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Departments of Pharmacology and Physiology (S.M., M.G., T.M.B., A.V.S.) and Biochemistry and Biophysics (A.V.S.), University of Rochester School of Medicine and Dentistry, Rochester, New York
Department of Pharmacology (G.G.T.), University of Texas Southwestern Medical Center, Dallas, Texas
Abstract
Peptides derived from a random-peptide phage display screen with purified G12 subunits as the target promote the dissociation of G protein heterotrimers in vitro and activate G protein signaling in intact cells. In vitro, one of these peptides (SIRKALNILGYPDYD; SIRK) promotes subunit dissociation by binding directly to G subunits and accelerating the dissociation of GGDP without catalyzing nucleotide exchange. The experiments described here were designed to test whether the mechanism of SIRK action in vitro is in fact the mechanism of action in intact cells. We created a mutant of G1 subunits (1W332A) that does not bind SIRK in vitro. Transfection of G1W332A mutant into Chinese hamster ovary cells blocked peptide-mediated activation of extracellular signal-regulated kinase (ERK), but it did not affect receptor-mediated G subunit-dependent ERK activation, indicating that G subunits are in fact the direct target in cells responsible for ERK activation. To determine whether free G subunits were released from G protein heterotrimers upon peptide treatment, cells were transfected with Ric-8A, a guanine nucleotide exchange factor for free GGDP, but not heterotrimeric G proteins. Ric-8A-transfected cells displayed enhanced myristoyl-SIRKALNILGYPDYD (mSIRK)-dependent inositol phosphate (IP) release and ERK activation. Ric-8A also enhanced ERK activation by the Gi-linked G protein coupled receptor agonist lysophosphatidic acid. Inhibitors of G subunit function blocked Ric-8-enhanced activation of ERK and IP release. These results suggest that one potential function of Ric-8 in cells is to enhance G protein G subunit signaling. Overall, these experiments provide further support for the hypothesis that mSIRK promotes G protein subunit dissociation to release free subunits in intact cells.
G protein-coupled receptors (GPCRs) comprise a large family of proteins that bind a diverse array of molecules and communicate this binding information to alterations of cell physiology (Gilman, 1987; Hamm, 1998). Activated GPCRs interact with heterotrimeric G proteins to catalyze the exchange of bound GDP for GTP. This process requires the presence of both G and G subunits, and there is evidence for direct binding of the receptor to both G and G subunits (Taylor et al., 1994, 1996). Binding of GTP to the G subunit activates the G protein and is thought to cause dissociation of G subunits from G subunits, liberating free GGTP and G subunits to interact with downstream target proteins and regulate their activities.
It has become apparent that receptor-independent mechanisms exist for G protein activation. AGS proteins, discovered in a yeast screen for activation of the pheromone pathway, all act to release subunits from subunits (Cismowski et al., 2001). GPR or GoLoco peptides derived from AGS proteins promote dissociation of GGDP subunits from G subunits, causing release of G (Peterson et al., 2000; Kimple et al., 2002; Ghosh et al., 2003). In addition, a novel protein, Ric-8, has been identified that binds specifically to free GGDP subunits and promotes GDP release (Miller et al., 2000; Tall et al., 2003). Thus, a system potentially exists outside of G protein-coupled receptors for G protein activation that involves sequential action of proteins to release GDP followed by Ric-8-catalyzed nucleotide exchange. Several recent publications suggest a relationship between AGS proteins and Ric-8 in unconventional G protein signaling during spindle pole positioning in the initial cell division events in Caenorhabditis elegans zygotes (Afshar et al., 2004; Couwenbergs et al., 2004; Hess et al., 2004).
We have identified a mechanism by which G binding peptides can activate G protein signaling by a receptor-independent mechanism. Cell-permeant versions of peptides identified by random-peptide phage display screening against G protein subunits promote activation of G protein subunit-dependent pathways, including mitogen-activated protein kinase and phospholipase C activation, in intact cells (Goubaeva et al., 2003). In vitro these peptides bind directly to G protein 12 subunits and accelerate dissociation of GiGDP subunits from Gi112 heterotrimers (Ghosh et al., 2003). The structure of G protein 12 subunits bound to one of the peptides (SIGKAFKILGYPDYD; SIGK) has been solved (T. L. Davis, T. M. Bonacci, S. R. Sprang, and A. V. Smrcka, submitted). In the structural model, the peptide is bound to a site on G12 subunits normally occupied by the switch II helix of G subunits (Wall et al., 1995; Lambright et al., 1996). These data suggest a molecular mechanism by which these peptides promote G protein subunit dissociation by interfering with G subunit interactions with G subunits.
