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. Author manuscript; available in PMC: 2012 May 10.
Published in final edited form as: Oncogene. 2011 Feb 14;30(23):2691–2696. doi: 10.1038/onc.2010.645

Molecular Basis of a Novel Oncogenic Mutation in GNAO1

Mikel Garcia-Marcos 1,*, Pradipta Ghosh 2, Marilyn G Farquhar 1
PMCID: PMC3349449  NIHMSID: NIHMS373477  PMID: 21317923

Abstract

Heterotrimeric G proteins are molecular switches that control signal transduction and their dysregulation can promote oncogenesis. Somatic mutations in GNAS, GNAI2 and GNAQ genes induce oncogenesis by rendering Gα-subunits constitutively activated. Recently the first somatic mutation, Arginine243 → Histidine (R243H) in the GNAO1 (Gαo) gene was identified in breast carcinomas and shown to promote oncogenic transformation when introduced into cells. Here we provide the molecular basis for the oncogenic properties of the Gαo R243H mutant. Using limited proteolysis assays, nucleotide binding assays, and single-turnover and steady-state GTPase assays, we demonstrate that the oncogenic R234H mutation renders Gαo constitutively active by accelerating the rate of nucleotide exchange; however, this mutation does not affect Gαo’s ability to become deactivated by GTPase Activating Proteins (GAPs) or by its intrinsic GTPase activity. This mechanism differs from that of previously reported oncogenic mutations which impair GTPase activity and GAP sensitivity without affecting nucleotide exchange. The constitutively active Gαo R243H mutant also enhances Src-STAT3 signaling in NIH-3T3 cells, a pathway previously shown to be directly triggered by active Gαo proteins to promote cellular transformation. Based on structural analyses we promose that the enhanced rate of nucleotide exchange in Gαo R243H results from loss of the highly conserved electrostatic interaction of R243 with E43, located in the in the P-loop which represents the binding site for the α and β-phosphates of the nucleotide. We conclude that the novel R234H mutation imparts oncogenic properties to Gαo by accelerating nucleotide exchange and rendering it constitutively active, thereby enhancing signaling pathways, e.g., src-STAT3, responsible for neoplastic transformation.

Keywords: Oncoprotein, somatic mutation, heterotrimeric G protein, GAIP/RGS19, neoplasia, STAT3

INTRODUCTION

Identification of oncogenic mutations in G proteins has provided insights into the molecular mechanisms that drive tumorigenesis and has also highlighted the importance of maintaining the critical balance between G protein activation and deactivation. Mutation of the small G protein Ras is very common in cancer, e.g., K-ras is mutated ~20% of all tumors (Forbes et al., 2008). Gα-subunits of heterotrimeric G proteins, which have structural similarities to the Ras superfamily of G proteins, have also been reported to be mutated in cancer. For instance, mutations in codons 201 and 227 of GNAS (Gαs) are found in ~25% of pituitary adenomas (Forbes et al., 2008; Landis et al., 1989) and mutation of the corresponding codons in GNAI2 (Gαi2) (Lyons et al., 1990) and GNAQ (Gαq) (Lamba et al., 2009; Van Raamsdonk et al., 2009) have also been reported in human tumors. All these mutations in Ras and heterotrimeric G proteins promote malignant transformation and tumorigenesis by rendering the G proteins constitutively active in the GTP-bound conformation (Downward, 2003; Farfel et al., 1999; Iiri et al., 1998; Landis et al., 1989). The molecular mechanism by which this is achieved is also highly conserved in that these oncogenic mutations block the deactivation step of the G protein by impairing its intrinsic ability to hydrolyze GTP (i.e., GTPase-deficient) (Landis et al., 1989) and its sensitivity to the action of GTPase Activating Proteins (GAPs) (Berman et al., 1996; Downward, 2003). No such mutations in other Gα-subunits (i.e., Gαo, Gα12, Gα13 and Gαz) have been described in carcinomas but they were found to be oncogenic when artificially introduced into cultured cells (Kroll et al., 1992; Marinissen & Gutkind, 2001; Radhika & Dhanasekaran, 2001; Vara Prasad et al., 1994; Voyno-Yasenetskaya et al., 1994; Wong et al., 1995; Xu et al., 1993; Xu et al., 1994).

