RGS16 Attenuates Galpha q-dependent p38 Mitogen-activated Protein Kinase Activation by Platelet-activating Factor*

Yi Zhang, Soek Ying Neo, Jiahuai HanDagger , Lai Ping Yaw, and Sheng-Cai Lin§

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 Dagger  Department of Immunology, The Scripps Research Institute, La Jolla, California 92037

    ABSTRACT
Top
Abstract
Introduction
References

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 Galpha 11 (Galpha 11Q209L) prevented the RGS16-mediated attenuation of p38 activity, suggesting that Galpha q/11 is involved in PAF activation of p38. The Galpha 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

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 alpha -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 Galpha 12 and Galpha 13 and regulates Rho, mediating cell morphology, adhesion, and cell proliferation (22, 23). In addition, recent studies have shown that RGS4 and Galpha -interacting protein block Galpha i-mediated inhibition of adenylyl cyclase (24), whereas RGS1, RGS2, RGS3T, and RGS4 attenuate Galpha i- or Galpha q-regulated activation of the ERK group of MAPK (25-27). Furthermore, RGS3, RGS4, and Galpha -interacting protein suppress Galpha q-mediated synthesis of inositol trisphosphate (24, 26-28).

We have previously identified and characterized RGS16 (also known as RGS-r; Ref. 29); and we have shown that RGS16 binds Galpha i2, Galpha i3, and Galpha 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 Galpha 11Q209L, which indicates an involvement of Galpha 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

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 Galpha 11 and its GTPase-deficient mutant Galpha 11Q209L were gifts from Dr. H. Itoh (Tokyo Institute of Technology, Yokohama, Japan). Gbeta 1 was amplified from rat pituitary cDNA, while Ggamma 2 and RGS1 (34) were amplified from human brain cDNA. The oligonucleotide sequences were as follows: 5'GAAGCTATGAGTGAGCTTGACCAGTTG-3' and 5'-GGGTTAGTTCCAGATCTTGAGGAA-3' (for Gbeta 1); 5'-CCATGGCCAGCAACAACACCGCCA-3' and 5'-TCAGAGACTTAAAGGATGGCGCAG-3' (for Ggamma 2); 5'-CATATGCCAGGAATGTTCTTCTC-3' and 5'-GTCACTTTAGGCTATTAGCCTG-3' (for RGS1). The Galpha i2 cDNA in pBluescript (30) was used to generate Galpha 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.

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 beta -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 beta -glycerolphosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate, 10 mM MgCl2), 5 µCi of [gamma -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.

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.

    RESULTS

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.


View larger version (23K):
[in this window]
[in a new window]
 
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.

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 Galpha 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.

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.


View larger version (25K):
[in this window]
[in a new window]
 
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.

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-Delta 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).


View larger version (36K):
[in this window]
[in a new window]
 
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.


View larger version (21K):
[in this window]
[in a new window]
 
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.

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 Galpha i.


View larger version (27K):
[in this window]
[in a new window]
 
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.

Galpha 11Q209L, but Not Galpha i2Q205L, Can Overcome the Suppression Effect of RGS16 on PAF-stimulated p38 Activity-- Mutations in the catalytic domain of Galpha subunits that inhibit their intrinsic GTPase activity are known to render these proteins constitutively active (40). To identify the Galpha 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 Galpha 11 (Galpha 11Q209L) or Galpha i2 (Galpha i2Q205L).

In the absence of PAF stimulation, the basal levels of p38 activities in cells transfected with Galpha i2, Galpha i2Q205L, or Galpha 11 were similar (Fig. 6). Stimulation by PAF in the Galpha i2-, Galpha i2Q205L-, or Galpha 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.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 6.   Galpha 11Q209L, but not Galpha 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 Galpha i2, Galpha i2Q205L, Galpha q/11, or Galpha 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.

