Ric-8B Stabilizes the α Subunit of Stimulatory G Protein by Inhibiting Its Ubiquitination

Abstract

The α subunit of stimulatory G protein (Gα_s) activates adenylyl cyclase, which catalyzes cAMP production, and regulates many physiological aspects, such as cardiac regulation and endocrine systems. Ric-8B (resistance to inhibitors of cholinesterase 8B) has been identified as the Gα_s-binding protein; however, its role in G_s signaling remains obscure. In this study, we present evidence that Ric-8B specifically and positively regulates G_s signaling by stabilizing the Gα_s protein. An in vitro biochemical study suggested that Ric-8B does not possess guanine nucleotide exchange factor activity. However, knockdown of Ric-8B attenuated β-adrenergic agonist-induced cAMP accumulation, indicating that Ric-8B positively regulates G_s signaling. Interestingly, overexpression and knockdown of Ric-8B resulted in an increase and a decrease in the Gα_s protein, respectively, without affecting the Gα_s mRNA level. We found that the Gα_s protein is ubiquitinated and that this ubiquitination is inhibited by Ric-8B. This Ric-8B-mediated inhibition of Gα_s ubiquitination requires interaction between Ric-8B and Gα_s because Ric-8B splicing variants, which are defective for Gα_s binding, failed to inhibit the ubiquitination. Taken together, these results suggest that Ric-8B plays a critical and specific role in the control of Gα_s protein levels by modulating Gα_s ubiquitination and positively regulates G_s signaling.

Introduction

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) transmit extracellular signals from the G protein-coupled receptor to effector proteins, controlling a wide variety of cellular processes. The G protein consists of α, β, and γ subunits and undergoes an activation-inactivation cycle dependent on bound guanine nucleotides. In the basal state, GDP-bound α subunit (Gα) and βγ subunits (Gβγ) are associated. Once the G protein-coupled receptor is stimulated by its specific ligand, the exchange reaction of GDP to GTP on Gα is promoted. The GTP-bound Gα dissociates from Gβγ, and both Gα and Gβγ independently or cooperatively modulate the activity of specific effectors. G protein signaling is terminated by GTP hydrolysis, returning the protein to the GDP-bound state and allowing reformation of the inactive heterotrimer (1, 2). Although G proteins are primarily regulated by G protein-coupled receptors, growing evidence demonstrates that non-receptor types of regulators, including RGS (regulators of G protein signaling) and AGS (activators of G protein signaling) proteins, also modulate G protein signaling (3, 4).

Ric-8 is a novel non-receptor type of the G protein regulator that was originally identified by a genetic screening of Caenorhabditis elegans mutants, which are resistant to inhibitors of acetylcholinesterase (5). Ric-8 functions as a guanine nucleotide exchange factor (GEF)² for Gα in vitro (6). Genetic studies indicate that Ric-8 is involved in asymmetric cell division in C. elegans embryos (6–8) and Drosophila melanogaster neuroblasts (9–11). In contrast to invertebrates, which have one Ric-8, in mammals, there are two homologues of C. elegans Ric-8, named Ric-8A and Ric-8B (12). Previous studies have indicated that Ric-8A also functions as a GEF for Gα_q, Gα_i, Gα_o, and Gα₁₃ in vitro (12) and potentiates G_q signaling (13). On the other hand, Ric-8B was shown to interact with Gα_s and Gα_q, and some evidence suggests that Ric-8B potentiates olfactory-specific G protein (G_olf)-mediated signaling (14, 15). Recently, a small pigment phenotype caused by a defect of the zebrafish synembryn-like protein, which is a homologue of mammalian Ric-8B, was rescued by treatment with forskolin, an activator of adenylyl cyclase (16). These findings collectively suggest that Ric-8B is involved in G_s signaling; however, whether and how Ric-8B regulates G_s signaling remain to be clarified.

In this study, we found a novel regulatory mechanism for G_s signaling by Ric-8B. The GEF activity of Ric-8B could not be observed in vitro; however, the knockdown of Ric-8B in NIH3T3 cells suppressed cellular cAMP accumulation in response to a β-adrenergic agonist. Surprisingly, knockdown of Ric-8B resulted in the reduction of the Gα_s protein but not of other Gα and Gβ proteins. In contrast, overexpression of Ric-8B increased the Gα_s protein without affecting Gα_s mRNA levels. These results suggest that Ric-8B specifically regulates Gα_s protein levels. Furthermore, we found that the Gα_s protein was covalently modified with ubiquitin and degraded by the proteasome. The ubiquitination of Gα_s was suppressed by the overexpression of Ric-8B. These results suggest that Ric-8B specifically regulates Gα_s protein levels by suppressing Gα_s ubiquitination and positively regulates G_s signaling.