Very recently, the first somatic mutation for GNAO1 (Gαo) has been described in breast cancer (Kan et al., 2010). This oncogenic mutation, i.e., R243H, is unique because it does not correspond to any of the previously described mutations in other G proteins. However, the molecular basis for its ability to induce oncogenic transformation but was not investigated. Here we studied the biochemical properties of Gαo R243H and its impact on cellular signaling. We found that Gαo R243H is rendered constitutively active by a molecular mechanism distinct from that reported for any of the previously characterized oncogenic mutations in G proteins and discuss the structural basis for this mechanism using insights from the available G protein crystal structures. Finally, we show that this mutant triggers a signaling program responsible for malignant transformation. These data advance our understanding of how heterotrimeric G proteins can be transformed from proto-oncogenes to oncogenes by naturally occurring mutations in key residues.

RESULTS AND DISCUSSION

Gαo R234H is capable of binding nucleotides and of changing conformation upon activation

To investigate the effect of the R234H mutation on the biochemical properties of Gαo and to gain insights into the molecular basis of its oncogenicity (Kan et al., 2010) we purified hexahistidine-tagged wild-type (wt) Gαo and R234H mutant from E. coli (>98%purity). Because some mutations in Gα-subunits affect their ability to fold properly, bind nucleotides and/or change conformation upon activation (Grishina & Berlot, 1998; Kleuss et al., 1994; Pereira & Cerione, 2005; Warner & Weinstein, 1999), we determined whether Gαo R234H retains the native properties of the wild-type protein by using a well established assay based on differential resistance to proteolysis (Kleuss et al., 1994). When Gα-subunits are in the inactive GDP-bound conformation, they are readily digested by trypsin whereas upon binding of either AlF4− (complexes with GDP to mimic GTP in the G protein active site) or GTPγS (a non-hydrolyzable GTP analog) and adoption of the active conformation only a short sequence can be cleaved, and the remainder of the protein remains trypsin-resistant. Gαo R234H behaved like wt Gαo in that it was hydrolyzed by trypsin when pre-loaded with GDP but generated a trypsin-resistant form when preloaded with GDP·AlF4− or GTPγS (Fig. 1A). Thus, Gαo R234H can efficiently adopt the active conformation upon AlF4− or GTPγS binding. Given that activation by AlF4− requires the nucleotide binding site to contain GDP and the G protein to be in an appropriate conformation, these results also indicate that Gαo R234H is properly folded and is capable of binding both GDP and GTP nucleotides.

Figure 1. The R243H mutation enhances the rate of nucleotide exchange by Gαo but has no effect on the intrinsic or GAP-enhanced GTPase activity of the G protein.

Figure 1

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A. The Gαo R243H mutant retains the ability to adopt a trypsin-resistant conformation upon activation. Rat Gαo (isoform 1; Gαo1, hereafter referred to as Gαo) cloned into pET28b (Garcia-Marcos et al., 2010) was used as template to generate the R243H mutant (Quikchange mutagenesis kit). Hexahistidine-tagged wt Gαo and the R243H mutant were purified from BL21(DE3) E. coli as previously described (Garcia-Marcos et al., 2010). A gel filtration chromatographic step (Superdex 200) was substituted for the final steps of dialysis and buffer exchange. Limited trypsin proteolysis was performed as previously described (Garcia-Marcos et al., 2010). Wt Gαo and R243H mutant (0.5 mg/ml) were incubated at 30°C in the presence of GDP (30 μM), GDP·AlF4− (30 μM AlCl3, 10 mM NaF) or GTPγS (30 μM) and treated or not with trypsin (12.5 μg/ml) for 10 min as indicated. The reaction was stopped by adding SDS sample buffer and boiling. Protein (~5 μg) were separated by SDS-PAGE and stained with Coomassie blue. One representative experiment out of 3 is shown. The arrowhead denotes the position of the non-trypsinized, full-length proteins loaded, and the starred arrowhead (*) denotes the trypsin-resistant form of the Gα-subunit in the active conformation. Both wt Gαo and R234H are digested when pre-loaded with GDP and adopt the trypsin-resistant conformation after incubation with GDP·AlF4− or GTPγS.