Although these in vitro data support a model for peptide-mediated dissociation of GGDP from G as the mechanism for (myristoyl-SIRKALNILGYPDYD) (mSIRK) activation of signaling pathways in intact cells, they do not directly demonstrate this. In this study, we set out to demonstrate that G protein subunits are the direct target of these cell-permeable peptides in cells and that interaction of these peptides with heterotrimeric G proteins results in release of free GGDP in intact cells. As part of our analysis, we studied the ability of Ric-8 proteins to affect peptide-mediated responses based on the ability of Ric-8 to selectively activate free GGDP subunits. We were surprised to find that Ric-8 can enhance G protein subunit-mediated responses, probably by a mechanism that involves sequestration of free G subunits.
Materials and Methods
Materials and Plasmids. GFP-G1, GFP-G1W332A, G2, and Ric-8A-3HA were in pCI-Neo. EE-i1 and -t were supplied from Guthrie cDNA Resource Center (Rolla, MO) in pcDNA 3.1+; ARKct, kindly supplied by Dr. Robert Lefkowitz (Duke University, Durham, NC), was in pRK5 and Ric-8A; and Ric-8B was in pCMV5. mSIRK and SIGK were synthesized and purified by Alpha Diagnostics International (San Antonio, TX). myo-[3H]Inositol (25 Ci/mmol) was from PerkinElmer Life and Analytical Sciences (Boston, MA). Pertussis toxin, lysophosphatidic acid (LPA), and ATP were from Sigma-Aldrich (St. Louis, MO). Rabbit anti-ERK and anti-phospho-ERK antisera were from Cell Signaling Technologies Inc. (Beverly, MA). Anti Ric-8A antiserum was generated in rabbits against holo-purified Ric-8A protein by Caprologics, Inc. (Hardwick, MA). Mouse anti-HA and anti-EE antisera were from Covance (Princeton, NJ). Mouse anti-GFP, goat anti-rabbit IgG-horseradish peroxidase conjugate (HRP) and goat anti-mouse IgG-HRP were from Roche Diagnostics (Indianapolis, IN).
Construction and Purification of Biotinylated G Subunits. Construction of baculovirus encoding biotinylated G1 (bG1) subunit in the baculovirus transfer vector PDW464 was described previously (Goubaeva et al., 2003). For other experiments, G protein 1 subunits were tagged at the amino terminus with GFP. We used GFP-tagged 1 subunits to monitor subunit transfection efficiency by epifluorescence microscopy and to monitor the level of expression of the transfected protein relative to endogenous subunits by immunoblotting. We (unpublished data) and others have shown that amino terminal modification of G with GFP does not alter G subunit functions (Azpiazu and Gautam, 2004). Mutants (W332A and K337A) were created by overlap extension polymerase chain reaction (PCR) using standard methods, and the entire protein coding region was sequenced to confirm the presence of the mutation and lack of additional mutations.
Phage ELISA. The phage used in this study was from the random-peptide phage display screen described previously (Scott et al., 2001). Phage were propagated and ELISA assays with bG12 subunits were performed as described previously (Smrcka and Scott, 2002).
Measurement of - Interactions via Flow Cytometry. The fluorescein-labeled i1 used in these experiments was prepared as described previously (Sarvazyan et al., 1998), and competition assays were performed as described in detail in Ghosh et al. (2003). In brief, for competition based assays, 100 to 200 pM fluorescein-labeled i1 and indicated concentrations of peptides were added to 50 pM bG12 immobilized on 105 beads per milliliter of buffer and incubated at room temperature for 30 min to reach equilibrium. The bead-associated fluorescence was then recorded in the flow cytometer. The data were corrected for nonspecific binding and fit with a sigmoid dose-response curve using Prism 4 (GraphPad Software Inc., San Diego, CA).
