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Department of Cell Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan
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subunits. However, the physiological role of a mammalian homolog Ric-8A on G protein-coupled receptor signaling in intact cells is largely unknown. We isolated Ric-8A using a yeast two-hybrid system with Gq and examined the role of Ric-8A on Gq-mediated signaling. The small interfering RNA of Ric-8A diminished the Gq-coupled receptor-mediated ERK activation and intracellular calcium mobilization in 293T cells. Ric-8A was translocated to the cell membrane in response to the Gq-coupled receptor stimulation. The expression of the myristoylation sequence-conjugated Ric-8A mutant was located in the membranes and shown to enhance the Gq-coupled receptor-mediated ERK activation. Moreover, this enhancement on ERK activation and the guanine nucleotide exchange activity of Ric-8A for Gq were inhibited by Gq selective inhibitor YM-254890. These results suggested that Ric-8A potentiates Gq-mediated signal transduction by acting as a novel-type regulator in intact cells. |
Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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subunit (G). The binding of GTP changes the conformation of G, allowing the G protein to release from a receptor and dissociate G-GTP from Gß
. G-GTP and Gß interact with downstream effectors, including adenylyl cyclase, phospholipase C, and ion channels. The signal is terminated by GTP hydrolysis, allowing G-GDP to associate with Gß and reform the inactive heterotrimer (Gilman 1987; Kaziro et al. 1991).
Recently, several proteins were identified to be involved in the regulation of the G protein cycle. The regulator of G protein signaling (RGS) proteins accelerate the GTPase activity of G, leading to the rapid termination of the signal (Ross & Wilkie 2000). RGS proteins comprise more than 20 members and are involved in diverse cellular responses, such as cell migration, growth, and differentiation (Hollinger & Hepler 2002). AGS3 and LGN, which contain the GoLoco motif, act as a guanine-nucleotide dissociation inhibitor (GDI) and activate the Gß-mediated signal transduction pathway (De Vries et al. 2000; Peterson et al. 2000). AGS3 was first identified in a functional screening on the basis of the receptor-independent activation of G protein signaling in yeast (Takesono et al. 1999), whereas LGN was found in a yeast two-hybrid screening using Gi as bait (Mochizuki et al. 1996). GPR-1/2 and Pins, which also contain the GoLoco motif, regulate G protein signaling in a receptor-independent manner during asymmetric cell division of the C. elegans embryo and D. melanogaster neuroblast, respectively (Schaefer et al. 2000; Willard et al. 2004). In mammalian cells, it has recently been reported that LGN is involved in spindle positioning during cell division (Du et al. 2001; Willard et al. 2004). Moreover, non-receptor proteins, which have a guanine nucleotide exchange (GEF) activity for Gi, have been characterized (Blumer et al. 2005). However, the physiological roles of these regulatory proteins on GPCR-mediated signaling have not been clarified.
RIC-8 (resistance to inhibitors of cholinesterase-8) is a cytoplasmic protein which was identified by a genetic screening of C. elegans mutants, which are resistant to inhibitors of acetylcholinesterase (Miller et al. 2000). Neurotransmitter release at the C. elegans neuromuscular junction is controlled by the Gq-Go signaling pathway. EGL-30 (Gq) activates EGL-8 (PLCß), leading to the production of diacylglycerol (DAG). On the other hand, the EGL-30 pathway is negatively regulated by GOA-1 (Go), which stimulates DAG kinase to reduce the DAG level (Lackner et al. 1999; Miller et al. 1999). Miller et al. (2000) have reported that ric-8 and egl-30 reduction-of-function mutants show similar phenotypes and RIC-8 functions up-stream of or parallel with EGL-30. Additionally, it has been reported that RIC-8 is involved in the asymmetric division of C. elegans embryos (Miller & Rand 2000; Afshar et al. 2004; Couwenbergs et al. 2004) and D. melanogaster neuroblasts (David et al. 2005; Hampoelz et al. 2005; Wang et al. 2005).