EXPERIMENTAL PROCEDURES

Molecular Cloning

The open reading frames of mouse full-length Ric-8B, Ric-8BΔ9, and Ric-8BΔ3Δ9 were amplified by PCR using forward primer 5′-GGCGGATCCATGGATGAAGAGCGCGCCCT-3′, reverse primer 5′-GGCGGATCCTCAGTCTGTGTCCGAGCTGG-3′, and cDNAs prepared from mouse brain (full-length Ric-8B) or heart (Ric-8BΔ9 and Ric-8BΔ3Δ9). The PCR products were cloned into the BamHI site of the pCMV5 expression vector. The cDNA encoding full-length Ric-8B was subcloned into the BglII site of pCMV5-FLAG or into the BamHI site of pCMV-Myc, pFASTBac-GST, and pGEX-6P-1 (GE Healthcare). The Ric-8BΔ9 and Ric-8BΔ3Δ9 cDNAs were subcloned into the BglII site of pCMV5-FLAG. pcDNA3.1-human Gα_olf was obtained from the Missouri S&T cDNA Resource Center and digested with KpnI and XhoI. The fragment was ligated into the KpnI and SalI sites of pCMV-FLAG. Short hairpin RNAs (shRNAs) directed against mouse Ric-8B (two different sequences) and Ric-8A were generated from the following annealed primers: Ric-8B2, 5′-GATCCCCACAGTTGGAAGGTGCATAATTCAAGAGATTATGCACCTTCCAACTGTTTTTTA-3′ (sense) and 5′-AGCTTAAAAAACAGTTGGAAGGTGCATAATCTCTTGAATTATGCACCTTCCAACTGTGGG-3′ (antisense); and Ric-8B3, 5′-GATCCCCGGCAGCAACTCTAGATGAATTCAAGAGATTCATCTAGAGTTGCTGCCTTTTTA-3′ (sense) and 5′-AGCTTAAAAAGGCAGCAACTCTAGATGAATCTCTTGAATTCATCTAGAGTTGCTGCCGGG-3′ (antisense). These annealed primers were inserted into the BglII and HindIII sites of pSUPER-retro-puro (Oligoengine, Seattle, WA). The Gα_s/i chimera constructs were generated by overlapping PCR (Gα_s/i-SWI_i, residues 1–185 of Gα_s-short, residues 177–195 of Gα_i, and residues 205–380 of Gα_s-short; Gα_s/i-SWII_i, residues 1–214 of Gα_s-short, residues 206–217 of Gα_i, and residues 227–380 of Gα_s-short; and Gα_s/i-SWIII_i, residues 1–232 of Gα_s-short, residues 224–240 of Gα_i, and residues 250–380 of Gα_s-short).

Cell Culture and Transfection

HEK293T and NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 units/ml penicillin at 37 °C with 5% CO₂. Plasmid DNAs were transfected into HEK293T cells using the calcium phosphate method or Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

Retroviral Production and Infection

HEK293T cells were transfected with ecotropic helper retroviral plasmid together with pSUPER-retro-puro vectors encoding shRNA directed against Ric-8B. Viruses harvested 24–60 h post-transfection were pooled. NIH3T3 cells (1 × 10⁵ cells/60-mm dish) were infected twice with 1.5 ml of retrovirus-containing supernatant supplemented with 8 μg/ml Polybrene at 2-h intervals. Twenty-four hours after infection, cells were selected in 7.5 μg/ml puromycin for 48 h.

