Peptides derived from a random-peptide phage display screen with purified Gβ1γ2 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 Gβγ subunits and accelerating the dissociation of GαGDP 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 Gβ1 subunits (β1W332A) that does not bind SIRK in vitro. Transfection of Gβ1W332A mutant into Chinese hamster ovary cells blocked peptide-mediated activation of extracellular signal-regulated kinase (ERK), but it did not affect receptor-mediated Gβγ subunit-dependent ERK activation, indicating that Gβγ subunits are in fact the direct target in cells responsible for ERK activation. To determine whether free Gα subunits were released from G protein heterotrimers upon peptide treatment, cells were transfected with Ric-8A, a guanine nucleotide exchange factor for free GαGDP, 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 Gβγ 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 Gβγ subunit signaling. Overall, these experiments provide further support for the hypothesis that mSIRK promotes G protein subunit dissociation to release free βγ 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 Gα and Gβγ subunits, and there is evidence for direct binding of the receptor to both Gα and Gβγ subunits (Taylor et al., 1994, 1996). Binding of GTP to the Gα subunit activates the G protein and is thought to cause dissociation of Gα subunits from Gβγ subunits, liberating free GαGTP and Gβγ 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 βγ subunits from α subunits (Cismowski et al., 2001). GPR or GoLoco peptides derived from AGS proteins promote dissociation of GαGDP subunits from Gβγ subunits, causing release of Gβγ (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 GαGDP 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 αGDP 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 Gβγ 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 βγ subunits promote activation of G protein βγ 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 β1γ2 subunits and accelerate dissociation of GαiGDP subunits from Gαi1β1γ2 heterotrimers (Ghosh et al., 2003). The structure of G protein β1γ2 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 Gβ1γ2 subunits normally occupied by the switch II helix of Gα 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 Gα subunit interactions with Gβγ subunits.
Although these in vitro data support a model for peptide-mediated dissociation of GαGDP from Gβγ 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 βγ 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 GαGDP 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 GαGDP subunits. We were surprised to find that Ric-8 can enhance G protein βγ subunit-mediated responses, probably by a mechanism that involves sequestration of free Gα subunits.
Materials and Methods
Materials and Plasmids. GFP-Gβ1, GFP-Gβ1W332A, Gγ2, and Ric-8A-3HA were in pCI-Neo. EE-αi1 and -αt were supplied from Guthrie cDNA Resource Center (Rolla, MO) in pcDNA 3.1+; βARKct, 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 Gβγ Subunits. Construction of baculovirus encoding biotinylated Gβ1 (bGβ1) subunit in the baculovirus transfer vector PDW464 was described previously (Goubaeva et al., 2003). For other experiments, G protein β1 subunits were tagged at the amino terminus with GFP. We used GFP-tagged β1 subunits to monitor β subunit transfection efficiency by epifluorescence microscopy and to monitor the level of expression of the transfected protein relative to endogenous β subunits by immunoblotting. We (unpublished data) and others have shown that amino terminal modification of Gβ with GFP does not alter Gβγ subunit functions (Azpiazu and Gautam, 2004). Mutants (βW332A 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 bGβ1γ2 subunits were performed as described previously (Smrcka and Scott, 2002).
Measurement of α-βγ Interactions via Flow Cytometry. The fluorescein-labeled αi1 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 αi1 and indicated concentrations of peptides were added to 50 pM bGβ1γ2 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 βARK-ct in pRK5, 800 ng of αt, 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 μl of 2× SDS sample buffer. The resulting suspension was boiled for 5 min, and 5 to 10 μl 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 μCi 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 μl 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 μM 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 μl of 2× 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.