Cell Culture and Transfection. All cell culture reagents were obtained from Invitrogen (Carlsbad, CA). Chinese hamster ovary cells obtained from American Type Culture Collection (Manassas, VA) were grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO2. Cells were grown in six-well dishes (35-mm wells) for ERK activation experiments. For these experiments, 200 ng of Ric-8A or Ric-8B was transfected with or without 800 ng of ARK-ct in pRK5, 800 ng of t, or pRK5 empty vector control, using LipofectAMINE Plus (Invitrogen) unless otherwise indicated. For inositol phosphate (IP) release measurements, cells were grown in 12-well plates and 200 ng of Ric-8A was transfected with 200 ng of the appropriate inhibitor with a total of 400 ng of DNA transfected in each well. Transfections were performed 48 h before the final treatment and when multiple plasmids were transfected, appropriate amounts of control cDNAs were added such that the total DNA transfected was constant in each experiment.
Measurement of ERK Activation and General Immunoblotting. For measurement of phospho-ERK, serum was removed from 50 to 80% confluent CHO cells 16 h before treatment. Peptides in dimethyl sulfoxide, dimethyl sulfoxide vehicle, or other agonists were diluted 100- to 400-fold into the medium and incubated at 37°C for the indicated times. For all immunoblotting: after treatment, cells were transferred to ice, and the medium was quickly aspirated and replaced with 100 e of 2x SDS sample buffer. The resulting suspension was boiled for 5 min, and 5 to 10 e was loaded onto a 12% SDS-polyacrylamide gel. After SDS-PAGE, the proteins were transferred to nitrocellulose for 16 h at 25 V. The transferred proteins were immunoblotted using standard protocols with 1:1000 dilution of primary antibody (unless otherwise indicated) and 1:1000 dilution of the appropriate IgG-horseradish peroxidase conjugate. The proteins were visualized by incubation with the chemiluminescence reagent "Pico" (Pierce Chemical, Rockford, IL) and exposure to film. Film was quantitated by densitometry. Film was quantitated at different levels of exposure to ensure linearity, and results presented are within the linear range.
Inositol Phosphate Assays. Cells in 12-well plates were labeled by adding 3 to 5 e藽i of [3H]inositol for 24 to 48 h in inositol-free DMEM. After labeling, the medium was removed and replaced with 1 ml of HEPES-buffered DMEM containing 10 mM LiCl and equilibrated for 20 min at 37°C. Ligands or peptides were added in a volume of 50 e for 45 min after which the medium was aspirated and replaced with 1 ml of ice-cold 50 mM formic acid and applied to Dowex AG1-X8 columns (Bio-Rad, Hercules, CA). The columns were washed with 50 and 100 mM ammonium formate, followed by elution of the IP-containing fraction with 1.2 M ammonium formate/0.1 M formic acid. The eluted fraction was mixed with scintillation fluid and analyzed by liquid scintillation counting.
Coimmunoprecipitation. CHO cells were plated on 35-mm dishes and transfected with 250 or 500 ng of each cDNA as indicated. Forty-eight hours after transfection, cells were lysed in 1% Nonidet P-40 lysis buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 e phenylmethylsulfonyl fluoride, and 1% Nonidet P-40). After sonication and centrifugation, the supernatant was incubated overnight with the antibody and protein G plus agarose beads (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 4°C with rocking. Beads were centrifuged for 1 min at 13,000 rpm, washed twice with 1.0 ml of lysis buffer, once with 1.0 ml of phosphate-buffered saline, boiled in 50 e of 2x SDS sample buffer, and loaded onto a 12% SDS polyacrylamide gel. After SDS-PAGE, proteins were transferred to nitrocellulose for 16 h at 25 V followed by immunoblotting as described above.