B. The R243H mutation does not affect either the intrinsic or the GAP-enhanced GTPase activity of Gαo. Single-turnover GTPase assays were performed exactly as previously described (Garcia-Marcos et al., 2010). Wt Gαo (blue circles) and Gαo R243H mutant (red triangles) were preloaded with 4 μM [γ-32P]GTP (~100 cpm/fmol) in the absence of Mg2+ to prevent GTP hydrolysis (Berman et al., 1996). The tubes were cooled on ice and the reaction started by diluting (10x) the GTP-loaded G proteins (50 nM final concentration) into buffer containing ~2 mM free Mg2+ and excess cold GTP in the presence (open symbols, dotted lines) or absence (closed symbols, solid lines) of GST-GAIP (1 μM). Aliquots were removed at the indicated time points, and reactions were stopped with ice-cold 5% (w/v) activated charcoal in 20 mM H3PO4, pH 3. The amount of [32P]Pi released was quantified by liquid scintillation counting after centrifugation. One representative experiment of 3 is shown. The rates of GTP hydrolysis by wt Gαo (blue closed circles) and Gαo R243H (red closed triangles) are virtually identical. GAIP enhances the rate of GTP hydrolysis by wt Gαo (blue open circles) or Gαo R243H (red open triangles) to a simlar extent.

C. Gαo R234H displays increased steady-state GTPase activity. Steady-state GTPase assays were performed as previously described (Garcia-Marcos et al., 2010; Garcia-Marcos et al., 2009). Wt Gαo (50 nM, blue circles) and Gαo R243H mutant (50 nM, red triangles) were incubated in the presence of 500 nM [γ-32P]GTP (~50 cpm/fmol) at 30°C. Duplicate aliquots were removed at the indicated time points, and reactions were stopped with ice-cold 5% (w/v) activated charcoal in 20 mM H3PO4, pH 3. The amount of [32P]Pi released was quantified by liquid scintillation counting after centrifugation. Results are expressed as mean ± SD of one representative experiment of three. The steady-state GTPase activity of Gαo R243H (red triangles) is increased ~4-fold compared to wt Gαo (blue circles) in the linear region (2–6 min) of the kinetic curve.

D. GTPγS binding to Gαo R234H is accelerated. GTPγS binding assays were performed as previously described (Garcia-Marcos et al., 2010; Sternweis & Robishaw, 1984). Wt Gαo (50 nM, blue circles) and Gαo R243H mutant (50 nM, red triangles) were incubated in the presence of 500 nM [35S]GTPγS (~50 cpm/fmol) at 30°C. Duplicate aliquots were removed at the indicated time points, 2 ml ice-cold wash buffer were added to stop binding of radioactive nucleotide and the quenched reactions were rapidly passed through BA-85 nitrocellulose filters. Filters were dried and counted. Radioactive counts were normalized to maximal binding and fitted to a non-linear, one phase exponential association curve (solid lines) using Prism 4.0 to determine the rate constant. Results are expressed as mean ± SD of one representative experiment of three. The rate of GTPγS binding by Gαo R243H is ~6-fold faster than wt Gαo.

The intrinsic GTPase activity of Gαo R234H is intact and is efficiently enhanced by GAPs

Previously reported oncogenic mutations in G proteins have been shown to be GTPase-deficient and GAP-insensitive (Berman et al., 1996; Iiri et al., 1998; Landis et al., 1989), thereby blocking the G protein deactivation step and rendering it constitutively active. We investigated whether the GTPase activity and GAP sensitivity of Gαo R243H was similarly affected. For this, we performed single-turnover GTPase assays (Berman et al., 1996; Garcia-Marcos et al., 2010) with purified wt Gαo and Gαo R243H alone or in the presence of GAIP/RGS19, a GAP for Gαi/o subunits (Berman et al., 1996; De Vries et al., 1995). The experimental conditions of the single-turnover assay allow direct measurement of the rate of GTP hydrolysis without any interference from the rate of nucleotide binding. We found that Gαo R243H hydrolyzed GTP virtually at the same rate (i.e., same Kcat) as wt Gαo and that GAIP/RGS19 accelerated the GTPase activity of Gαo R243H or wt Gαo equally (Fig 1B). These results demonstrate that the R243H mutation does not affect the basal or GAP-enhanced GTPase activity of Gαo, indicating that the deactivation step in the G protein cycle of Gαo R243H is intact. This property of Gαo R234H differs from the previously reported oncogenic mutations in GNAS, GNAI2 and GNAQ which impair the intrinsic GTPase activity of the G protein (Lyons et al., 1990) and its deactivation by GAPs (Berman et al., 1996).