Protein Purification

Escherichia coli Rosetta(DE3) pLysS strain (Novagen) cells harboring pGEX6P-1-mouse Ric-8B were incubated in LB medium containing 100 μg/ml ampicillin and 34 μg/ml chloramphenicol at 37 °C. When A₆₀₀ was between 0.5 and 0.6, isopropyl 1-thio-β-d-galactopyranoside was added to a final concentration of 0.4 mm. Cells were incubated for 8 h at 20 °C and collected by centrifugation. Pelleted cells were suspended in extraction buffer (50 mm Tris-HCl, pH 7.5, 5 mm MgCl₂, 150 mm NaCl, 1 mm dithiothreitol (DTT), 1 mm EDTA, 20% glycerol, and 0.5% sodium cholate) including protease inhibitors (16 μg/ml phenylmethylsulfonyl fluoride, 16 μg/ml N-tosyl-l-phenylalanine chloromethyl ketone, 16 μg/ml N^α-tosyl-l-lysine chloromethyl ketone, 3.2 μg/ml leupeptin, and 3.2 μg/ml lima bean trypsin inhibitor) and disrupted by sonication. After the lysate was clarified by centrifugation, glutathione-Sepharose 4B (1-ml bed volume/liter of culture) was added to the lysate and gently agitated for 1 h at 4 °C. The resin was washed with extraction buffer containing 300 mm NaCl and subsequently wash buffer (50 mm Tris-HCl, pH 7.5, 5 mm MgCl₂, 150 mm NaCl, 1 mm DTT, 1 mm EDTA, 10% glycerol, and 0.7% CHAPS). Glutathione S-transferase (GST)-Ric-8B was eluted with 20 mm glutathione in wash buffer. To separate the aggregated proteins, the eluate was loaded onto HiLoad 16/60 Superdex 200 pg (GE Healthcare) pre-equilibrated in 50 mm Tris-HCl, pH 7.5, containing 5 mm MgCl₂, 1 mm DTT, 150 mm NaCl, and 10% glycerol at 0.5 ml/min. Fractions containing non-aggregated GST-Ric-8B were pooled and concentrated in a 30,000 molecular weight cut-off Amicon Ultra filter unit (Millipore). Purification of GST-Ric-8A was performed as described previously (13) with some modification. Briefly, the proteins eluted from glutathione-Sepharose were loaded onto HiLoad 16/60 Superdex 200 pg pre-equilibrated in 20 mm HEPES-NaOH, pH 8.0, containing 100 mm NaCl and 1 mm DTT at 0.5 ml/min. Peak fractions containing non-aggregate GST-Ric-8A were pooled and concentrated in a 30,000 molecular weight cut-off Amicon Ultra filter unit. Baculoviruses encoding Gα_q, Gβ₁, and His-Gγ₂ were kindly provided by Dr. Tohru Kozasa (University of Illinois at Chicago). Purification of Gα_q was performed as previously described (17). To prepare recombinant Gα_s-short, the cDNA encoding bovine Gα_s-short (spliced variant 4) was subcloned into the XbaI and HindIII sites of pQE60 (Qiagen). The expression and purification of Gα_s proteins were performed as described previously (18, 19).

In Vitro Binding Assay

The in vitro binding assay of GST-Ric-8B and Gα_s or Gα_q was performed as described previously (12). Briefly, 100 nm Gα_s or Gα_q was incubated with 100 nm GST or GST-Ric-8B in binding buffer (20 mm HEPES-NaOH, pH 8.0, 100 mm NaCl, 10 mm MgSO₄, 1 mm EDTA, 1 mm DTT, and 0.05% Lubrol PX) for 1 h at 25 °C. Glutathione-Sepharose 4B (GE Healthcare) was added to the reaction mixture and gently agitated for 1 h at 4 °C. The resins were washed three times with binding buffer and treated with SDS-PAGE sample buffer. The eluted proteins were resolved by SDS-PAGE, stained with Coomassie Blue, and immunoblotted with anti-Gα_s or anti-Gα_q antibodies.

GTPγS Binding Assays

GTPγS binding reactions were initiated by the addition of 5 pmol (50 nm) of Gα_s or Gα_q to reaction buffer (20 mm HEPES-NaOH, pH 8.0, 100 mm NaCl, 10 mm MgSO₄, 1 mm EDTA, 1 mm DTT, and 0.05% C₁₂E₁₀) containing 20 pmol (200 nm) of GST, GST-Ric-8A, or GST-Ric-8B and 10 μm [³⁵S]GTPγS (10,000 cpm/pmol) in a total volume of 100 μl at 20 °C. The reaction buffer for Gα_q was identical to that for Gα_s except that 0.05% Genapol C-100 detergent was used instead of C₁₂E₁₀. Aliquots (20 μl) were removed at the indicated times, and ice-cold buffer containing 20 mm Tris-HCl, pH 7.7, 100 mm NaCl, 2 mm MgSO₄, 0.05% C₁₂E₁₀, and 1 mm GTP was added before filtration through BA85 nitrocellulose membranes. The membranes were washed twice with an ice-cold wash buffer (20 mm Tris-HCl, pH 7.7, 100 mm NaCl, and 2 mm MgSO₄) and dried. The radioactivity of each membrane was measured using an LS6500 liquid scintillation counter (Beckman Coulter).