Results
Mutation of W332 to Alanine Inhibits Interaction of Peptides with G. These experiments were designed to determine whether G protein subunits were indeed the direct target of mSIRK in intact cells responsible for ERK activation. We hypothesized that a transfected mutant G subunit that could not bind SIRK would not be responsive to mSIRK treatment and thus not promote ERK activation. We made single alanine substitutions in the G1 subunit to identify amino acids important for interaction with SIRK. We chose to mutate W332 to A because this mutation had been shown previously to inhibit activation of PLC and not affect inhibition of adenylyl cyclase (Li et al., 1998), and both of these properties were consistent with the ability of SIRK to inhibit -dependent activation of PLC but not -dependent inhibition of adenylyl cyclase (Scott et al., 2001). Single alanine-substituted mutant biotinylated-G1 subunits were expressed with 2 and 6his-i1 subunits in Sf9 insect cells and partially purified by nickel-agarose chromatography. That the G subunits bound to the nickel column and eluted with AlFeC4 indicates that these mutants folded and assembled properly with and subunits.
We used a phage ELISA assay to examine peptide binding to the partially purified bG12 mutant. In this assay, we used a peptide closely related to SIRK, SIGK, that gives a greater ELISA signal and has a higher affinity for G subunits than SIRK. Here, SIGK displayed on the surface of an M13-derived phage (f88) was tested for binding to immobilized wt or mutant bG12 subunits. As previously demonstrated, these phages do not give an appreciable binding signal in the absence of bG12, and phages that do not display peptide (f88 control) also do not bind bG12. SIGK-displaying phages bound strongly to wild-type bG12 and bG1K337A2, whereas binding to bG1W332A2 was negligible (Fig. 1A).
To more quantitatively evaluate the decrease in apparent affinity of SIGK for G1W332A, the ability of SIGK to compete for G-G interactions was tested in a flow cytometry assay (Fig. 1B). The G1W332A mutation decreased the apparent affinity of peptide for bG12 by approximately 40-fold. Previous reports indicate that heterotrimers containing GW332A are still capable of interacting with receptors, G protein subunits, and some effectors (Ford et al., 1998; Li et al., 1998; Myung and Garrison, 2000). The three-dimensional crystal structure of G12 subunits bound to SIGK has been solved, demonstrating a direct interaction of this peptide with W332 on G1 (T. L. Davis, T. M. Bonacci, S. R. Sprang, and A. V. Smrcka, submitted). Thus, the results showing that G1W332A binds to subunits in the flow cytometry assay, yet has a decreased affinity for SIGK, are consistent with previously published data and the crystal structure.
Transfection of W332A into Intact Cells Inhibits mSIRK-Dependent G Protein Activation. Gi1 and G2 were cotransfected into CHO cells with either GFP-G1 or GFP-G1W332A. We expected that transfection of the wt heterotrimer would enhance mSIRK-mediated ERK phosphorylation, but it did not (Fig. 2A, lanes 1eC4). We were surprised to find that transfection of the trimer containing GFP-1W332A significantly inhibited the response of these cells to mSIRK (Fig. 2A, compare lanes 1 and 2 with lanes 5 and 6). It is possible that the subunit transfected with G1W332A was weakened in its interaction with 12 containing this mutation and that the excess free G subunits could sequester endogenous G subunits released upon peptide addition. To test this, we transfected G1W332A with 2 subunits without subunits. Transfected G1W332A with G2 also inhibited mSIRK-dependent ERK phosphorylation, whereas transfection of wild-type G1 and G2 did not (Fig. 2B).
If G1W332A is acting as a dominant negative inhibitor of peptide-mediated ERK activation, then transfection of cells with excess wild-type GFP-1 should overcome the inhibition by G1W332A. Cells were transfected with either GFP-G1W332A2, GFP-G12, or GFP-G1W332A2 cotransfected with a 2-fold excess of GFP-G12. mSIRK-dependent ERK activation (lanes 1 and 2) was strongly inhibited by transfection of mutant 1W332A2 (lanes 3 and 4), and this was largely rescued by the cotransfection of the wild-type 12 subunit (lanes 5 and 6).