The nucleotide exchange rate of Gαo R243H is accelerated

Because G proteins are activated by exchanging GDP for GTP we hypothesized that the R243H mutation might render Gαo contitutively active by increasing the rate of nucleotide exchange. To investigate the effect of the R243H mutation on the rate on nucleotide exchange by Gαo we used two well established assays-- steady-state GTPase and GTPγS binding assays. Steady-state GTP hydrolysis by Gα-subunits is a reaction with two major steps -- nucleotide exchange (release of GDP and GTP loading) and GTP hydrolysis to GDP. The nucleotide exchange step is rate limiting for the steady-state GTPase reaction because it is 10 to 100 times slower than the GTP hydrolysis step (Mukhopadhyay & Ross, 2002). For this reason, steady-state GTP hydrolysis normally reflects the rate of nucleotide exchange. We found that the steady-state rate of GTP hydrolysis (Kss) of Gαo R243H was ~4-fold faster than wt Gαo (Fig. 1C). The rate of nucleotide exchange (KGTP) for Gαo R243H was calculated based on the approximation KGTP = 1/Kss − 1/Kcat (Higashijima et al., 1987; Makita et al., 2007) assuming that Kcat remains unchanged (Fig. 1B) and Kss is increased 4-fold (Fig. 1C) compared to the constant values reported for wt Gαo (Linder et al., 1990). This analysis predicts that KGTP for Gαo R243H is ~6-times faster than wt Gαo. To validate this result we performed GTPγS binding experiments, which is a direct measure of nucleotide exchange activity and found that the rate of GTPγS binding to Gαo R243H is ~6 times faster than to wt Gαo (Fig. 1D). This result demonstrates that the R243H mutation accelerates the rate of nucleotide exchange by Gαo. The fractional occupancy of GTP in Gαo R243H was estimated based on the approximation 100(KGTP/(KGTP + Kcat) (Higashijima et al., 1987; Johnston et al., 2007) considering that Kcat remains unchanged (Fig. 1B), and KGTP is increased 6-fold (Fig 1D) compared to wt Gαo. This analysis predicts that >60% of Gαo R243H is loaded with GTP (active) at steady-state whereas the GTP occupancy in wt Gαo under the same conditions is predicted to be only 10%. These results suggest that Gαo R243H would behave as a constitutively active protein in vivo because its accelerated rate of nucleotide exchange along with the high intracellular GTP:GDP ratio would promote spontaneous activation of the G protein without the need for GEFs. Thus, Gαo R243H shares in common with other G protein oncogenic mutants that it is constitutively active but differs in that increased activation arises by accelerated nucleotide exchange rather than decreased inactivation by impaired GTPase activity.

Gαo R243H enhances signaling pathways required for Gαo-mediated cellular transformation

Although no direct effector for Gαo has been characterized to date, previous reports have demonstrated that active Gαo (i.e. constitutively active Q205L mutant) enhances Src-dependent activation of STAT3 to promote neoplastic transformation of NIH3T3 fibroblasts (Ram et al., 2000). Next we investigated whether constitutively active Gαo R243H similarly activates the STAT3/Src signaling pathway because it also induces oncogenic transformation (Kan et al., 2010). NIH3T3 cells were transfected with plasmids encoding for wt Gαo, Gαo R243H or Gαo Q205L (as a positive control) and the levels of STAT3/Src activation were determined by quantifying the extent of phosphorylation at Y705 of STAT3 and Y416 of c-Src. Expression of similar amounts of Gαo R243H or Gαo Q205L enhanced the levels of pSTAT3 and pSrc approximately three-fold whereas expression of wt Gαo had no significant effect and was similar to vector control (Fig. 2A). Phosphorylation of Akt and ERK1/2 was unaffected (Fig. 2A), indicating that the effect of Gαo R243H and Gαo Q205L on signaling was specific for the Src-STAT3 pathway. Identical results were obtained in MDA-MB 231 breast carcinoma cells (data not shown). Thus, Gαo R243H triggers activation of the Src-STAT3 pathway and behaves like constitutively active Gαo in vivo. Because blocking Src-STAT3 activation abolishes neoplastic transformation by constitutively active Gαo Q205L (Ram et al., 2000), we propose that the constitutively active Gαo R243H mutant is likely to promote oncogenic transformation (Kan et al., 2010) similarly by activating the Src-STAT3 pathway. Further investigation is required to unequivocally resolve this point.