Intracellular cAMP Accumulation

NIH3T3 cells infected with retroviruses that express shRNA directed against mouse Ric-8B or HEK293T cells were pretreated with 0.5 mm 3-isobutyl-1-methylxanthine for 1 h and subsequently stimulated with 10 μm isoproterenol or 10 nm pituitary adenylate cyclase-activating polypeptide for the indicated times, respectively. Cyclic AMP was measured using the AlphaScreen cAMP assay kit (PerkinElmer Life Sciences) according to the manufacturer's protocol.

Degradation of Gα_s after Inhibition of Protein Synthesis (Cycloheximide (CHX) Chase)

HEK293T cells transfected with an empty vector or FLAG-Ric-8B were grown to 80% confluence in 60-mm dishes and treated with 100 μg/ml CHX for the indicated times. Cells were harvested and subsequently lysed with lysis buffer (20 mm HEPES-NaOH, pH 7.5, 100 mm NaCl, 3 mm MgCl₂, 1 mm EDTA, 1 mm DTT, 0.5% Nonidet P-40, 1 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride). Cell lysates were analyzed by immunoblotting using anti-Gα_s, anti-FLAG, and anti-actin antibodies.

In Vivo Ubiquitination Assay

An in vivo ubiquitination assay was performed as described previously (20). HEK293T cells were transfected with pMT107-6xHis-ubiquitin (21) and other expression plasmids. Twenty-four hours post-transfection, cells were treated with 10 μm MG132 for 12 h, and cells were harvested by centrifugation. Cells were lysed with urea lysis buffer (10 mm Tris-HCl, pH 8.0, 10 mm NaH₂PO₄, 8 m urea, 10% glycerol, 0.1% Triton X-100, 0.5 m NaCl, 10 mm imidazole, and 10 mm 2-mercaptoethanol) and disrupted by sonication. Lysates were centrifuged at 15,000 × g for 5 min at room temperature, and supernatants were collected. For purification of His₆-tagged ubiquitinated proteins, nickel-nitrilotriacetic acid-agarose (Qiagen) was added to the supernatant and gently agitated for 4 h at room temperature. The resins were washed five times with 20 mm imidazole in urea lysis buffer and treated with 150 mm Tris-HCl, pH 6.8, containing 200 mm imidazole, 5% SDS, 30% glycerol, and 0.72 m 2-mercaptoethanol. Ubiquitinated Gα_s, FLAG-Gα_s, or FLAG-Gα_olf proteins were detected by immunoblotting using anti-Gα_s or anti-FLAG antibodies.

Reverse Transcription-PCR

Total RNAs were prepared using TRIzol reagent (Invitrogen) from HEK293T cells transfected with FLAG-Ric-8B. First-strand cDNAs were synthesized from 2 μg of total RNA with SuperScript II (Invitrogen). PCR mixtures (50 μl) containing 0.5 μl of cDNA and 0.5 μm each forward and reverse primers were heated at 94 °C for 2 min, followed by 25 (Gα_s) or 20 (glyceraldehyde-3-phosphate dehydrogenase) cycles of 94 °C for 1 min, 61 °C for 1 min, and 72 °C for 30 s. PCR products were analyzed in 2% agarose gels stained with ethidium bromide. The following primers were used: Gα_s, 5′-GCACCATTGTGAAGCAGATG-3′ (forward) and 5′-TCATCCTCCCACAGAGCCTT-3′ (reverse); and glyceraldehyde-3-phosphate dehydrogenase, 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-TCCACCACCCTGTTGCTGTA-3′ (reverse).

