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Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gαi/o-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive Gαi1/3-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive Gαi1-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive Gαi1-GDP but not active Gαi1-GTP. Active H-Ras(G/V) interacts with a native RGS14·Gαi1 complex in brain lysates, and co-expression of RGS14 and Gαi1 in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the Gαi-linked α2A-adrenergic receptor induces a conformational change in the Gαi1·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive Gαi1-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.
Canonical G protein signaling pathways include a G protein-coupled receptor (GPCR)2 coupled to a heterotrimeric G protein (Gαβγ) that acts as a GTPase timing switch. Upon GPCR activation, the receptor acts as a guanine nucleotide exchange factor (GEF) and facilitates GDP release and subsequent GTP binding to the Gα subunit that is followed by Gβγ dissociation/rearrangement from Gα-GTP. Free Gβγ and Gα-GTP are then able to engage downstream effectors and regulate signaling events (1, 2). Recent studies have examined the function of the regulators of G protein signaling (RGS) proteins in conventional G protein signaling, specifically how they act as GTPase accelerating proteins (GAPs) toward activated Gα subunits. The conserved RGS domain binds and enhances the intrinsic rate of Gα nucleotide hydrolysis, resulting in GPCR/G protein signal termination (3–5).
Regulator of G protein signaling 14 (RGS14) is a complex RGS protein grouped in the R12 subfamily of RGS proteins along with its closest relatives, RGS10 and RGS12 (3, 6). Predominately expressed in the hippocampus of brain (7, 8), RGS14 has been implicated in hippocampal-based learning, memory, and cognition (7, 9). The molecular mechanisms underlying these central nervous system (CNS) actions of RGS14, however, remain largely unknown. The highly unusual sequence and domain structure of RGS14 suggests it serves as a multifunctional scaffold that integrates G protein and H-Ras/MAPK signaling (10). In addition to the conserved RGS domain that confers GAP activity toward Gαi/o subunits (8, 11, 12), RGS14 also possesses two tandem Ras/Rap binding domains (RBDs) and a G protein regulatory (GPR) motif that binds selectively to inactive Gαi1 and Gαi3 subunits to form a stable complex at intracellular membranes (13–15). Recent work has shown that RGS14 participates in newly appreciated unconventional G protein signaling networks, which involve G protein activation in the absence of GPCRs (16–23). Specifically, the RGS14·Gαi1-GDP complex is regulated by the non-receptor GEF Ric-8A (24) both in the absence and presence of a coupled GPCR (25). This finding highlights a novel mechanism of action for an RGS protein, providing insight on how RGS14 may function within hippocampal neurons to regulate their signaling.
Although RGS14 may function within neurons by binding Gαi1/3 and participating in unconventional G protein signaling pathways, evidence also suggests that RGS14 regulates MAPK signaling through binding H-Ras and Raf-1 via its RBDs (26). RGS14 binds directly to H-Ras via its first RBD (27), preferring to bind the activated form of H-Ras (26). By binding activated H-Ras, RGS14 inhibits PDGF-mediated ERK activation, an effect that is dependent on the presence of Gαi1. When RGS14 is bound to Gαi1, it can no longer bind Raf-1 and, therefore, can no longer regulate PDGF signaling (26). These results suggest that RGS14 may promote formation of a Gαi1·RGS14·H-Ras ternary complex that serves as a molecular switch to regulate H-Ras/Raf-1 signaling on the one hand and Gαi signaling on the other. This would be similar to, and yet mechanistically distinct from GPCR cross-talk with growth factor stimulated Ras-mediated MAPK signaling (28–32). This idea of an RGS14 signaling switch mechanism has not yet been tested directly, and whether a GPCR may be involved in promoting this proposed switch mechanism is also unknown.
The goals of these studies were to investigate how RGS14/H-Ras interactions are regulated in live cells, specifically examining the effects of both active and inactive Gαi and GPCRs on this interaction. Using bioluminescence resonance energy transfer (BRET), we show that RGS14 binds preferentially to activated H-Ras in live cells and that this interaction and downstream cellular actions are greatly facilitated by complex formation with inactive Gαi1 at the plasma membrane. In addition, activation of the Gαi-linked α2A-adrenergic receptor induces conformational changes within the Gαi1·RGS14·H-Ras complex, supporting a functional link between GPCR/G protein coupling and H-Ras signaling. These findings indicate that GPCR activation may promote the switch mechanism of RGS14 and allow it to participate in both G protein and H-Ras signaling, which may ultimately underlie the function of RGS14 in regulating synaptic plasticity within hippocampal neurons (7).