It has been demonstrated that the mammalian homolog of RIC-8, Ric-8A, has GEF activity for Gi1, Go, and Gq but not Gs in vitro (Tall et al. 2003). The expression of the Ric-8A gene in the neuronal system of the developing and adult mouse has been investigated using lacZ transgenic mice (Tonissoo et al. 2003). Although the positive effect of RIC-8 on Gq-mediated neurotransmitter release in C. elegans has been postulated, the role of Ric-8A in the Gq-coupled receptor-mediated signal transduction pathway in vertebrates has not been well elucidated.
To identify a novel effector or regulator of Gq, we performed a yeast two-hybrid screening using Gq as bait and obtained mouse Ric-8A. In this study, we demonstrate that the N-terminal region of Ric-8A interacts with Gq and the siRNA-mediated gene silencing of Ric-8A reduces Gq-coupled receptor-mediated ERK activation and intercellular calcium mobilization. Ric-8A is located in the cytosol and partly translocates to the plasma membranes in response to Gq-coupled receptor stimulation. Additionally, the membrane-targeted Ric-8A mutant enhances Gq-mediated ERK activation. Taken together, our results indicate that Ric-8A enhances the Gq-mediated signal transduction pathway by acting as a novel type G protein regulator in vertebrates.
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Results |
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q
To isolate Gq-interacting proteins, we performed a yeast two-hybrid screening of a mouse brain cDNA library with GqQL, a GTPase-deficient Q209L mutant, as bait. Approximately 3.5 x 106 independent clones were screened as described under Experimental procedures. We obtained 13 positive clones and analyzed their sequences. Three of these clones contained Ric-8A cDNA, which encodes a 530 amino acid protein. To test the direct interaction of Ric-8A with Gq, we prepared recombinant proteins of Gq and GST-tagged Ric-8A from Sf9 cells infected with the baculovirus. For the in vitro binding assay, GST or GST-Ric-8A was incubated with Gq and precipitated with Glutathione-Sepharose. Bounded Gq was detected by immunoblotting with an anti-Gq antibody. In agreement with our results and those from other yeast two-hybrid studies (Tall et al. 2003), GST-Ric-8A proteins interacted with Gq in vitro (Fig. 1A).
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i1 in vitro (Tall et al. 2003). We examined whether Ric-8A also binds an inactive form of Gq as well as Gi in intact cells. We transfected FLAG-tagged Ric-8A and Gq in 293T cells and then treated 293T cells with or without 
for 3 h. The lysates were incubated with an anti-FLAG antibody and Protein G-Sepharose, and Ric-8A proteins were then immunoprecipitated. Gq was co-immunoprecipitated with FLAG-Ric-8A in the absence of , whereas its interaction was attenuated in the presence of (Fig. 1B). These results suggested that Ric-8A binds to an inactive form of Gq in intact cells.
To identify the region of Ric-8A required for the interaction with Gq, we constructed two truncated forms of Ric-8A and examined their binding to Gq using a co-immunoprecipitation study. FLAG-tagged full-length Ric-8A (Ric-8A-FL), the N-terminal region of Ric-8A (Ric-8A-N301, amino acids 1-301), or the C-terminal region of Ric-8A (Ric-8A-C302, amino acids 302-530) was co-transfected with Gq in 293T cells. Gq was co-immunoprecipitated with FLAG-Ric-8A-N301 as well as FLAG-Ric-8A-FL (Fig. 1C). However, the co-immunoprecipitation of Gq with FLAG-Ric-8A-C302 was not detected, indicating that the N-terminal region of Ric-8A is necessary for interaction with Gq.
Ric-8A is ubiquitously expressed in mouse tissues and during rat brain development
To examine the expression pattern of Ric-8A in mouse adult tissues, lysates prepared from ten different adult tissues were separated by SDS-PAGE, and immunoblot analysis was performed using an anti-Ric-8A-specific antibody. The Ric-8A protein, with a molecular weight of approximately 63 kDa, was detected in various tissues (Fig. 2A). Because it has been reported that the Ric-8A gene is expressed in the nervous system of the early mouse embryo (Tonissoo et al. 2003), we investigated the expression level of Ric-8A during rat brain development. Lysates of whole brain from embryonic day 15 until adulthood were prepared and immunoblotted. Ric-8A was expressed at similar levels during brain development (Fig. 2B). Moreover, the expression of Ric-8A in various human cell lines was investigated. Most of the cell lines, except for A549 and HT1080 cells, express Ric-8A similarly to Gq (Fig. 2C).
