, "The Endogenous G Protein-coupled Receptor List" on the World Wide Web at http://www.biomedcomp.com/GPCR.html.
3 C. Chen and S-C. Lin, unpublished observations.
4 C. Chen and S.-C. Lin, unpublished results.
5 Y. Zhang, S. Y. Neo, and S.-C. Lin, unpublished observations.
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