RESULTS

Effect of Ric-8B on GTPγS Binding to Gα_s and Gα_q in Vitro

Previously, it has been demonstrated that C. elegans Ric-8 (6) and mammalian Ric-8A (12) possess GEF activity for Gα in vitro. In addition, it has been reported that Ric-8B potentiates Gα_olf-mediated signaling in HEK293 cells (14, 15). These findings suggest that Ric-8B might have GEF activity for Gα; however, no data for the GEF activity of Ric-8B have been reported so far. First, we confirmed the binding of Ric-8B to Gα_s and Gα_q. GST-Ric-8B, which was prepared from E. coli in the presence of glycerol using gel filtration chromatography as non-aggregate, was incubated with Gα_s or Gα_q, and the protein complexes were then precipitated with glutathione-Sepharose. Gα_q and Gα_s were co-precipitated with GST-Ric-8B, indicating that Ric-8B directly binds to Gα_q and Gα_s (Fig. 1A). Next, the kinetics of GTPγS binding to Gα_s or Gα_q proteins was investigated in the presence of GST-Ric-8B or GST-Ric-8A. GST-Ric-8A dramatically increased the rate of GTPγS binding to Gα_q but not Gα_s, as reported previously (12). In contrast, GST-Ric-8B did not affect the GTPγS-binding activity of Gα_q and Gα_s (Fig. 1, B and C). In these experiments, we used recombinant Gα_s and Gα_q proteins that were expressed in E. coli and Sf9 cells, respectively. Because N-terminal myristoylation of Gα_i1 greatly improves the ability of the protein to serve as a substrate for Ric-8A-stimulated guanine nucleotide exchange (22), we examined the effect of Ric-8B on palmitoylated Gα_s. Palmitoylated Gα_s was purified from a membrane fraction of Sf9 cells that were infected with baculoviruses encoding Gα_s-short, Gβ₁, and His-Gγ₂. However, we could not observe the effect of GST-Ric-8B on the guanine nucleotide exchange reaction for palmitoylated Gα_s (data not shown). These results suggest that Ric-8B alone does not possess GEF activity for Gα_s and Gα_q in vitro.

FIGURE 1.

Effect of Ric-8B on guanine nucleotide exchange. A, GST or GST-Ric-8B (100 nm each) was incubated with Gα_s or Gα_q (100 nm each) for 1 h at 25 °C. These mixtures were bound to glutathione-Sepharose and washed extensively with a buffer. The proteins were eluted with an SDS-PAGE sample buffer. Co-precipitation of Gα proteins was detected by immunoblotting (IB). B and C, Gα_q (B) and Gα_s (C) (5 pmol each) were incubated with [³⁵S]GTPγS in reaction mixture (100 μl) containing GST (●), GST-Ric-8B (▲), or GST-Ric-8A (▼) (20 pmol each). Aliquots (20 μl) of these reaction mixtures were taken at the indicated time points and filtered to absorb nucleotide-bound protein. The amount of G protein-bound [³⁵S]GTPγS was determined by scintillation counting.

Knockdown of Ric-8B Reduces Isoproterenol-induced cAMP Accumulation

Next, to test the involvement of Ric-8B in G_s signaling, we prepared two retroviral constructs expressing the shRNA directed against mouse Ric-8B and infected NIH3T3 cells with them. As shown in Fig. 2A, both shRNAs showed effective reduction of the Ric-8B protein, and their efficiency of knockdown was above 80%. In control cells, cAMP accumulation was promoted in response to isoproterenol. In cells expressing shRNA directed against Ric-8B, isoproterenol-induced cAMP accumulation was greatly reduced (Fig. 2B). The extent of the inhibition of cAMP accumulation was correlated with the protein reduction of Ric-8B (Fig. 2, A and C). These results indicate that Ric-8B positively regulates G_s signaling and are consistent with previous observations reporting the functional requirement of Ric-8B in Gα_olf-mediated signaling in mammals (14) and pigment dispersion in zebrafish (16). In addition, Ric-8B seems to be specifically involved in G_s signaling because we did not observe any significant effect of knockdown of Ric-8B on UTP-stimulated (Gα_q-coupled) intracellular calcium mobilization and platelet-derived growth factor-induced extracellular signal-regulated kinase (ERK) activation (supplemental Fig. S1, A and B).

FIGURE 2.

Knockdown of Ric-8B decreases isoproterenol-induced cAMP accumulation. A, cell lysates from NIH3T3 cells infected with retroviruses encoding control shRNA or two different sequences of shRNAs directed against Ric-8B were analyzed by immunoblotting (IB) using anti-Ric-8B and anti-tubulin antibodies. B, shown is the time course of cAMP accumulation. cAMP accumulation of NIH3T3 cells infected with retroviruses encoding control (○) or Ric-8B2 (shRic-8B#2; ●) shRNA was measured following exposure to 10 μm isoproterenol for the indicated times. C, NIH3T3 cells infected with retroviruses were exposed to 10 μm isoproterenol for 20 min, and cAMP accumulation was measured. The data are expressed as the mean ± S.D. from three independent experiments. CTL, control.