To determine whether this dominant negative effect was specific to mSIRK-mediated ERK activation, we tested whether transfection of G1W332A2-affected LPA receptor-dependent ERK activation. In contrast to its effects on mSIRK-mediated ERK activation, transfection of G1W332A2 had no effect on LPA-mediated ERK activation (Fig. 3A). To confirm that LPA-dependent ERK activation in CHO cells was mediated by G subunits, we tested the effects of pertussis toxin (PTX) pretreatment. PTX is thought to inhibit GPCR-dependent ERK activation by preventing the release of free G from Gi heterotrimers (Luttrell et al., 1997). PTX strongly inhibited LPA-mediated responses, indicating that ERK activation by LPA in these cells is through a G subunit-dependent pathway (Fig. 3B). mSIRK-dependent ERK activation was not inhibited by PTX because mSIRK works through a noneCreceptor-dependent mechanism. These data indicate that the W332A mutation does not affect the ability of the subunit to activate ERK through GPCRs and that it is specific for peptide-mediated ERK activation. Thus, a binding site containing W332 on the G subunit is probably the direct target of peptide-mediated ERK activation in intact cells. The exact mechanism for the dominant negative effect of GW332A on peptide-mediated activation of endogenous ERK pathways is unknown, but we suspect that the overexpressed mutant replaces endogenous G subunits in endogenous G protein heterotrimers and these heterotrimers are resistant to mSIRK activation but not to receptor-mediated activation.
Ric-8A Enhances mSIRK-Mediated IP Release. Ric-8A, a recently described G protein guanine nucleotide exchange factor (GEF) for Gq, 11, i, o, and 12/13, exchanges GDP for GTP on free GGDP but not GGDP (Tall et al., 2003). We reasoned that if free GGDP subunits were released from G protein heterotrimers by mSIRK in cells transfected with Ric-8A, the GGTP subunit-mediated responses to mSIRK would be enhanced. Because GqGDP is a substrate for Ric-8A in vitro, we predicted that cells expressing Ric-8A would have enhanced mSIRK-dependent IP production because of an increased level of GqGTP. mSIRK alone causes a small but reproducible increase in IP release in cells transfected with vector control DNA, similar to what we have reported previously (Goubaeva et al., 2003). mSIRK-dependent IP production was enhanced in a dose-dependent manner with transfection of increasing amounts of Ric-8A cDNA (Fig. 4, A and B). On the other hand, Ric-8A had no significant effect on basal IP release (data not shown) or IP release mediated by the GPCR agonists ATP or LPA (Fig. 4, C and D) consistent with previous reports (Tall et al., 2003). Pretreatment with PTX inhibited ATP-dependent IP release by 50% and LPA-dependent IP release by 80% (data not shown), indicating that ATP activates PLC through a combination of Gq and Gi/ pathways, whereas LPA is entirely through Gi/ in these CHO cells.
Ric-8A Enhances mSIRK-Dependent IP Production and ERK Activation through a -Dependent Mechanism. To test whether the Ric-8A enhancement of mSIRK-dependent IP production was through qGTP or subunits, we determined whether Ric-8A-enhanced IP production could be suppressed by inhibitors of G protein subunit signaling. CHO cells were transfected with Ric-8A or Ric-8A and either the C terminus from ARK (ARK-ct) or the G subunit of transducin, t. These reagents have been extensively used to sequester free G subunits without interfering directly with receptor catalyzed G protein activation (Koch et al., 1994). Both transducin and the ARK-ct inhibited responses by mSIRK and mSIRK/Ric-8A to similar levels (Fig. 5, A and B). This indicates that mSIRK-mediated IP release is through free G subunits and that Ric-8A enhances this -dependent response.
We had previously shown that mSIRK peptides activate ERK in a manner that was blocked by the ARK-ct, strongly suggesting that this response was dependent upon the release of free G subunits in rat arterial smooth muscle cells (Goubaeva et al., 2003; data not shown). Here, we tested whether G-dependent ERK activation in CHO cells could be enhanced by transfection of Ric-8A to further explore the idea that Ric-8A can enhance G-mediated responses. As shown in Fig. 6, A and B, ERK phosphorylation was increased in the presence of mSIRK, and the response was significantly enhanced in cells transfected with Ric-8A or Ric-8B. mSIRK/Ric-8A-dependent ERK activation was significantly attenuated by transducin (Fig. 6, A and B) and ARK-ct expression (data not shown), indicating that Ric-8A enhancement of mSIRK-dependent ERK activation is mediated by G subunits and not G subunits.