Mutation of βW332 to Alanine Inhibits Interaction of Peptides with Gβγ. These experiments were designed to determine whether G protein βγ subunits were indeed the direct target of mSIRK in intact cells responsible for ERK activation. We hypothesized that a transfected mutant Gβ 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 Gβ1 subunit to identify amino acids important for interaction with SIRK. We chose to mutate βW332 to A because this mutation had been shown previously to inhibit activation of PLCβ 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 βγ-dependent activation of PLCβ but not βγ-dependent inhibition of adenylyl cyclase (Scott et al., 2001). Single alanine-substituted mutant biotinylated-Gβ1 subunits were expressed with γ2 and 6his-αi1 subunits in Sf9 insect cells and partially purified by nickel-agarose chromatography. That the Gβγ subunits bound to the nickel column and eluted with AlF–4 indicates that these mutants folded and assembled properly with γ and α subunits.
We used a phage ELISA assay to examine peptide binding to the partially purified bGβ1γ2 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 Gβγ subunits than SIRK. Here, SIGK displayed on the surface of an M13-derived phage (f88) was tested for binding to immobilized wt or mutant bGβ1γ2 subunits. As previously demonstrated, these phages do not give an appreciable binding signal in the absence of bGβ1γ2, and phages that do not display peptide (f88 control) also do not bind bGβ1γ2. SIGK-displaying phages bound strongly to wild-type bGβ1γ2 and bGβ1K337Aγ2, whereas binding to bGβ1W332Aγ2 was negligible (Fig. 1A).
To more quantitatively evaluate the decrease in apparent affinity of SIGK for Gβ1W332A, the ability of SIGK to compete for Gα-Gβγ interactions was tested in a flow cytometry assay (Fig. 1B). The Gβ1W332A mutation decreased the apparent affinity of peptide for bGβ1γ2 by approximately 40-fold. Previous reports indicate that heterotrimers containing GβW332A are still capable of interacting with receptors, G protein α subunits, and some effectors (Ford et al., 1998; Li et al., 1998; Myung and Garrison, 2000). The three-dimensional crystal structure of Gβ1γ2 subunits bound to SIGK has been solved, demonstrating a direct interaction of this peptide with W332 on Gβ1 (T. L. Davis, T. M. Bonacci, S. R. Sprang, and A. V. Smrcka, submitted). Thus, the results showing that Gβ1W332A binds to α 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 βW332A into Intact Cells Inhibits mSIRK-Dependent G Protein Activation. Gαi1 and Gγ2 were cotransfected into CHO cells with either GFP-Gβ1 or GFP-Gβ1W332A. We expected that transfection of the wt heterotrimer would enhance mSIRK-mediated ERK phosphorylation, but it did not (Fig. 2A, lanes 1–4). We were surprised to find that transfection of the trimer containing GFP-β1W332A 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 α subunit transfected with Gβ1W332A was weakened in its interaction with β1γ2 containing this mutation and that the excess free Gα subunits could sequester endogenous Gβγ subunits released upon peptide addition. To test this, we transfected Gβ1W332A with γ2 subunits without α subunits. Transfected Gβ1W332A with Gγ2 also inhibited mSIRK-dependent ERK phosphorylation, whereas transfection of wild-type Gβ1 and Gγ2 did not (Fig. 2B).
If Gβ1W332A is acting as a dominant negative inhibitor of peptide-mediated ERK activation, then transfection of cells with excess wild-type GFP-β1 should overcome the inhibition by Gβ1W332A. Cells were transfected with either GFP-Gβ1W332Aγ2, GFP-Gβ1γ2, or GFP-Gβ1W332Aγ2 cotransfected with a 2-fold excess of GFP-Gβ1γ2. mSIRK-dependent ERK activation (lanes 1 and 2) was strongly inhibited by transfection of mutant β1W332Aγ2 (lanes 3 and 4), and this was largely rescued by the cotransfection of the wild-type β1γ2 subunit (lanes 5 and 6).