The rat RGS14 cDNA used in this study (GenBankTM accession number U92279) was acquired as described (8). Wild-type (WT) and GPR-null rat RGS14-Luciferase (Luc) constructs were generated as previously described (25) using the phRLucN2 vector graciously provided by Dr. Michel Bouvier (University of Montreal). Venus-tagged H-Ras constructs were made from the parental H-Ras cDNA purchased from the UMR cDNA Resource Center (Rolla, MO). The Gαi1-Q204L plasmid was also purchased from the UMR cDNA Resource Center. Venus-tagged wild-type H-Ras (H-Ras-WT-Venus) was generated by digesting the parental H-Ras-WT plasmid at EcoRI and SacII restriction sites and ligating the resulting product into Venus-C1 vector (graciously provided by Brandon Stauffer, Stephen Ikeda, and Steven Vogel, National Institutes of Health). Constitutively activated H-Ras(G/V)-Venus was generated by mutating the Gly-12 residue of H-Ras-WT-Venus to Val-12 using the QuikChange kit (Stratagene) and the following oligonucleotide primers: forward primer, 5′-AAT ATA AGC TGG TGG TGG TGG GCG CCG TCG GTG TGG GCA AGA GT-3′; reverse primer, 5′-ACT CTT GCC CAC ACC GAC GGC GCC CAC CAC CAC CAG CTT ATA TT-3′. The H-Ras CAAX box mutants (C186S mutation) were made using the QuikChange kit (Stratagene) and the following oligonucleotide primers: forward primer, 5′-GGC TGC ATG AGC TGC AAG TCT GTG CTC TCC-3′; reverse primer, 5′-GGA GAG CAC AGA CTT GCA GCT CAT GCA GCC-3′. The RGS14-R333L-Luc mutant was constructed using the QuikChange kit (Stratagene) and the following oligonucleotide primers: forward primer, 5′-GGC ATC TGT GAG AAG CTA GGC CTC TCT CTA CCT G-3′; reverse primer, 5′-CAG GTA GAG AGA GGC CTA GCT TCT CAC AGA TGC C-3′. Rat Gαi1-EYFP (Gαi1-YFP) in pcDNA3.1 was generated by Dr. Scott Gibson (University of Texas Southwestern) (33). α2A-adrenergic receptor (α2A-AR)-Venus and β2-adrenergic receptor (β2-AR)-Venus plasmids were generated as described and provided by Dr. Michel Bouvier (University of Montreal) (34, 35). Anti-sera used include Alexa 546 goat anti-rabbit secondary IgG (Invitrogen), Alexa 633 goat anti-mouse secondary IgG (Invitrogen), peroxidase-conjugated goat anti-mouse IgG (Rockland Immunochemicals, Inc.), peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad), anti-Gαi1 (Santa Cruz Biotechnologies, Inc.), anti-H-Ras (Abcam), anti-FLAG (Sigma), and anti-RGS14 (Antibodies, Inc.) antibodies.
HEK293 cells were maintained in Dulbecco's minimal essential medium (without phenol red) containing 10% fetal bovine serum (5% after transfection), 2 mm glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. PC12 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum (2.5% after transfection), 10% horse serum (5% after transfection), 100 units/ml penicillin, and 100 mg/ml streptomycin. Cells were incubated at 37 °C with 5% CO2 in a humidified environment. Transfections were performed using previously described protocols with polyethyleneimine (Polysciences, Inc.) (36).
BRET experiments were performed as previously described (36, 37). Briefly, HEK293 cells were transiently transfected with BRET donor and acceptor plasmids using polyethyleneimine. Twenty-four to 48 hours after transfection, the culture medium was removed, and cells were harvested with Tyrode solution (140 mm NaCl, 5 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 0.37 mm NaH2PO4, 24 mm NaHCO3, 10 mm HEPES, and 0.1% glucose (w/v), pH 7.4). Cells were seeded in triplicate into gray 96-well OptiPlates (PerkinElmer Life Sciences) with each well containing 1 × 105 cells. The acceptor (YFP/Venus-tagged) protein expression levels were monitored by measuring total fluorescence using the TriStar LB 941 plate reader (Berthold Technologies) with excitation and emission filters at 485 and 535 nm, respectively. After fluorescence measurement, coelenterazine H (Nanolight Technology; 5 μm final concentration) was added, and luminescence was detected by the TriStar LB 941 plate reader in the 480 ± 20- and 530 ± 20-nm ranges for donor (Luc) and acceptor (YFP/Venus), respectively. Samples of cells overexpressing α2A-AR or β2-AR were treated with either vehicle (Tyrode solution) or a final concentration of 10 μm UK14304 (Sigma) or 100 μm isoproterenol (Sigma), respectively, before the addition of coelenterazine. BRET signals were obtained by calculating the ratio of the light intensity emitted by the YFP/Venus divided by the light intensity emitted by Luc. Net BRET values were derived from subtracting the background BRET signal detected from the expression of the donor fusion protein (Luc) alone. All data were collected with MikroWin 2000 software and analyzed using Graphpad Prism and Microsoft Excel. Statistical analysis was performed using Student's t test, and immunoblots were performed as described previously (38).