Ric-8B Positively Regulates Gα_s Protein Levels

To investigate how Ric-8B is involved in G_s signaling, we first examined the effect of knockdown of Ric-8B on the protein levels of α and β subunits of G proteins. Surprisingly, knockdown of Ric-8B dramatically reduced Gα_s protein levels (Fig. 3A). This effect was not observed in other Gα proteins or the Gβ subunit. In contrast, overexpression of Ric-8B greatly increased the Gα_s protein level (Fig. 3B). These results indicate that Ric-8B specifically and positively regulates Gα_s protein levels. Next, we performed a reverse transcription-PCR analysis using HEK293T cells transfected with Ric-8B. Overexpression of Ric-8B increased the Gα_s protein but had little effect on its transcriptional level (Fig. 3B), suggesting that Ric-8B affects Gα_s protein stability.

FIGURE 3.

Ric-8B positively regulates Gα_s protein levels. A, cell lysates were prepared from NIH3T3 cells infected with retroviruses encoding control shRNA or two different sequences of shRNAs directed against Ric-8B (shRic-8B) and were analyzed by immunoblotting (IB) using the indicated antibodies. The Gα_s protein exists as two spliced forms, short and long. The ratio of the spliced forms varies in different cell types. The positions of the long and short Gα_s variants are indicated by lines. B, HEK293T cells were transfected with FLAG-Ric-8B and harvested for RNA and protein preparations. Semiquantitative reverse transcription-PCR (RT-PCR) and immunoblotting were performed to assess the effect of Ric-8B on the Gα_s mRNA and protein levels, respectively. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

To examine whether Ric-8B knockdown affects the expression and function of other G_s signaling components, G_s-coupled receptor, and adenylyl cyclase, we evaluated the β-adrenergic receptor expression and adenylyl cyclase activity. Ligand binding assays using a radiolabeled β-adrenergic receptor antagonist, [¹²⁵I]iodocyanopindolol, were carried out. The numbers of endogenous β-adrenergic receptors were determined by Scatchard analysis of [¹²⁵I]iodocyanopindolol saturation binding. The level of β-adrenergic receptor expression was not affected by Ric-8B knockdown (supplemental Fig. S2A). Intrinsic adenylyl cyclase activity was evaluated by Gα_s reconstitution assay. Membranes of NIH3T3 cells expressing shRNA directed against Ric-8B were incubated with GTPγS-preloaded Gα_s, and then cAMP production was measured. No effect of Ric-8B knockdown on intrinsic adenylyl cyclase activity was observed (supplemental Fig. S2B).

Ric-8B Inhibits Gα_s Degradation and Ubiquitination

We focused on the ubiquitin-proteasome pathway, which is involved in protein stability (23). We first investigated whether the Gα_s protein is controlled by the ubiquitin-proteasome pathway. HEK293T cells were treated with a potent proteasome inhibitor, MG132. Interestingly, treatment with MG132 increased the Gα_s protein and potentiated the pituitary adenylate cyclase-activating polypeptide-induced cAMP accumulation (Fig. 4, A and B). These results suggest that the Gα_s protein is controlled by proteasomal degradation. Next, we monitored the endogenous Gα_s protein level after treatment with a protein synthesis inhibitor, CHX. HEK293T cells transfected with FLAG-Ric-8B or an empty vector were treated with CHX for 0–5 h. In control cells, the levels of the Gα_s protein decreased after exposure to CHX. In cells transfected with FLAG-Ric-8B, the degradation of the Gα_s protein was inhibited (Fig. 4C), indicating that Ric-8B enhances Gα_s stability. These results suggested that Ric-8B modulates the post-translational modification, such as ubiquitination, of Gα_s. Therefore, we performed an in vivo ubiquitination assay. HEK293T cells were transfected with Gα_s together with His-tagged ubiquitin and treated with MG132 for 12 h. These cells were subsequently lysed under denaturing conditions. His-tagged ubiquitinated proteins were collected with nickel-agarose resin, and the precipitated Gα_s was detected by immunoblotting. We detected a ladder of ubiquitinated Gα_s proteins whose intensity increased with MG132 treatment (Fig. 4D). To examine the effect of Ric-8B on Gα_s ubiquitination, we expressed Ric-8B in HEK293T cells together with Gα_s and His-tagged ubiquitin. Overexpression of Ric-8B increased the Gα_s protein, as shown in Fig. 3B; however, the amount of ubiquitinated Gα_s proteins was reduced. These findings strongly suggest that Gα_s is a novel substrate for ubiquitination and that Ric-8B regulates Gα_s protein stability by suppression of the ubiquitination and degradation of Gα_s in mammalian cells.

FIGURE 4.