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To investigate the role of Ric-8A on the Gq-mediated signal pathway in intact cells, we performed the siRNA-mediated gene silencing of Ric-8A in 293T cells. We designed a pair of oligonucleotides (siRic-8A), as shown in Fig. 3A. The expression of Ric-8A, but not tubulin, was remarkably suppressed by the transfection of siRic-8A. It has been reported that Gq-coupled P2Y1 and P2Y2 purinergic receptors are natively expressed and induce ERK activation in HEK293 cells (Schachter et al. 1997). On the other hand, it has also been reported that the Gi-coupled P2Y12 purinergic receptor and some P2X purinergic receptor channels induce ERK activation in several cells (Amstrup & Novak 2003; Czajkowski et al. 2004). Therefore, we first examined whether ATP, a purinergic receptor agonist, induces ERK activation through Gq. ERK activation was assessed by immunoblotting of cell lysates using an anti-phospho-ERK antibody. ATP-stimulated ERK phosphorylation was completely inhibited by pretreatment with YM-254890, a specific inhibitor of Gq/11 (Takasaki et al. 2004), but not with pertussis toxin (PTX), a Gi-family inhibitor (Fig. 3B). Additionally, transfection of the LSC-RGS domain, which acts as GAP for the G12 family, and ßARKct, which inhibits the Gß-mediated signal, had no effect on ATP-induced ERK phosphorylation (Fig. 3B). These results indicated that ATP-induced ERK activation in 293T cells was mediated through the Gq-coupled purinergic receptor. Endothelin-1 (ET-1)-induced ERK phosphorylation was partially blocked by pretreatment with YM-254890 and PTX (Fig. 3C). LSC-RGS did not inhibit ET-1-induced ERK phosphorylation. Therefore, it appeared that ET-1 activates ERK through Gq and Gi. Next, 293T cells were transfected with siGFP (control) or siRic-8A. At 48 h post-transfection, the cells were stimulated with ATP or ET-1, and ERK activation was analyzed. Ric-8A depletion diminished ATP- and ET-1-induced ERK phosphorylation by approximately 30% (Fig. 3D). In contrast, siRic-8A had no effect on ERK phosphorylation induced by m-3M3FBS, a PLC activator (Fig. 3D) (Bae et al. 2003). These results indicated that Ric-8A is involved in Gq-mediated ERK activation.
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Next, we conducted tests to determine whether or not the knock-down of Ric-8A reduces [Ca2+]i mobilization induced by ET-1. We attempted to confirm that ET-1 induces [Ca2+]i mobilization through Gq because both YM-254890 and PTX partially inhibited ET-1 activation of ERK. Pretreatment with YM-254890 significantly blocked [Ca2+]i elevation in response to ET-1 (Fig. 4A), indicating that ET-1-induced [Ca2+]i mobilization is mediated by Gq. The peak height of the [Ca2+]i increase upon ET-1 but not m-3M3FBS stimulation was reduced modestly and reproducibly by siRic-8A (Fig. 4B–D). Taken together, these results suggested that Ric-8A is a positive regulator of the Gq-mediated signal transduction pathway and functions upstream of PLCß activation.
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To clarify the mechanism by which Ric-8A enhances the Gq signal pathway, the subcellular localization of Ric-8A was examined in 293T cells. For the biochemical fractionation study, we prepared cytoplasmic soluble and membrane-rich particular fractions from the homogenates of 293T cells transfected with or without FLAG-Ric-8A. The distribution of Ric-8A and Gq was analyzed by immunoblotting. Endogenous Ric-8A and FLAG-Ric-8A were detected in the cytoplasmic soluble fraction (Fig. 5A). In contrast, endogenous Gq was detected in the membrane-rich particular fractions, suggesting that Ric-8A does not co-localize with Gq under the non-stimulated condition.