Figure 2. Gαo R243H enhances STAT3 and Src activation in NIH3T3 cells.

Figure 2

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Rat Gαo was cloned into pcDNA3.1(+) (Eco RI/Not I), and R243H and Q205L mutants were generated using a Quikchange mutagenesis kit. NIH3T3 cells were transfected with the indicated plasmids using Genejuice as previously described (Ghosh et al., 2010; Ghosh et al., 2008) and maintained in DMEM media supplemented with 10% FBS. Approximately 32 h after transfection the cells were switched to DMEM media supplemented with 2% FBS and cultured overnight. Cells were lysed in sample buffer, boiled and proteins separated by SDS-PAGE. Samples were analyzed by immunobloting (IB) for Y705 phospho-STAT3 (pSTAT3, rabbit pAb, Cell Signaling), total STAT3 (tSTAT3 mouse mAb, Santa Cruz Biotechnology), Y416 phospho-Src (pSrc, rabbit pAb, Cell Signaling), total Src (tSrc, mouse mAb, Santa Cruz Biotechnology), phospho-ERK1/2 (pERK1/2, rabbit pAb, Cell Signaling), S473 phospho-Akt (pAkt, rabbit pAb, Cell Signaling) and Gαo (rabbit pAb, Santa Cruz Biotechnology). When the Gαo R243H (lane 3) and Gαo Q205L (lane 4) mutants were expressed the levels of pSTAT3 and pSrc increased approximately 3 times compared to vector transfected controls (lane 1), whereas wt Gαo (lane 2) had no effect. There was no effect on Akt and ERK1/2 phosphorylation when the different Gαo constructs were expressed.

Structural basis for the constitutive activation promoted by the R243H mutation

Structural and functional analysis of oncogenic mutants have unraveled the similarities and differences between heterotrimeric G proteins and the Ras superfamily of G proteins (Sprang, 1997). The Q227L mutation in GNAS (or the equivalent mutation in other heterotrimeric G proteins) is fully analogous to the oncogenic mutation Q61L found in Ras (Coleman et al., 1994; Kleuss et al., 1994) whereas the R201C mutation in GNAS (or the equivalent in other Gα subunits) is unique to heterotrimeric G proteins because this residue is not shared with G proteins of the Ras superfamily (Coleman et al., 1994). It has been proposed (Bourne, 1997) that this residue works as an “arginine finger”, i.e., interacts with GTP to facilitate nucleotide hydrolysis, because it structural and functionally resembles the “arginine finger” found in GAPs for G proteins of the Ras superfamily. We propose that the heterotrimeric G protein residue corresponding to R243 in Gαo behaves as yet another “arginine finger” whose primary function is to stabilize nucleotide binding. This R243 “finger” points at E43, which is located in the highly conserved P-loop responsible for binding of the α and β phosphates of the nucleotide (Fig. 3A). Based on our finding that the R243H mutation facilitates nucleotide exchange without affecting GTP hydrolysis, we propose that this mutation disrupts the electrostatic contact of R243 with E43 by loss of its negative charge when mutated to H and that by doing so destabilizes the interaction of the P-loop with the nucleotide which is then released more easily. Importantly, R243 and E43 are highly conserved across Gα-subunits but not among members of the Ras superfamily (Fig. 3B, left panel), and the electrostatic contact between them is observed in the crystal structures of all Gα-subunits resolved to date (Fig. 3B, right panel). Thus, much like the previously described “arginine finger” in Gα-subunits (Bourne, 1997), the R234 “arginine finger” seems to be a unique and conserved feature of heterotrimeric G proteins that is not shared with members of the Ras superfamily.

Figure 3. Structural basis for Gαo R243H properties.