Ric-8B inhibits Gα_s degradation and ubiquitination. A, HEK293T cells were treated with 10 μm MG132 for 12 h, and cell lysates were analyzed by immunoblotting (IB) using anti-Gα_s and anti-actin antibodies. B, HEK293T cells were pre-incubated with 10 μm MG132 for 8 h, and cAMP accumulation was then measured following exposure to 10 nm pituitary adenylate cyclase-activating polypeptide (PACAP) for 15 min. C, HEK293T cells transfected with an empty vector or FLAG-Ric-8B were treated with 100 μg/ml CHX for the indicated times. Cell lysates were analyzed by immunoblotting using anti-Gα_s, anti-FLAG, and anti-actin antibodies. D, HEK293T cells were transfected with Gα_s-short, His-ubiquitin (His-Ub), and FLAG-Ric-8B. Twenty-four hours after transfection, cells were treated with 10 μm MG132 for 12 h and subsequently lysed with an 8 m urea-containing buffer, and His-tagged ubiquitinated proteins were precipitated with nickel-agarose resin. Ubiquitinated Gα_s was detected by immunoblotting using the anti-Gα_s antibody.

Interaction of Ric-8B with Gα_s Is Important for Inhibiting Gα_s Ubiquitination

To understand in detail the mechanism whereby Ric-8B inhibits Gα_s ubiquitination, we hypothesized that interaction of Ric-8B with Gα_s is required for Ric-8B-mediated inhibition of Gα_s ubiquitination. To examine this hypothesis, we used two spliced variants of Ric-8B (supplemental Fig. S3A). Ric-8BΔ9, which lacks exon 9, was previously reported (14). In addition, we identified another novel spliced variant, Ric-8BΔ3Δ9, which lacks both exons 3 and 9. HEK293T cells were transfected with FLAG-tagged full-length Ric-8B and its variants. Endogenous Gα_s protein was co-immunoprecipitated with FLAG-tagged full-length Ric-8B but not with Ric-8BΔ9 or Ric-8BΔ3Δ9 (supplemental Fig. S3B). As expected, these spliced variants failed to inhibit Gα_s ubiquitination (Fig. 5A), suggesting that the suppressive effect of Ric-8B on the ubiquitination of Gα_s requires the interaction between Gα_s and Ric-8B.

FIGURE 5.

Interaction of Ric-8B with Gα_s is important for inhibiting Gα_s ubiquitination. HEK293T cells were transfected with Gα_s-short, His-ubiquitin (His-Ub), and FLAG-tagged full-length Ric-8B (FL) or spliced variants (A) or with His-ubiquitin, FLAG-Ric-8B, and wild-type Gα_s-short (Gα_sWT) or Gα_s/i-SWII_i (B). The ubiquitinated Gα_s was detected as described in the legend to Fig. 4D.

Next, we generated a series of the Gα_s/i chimeric proteins because the Gα_i protein does not interact with Ric-8B in cells (supplemental Fig. S4) (12). Gα contains three switch regions (SWI, SWII, and SWIII), and each switch region of Gα_s was replaced with that of Gα_i (supplemental Fig. S5A). These constructs retain proper conformation because chimeric α subunits were dissociated from the Gβγ complex in the presence of AlF₄⁻ (supplemental Fig. S5B). Among these chimeric proteins, only Gα_s/i-SWII_i dramatically reduced the affinity for FLAG-Ric-8B in HEK293T cells (supplemental Fig. S5C), suggesting that the switch II region of Gα_s is required for interaction with Ric-8B. Similarly to the wild-type Gα_s protein, the Gα_s/i-SWII_i chimeric protein was also ubiquitinated; however, the ubiquitination of Gα_s/i-SWII_i was not inhibited by the overexpression of Ric-8B (Fig. 5B). Taken together, these results strongly support our hypothesis that the interaction of Ric-8B with Gα_s is important for inhibiting Gα_s ubiquitination.

DISCUSSION

Previously, Tall et al. (12) and our group (13) reported that Ric-8A, another mammalian homologue of Ric-8, exhibits GEF activity for Gα_q and contributes to the G_q signaling pathway. According to the analogy to Ric-8A, it was speculated that Ric-8B would also harbor GEF activity for Gα_s because Ric-8B showed potent ability to interact with Gα_s (12). However, we could not observe the GEF activity of Ric-8B in vitro (Fig. 1). On the other hand, our analysis utilizing shRNA against Ric-8B clearly demonstrated the functional involvement of Ric-8B in ligand-induced cAMP accumulation (Fig. 2). Our observation suggested that Ric-8B plays an essential role in G_s signaling without its GEF activity. Furthermore, we found that the expression level of Ric-8B apparently affected the expression level of Gα_s without any changes in the amount of Gα_s mRNA (Fig. 3).