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q. Membrane localization of Ric-8A is important for Gq signal enhancement by Ric-8A
Figure 5 indicated that the membrane localization of Ric-8A should be important for its function. Therefore, we constructed the membrane-bound Ric-8A mutant by the addition of the myristoylation signal sequence of c-Src to the N terminus of Ric-8A-FLAG (Myr-Ric-8A-FLAG) (Fig. 6A). We confirmed that Myr-Ric-8A-FLAG was localized to the plasma membrane (Fig. 6Bb). As shown in Fig. 6C, Myr-Ric-8A-FLAG significantly promoted ERK activation induced by ATP. Conversely, Myr-Ric-8A-FLAG had no effect on m-3M3FBS-induced ERK activation (Fig. 6D). Furthermore, ERK signal enhancement by Myr-Ric-8A-FLAG in response to ATP was blocked with preincubation with YM-254890 (Fig. 6E). These results indicated that the membrane localization of Ric-8A is necessary for the positive effect of Ric-8A on the Gq-mediated signal pathway.
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q in vitro
It has been reported that YM-254890 inhibits Gq-coupled receptor-mediated calcium mobilization and [35S]GTPS binding to Gq (Takasaki et al. 2004). We tested whether YM-254890 blocks the GEF activity of Ric-8A for Gq in vitro. We measured [35S]GTPS binding to Gq in vitro. As shown in Fig. 7, GST-Ric-8A markedly stimulated [35S]GTPS binding to purified Gq, as previously described (Tall et al. 2003). Interestingly, YM-254890 inhibited GST-Ric-8A-induced [35S]GTPS binding to Gq. These results demonstrated that YM-254890 directly inhibits the guanine nucleotide exchange of Gq stimulated by Ric-8A.
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Discussion |
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is mainly controlled by two classes of regulators, GEFs and GAPs. GPCRs stimulate the exchange of bound GDP for GTP on G in response to ligand binding, acting as ligand-dependent GEFs. In contrast, RGSs accelerate the GTP hydrolysis activity of G, behaving as GAPs. However, the additional regulatory proteins are thought to operate in the GPCR-mediated signaling under a variety of physiological conditions. To search for regulatory proteins for Gq, we used a yeast two-hybrid system to screen a mouse brain cDNA library using Gq as bait and obtained Ric-8A. We could demonstrate the interaction of Gq with Ric-8A through its N-terminal region and the enhancement of Gq-coupled receptor-mediated signaling by Ric-8A. Tall et al. (2003) have reported that Ric-8A has GEF activity for Gq, Gi, and Go and functions as a GEF only for monomeric Gin vitro. They proposed that Ric-8A may function as a signal amplifier in intact cells. If G-GDP associates with Ric-8A before rebinding to Gß, Ric-8A would be able to reactivate G, making an amplification circuit.
In this paper, we demonstrated that Ric-8A has the ability to potentiate Gq signaling dependently on GPCR activation. First, we showed that the suppression of endogenous Ric-8A by siRNA reduced Gq-mediated ERK activation and [Ca2+]i mobilization. Second, Ric-8A is translocated from the cytosol to the plasma membrane in response to Gq-coupled receptor activation, and membrane-bound Ric-8A potentiates ERK activation stimulated by the Gq-coupled receptor. Moreover, a Gq-specific inhibitor YM-254890 blocked the Ric-8A-induced ERK potentiation and guanine nucleotide exchage of Gq. These findings indicate that Ric-8A positively regulates Gq-coupled receptor-mediated signaling in the membrane and functions as a signal amplifier.