We also examined whether Ric-8A or Ric-8B could alter ERK activation in CHO cells in response to the GPCR agonists LPA or ATP. LPA is coupled to ERK activation primarily through Gi/G, whereas ATP is coupled partially through Gi/ and partially through a PTX-insensitive G protein, presumably Gq. Ric-8A and Ric-8B both enhanced ERK activation in response to LPA (Fig. 7, A and B) and ATP (Fig. 8, A and B). LPA-dependent ERK activation was completely blocked by PTX (Fig. 7, A and B), whereas ATP-dependent ERK activation was partially inhibited by PTX (Fig. 8, A and B). These data are consistent with a partial and complete dependence on Gi/ pathways for ATP- and LPA-dependent ERK activation, respectively. The enhancement of mSIRK-, LPA-, and ATP-dependent ERK activation by Ric-8A or Ric-8B is modest (a 50eC100% increase). For this reason, the results from multiple experiments were quantitated, pooled, and presented in Figs. 6B, 7B, and 8B with analysis for statistical significance. For mSIRK and LPA, the data clearly show a significant enhancement of ERK activation by Ric-8A and Ric-8B. For ATP, there is a trend toward enhancement that it is not statistically significant. This could be because not all of the ATP-dependent ERK activation is mediated by G subunits. Overall, these data suggest that Ric-8A enhances the responses to these agonists by enhancing G protein -dependent signaling.
Ric-8A Binds Subunits in Transfected CHO Cells. We were surprised that Ric-8A enhanced -dependent rather than subunit-dependent responses. To explain this, we hypothesized that excess Ric-8A transfected in cells could bind and sequester the endogenous subunits, thereby enhancing signaling by subunits. To determine whether Ric-8A stably binds subunits in CHO cells, we transfected the cells with HA-tagged Ric-8A and either EE-i1 or the empty vector. Cell lysates were prepared, followed by immunoprecipitation with anti-EE antibody. The immunoprecipitate was probed with anti-HA antibody (Fig. 9). Ric-8A-3HA only coimmunoprecipitated from cell lysates containing expressed EE-i1 subunits. Similar results were seen when Ric-8A-3HA was cotransfected with EE-Gq (data not shown). Together, these results show that in CHO cells Ric-8A can efficiently bind and sequester G subunits.
Discussion
We have previously shown that phages display derived peptides that bind to G protein subunits that can activate several signaling pathways in intact cells and promote G protein subunit dissociation in vitro. The cocrystal structure of the peptide bound to G protein subunits was recently solved, with the peptide bound at a position occupied by the switch II helix of Gi1 (T. L. Davis, T. M. Bonacci, S. R. Sprang, and A. V. Smrcka, submitted). This provides a plausible explanation at the molecular level of how the peptide causes G protein activation. Here, we present evidence that the peptide binds directly to G subunits in intact cells and causes subunits to dissociate from G subunits to promote G-dependent signaling.
First, the W332A mutant of G1, but not wt G1, blocked mSIRK-dependent ERK activation in intact cells. G1W332A does not bind to SIRK and should not respond to mSIRK treatment. We expected the GW332A mutation would alter the behavior of the transfected G protein heterotrimer (both G and G transfected) and were surprised to find that it behaved as a dominant negative inhibitor of peptide-dependent activation of endogenous G protein signaling. We do not fully understand the mechanism of action of this dominant negative inhibition but hypothesize that the overexpressed G1 mutant incorporates into and replaces at least part of the endogenous G protein signaling pool. Regardless of the mechanism, it is clear that transfection of this mutant G1 subunit specifically inhibits ERK activation by mSIRK but not by LPA. The fact that signaling to ERK by endogenous GPCRs remains intact indicates that the ability of G1W332A to activate ERK is not impaired. This is not entirely surprising because this is a binding site for SIRK, and SIRK does not inhibit ERK activation in cells (Goubaeva et al., 2003). In addition, mutation of W332 to A has previously been shown to selectively inhibit its ability to interact with effectors and does not interfere with its ability to interact with certain receptors (Ford et al., 1998; Li et al., 1998; Myung and Garrison, 2000). This demonstrates that direct binding of mSIRK to G subunits is required for mSIRK to activate ERK in transfected cells.
Although this result strongly supports the idea that the G subunits of G protein heterotrimers are the target of these peptides in intact cells, it does not necessarily indicate that binding of the peptide to G causes subunit dissociation in intact cells. To test this, cells were transfected with Ric-8A, with the idea that it would convert free GGDP released by mSIRK to GGTP, which could then activate signal transduction pathways downstream of GGTP. We had previously shown that mSIRK causes increases in IP production in RASM cells. It was not clear whether this was caused by free G subunits or by free GqGDP released that spontaneously exchanged GDP for GTP (Higashijima et al., 1987). If free qGDP was released by mSIRK and this was a potential substrate for Ric-8A, then we predicted Ric-8A would enhance mSIRK-mediated IP release. This is in fact what was observed; to our surprise, however, the enhanced IP release seems to be dependent on G rather than Gq. This is based on the observation that the IP production in response to mSIRK/Ric-8A can be almost completely abrogated by treatment with transducin and the ARK-ct.
The surprising result that Ric-8A can enhance G-dependent responses is supported by the observation that Ric-8A also enhances mSIRK-dependent ERK activation. We had reported previously, and confirm here in CHO cells, that mSIRK-dependent ERK activation is entirely dependent on G subunits. Similar results were seen with activation of G protein-coupled receptor agonists where Ric-8A or Ric-8B enhanced the ligand-dependent ERK activation. The enhancement in these cases is modest yet reproducible. For LPA in particular, the entire response was blocked by PTX, indicating that Ric-8A enhanced a G-dependent pathway.
These data are among the first to show that transfected Ric-8 has a biological effect. Previous work noted that transfected Ric-8A had no effect on Gq-dependent signaling in intact cells (Tall et al., 2003). In those studies, there were multiple possible reasons that transfected Ric-8 was either inactive or unable to access the G protein. The studies presented here show that that Ric-8A binds G protein subunits in cells and enhances subunit-dependent signaling, yet does not seem to enhance subunit-mediated responses. If the Ric-8 can access and bind to endogenous G protein subunits, why is no GTP subunit-dependent signaling observed A possibility is that at the high concentrations of Ric-8 expressed in these cells, the excess Ric-8 can bind GGTP attenuating GGTP-dependent signaling. Such a possibility is suggested by the observation that Ric-8A stimulates steady-state GTP hydrolysis at low concentrations of Ric-8A, but it inhibits at higher concentrations (G. G. Tall and A. G. Gilman, unpublished observations).
Demonstration that Ric-8 can enhance G-dependent pathways was unexpected but not entirely inconsistent with its known function. Ric-8A binds to Gi, Go, G12/13, and Gq GDP subunits and catalyzes exchange of GDP for GTP. After hydrolysis of GGTP to GGDP, the GGDP might preferentially bind to the expressed Ric-8 over free G and another round of exchange could occur. Neither free GGTP, Ric-8:GGTP, or Ric-8:GGDP would be expected to rebind to G protein subunits, thus the presence of excess Ric-8 would extend the lifetime of free G protein subunits in the cell. Overall, the data support the notion that free GGDP subunits are generated in the cell upon treatment with mSIRK because Ric-8 enhances the mSIRK effects.
Several articles have been published suggesting a role for Ric-8 in asymmetric cell division in C. elegans (Afshar et al., 2004; Couwenbergs et al., 2004; Hess et al., 2004). Because deletion of G subunits in these animals enhances the G protein-dependent effects on spindle positioning, presumably by raising the level of free G subunits in cells, it is unlikely that G is directly involved in this process. Thus, it is also unlikely that there is a role for Ric-8 in generating free G subunits in this system. Although it is not entirely clear that release of free G subunits is a mechanism that occurs with these endogenous Ric-8/G protein signaling systems, our data suggest the possibility that Ric-8 may enhance G effects through a novel mechanism in more conventional G protein signaling.
doi:10.1124/mol.104.010116.
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