HEK293 cells were transiently transfected with H-Ras(G/V). Eighteen hours post-transfection, cells were lysed in buffer containing 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EGTA, 1 mm EDTA, 2 mm dithiothreitol, 5 mm MgCl2, phosphatase inhibitor mixture (Sigma), protease inhibitor mixture (Roche Applied Science), and 1% Nonidet P-40. In parallel, two brains from C57BL6 wild-type mice were isolated and homogenized by a Dounce tissue grinder in the same lysis buffer stated above. Both cell and brain lysates were incubated on a 4 °C rotator for 1 h and then cleared by centrifugation at 100,000 × g for 30 min at 4 °C. Lysates were precleared by incubating with 50 μg of protein G-sepharose resin for 1 h on a 4 °C rotator. After preclearing, lysates were incubated in the absence or presence of a 1:50 dilution of anti-RGS14 antibody (Antibodies, Inc.) on a 4 °C rotator overnight. Next, 50 μg of BSA-preblocked protein G-sepharose resin was added to each sample. Samples were then rotated at 4 °C for 1.5 h. Resin was washed with ice-cold TBS four times, and proteins were eluted by the addition of Laemmli sample buffer and subsequent boiling for 5 min. Samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-RGS14, anti-Gαi1, and anti-H-Ras antibodies followed by appropriate secondary antibodies. Proteins were detected by enhanced chemiluminescence.
Transfected HEK293 or PC12 cells were fixed at room temperature for 15 min in buffer containing 3.7% paraformaldehyde diluted in PBS. Cells were washed in PBS and incubated for 8 min with 0.4% Triton X-100 diluted in PBS. Cells were then blocked for 1 h at room temperature in PBS containing 10% goat serum and 3% BSA. Next, cells were incubated in this same buffer with a 1:1000 dilution of rabbit anti-FLAG and/or 1:300 dilution of mouse anti-Gαi1 antibodies at 37 °C for 1.5 h. Cells were washed with PBS (3×) and incubated with 1:300 dilutions of Alexa 546 goat anti-rabbit and Alexa 633 goat anti-mouse secondary antibodies at 37 °C for 1 h. Cells were washed with PBS again (3×) and mounted with ProLong Gold Antifade Reagent (Invitrogen). Confocal images were taken using a 63× oil immersion objective from an LSM510 laser scanning microscope with AxioObserver Stand (Zeiss). Images were processed using the ZEN 2009 Light Edition software and Adobe Photoshop 7.0 (Adobe Systems). Measurement and quantification of RGS14, Gαi1, and H-Ras subcellular co-localization within cells was measured and plotted as relative fluorescence intensity using ImageJ software and analysis.
Twenty-four hours after transfection, cells were serum-starved overnight. Twenty-four hours later, cells were subjected to immunofluorescence and confocal imaging. Neurites were then counted and analyzed. Scored neurites were identified as cell protrusions having lengths of at least one cell body. In addition to the number of neurites, the percentage of cells with neurites at least two times longer than one cell body was also determined. Only cells confirmed to express all proteins of interest were analyzed. Statistical analysis was performed using one-way analysis of variance with Tukey's post-hoc test.
Because RGS14 has been shown to bind H-Ras both in vitro (27) and in cells through co-immunoprecipitation (26), we sought to quantitatively measure this interaction in live cells using BRET analysis. We first measured the magnitude and selectivity of a BRET signal between RGS14-Luc and either H-Ras-WT-Venus or constitutively activated H-Ras(G12V)-Venus (referred to as H-Ras(G/V)-Venus) (Fig. 1A). Transfection of HEK cells with increasing amounts of Venus-tagged H-Ras plasmids and a fixed amount (5 ng) of RGS14-Luc plasmid showed a robust BRET signal in the presence of H-Ras(G/V)-Venus, whereas an approximate 3-fold decrease in the maximal signal (BRETmax) was observed in the presence of H-Ras-WT-Venus (Fig. 1A). To determine the specificity of this interaction, H-Ras(G/V) was co-expressed with either WT RGS14 or the R333L mutant of RGS14 (Fig. 1B), which is reported not to bind H-Ras(G/V) in cell lysates (26). As in Fig. 1A, there was a strong BRET signal between RGS14-WT and H-Ras(G/V). This signal was reduced nearly 3-fold in the presence of RGS14-R333L but was not completely eliminated, consistent with our observation that this mutant retains a limited capacity (albeit greatly reduced) to bind H-Ras in co-immunoprecipitation studies from these cells.3 Furthermore, the observed large acceptor/donor ratio (H-Ras(G/V) to RGS14) that was required to generate these BRET signals indicates that this interaction is relatively weak, suggesting the requirement for other cellular binding partners and/or modifications that may optimize RGS14/H-Ras(G/V) interactions in cells.