Ric-8A can interact with Gq, Gi, and Go (Tall et al. 2003). To investigate which G protein is involved in the GPCR-mediated signal amplification by Ric-8A, we used a Gq-specific inhibitor, YM-254890 (Takasaki et al. 2004). As expected, YM-254890 inhibited ATP- and ET-1-induced ERK activation in 293T cells (Fig. 3). ET-1-induced [Ca2+]i mobilization was also inhibited by YM-254890 (Fig. 4). Similarly, siRNA-mediated Ric-8A knock-down inhibited these Gq-coupled receptor-mediated cellular responses. Moreover, we found that the membrane-localized Ric-8A-induced ERK activation in cells and the Ric-8A-induced guanine nucleotide exchange of Gq in vitro were blocked by YM-254890 (Figs 6 and 7). In vitro GTPS-binding experiments demonstrated that the spontaneous GDP release from the Gq protein is very slow when compared with those from Gs, Gi, and Go and that Ric-8A intensely stimulates the guanine nucleotide exchange of Gq (Fig. 7). It is possible that the activation of Gq is strictly regulated by GEF(s) in intact cells. Therefore, the effect of Ric-8A on Gq-mediated signaling is prominent, as shown in this study. Ric-8A may be involved in the signaling via the Gi-coupled receptor as well as the Gq-coupled receptor, since Ric-8A has GEF activity for Gi in vitro (Tall et al. 2003). We obtained the result that the suppression of endogenous Ric-8A by siRNA reduced the Gi-mediated ERK activation in m2 muscarinic acetylcholine receptor-over-expressed 293T cells (data not shown). Malik et al. (2005) have recently reported that the over-expression of wild-type Ric-8A enhances ERK activation induced by the Gß-binding peptides in CHO cells. LPA- and ATP-induced ERK activation was also enhanced by the over-expression of Ric-8A. Both enhancements were inhibited by the co-expression of ßARKct and the trasducin subunit, which were used as Gß sequesters (Malik et al. 2005). In contrast, in 293T cells, ATP-induced ERK activation was not inhibited by ßARKct (Fig. 3). This difference may be due to the cell types. More recently, Von Dannecker et al. (2005) have reported that Ric-8B binds to Golf and enhances Golf-dependent cAMP accumulation. Taken together, these reports and the present study indicate that Ric-8 proteins in vertebrates positively regulate G protein signaling downstream of GPCR.
In the non-stimulated condition, Ric-8A locates in the cytosol and would not effectively interact with G. Stimulation with carbachol rapidly induced translocation of a part of Ric-8A from the cytosol to the cell periphery (Fig. 5). Moreover, although the over-expression of Ric-8A did not affect ATP-induced ERK activation, the expression of the membrane-bound Ric-8A mutant potentiated ERK activation. Therefore, it is suggested that the membrane translocation of Ric-8A is important for its function as a G protein signal amplifier. However, the mechanism of the membrane translocation of Ric-8A still remains unclear. Ric-8A does not contain a cationic amphipathic helix, a lipid modification site, or a membrane-targeting domain. Further studies focusing on the investigation of Ric-8A-interacting molecules may help to understand the mechanism of Ric-8A translocation and activation.
We demonstrated that the membrane distribution of Ric-8A in response to GPCR activation is not uniform but local in the lamellipodia-like structure (Fig. 5). This result suggests that Ric-8A may amplify the GPCR-mediated signal pathway in a region-specific manner and play a role in cell polarization. Cell migration regulated by GPCR ligands is one of the most important physiological events in immunological cells, neuronal cells, and endothelial cells. When presented with a gradient of a chemoattractant, neutrophils respond with highly oriented polarity and motility towards the chemoattractant (Van Haastert & Devreotes 2004). However, GPCRs and G protein subunits are uniformly distributed on the membrane (Servant et al. 1999; Jin et al. 2000), and the difference in chemoattractant concentration between the front and back of the cell is very small (Zigmond 1977). In contrast, the downstream components of the cell migration signal, such as PtdIns(3,4,5)P3, activated small GTPases, and actin, are strongly polarized in response to the shallow gradient of the chemoattractant (Gardiner et al. 2002; Itoh et al. 2002). To cause an asymmetry signal for cell migration, it would be necessary for the difference in the signal intensity from the receptor to be amplified. Ric-8A may be distributed in the lamellipodia at the cell front in response to a chemoattractant and may amplify the signaling to polarize the downstream components. It would be interesting to investigate whether Ric-8A is involved in cell polarization induced by a chemoattractant.