In contrast, in the absence of PAF treatment, cells overexpressing the GTPase-deficient mutant Galpha 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 Galpha 11Q209L-transfected cells did not further increase the activity of p38, suggesting that PAF stimulation of p38 MAPK occurs via Galpha q/11 and that RGS16 interacts with Galpha q/11 to attenuate p38 activation induced by PAF.

RGS16 Does Not Affect Gbeta 1gamma 2-mediated p38 Activation-- It has been reported that overexpression of Gbeta 1gamma 2 can stimulate p38 MAPK activity (16). We asked whether accelerated Galpha inactivation by RGS16 could also lead to suppression of the Gbeta gamma -stimulated p38 activity. We included Gbeta 1 and Ggamma 2 in the cotransfections and assayed for p38 activity in the presence or absence of overexpressed RGS16. Cells cotransfected with Gbeta 1gamma 2 showed approximately 3-fold higher p38 activity than the control (Fig. 7). Overexpression of either RGS1 or RGS16 did not affect the Gbeta 1gamma 2-stimulated p38 activity. Furthermore, PAF treatment in the Gbeta 1gamma 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 Gbeta 1gamma 2 is different from that activated by PAF.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 7.   RGS16 does not affect Gbeta 1gamma 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 Gbeta 1 and Ggamma 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

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 Galpha 11Q209L, but not Galpha i2Q205L, indicating that Galpha q/11 mediates signaling between the G protein-coupled PAF receptor and p38 MAPK.

We and others have shown that RGS proteins are rather promiscuous with respect to various G protein alpha -subunits in manifesting their GTPase-activating activity. RGS4, for instance, binds to and serves as a GTPase-activating protein for, Galpha i1, Galpha i2, Galpha i3, Galpha o, Galpha t, Galpha z, and Galpha q (41-44). Similarly, RGS16 binds to Galpha i2, Galpha i3, Galpha o, Galpha t (29, 30), and Galpha 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 Delta 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.

Several lines of evidence suggest that p38 activation by G protein-coupled receptor can be mediated by Galpha q/11 and Gbeta gamma (16, 48, 49). Galpha 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 Galpha 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 Galpha q/11 and Gbeta gamma , while m2 muscarinic acetylcholine and beta -adrenergic receptors act through Gbeta gamma in human embryonic kidney 293 cells (16). It is intriguing that RGS4 can inhibit Gbeta gamma -activated inwardly rectifying potassium channels (47), whereas RGS16 appears not to play a role in the modulation of Gbeta 1gamma 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 Gbeta gamma dimers.

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.

    ACKNOWLEDGEMENTS

We thank Dr. H. Itoh (Tokyo Institute of Technology, Yokohama, Japan) for Galpha 11 and Galpha 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.

    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.