Because our previous work suggests that RGS14 acts as a molecular switch for regulating MAPK and G protein signaling (26), we next tested the effects of Gαi1 on RGS14/H-Ras(G/V) BRET signals. The BRET signal between RGS14-Luc and increasing amounts of H-Ras(G/V)-Venus was measured in the absence or presence of Gαi1 (Fig. 2A). The observed RGS14/H-Ras(G/V) BRET signal (BRETmax) increased slightly in the presence of overexpressed Gαi1, whereas the BRET50 was significantly reduced (acceptor/donor ratio of 2 ± 0.732 versus 24 ± 4.8; p < 0.05) (Fig. 2A), indicating Gαi1-mediated regulation of this complex. This effect was concentration-dependent, as increasing amounts of Gαi1 enhanced RGS14/H-Ras(G/V) BRET signals (Fig. 2B). Little detectable BRET signal was observed between membrane associated Gαi1-YFP and H-Ras(G/V)-Luc (Fig. 2C). However, BRET activity between these two proteins was readily observed upon the addition of untagged RGS14 (Fig. 2C), strongly supporting the idea that RGS14 promotes assembly of a Gαi1·RGS14·H-Ras ternary complex. This BRET signal was comparatively small, as would be expected because Gαi1-YFP and H-Ras(G/V)-Luc are physically separated by RGS14. To test whether Gαi1 remained bound to RGS14 in the presence of H-Ras(G/V), the BRET signals between RGS14-Luc and Gαi1-YFP were measured in the absence or presence of untagged H-Ras(G/V) (Fig. 2D). The BRET signals between RGS14 and Gαi1 remained relatively unchanged in the presence of H-Ras(G/V), suggesting that binding of Gαi1 and H-Ras are not mutually exclusive and further supporting the data from Fig. 2C that RGS14 may form a trimeric complex with Gαi and H-Ras. We note that the YFP tag did not alter the function of Gαi1, as insertion of the YFP tag into the loop joining the αB and αC helices (33, 39, 40) retained nucleotide binding and hydrolysis properties similar to wild-type Gαi1 (33).
Supporting these BRET results, we observed that RGS14 translocated from the cytosol to the plasma membrane in the presence of either H-Ras(G/V), Gαi1, or both (Fig. 3). When expressed alone in cells, RGS14 localized within the cytosol, whereas both Gαi1 and H-Ras(G/V) localized at the plasma membrane. However, when RGS14 was co-expressed with either Gαi1, H-Ras(G/V), or both, RGS14 translocated to the plasma membrane and co-localized with Gαi1 and/or H-Ras(G/V). Given that there was little observable BRET signal between Gαi1 and H-Ras(G/V) (Fig. 2C) despite both being at the plasma membrane (Fig. 3), these findings suggest that RGS14 promotes formation of a heterotrimeric complex with both Gαi1 and activated H-Ras(G/V) at the plasma membrane.
Because Gαi1 enhanced RGS14/H-Ras(G/V) BRET signals (Fig. 2, A and B) and the R333L mutation in RGS14 decreased these signals (Fig. 1B), we next determined the effects of Gαi1 expression on the BRET signals between H-Ras(G/V) and the RGS14-R333L mutant. In the presence of WT RGS14, the BRET signal between RGS14 and H-Ras(G/V) was enhanced by co-expressed Gαi1 (Fig. 2E). The RGS14-R333L mutant exhibited a 50% reduction in BRET signal with H-Ras(G/V) compared with RGS14-WT; however, this signal was still enhanced in the presence of co-expressed Gαi1 (Fig. 2E). The presence of Gαi1 induced an approximate 80% increase in RGS14/H-Ras(G/V) BRET signals in the presence of RGS14-WT as compared with only a 35% increase in the presence of RGS14-R333L. These findings illustrate the specificity of this Gαi1-mediated effect. The observed BRET signal between RGS14-R333L and H-Ras(G/V) (Figs. 1B and and22E) likely reflects residual specific H-Ras binding to RGS14 as the R333L mutation fails to completely abolish H-Ras(G/V) binding.3
All of our data thus far suggest that H-Ras only interacts with RGS14 at the plasma membrane. To test this idea, the C186S mutation within the CAAX boxes of both wild-type H-Ras and H-Ras(G/V) was generated to prevent membrane localization (41–43). Specifically, this mutation prohibits the addition of lipid modifications that target H-Ras to the plasma membrane. The observed BRET signals between RGS14 and both H-Ras-WT and H-Ras(G/V) were almost completely ablated in the presence of the C186S H-Ras mutants (Fig. 4). Even the co-expression of Gαi1 could not overcome the loss of BRET signal generated by the H-Ras-C186S mutants (Fig. 4B), indicating that RGS14/H-Ras interactions and all generated BRET signals are specific and dependent on plasma membrane localization of H-Ras.