In conclusion, we demonstrated that Ric-8A positively modulates Gq-coupled receptor-mediated calcium mobilization and ERK activation by acting as a novel type G protein regulator in membranes. Further studies are necessary to clarify how Ric-8A localization and activity are regulated. Such studies will contribute to our understanding of G protein signaling and its physiological role in the development and cell response to extracellular stimuli.
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Experimental procedures |
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A rabbit polyclonal antibody against Ric-8A was a gift from Dr Mitsuo Tagaya (Tokyo University of Pharmacy and Life Science). A mouse monoclonal antibody against GST and a rabbit polyclonal antibody against Gq/11 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). A mouse monoclonal antibody against phospho-p44/42 MAPK (Thr202/Tyr204) and a rabbit polyclonal antibody against p44/42 MAPK were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Mouse monoclonal antibodies against FLAG (M2) and tubulin, ATP, GTPS, and m-3M3FBS were purchased from Sigma. YM-254890 was a gift from Jun Takasaki (Astellas Pharma Inc.). ET-1 was purchased from the Peptide Institute, Inc. (Osaka, Japan). [35S]GTPS was from NEN Life Sciences Products (Boston, MA, USA).
Plasmid construction
The cDNA of a GTPase-deficient Q209L mutant of Gq (GqQL) was subcloned into a yeast two-hybrid bait vector, pGBKT7. The mouse Ric-8A cDNA was amplified from the total RNA of mouse brain using reverse transcription polymerase chain reaction and then cloned into the mammalian FLAG tag expression vector, pCMV5-FLAG, the mammalian GST tag expression vector, pCMV5-GST, and the baculovirus transfer vector, pFastBac-GST. The fragments of Ric-8A-N301 (1-301 amino acids) and -C302 (302-530 amino acids) were amplified from Ric-8A cDNA as a template and ligated into pCMV5-FLAG. For the myristoylation sequence-conjugated Ric-8A mutant, Ric-8A cDNA was ligated into the pEGFP-myr-N3 vector (Miyamoto et al. 2004). The region encoding EGFP was then deleted, and the FLAG tag sequence was inserted into the same site to make pMyr-Ric-8A-FLAG. The RGS domain of human LSC (1-252 amino acids) was subcloned into pCMV5-FLAG.
Yeast two-hybrid analysis
MATCHMAKER Two-Hybrid System 3 (Clontech) was used to screen for GqQL interacting proteins according to the manufacturer's protocol. A mouse brain cDNA library fused to the GAL4 activation domain of the pACT2 vector was screened using pGBKT7-GqQL as bait in the AH109 yeast strain. From the transformants, positive clones were selected on Synthetic Dropout (SD) (-Trp, -Leu, -His, -Ade) plates. Plasmids were recovered from positive clones and sequenced.
Cell culture and transfection
Human embryonic kidney 293T cells (293T cells) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum and 100 µg/mL kanamycin at 37 °C and 5% CO2. Plasmid DNAs were transfected into cells using the calcium-phosphate method or LipofectAMINETM 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol. For the calcium-phosphate method, the total amount of transfected DNA was adjusted to 6 µg for a 35-mm dish or 12 µg for a 60-mm dish with an empty vector.
Immunoprecipitation and immunoblotting
293T cells transfected with each plasmid were lyzed with a lysis buffer (20 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 3 mM MgCl2, 1 mM DTT, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 20 mMß-Glycerophosphate, 0.5% Lubrol-PX, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 µg/mL leupeptin). In the case of the 
treatment, 293T cells treated with (10 mM MgCl2, 5 mM NaF, and 50 µM AlCl3) for 3 h were lyzed with a lysis buffer containing . The lysates were centrifuged at 14 000 g for 10 min, and the supernatants were then incubated with an anti-FLAG antibody and protein G-Sepharose for 1 h. Immunoprecipitates were washed 3 times with a lysis buffer and eluted with a sample buffer. Samples were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 5% skim milk in TBS-T (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, and 0.1% Tween-20) for 1 h, followed by incubation with a primary antibody for 2 h. After three washes in TBS-T, the membrane was incubated with horseradish peroxidase-linked anti-mouse or rabbit IgG for 30 min, and signals were detected using ECL (Amersham).