Figure 3

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A. View of the nucleotide binding site of Gαo. The coordinates of Gαo were extracted from the Protein Data Bank ID number 3CK7 and visualized by Molsoft ICM Browser. Only key residues and their corresponding segments of the backbone chain are depicted for clarity. R179 and Q205 are positioned toward AlF4− (small green spheres), which mimics the γ phosphate of GTP in the transition state, in an orientation required to catalyze the GTP to GDP + Pi reaction. R243 points toward E43, which is located in the P-loop (yellow) known to make contact with α and β phosphates of GDP. The negatively charged guanidino group of R243 interacts with the negatively charged carboxyl group of E43 located in close proximity (~4 A). Disruption of this electrostatic interaction by loss of the negative charge in the R243H mutant is predicted to affect the interaction between the P-loop and the nucleotide, thereby affecting the rate of nucleotide exchange. The large blue sphere represents Mg2+ and the red ribbon corresponds to the α3 helix.

B. The electrostatic contact between R243 and E43 is conserved across Gα-subunits but not among members of the Ras superfamily. Left, sequence alignment of Gα-subunits and Ras-related G proteins. Sequences of the indicated proteins were obtained form the NCBI database and aligned using CLUSTAL W. Conserved identical residues are shaded in black; similar residues in grey. Structural elements (P-loop, SwIII = switch III, α3 = α-helix 3) indicated above the alignment are named according to the Gαo crystal structure. The positions corresponding to E43 (in red) and R243 (in green) of Gαo are highly conserved across Gα-subunits (with the exception of Gαz, which has biochemical properties different from other Gα-subunits (Casey et al., 1990)) but are not present in Ras and other members of the Ras superfamily. Right, structural view of the electrostatic contact between E43 and R243 in different Gα-subunits. The coordinates of different Gα-subunits crystallized to date were extracted from the following ID numbers of the Protein Data Bank: Gαo, 3CK7; Gαi1, 1GDD; Gαt, 1TAG; Gαs, 1AZT; Gα13, 2BCJ; Gαq, 1SHZ. These structures were overlapped and displayed using Molsoft ICM Browser following the color code indicated in the black box on the bottom right corner. Only key residues with their corresponding segments of the backbone chain are depicted for clarity. The residues corresponding to R243 and E43 in Gαo are positioned in close proximity in all the other Gα-subunit crystal structures, indicating that this electrostatic interaction is highly conserved in heterotrimeric G proteins.

This conserved “arginine finger” has also been investigated in Gα subunits other than Gαo. For example, its mutation to Q in the Gαq homolog of C. elegans is known to promote a gain-of-function phenotype (Doi & Iwasaki, 2002). Subsequent studies have revealed that mutation of the corresponding arginine to E in two different Gαt/i chimeras or Gαs gives rise to G proteins with different biochemical properties (GTPase deficient (Barren et al., 2006), incompetence for nucleotide binding (Pereira & Cerione, 2005), or facilitated activation by GTPγS (Zurita & Birnbaumer, 2008)) which was attributed to subtle differences in the molecular environment of the arginine in the different G proteins (Zurita & Birnbaumer, 2008). It is therefore not surprising that substitution of that same critical arginine by a different amino acid (R→H), in a different G protein (Gαo) in this current study reveals a completely different set of biochemical properties compared to the above mentioned studies, i.e., unaltered GTP hydrolysis and enhanced nucleotide exchange. Although the net result of mutating this conserved “arginine finger” varies the findings highlight its importance in controlling the activation-deactivation kinetics of Gα-subunits.

Conclusions

Taken together our results provide the molecular basis for the oncogenic properties of Gαo R243H. Gαo R234H behaves like a constitutively active protein in vitro and in vivo because of the accelerated rate of nucleotide exchange and enhances cellular signaling responsible for oncogenic transformation. This work reveals a new mechanism by which heterotrimeric G proteins can be turned into oncoproteins by somatic mutations.

Acknowledgments

This work was funded by NIH grants CA100768 and DKI7780 (to M.G.F.). M.G.-M. was supported by a Susan G. Komen postdoctoral fellowship KG080079 and P.G. by Burroughs Wellcome Fund and Research ScholarAward (American Gastroenterology Association FDN).

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