To determine whether Gαi1-mediated effects on RGS14/H-Ras(G/V) interactions were dependent on the Gαi1 activation state, the BRET signals were measured between H-Ras(G/V) and RGS14-Q515A/R516A (referred to as RGS14(GPR-null)), which cannot bind inactive Gαi1 (25, 44, 45) (Fig. 5A). The Gαi1-facilitated BRET between H-Ras(G/V) and RGS14-WT was abolished in the presence of RGS14(GPR-null), indicating that Gαi1 effects on these BRET signals are dependent on binding to the GPR motif. The effects of mutating Gαi1 in the presence of H-Ras(G/V) and wild-type RGS14 were also examined (Fig. 5B). The BRET signals observed between RGS14 and H-Ras(G/V) were enhanced by wild-type Gαi1 (WT) but remained unchanged in the presence of constitutively active Gαi1-Q204L (Fig. 5B), further supporting a role for inactive Gαi1 in regulating RGS14 interactions with H-Ras in live cells.
We previously reported that the α2A-AR functionally associates with the Gαi1·RGS14 complex (25). Therefore, we next examined whether GPCRs could influence the RGS14·H-Ras(G/V) complex. For these studies, the BRET signals between RGS14-Luc and H-Ras(G/V)-Venus were analyzed in the presence of both Gαi1 and GPCRs, specifically the Gαi-linked α2A-AR and the Gαs-linked β2-AR (Fig. 6). In the absence of α2A-AR receptor agonist, RGS14/H-Ras(G/V) BRET signals were similar to those seen in the presence of Gαi1 only (Figs. 2A and and66A). However, these signals decreased by ~35% in the presence of the α2A-AR agonist UK14304 (UK) (Fig. 6A). Even with this decrease in signal (BRETmax), the BRET50 values remained the same (acceptor/donor ratio of ~3.1), suggesting that activation of the GPCR induced a conformational change within the RGS14·H-Ras(G/V) complex rather than a dissociation of the complex. To expand on this idea, BRET signals were measured between RGS14-Luc and the α2A-AR-Venus either in the absence or presence of untagged Gαi1 and H-Ras(G/V) (Fig. 6B; left panel) to determine if H-Ras(G/V) could influence RGS14 proximity to the receptor. As previously observed (25), there was little detectable BRET signal generated between RGS14 and the α2A-AR when these two proteins were co-expressed in cells in the absence of Gαi1. The addition of H-Ras(G/V) alone had little effect on RGS14/α2A-AR BRET signals regardless of the presence of receptor agonist. However, a 4-fold increase in the BRET signal was observed between the α2A-AR and RGS14 in the presence of co-expressed Gαi1 alone (Fig. 6B), as previously observed (25). This signal was reduced by >50% in the presence of UK14304, as previously observed (25). These Gαi1 effects were partially blocked in the presence of untagged H-Ras(G/V). In the absence of agonist, H-Ras(G/V) inhibited the Gαi1-mediated BRET signal between RGS14 and the α2A-AR by ~30%. In addition, H-Ras(G/V) blocked the agonist-induced decrease in RGS14/α2A-AR BRET signals that was observed in the presence of Gαi1 only. As a negative control, the Gαs-linked β2-AR failed to recruit the Gαi1·RGS14 complex (Fig. 6B; right panel). Together, these results suggest that H-Ras(G/V) and the α2A-AR regulate one another's association with RGS14 in a Gαi1-dependent manner.