MAP kinase assay
293T cells were transfected with pCMV5-FLAG-Ric-8A or pMyr-Ric-8A-FLAG. The medium was changed 24 h after transfection, and the cells were starved in serum-free DMEM for an additional 24 h. The cells were pretreated with PTX (200 ng/mL for 18 h) or YM-254890 (10 µM for 1 h) and stimulated with 100 µM ATP, 100 nM ET-1, or 100 µM m-3M3FBS for 5 min. Lysates were immunoblotted with an anti-phospho-ERK antibody. Signal intensities were quantified using an LAS-1000 image analyzer (Fujifilm). After quantification, the membrane was reblotted with an anti-ERK antibody. The ERK phosphorylation level was normalized to the total ERK level.
Immunofluorescence microscopy
At 24 h after transfection, 293T cells were replaced on collagen-coated coverslips and incubated for an additional 20 h. The cells were starved in Krebs-Ringer-HEPES buffer (20 mM HEPES-NaOH (pH 7.5), 136 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 1.25 mM MgSO4, and 1% BSA for 3 h). The cells pretreated with or without 10 µM pirenzepine for 10 min were then stimulated with 10 µM carbachol for the indicated time and fixed with 4% paraformaldehyde in PBS for 20 min. After 5 min permeabilization with 0.1% Triton X-100 in PBS, the cells were incubated with a blocking buffer (10% fetal bovine serum in PBS) for 1 h, followed by incubation with an anti-FLAG monoclonal antibody (2.5 µg/mL) in a blocking buffer for 2 h. After washing in PBS, the cells were incubated with an Alexa Fluor 488-conjugated anti-mouse IgG antibody (Molecular Probes) 1 : 1000 dilution and mounted with Perma Fluor (Shandon). They were imaged on an LSM510 laser-scanning confocal microscope (Zeiss) equipped with a Plan-Apo 100 x 1.3 oil immersion objective lens.
Intracellular calcium measurement
The intracellular free Ca2+ concentration ([Ca2+]i) was measured using the fluorescent Ca2+ indicator Fura-2 acetoxymethyl ester (Fura-2/AM). Briefly, the cells were washed with a suspension buffer (20 mM HEPES-NaOH (pH 7.5), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, and 5.6 mM glucose) and detached using a buffer stream. Suspended cells were loaded with 2 µM Fura-2/AM for 40 min at 30 °C and then washed with a suspension buffer to remove the extracellular dye. For the fluorimetric measurement of [Ca2+]i, 8 x 105 cells were placed into a cuvette in a thermostatically controlled cell holder at 37 °C with continuous stirring. The cells were pretreated with or without 10 µM YM-254890 for 5 min and then stimulated with 100 nM ET-1 or 100 µM m-3M3FBS. Fluorescence was monitored and measured using an F-2000 fluorescence spectrophotometer (Hitachi).
Protein expression and purification
GST-Ric-8A was purified from baculovirus-infected Sf9 insect cells. A baculovirus encoding GST-Ric-8A was generated using the Bac-to-Bac baculovirus expression system (Invitrogen, CA, USA). Briefly, Sf9 cells expressing GST-Ric-8A were lyzed using an Sf9 lysis buffer (20 mM HEPES-NaOH (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.5% Nonidet P40) including protease inhibitors (16 µg/mL PMSF, 16 µg/mL N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK), 16 µg/mL N-p-tosyl-L-lysine chloromethyl ketone (TLCK), 3.2 µg/mL leupeptin, and 3.2 µg/mL lima bean trypsin inhibitor). Lysates were centrifuged for 10 min. Supernatants were then loaded on to a Glutathione-Sepharose 4B column. GST-Ric-8A was eluted using an elution buffer (20 mM HEPES-NaOH (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 20 mM glutathione), buffer-changed into a storage buffer (20 mM HEPES-NaOH (pH 8.0), 150 mM NaCl, and 1 mM DTT, and concentrated using Centricon YM-30 Millipore. Baculoviruses encoding Gq, Gß1, and His-G2 were gifts from Dr Tohru Kozasa (University of Illinois at Chicago, IL, USA), and the purification of Gq was performed as previously described (Kozasa 2004).