We demonstrated that Gα_s is ubiquitinated and that both its ubiquitination and degradation are suppressed by Ric-8B (Fig. 4). Combining all of our current data, we propose a new mode of regulatory mechanism whereby Ric-8B stabilizes G_s signaling through suppressing the ubiquitination and degradation of Gα_s. According to a previous report, Gα_olf-mediated cAMP accumulation is also potentiated by Ric-8B (14). However, the mechanism whereby Ric-8B emphasizes olfactory signaling remained obscure. In this study, we demonstrated that overexpression of Ric-8B increased the protein amount of Gα_olf and inhibited its ubiquitination similarly to Gα_s (supplemental Fig. S6). These findings raise the possibility that Ric-8B may also amplify Gα_olf signaling by the stabilization of Gα_olf. However, it is still possible that Ric-8B harbors Gα_s/Gα_olf-specific GEF activity with the additional factors in the cells. Detailed biochemical analysis for the Ric-8B-interacting proteins that may be critical for GEF activity would prove this possibility.

Several observations in this study suggest that the interaction between Ric-8B and Gα_s is important for the suppression of ubiquitination of Gα_s (Fig. 5). The Gα_s protein preferentially locates in the plasma membrane, whereas Ric-8B mostly localizes in the cytoplasm in quiescent cells. Klattenhoff et al. (24) reported that isoproterenol induces the translocation of Ric-8B into the plasma membrane and the co-localization of Ric-8B with Gα_s. Consistent with these findings, another group reported that the overexpression of Ric-8B increases the amount of Gα_olf protein on the plasma membrane (15, 25). In addition, we observed that the polyubiquitinated Gα_s protein seemed to localize in the plasma membrane and that MG132 treatment resulted in the accumulation of Gα_s in the plasma membrane (supplemental Fig. S7). Although the manner in which a ligand induces membrane localization of Ric-8B is unclear, it allows us to provide a novel regulatory mechanism whereby Ric-8B may interact with Gα_s in response to G_s activation and to stabilize Gα_s through the suppression of the ubiquitination of Gα_s. Interestingly, several reports suggest that the activation of Gα_s shortens the half-life of Gα_s (26, 27).

Our observation provides a model of how Ric-8B stabilizes the Gα_s protein and enhances its signals; Ric-8B may mask the ubiquitination site of Gα_s and then perturb the accessibility of Gα_s-specific E3 ubiquitin ligase to the Gα_s protein. Although E3 ubiquitin ligase for Gα_s has not been identified yet, several observations provide some clues to explore the Gα_s-specific E3 ubiquitin ligase. A recent study described an RGS-GAIP-interacting protein, GIPN, which possesses E3 ubiquitin ligase activity and promotes the proteasome-dependent degradation of Gα_i3 (28). There are two main classes of E3 ubiquitin ligases: RING finger and HECT E3 ubiquitin ligases (29). RING finger E3 ubiquitin ligases bind to a specific E2 ubiquitin-conjugating enzyme through their RING finger domain, which is prerequisite for ubiquitination of the substrate protein. It has been reported that GIPN harbors the RING finger-like motif, suggesting that GIPN may exhibit E3 ubiquitin ligase activity through this non-canonical RING finger domain. Similarly, Gα_s-specific E3 ubiquitin ligase may be an interacting protein of RGS-PX, which has been reported as a sole RGS molecule for Gα_s so far (30). A recent genetic and biochemical study indicated that Rsp5 is an E3 ubiquitin ligase for yeast Gα Gpa1 (31). Although Gpa1 is ubiquitinated in a region that is absent in mammalian Gα (32), NEDD4 proteins, which are mammalian homologues of yeast Rsp5, might have the E3 ubiquitin ligase activity for mammalian G protein α subunits. More recently, it was reported that MGRN1 (Mahogunin ring finger-1) attenuates melanocortin receptor-mediated cAMP accumulation by competing the interaction between Gα_s and the melanocortin receptor (33). MGRN1 contains a RING finger domain and has been shown to display ubiquitin ligase activity for some proteins other than Gα_s (34, 35). MGRN1 might also function as E3 ubiquitin ligase for Gα_s and might attenuate G_s signaling. The determination of E3 ubiquitin ligase for Gα_s and analysis of the mechanism whereby E3 ubiquitin ligase regulates G_s signaling will be the focus of a future study.