To highlight the physiological relevance of our BRET data showing that recombinant RGS14 binds to both exogenously expressed Gαi1 and activated H-Ras in live cells (Figs. 11–6), we next examined whether native RGS14 could interact with native Gαi1 and H-Ras in brain lysates. For these studies, we immunoprecipitated RGS14 out of mouse brain and measured whether native RGS14 bound to endogenous Gαi1 and H-Ras (Fig. 7). We observed that native RGS14 bound to native Gαi1 in brain lysates; however, it did not bind to native H-Ras. This is most likely due to the fact that native H-Ras in brain exists predominately in an inactive state. To ensure that H-Ras was activated, we incubated the brain lysates with HEK293 cell lysates overexpressing H-Ras(G/V). In combination with these HEK293 cell lysates, native RGS14 bound to both native Gαi1 and the exogenous H-Ras(G/V), indicating formation of a heterotrimeric protein complex that may link G protein and H-Ras signaling pathways within brain.
Because activated H-Ras has been shown to induce differentiation of and neurite outgrowth in PC12 cells (46) and because RGS14 has been linked to regulating growth factor-mediated neuritogenesis (47), we examined how RGS14 and Gαi1 affected H-Ras-mediated PC12 cell differentiation and neurite outgrowth. Expression of H-Ras(G/V) alone in PC12 cells promoted greater neurite outgrowth compared with Venus vector only control (Fig. 8), inducing cells to acquire small, flat, thin cell bodies (Fig. 8A). Cells expressing both H-Ras(G/V) and Gαi1 had smaller cell bodies and short neurites, whereas cells overexpressing H-Ras(G/V) and RGS14 had large, flat cell bodies and more neurites/cell compared with cells overexpressing H-Ras(G/V) alone (Fig. 8, A and B). Cells overexpressing H-Ras(G/V), RGS14, and Gαi1 together had very complex morphologies, with longer neurites and filopodia-like extensions (Fig. 8, A and C). These cells had both a greater number of neurites/cell and longer neurites, with lengths twice as long as one cell body (Fig. 8C). Of note, neither RGS14 alone nor Gαi1 alone promoted differentiation and neurite outgrowth (Fig. 8A).
Recent evidence suggests that RGS14 switches between regulating G protein signaling and regulating H-Ras signaling (26); however, the mechanism(s) behind this molecular switch remains uncertain. Studies showing that the RGS14·Gαi1 complex associates with GPCRs (25) and that GPCRs and Gαi are critical regulators of growth factor receptor signaling (28, 29, 48, 49) suggest that multiple non-canonical G protein signaling pathways may be involved in promoting this cross-talk. Our results with RGS14 support this idea and highlight a specific Gαi1-directed mechanism underlying RGS14 interactions with H-Ras.
Overall, our findings here indicate the following: 1) RGS14 selectively interacts with activated H-Ras via the first RBD in live cells, 2) RGS14 interactions with activated H-Ras depend on the plasma membrane localization of H-Ras, 3) inactive Gαi1 greatly facilitates RGS14/H-Ras(G/V) interactions, 4) activation of the α2A-AR receptor promotes rearrangement of the Gαi1·RGS14·H-Ras(G/V) complex, 5) activated H-Ras regulates association/proximity of the Gαi1·RGS14 complex with the α2A-AR, 6) native RGS14 binds to Gαi1 and activated H-Ras in mouse brain lysates, and 7) RGS14 greatly facilitates H-Ras actions on neurite outgrowth in PC12 cells when in the presence of Gαi1. Taken together, these findings suggest that RGS14 integrates both G protein signaling and H-Ras signaling through a unique mechanism that may include GPCRs to facilitate H-Ras cellular functions.
Our BRET analysis indicates that RGS14 preferentially binds the active GTP-bound form of H-Ras in live cells and not the inactive GDP-bound form of the protein (Fig. 1A). Consistent with previous studies (26, 27), this interaction takes place via the first RBD of RGS14 (Fig. 1B). Surprisingly, this interaction was greatly facilitated by Gαi1 (Fig. 2, A and B), as the presence of overexpressed Gαi1 induced a marked decrease in the BRET50, indicating that Gαi1 may either enhance the affinity of RGS14 for H-Ras or increase the number of RGS14·H-Ras(G/V) complexes. The fact that Gαi1 remained bound to RGS14 in the presence of H-Ras(G/V) (Fig. 2D) indicates that RGS14 can bind activated H-Ras and Gαi1 at the same time in live cells as has been postulated (26). This idea is most profoundly illustrated by our observation that RGS14 promotes the generation of BRET signals between Gαi1-YFP and H-Ras(G/V)-Luc (Fig. 2C). Importantly, activated H-Ras and Gαi1 did not interact at the plasma membrane when co-expressed alone, indicating the absence of nonspecific by-stander BRET between these two proteins.