    REFERENCES
Top
Abstract
Introduction
References

  1. Hepler, J. R., and Gilman, A. G. (1992) Trends Biochem. Sci. 17, 383-387[CrossRef][Medline] [Order article via Infotrieve]
  2. Neer, E. J. (1995) Cell 80, 249-257[Medline] [Order article via Infotrieve]
  3. Gutkind, J. S. (1998) J. Biol. Chem. 273, 1839-1842[Free Full Text]
  4. Fields, T. A., and Casey, P. J. (1997) Biochem. J. 321, 561-571[Medline] [Order article via Infotrieve]
  5. Schneider, T., Igelmund, P., and Hescheler, T. (1997) Trends Pharmacol. Sci. 18, 8-11[CrossRef][Medline] [Order article via Infotrieve]
  6. Miller, B. A., Bell, L., Hansen, C. A., Robishaw, J. D., Linder, M. E., and Cheung, J. Y. (1996) J. Clin. Invest. 98, 1728-1736[Abstract/Free Full Text]
  7. Honda, Z., Takano, T., Hirose, N., Suzuki, T., Muto, A., Kume, S., Mikoshiba, K., Itoh, K., and Shimizu, T. (1995) J. Biol. Chem. 270, 4840-4844[Abstract/Free Full Text]
  8. Buhl, A. M., Johnson, N. L., Dhanasekaran, N., and Johnson, G. L. (1995) J. Biol. Chem. 270, 24631-24634[Abstract/Free Full Text]
  9. Fromm, C., Coso, O. A., Montaner, S., Xu, N., and Gutkind, J. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10098-10103[Abstract/Free Full Text]
  10. Rhee, S. G., and Bae, Y. S. (1997) J. Biol. Chem. 272, 15045-15048[Free Full Text]
  11. Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267[Medline] [Order article via Infotrieve]
  12. Vanhaesebroeck, B., Leevers, S. J., Panayotou, G., and Waterfield, M. D. (1997) Trends Biochem. Sci. 22, 267-272[CrossRef][Medline] [Order article via Infotrieve]
  13. Robinson, M. J., and Cobb, M. H. (1997) Curr. Opin. Cell Biol. 9, 180-186[CrossRef][Medline] [Order article via Infotrieve]
  14. Coso, O. A., Teramoto, H., Simonds, W. F., and Gutkind, J. S. (1996) J. Biol. Chem. 271, 3963-3966[Abstract/Free Full Text]
  15. Nahas, N., Molski, T. F. P., Fernandez, G. A., and Sha'afi, R. I. (1996) Biochem. J. 318, 247-253[Medline] [Order article via Infotrieve]
  16. Yamauchi, J., Nagao, M., Kaziro, Y., and Itoh, H. (1997) J. Biol. Chem. 272, 27771-27777[Abstract/Free Full Text]
  17. Reiser, C. O. A., Lanz, T., Hofmann, F., Hofer, G., Rupprecht, H. D., and Goppelt-Struebe, M. (1998) Biochem. J. 330, 1107-1114[Medline] [Order article via Infotrieve]
  18. Dohlman, H. G., and Thorner, J. (1997) J. Biol. Chem. 272, 3871-3874[Free Full Text]
  19. Koelle, M. R. (1997) Curr. Opin. Cell Biol. 9, 143-147[CrossRef][Medline] [Order article via Infotrieve]
  20. Berman, D. M., and Gilman, A. G. (1998) J. Biol. Chem. 273, 1269-1272[Free Full Text]
  21. Kehrl, J. H. (1998) Immunity 8, 1-10[Medline] [Order article via Infotrieve]
  22. Kozasa, T., Jiang, X., Hart, M. J., Sternweis, P. M., Singer, W. D., Gilman, A. G., Bollag, G., and Sternweis, P. C. (1998) Science 280, 2109-2111[Abstract/Free Full Text]
  23. Hart, M. J., Jiang, X., Kozasa, T., Roscoe, W., Singer, W. D., Gilman, A. G., Sternweis, P. C., and Bollag, G. (1998) Science 280, 2112-2114[Abstract/Free Full Text]
  24. Huang, C., Hepler, J. R., Gilman, A. G., and Mumby, S. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6159-6163[Abstract/Free Full Text]
  25. Druey, K. M., Blumer, K. J., Kang, V. H., and Kehrl, J. H. (1996) Nature 379, 742-746[CrossRef][Medline] [Order article via Infotrieve]
  26. Yan, Y., Chi, P. P., and Bourne, H. R. (1997) J. Biol. Chem. 272, 11924-11927[Abstract/Free Full Text]
  27. Chatterjee, T. K., Eapen, A. K., and Fisher, R. A. (1997) J. Biol. Chem. 272, 15481-15487[Abstract/Free Full Text]