To determine whether this dominant negative effect was specific to mSIRK-mediated ERK activation, we tested whether transfection of Gβ1W332Aγ2-affected LPA receptor-dependent ERK activation. In contrast to its effects on mSIRK-mediated ERK activation, transfection of Gβ1W332Aγ2 had no effect on LPA-mediated ERK activation (Fig. 3A). To confirm that LPA-dependent ERK activation in CHO cells was mediated by Gβγ 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 Gβγ 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 Gβγ subunit-dependent pathway (Fig. 3B). mSIRK-dependent ERK activation was not inhibited by PTX because mSIRK works through a non–receptor-dependent mechanism. These data indicate that the W332A mutation does not affect the ability of the β subunit to activate ERK through GPCRs and that it is specific for peptide-mediated ERK activation. Thus, a binding site containing W332 on the Gβ subunit is probably the direct target of peptide-mediated ERK activation in intact cells. The exact mechanism for the dominant negative effect of GβW332A on peptide-mediated activation of endogenous ERK pathways is unknown, but we suspect that the overexpressed mutant replaces endogenous Gβγ 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 Gαq, 11, i, o, and 12/13, exchanges GDP for GTP on free GαGDP but not GαGDPβγ (Tall et al., 2003). We reasoned that if free GαGDP subunits were released from G protein heterotrimers by mSIRK in cells transfected with Ric-8A, the GαGTP subunit-mediated responses to mSIRK would be enhanced. Because GαqGDP 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 GαqGTP. 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 PLCβ through a combination of Gq and Gi/βγ pathways, whereas LPA is entirely through Gi/βγ in these CHO cells.
Ric-8A Enhances mSIRK-Dependent IP Production and ERK Activation through a βγ-Dependent Mechanism. To test whether the Ric-8A enhancement of mSIRK-dependent IP production was through αqGTP or βγ subunits, we determined whether Ric-8A-enhanced IP production could be suppressed by inhibitors of G protein βγ subunit signaling. CHO cells were transfected with Ric-8A or Ric-8A and either the C terminus from βARK (βARK-ct) or the Gα subunit of transducin, αt. These reagents have been extensively used to sequester free Gβγ subunits without interfering directly with receptor catalyzed G protein activation (Koch et al., 1994). Both transducin and the βARK-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 Gβγ subunits and that Ric-8A enhances this βγ-dependent response.
We had previously shown that mSIRK peptides activate ERK in a manner that was blocked by the βARK-ct, strongly suggesting that this response was dependent upon the release of free Gβγ subunits in rat arterial smooth muscle cells (Goubaeva et al., 2003; data not shown). Here, we tested whether Gβγ-dependent ERK activation in CHO cells could be enhanced by transfection of Ric-8A to further explore the idea that Ric-8A can enhance Gβγ-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 βARK-ct expression (data not shown), indicating that Ric-8A enhancement of mSIRK-dependent ERK activation is mediated by Gβγ subunits and not Gα 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/Gβγ, whereas ATP is coupled partially through Gi/βγ and partially through a PTX-insensitive G protein, presumably Gαq. 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/βγ 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 50–100% 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 Gβγ subunits. Overall, these data suggest that Ric-8A enhances the responses to these agonists by enhancing G protein βγ-dependent signaling.
Ric-8A Binds α Subunits in Transfected CHO Cells. We were surprised that Ric-8A enhanced βγ-dependent rather than α subunit-dependent responses. To explain this, we hypothesized that excess Ric-8A transfected in cells could bind and sequester the endogenous α subunits, thereby enhancing signaling by βγ subunits. To determine whether Ric-8A stably binds α subunits in CHO cells, we transfected the cells with HA-tagged Ric-8A and either EE-αi1 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-αi1 subunits. Similar results were seen when Ric-8A-3HA was cotransfected with EE-Gαq (data not shown). Together, these results show that in CHO cells Ric-8A can efficiently bind and sequester Gα subunits.
We have previously shown that phages display derived peptides that bind to G protein βγ 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 βγ subunits was recently solved, with the peptide bound at a position occupied by the switch II helix of Gαi1 (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 Gβγ subunits in intact cells and causes α subunits to dissociate from Gβγ subunits to promote Gβγ-dependent signaling.
First, the W332A mutant of Gβ1, but not wt Gβ1, blocked mSIRK-dependent ERK activation in intact cells. Gβ1W332A does not bind to SIRK and should not respond to mSIRK treatment. We expected the GβW332A mutation would alter the behavior of the transfected G protein heterotrimer (both Gα and Gβγ 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 Gβ1 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 Gβ1 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 Gβ1W332A 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 βW332 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 Gβγ subunits is required for mSIRK to activate ERK in transfected cells.
Although this result strongly supports the idea that the Gβγ 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 Gβγ causes subunit dissociation in intact cells. To test this, cells were transfected with Ric-8A, with the idea that it would convert free GαGDP released by mSIRK to GαGTP, which could then activate signal transduction pathways downstream of GαGTP. We had previously shown that mSIRK causes increases in IP production in RASM cells. It was not clear whether this was caused by free Gβγ subunits or by free GαqGDP released that spontaneously exchanged GDP for GTP (Higashijima et al., 1987). If free αqGDP 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 Gβγ rather than Gαq. 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 βARK-ct.
The surprising result that Ric-8A can enhance Gβγ-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 Gβγ 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 Gβγ-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 Gαq-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 α subunits in cells and enhances βγ subunit-dependent signaling, yet does not seem to enhance α subunit-mediated responses. If the Ric-8 can access and bind to endogenous G protein α subunits, why is no αGTP 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 GαGTP attenuating GαGTP-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 Gβγ-dependent pathways was unexpected but not entirely inconsistent with its known function. Ric-8A binds to Gαi, Gαo, Gα12/13, and Gαq GDP subunits and catalyzes exchange of GDP for GTP. After hydrolysis of GαGTP to GαGDP, the GαGDP might preferentially bind to the expressed Ric-8 over free Gβγ and another round of exchange could occur. Neither free GαGTP, Ric-8:GαGTP, or Ric-8:GαGDP would be expected to rebind to G protein βγ subunits, thus the presence of excess Ric-8 would extend the lifetime of free G protein βγ subunits in the cell. Overall, the data support the notion that free GαGDP 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 Gβ subunits in these animals enhances the G protein-dependent effects on spindle positioning, presumably by raising the level of free Gα subunits in cells, it is unlikely that Gβγ is directly involved in this process. Thus, it is also unlikely that there is a role for Ric-8 in generating free Gβγ subunits in this system. Although it is not entirely clear that release of free Gβγ 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 Gβγ effects through a novel mechanism in more conventional G protein signaling.
This work was supported by National Institutes of Health grants GM60286 (to A.V.S.) and GM34497 (to G.G.T. and A.G.G.) and National Institutes of Health Predoctoral Training grant in Cardiovascular Biology HLT3207949 (to T.M.B.).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: GPCR, G protein-coupled receptor; AGS, activator of G protein signaling; SIGK, SIGKAFKILGYPDYD; SIRK, SIRKALNILGYPDYD peptide; mSIRK, myristoyl-SIRKALNILGYPDYD peptide; GFP, green fluorescent protein; LPA, lysophosphatidic acid; ERK, extracellular signaling-regulated kinase; HA, hemagglutinin; HRP, horseradish peroxidase; bGβ1, biotinylated Gβ1; ELISA, enzyme-linked immunosorbent assay; DMEM, Dulbecco's modified Eagle's medium; IP, inositol phosphate; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; PLC, phospholipase C; wt, wild-type; PTX, pertussis toxin; GEF, guanine nucleotide exchange factor; βARK, β adrenergic receptor kinase; ct, C terminus; EE, EYMPTE.
- Received December 7, 2004.
- Accepted March 31, 2005.
- The American Society for Pharmacology and Experimental Therapeutics