These findings highlight mechanisms underlying the formation and regulation of the Gαi1·RGS14·H-Ras(G/V) complex. Like other GPR proteins (50), RGS14 may form a clamshell-like structure that is regulated by its binding partners. Gαi1 binding to RGS14 may promote a conformational change in RGS14 that allows it to bind activated H-Ras more readily, thereby promoting a platform where RGS14 can switch from regulating G protein signaling to regulating H-Ras signaling. How or if Gαi1 ever dissociates from RGS14 upon H-Ras binding remains undetermined; however, our results indicate that regardless of the temporal aspects of the regulation, RGS14 binds both Gαi1 and activated H-Ras simultaneously.
A key component of this proposed switch mechanism is that only inactive Gαi1 can facilitate H-Ras(G/V) binding to RGS14. It is possible that RGS14 can only assume a conformation favorable for binding active H-Ras when it is bound to Gαi via its GPR motif. The importance of the GPR motif in promoting RGS14 interactions with other non-Gα binding partners is also highlighted by our observation that the GPR motif is critical in promoting association between RGS14 and Gαi1-linked GPCRs (Fig. 6 and Ref 25). Therefore, it is likely that Gαi1 bound to the GPR motif may promote a stabilized and open conformation of RGS14 at the plasma membrane. This conformational switch may be a target for regulation by other signaling pathways because RGS14 is phosphorylated by PKA at Thr-494, a modification that enhances RGS14 affinity for Gαi1 (51).
H-Ras(G/V) interacts with the Gαi1-GDP·RGS14 complex via the first RBD of RGS14 because a mutant that strongly inhibits H-Ras binding to the RBD (R333L) (26) greatly reduces the H-Ras/RGS14 BRET signal (Figs. 1B and and22E). Our data with the H-Ras CAAX box mutants that are not lipid-modified (Fig. 4) clearly demonstrates that H-Ras only interacts with RGS14 when it is at the plasma membrane. Functionally, this is similar to H-Ras interactions with other effectors such as Raf-1, where H-Ras lipid modification and membrane association are essential for its capacity to activate Raf-1 (summarized in Ref. 52). Our findings indicate that Gαi1 is necessary for recruiting RGS14 to the plasma membrane and anchoring it there (15, 24) and that this complex is what membrane-bound H-Ras recognizes. Both Gαi1 and H-Ras are lipid-modified to anchor them to the membrane, whereas RGS14 is not,4 suggesting that RGS14 can be recruited to different subcellular membranes depending on the presence/absence of membrane-bound binding partners. Indeed, recombinant RGS14 is reported to localize to centrosomes with Gαi1 in non-neuronal cells (15, 53). When in complex with Gαi1 at the plasma membrane, RGS14 may reside within specific lipid microdomains and serve to sequester active H-Ras away from its other downstream effectors and/or redirect H-Ras signaling to a different effector. At the same time, RGS14 when in complex with Gαi1 and H-Ras may itself serve as a novel effector to recruit other (yet unknown) downstream signaling partners. Further studies are in progress to distinguish between these possibilities.
To determine a possible functional effect of RGS14 and Gαi1 on H-Ras signaling, we examined the role of both on regulating H-Ras-mediated neurite outgrowth in PC12 cells where RGS14 mRNA is reported to be expressed (47). H-Ras-induced cell differentiation is significantly enhanced in the presence of both RGS14 and Gαi1, with cells exhibiting both a greater number and length of neurites (Fig. 8). Inactive Gαi1 recruits RGS14 to the membrane, where the RGS14·Gαi1 complex has a greater affinity for H-Ras (see Fig. 2A). Formation of this ternary protein complex may allow activated H-Ras and/or RGS14 to engage more efficiently with downstream effectors that promote changes in cytoskeleton filament length and organization that enhance neurite outgrowth. RGS14 may form a complex with Gαi1 and activated H-Ras to mediate signaling through one or more cell surface receptors, although further studies are needed to confirm this idea. By binding Gαi1 and/or switching to bind activated H-Ras in hippocampal neurons, RGS14 may play a significant role in postsynaptic signaling responsible for dendrite and spine remodeling.