  28. Neill, J. D., Duck, L. W., Sellers, J. C., Musgrove, L. C., Scheschonka, A., Druey, K. M., and Kehrl, J. H. (1997) Endocrinology 138, 843-846[Abstract/Free Full Text]
  29. Chen, C. K., Wieland, T., and Simon, M. I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12885-12889[Abstract/Free Full Text]
  30. Chen, C., Zheng, B., Han, J., and Lin, S-C. (1997) J. Biol. Chem. 272, 8679-8685[Abstract/Free Full Text]
  31. Chen, C., and Lin, S-C. (1998) FEBS Lett. 422, 359-362[CrossRef][Medline] [Order article via Infotrieve]
  32. Kravchenko, V. V., Pan, Z., Han, J., Herbert, J-M., Ulevitch, R. J., and Ye, R. D. (1995) J. Biol. Chem. 270, 14928-14934[Abstract/Free Full Text]
  33. Han, J., Lee, J-D., Jiang, Y., Li, Z., Feng, L., and Ulevitch, R. J. (1996) J. Biol. Chem. 271, 2886-2891[Abstract/Free Full Text]
  34. Hong, J. X., Wilson, G. L., Fox, C. H., and Kehrl, J. H. (1993) J. Immunol. 150, 3895-3904[Abstract/Free Full Text]
  35. Lin, S-C., and Morrison-Bogorad, M. (1991) J. Biol. Chem. 266, 23347-23353[Abstract/Free Full Text]
  36. Koike, H., Imanishi, N., Natsume, Y., and Morooka, S. (1994) Eur. J. Pharmacol. 269, 299-309[CrossRef][Medline] [Order article via Infotrieve]
  37. Lin, A-Y., and Rui, Y-C. (1994) Biochim. Biophys. Acta 1224, 323-328[Medline] [Order article via Infotrieve]
  38. Nick, J. A., Avdi, N. J., Young, S. K., Knall, C., Gerwins, P., Johnson, G. L., and Worthen, G. S. (1997) J. Clin. Invest. 99, 975-986[Abstract/Free Full Text]
  39. Honda, Z., Takano, T., Gotoh, Y., Nishida, E., Ito, K., and Shimizu, T. (1994) J. Biol. Chem. 269, 2307-2315[Abstract/Free Full Text]
  40. Bourne, H. R., Sanders, D. A., and McCormick, F. (1991) Nature 349, 117-127[CrossRef][Medline] [Order article via Infotrieve]
  41. Berman, D. M., Wilkie, T. M., and Gilman, A. G. (1996) Cell 86, 445-452[Medline] [Order article via Infotrieve]
  42. Berman, D. M., Kozasa, T., and Gilman, A. G. (1996) J. Biol. Chem. 271, 27209-27212[Abstract/Free Full Text]
  43. Watson, N., Linder, M. E., Druey, K. M., Kehrl, J. H., and Blumer, K. J. (1996) Nature 383, 172-175[CrossRef][Medline] [Order article via Infotrieve]
  44. Hepler, J. R., Berman, D. M., Gilman, A. G., and Kozasa, T. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 428-432[Abstract/Free Full Text]
  45. Dohlman, H. G., Song, J., Ma, D., Courchesne, W. E., and Thorner, J. (1996) Mol. Cell. Biol. 16, 5194-5209[Abstract]
  46. Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212[Medline] [Order article via Infotrieve]
  47. Doupnik, C. A., Davidson, N., Lester, H. A., and Kofuji, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10461-10466[Abstract/Free Full Text]
  48. Bence, K., Ma, W., Kozasa, T., and Huang, X-Y. (1997) Nature 389, 296-299[CrossRef][Medline] [Order article via Infotrieve]
  49. Lazou, A., Sugden, P. H., and Clerk, A. (1998) Biochem. J. 332, 459-465[Medline] [Order article via Infotrieve]
  50. Chao, W., and Olson, M. S. (1993) Biochem. J. 292, 617-629[Medline] [Order article via Infotrieve]
  51. Han, J., Lee, J-D., Bibbs, L., and Ulevitch, R. J. (1994) Science 265, 808-811[Medline] [Order article via Infotrieve]
  52. Lee, J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F., Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W., Strickler, J. E., McLaughlin, M. M., Siemens, I. R., Fisher, S. M., Livi, G. P., White, J. R., Adams, J. L., and Young, P. R. (1994) Nature 372, 739-746[CrossRef][Medline] [Order article via Infotrieve]
  53. Han, J., Jiang, Y., Li, Z., Kravchenko, V. V., and Ulevitch, R. J. (1997) Nature 386, 296-299[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.