In vitro binding assay
The in vitro binding assay of GST-Ric-8A and Gq was performed as previously described (Tall et al. 2003). Briefly, purified Gq (50 nM) was incubated with purified GST-Ric-8A (100 nM) in a binding buffer (20 mM HEPES-NaOH (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 2 mM MgSO4, and 0.05% Genapol C-100) for 1 h at 20 °C. Glutathione-Sepharose 4B was added to the reaction mixture and gently agitated for 1 h at 4 °C. The resins were washed three times with a binding buffer and eluted with a sample buffer. The eluted proteins were stained with Coomassie Blue and immunoblotted with an anti-Gq antibody.
GTPS binding assay
The kinetics of GTPS binding of recombinant Gq were measured using a filter binding method as previously described (Tall et al. 2003). Briefly, purified Gq (100 nM) was preincubated with DMSO or 10 µM YM-254890 for 3 min at 20 °C in an assay buffer (20 mM HEPES-NaOH (pH 8.0), 100 mM NaCl, 10 mM MgSO4, 1 mM EDTA, 1 mM DTT, and 0.05% Genapol C-100). Two hundred nanomolar GST or GST-Ric-8A and 10 µM[35S]GTPS (10 000 cpm/pmol) were added to the reaction mixtures. The reactions were performed at 20 °C. The reactions were stopped by the addition of an ice-cold stop buffer (20 mM Tris-HCl (pH 7.7), 100 mM NaCl, 2 mM MgSO4, 0.05% Genapol C-100, and 1 mM GTP, and the mixtures were filtrated through nitrocellulose membranes). The membranes were washed twice with an ice-cold wash buffer (20 mM Tris-HCl (pH 7.7), 100 mM NaCl, 2 mM MgSO4) and air-dried. The radioactivity of each membrane was measured using an LS6500 liquid scintillation counter (Beckman Coulter Inc., Palo Alto, CA, USA).
Subcellular fractionation
293T cells were plated in a 60-mm dish and transfected with expression vectors. Forty-eight hours post-transfection, the cells were homogenized in ice-cold buffer A (20 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM EDTA, and 250 mM sucrose) containing 1 µg/mL leupeptin and 1 mM PMSF using a Potter-Elvehjem homogenizer. Nuclei and unbroken cells were separated from the cell lysate by centrifugation for 10 min at 4 °C. The supernatants were subjected to ultracentrifugation at 100 000 g for 45 min at 4 °C. The resulting supernatants and pellets were designated cytoplasmic soluble (S) and membrane-rich particulate (P) fractions.
siRNA preparation and transfection
RNA oligonucleotides were synthesized by Dharmacon, Inc. (Lafayette, CO, USA). The synthetic siRNA sequence for Ric-8A was 5'-GCUUGUCCGCCUCAUGACAdTdT. dTdT indicates the 2-nucleotide 3' overhang of 2'-deoxythymidine. The siRNA was transfected using the LipofectAMINETM 2000 transfection reagent.
Statistical analysis
The values shown in the figures represent the mean ± S.D. from at least three independent experiments. Statistical significance was determined using a Student's t-test, and significance was defined as P < 0.01.
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Acknowledgements |
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q, Gß1, and His-G2. We also thank Y. Miyamoto and the members of our laboratory for helpful discussions. This work was partially supported by Grants-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (17048015, 17079006 and 17370051) and by grants from the Yamanouchi Foundation, the Cell Science Research Foundation, and the Ono Medical Research Foundation. |
Footnotes |
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a Present address: Department of Pharmocology, National Research Institute for Child Health and Development, Setagaya, Tokyo 157-8535, Japan
* Correspondence: E-mail: hitoh{at}bs.naist.jp |
References |
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) or 10 µM YM-254890 (
) for 3 min, and the GTP