Our findings here, when combined with our earlier findings (9, 25, 26), highlight a novel mechanism of action for RGS14 where it sits at the interface of both G protein and H-Ras signaling. One potential model to explain this mechanism is consistent with two cellular pools of RGS14·Gαi1 complexes, as has been postulated (25). In this model one pool of RGS14·Gαi1 complexes may be localized at the plasma membrane and functionally linked to GPCRs, as implied by the Gαi1-dependent BRET signal observed between RGS14 and the α2A-AR (Fig. 6; Ref. 25). This may involve direct coupling to a Gαi1·RGS14 complex or merely close proximity as BRET does not distinguish between these two scenarios. The other pool of RGS14·Gαi1 complexes may localize at the plasma membrane but not associate with GPCRs, allowing these complexes to bind other proteins such as activated H-Ras. This is supported by the decrease in Gαi1-dependent RGS14/α2A-AR BRET signals observed in the presence of untagged activated H-Ras (Fig. 6B). In the absence of overexpressed H-Ras(G/V), there may be an abundance of RGS14·Gαi1 complexes that couple with the GPCR. When H-Ras(G/V) is introduced into cells, some of these RGS14·Gαi1 complexes shift away from receptors and bind to activated H-Ras. We should note, however, that activated H-Ras only induces a modest decrease in the Gαi1-dependent RGS14/α2A-AR BRET signals, which suggests that binding of activated H-Ras to a putative GPCR·Gαi1·RGS14 complex may only induce conformational rearrangement. In this case, activated H-Ras would not compete for RGS14 binding.
An attractive alternative model that is supported by the data presented here suggests that a preformed GPCR·Gαi1·RGS14 complex can be regulated by activated H-Ras (see Fig. 9). Gαi1 is bound to both the GPCR and RGS14, thereby bringing RGS14 in close proximity but not necessarily in direct contact with the receptor. In this model stimulation of a Ras-GEF promotes activation of H-Ras, which then localizes to the plasma membrane adjacent to the RGS14·Gαi1 complex (Fig. 9; steps 1 and 2). Activation of H-Ras presents a high affinity substrate for the preformed RGS14·Gαi1 complex, allowing RGS14 to bind H-Ras-GTP very tightly via its first RBD (Fig. 9; steps 2 and 3). Activation of the coupled GPCR induces a conformational change in RGS14 (see Fig. 6A), which alters the proximity of RGS14 with both the GPCR and activated H-Ras (Fig. 9; step 4). Because the non-receptor GEF Ric-8A has been shown to induce dissociation of the RGS14·Gαi1 complex (24, 25), we propose that GPCR·Gαi·RGS14 complex rearrangement after GPCR stimulation may allow such a non-receptor GEF to act on the available Gαi (Fig. 9; step 5). In this case the GEF catalyzes nucleotide exchange to activate Gαi1. The RGS domain of RGS14, being in close proximity, may then recognize and act as a GTPase-accelerating protein toward the activated Gαi1 to restore Gαi1-GDP (Fig. 9; step 6). The GPR motif would then rebind the now inactive Gαi to reform a GPCR·Gαi1·RGS14 complex (Fig. 9; step 1). The workings of this model may take place within hippocampal neurons, with RGS14 serving to localize H-Ras to the correct place in the cell for interaction with an effector that promotes neurite outgrowth (Fig. 8) or dendrite/spine remodeling associated with synaptic plasticity (54). Taken together, this model highlights a potential mechanism for RGS14 involvement in GPCR cross-talk with H-Ras-mediated signaling.
Collectively, our findings show that RGS14 functions to integrate both G protein and Ras-mediated signaling. We showcase newly appreciated roles for RGS14 at the interface of G protein and H-Ras signaling pathways, in particular its capacity to act as a molecular switch between regulating these two pathways. The fact that native RGS14 binds to native Gαi1 and H-Ras(G/V) in brain (Fig. 7) suggests that RGS14 may function as a switch to regulate both GPCR/G protein and H-Ras-mediated signaling in hippocampal neurons. Because H-Ras, Gαi1, and the non-receptor GEF Ric-8A have been implicated in regulating similar hippocampal signaling pathways and functions (55–59), the molecular mechanisms we observe here may provide insight on how RGS14 functions physiologically within the brain to regulate synaptic plasticity and hippocampal-based learning, memory, and cognition (7, 9).
We thank Dr. Oskar Laur of the Custom Cloning Core Facility (Emory University School of Medicine) for generating H-Ras-Venus plasmids and Brandon Stauffer, Dr. Stephen Ikeda, and Dr. Steven Vogel (National Institutes of Health) for providing the Venus-C1 vector. We also thank Dr. Michel Bouvier (Universite de Montreal) for the α2A-AR-Venus, β2-AR-Venus, and the phRLucN vectors.
*This work was supported, in whole or in part, by National Institutes of Health Grants R01NS049195 and R01NS037112 (to J. R. H.) and GM086510 (to J. B. B.) and by Microscopy Core of the Emory Neuroscience NINDS Core Facilities Grant P30NS055077. This work was also supported by a PhRMA Foundation Pre-doctoral Pharmacology/Toxicology Fellowship (to C. P. V).
3F. J. Shu and J. R. Hepler, unpublished data.
4K. H. Pedone and J. R. Hepler, unpublished data.
2The abbreviations used are: