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Activation mechanism of Gi and Go by reactive oxygen species.

Reactive oxygen species are proposed to work as intracellular mediators. One of their target proteins is the alpha subunit of heterotrimeric GTP-binding proteins (Galpha(i) and Galpha(o)), leading to activation. H(2)O(2) is one of the reactive oxygen species and activates purified Galpha(i2). However, the activation requires the presence of Fe(2+), suggesting that H(2)O(2) is converted to more reactive species such as c*OH. The analysis with mass spectrometry shows that seven cysteine residues (Cys(66), Cys(112), Cys(140), Cys(255), Cys(287), Cys(326), and Cys(352)) of Galpha(i2) are modified by the treatment with *OH. Among these cysteine residues, Cys(66), Cys(112), Cys(140), Cys(255), and Cys(352) are not involved in *OH-induced activation of Galpha(i2). Although the modification of Cys(287) but not Cys(326) is required for subunit dissociation, the modification of both Cys(287) and Cys(326) is necessary for the activation of Galpha(i2) as determined by pertussis toxin-catalyzed ADP-ribosylation, conformation-dependent change of trypsin digestion pattern or guanosine 5'-3-O-(thio)triphosphate binding. Wild type Galpha(i2) but not Cys(287)- or Cys(326)-substituted mutants are activated by UV light, singlet oxygen, superoxide anion, and nitric oxide, indicating that these oxidative stresses activate Galpha(i2) by the mechanism similar to *OH-induced activation. Because Cys(287) exists only in G(i) family, this study explains the selective activation of G(i)/G(o) by oxidative stresses.

Reactive oxygen species (ROS) 1 have been shown to work as mediators of intracellular signal transduction (1, 2). ROS consist of several species such as H 2 (3,4). The increase in H 2 O 2 by platelet-derived growth factor receptor stimulation requires the activation of phosphatidylinositol 3-kinase (PI3K) and is possibly mediated by PI3K/Rac/NADPH oxidase pathway (3). The increase in H 2 O 2 was also observed by stimulation of G protein-coupled receptors such as angiotensin II (5), lysophosphatidic acid (6), and thrombin receptors (7). The generated H 2 O 2 is found to inactivate protein-tyrosine phosphatase 1B (PTP-1B) by modifying the cysteine residue located at catalytic moiety (8). However, the analysis of PTP-1B from H 2 O 2treated cells revealed that the cysteine residue of PTP-1B is not sulfenic acid-but glutathione-conjugated cysteine (9). Therefore, the cysteine residue of PTP-1B is at first modified by H 2 O 2 and then reacts with glutathione. This modification of cysteine leads to the inactivation of phosphatase activity of PTP-1B. Thus, the increase in H 2 O 2 indirectly changes the phosphorylation-dephosphorylation state on the tyrosine residue of proteins implicated in signal transduction (10). ROS can also be generated upon pathophysiological conditions. For instance, ROS are generated in a large amount on ischemia/reperfusion and can influence many intracellular signaling processes (11). Because the inclusion of superoxide dismutase mimics or antioxidant attenuates the cellular injury caused by ischemia/reperfusion, the prevention and mechanism of ROS generation is critical for the understanding of ischemia/reperfusion injury. The treatment with H 2 O 2 is frequently used for mimicking oxidative stress such as ischemia/ reperfusion, since H 2 O 2 freely enters cells like H 2 O (1, 2). The treatment with H 2 O 2 activates mitogen-activated protein kinase (MAPKs) such as ERK, c-Jun NH 2 -terminal kinase, and p38 MAPK and promotes or inhibits cellular injury (12).
We have previously reported that the treatment of rat neonatal myocytes with H 2 O 2 activates G i and G o in a receptorindependent manner, leading to the increased activity of ERK (13). However, the molecular mechanism of G i and G o activation by H 2 O 2 is still not clear, especially about the modified amino acids. We demonstrate in the present study that H 2 O 2 is converted to more reactive species in the presence of Fe 2ϩ and modifies specific cysteine residues that exist only in G␣ i and G␣ o . The modification of two cysteine residues results in subunit dissociation of G i and increase in GTP␥S binding. PMSF were purchased from Sigma, and a singlet oxygen generator * This research was supported in part by a grant from the Ministry of Education, Science, Sports, and Culture of Japan (to T. Nagao and H. Kurose). M. Nishida is a recipient of a grant from the Japan Society for the Promotion of Science for Young Scientists. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Materials
(EP-1) and a NO generator (NOC13) were synthesized as described (14,15). Anti-phospho-specific ERK or Akt antibodies were from Promega or New England Biolabs, respectively. pTrcHisB was from Invitrogen. Pfu DNA polymerase was from Stratagene. Nitrocellulose filters (0.45 m) were from Millipore. PTX was from Invitrogen.
Purification of G␣ i2 -G␣ i2 was purified by a similar method to Lee et al. (16). Six histidine residues were added to the amino terminus of rat G␣ i2 by PCR and inserted into NheI and HindIII sites of pTrcHisB. The resulting G␣ i2 was expressed in BL21(DE3)pLys by induction of 30 M isopropylthio-␤-D-galactoside for 16ϳ18 h at 30°C. The Escherchia coli extract was prepared with freeze-thawing in 50 mM Tris, pH 8.0, 20 mM ␤-mercaptoethanol, 0.1 mM PMSF. After centrifuging at 18,000 rpm for 30 min at 4°C, the supernatant was applied to a nickel nitrilotriacetic acid-agarose resin. The resin was sequentially washed with 50 mM Tris, pH 8.0, 20 mM ␤-mercaptoethanol, 0.1 mM PMSF, 100 mM NaCl and 50 mM Tris, pH 8.0, 20 mM ␤-mercaptoethanol, 0.1 mM PMSF, 500 mM NaCl, 10 mM imidazole. The G␣ i2 was eluted with a linear gradient from 10 to 150 mM imidazole in 50 mM Tris, pH 8.0, 20 mM ␤-mercaptoethanol, 0.1 mM PMSF, 100 mM NaCl, 10% glycerol. The fractions containing GTP␥S binding activity were pooled, diluted, and applied to Mono Q column. After washing with 50 mM Tris, pH 8.0, 1 mM EDTA, 10% glycerol, G␣ i2 was eluted with a linear gradient of 100ϳ300 mM NaCl. Although purified G␣ i2 showed a single band on Coomassie staining, the final purity of G␣ i2 was 20ϳ30% based on the comparison of protein content with [ 35 S]GTP␥S binding activity. This purity was almost the same as the value reported by Lee et al. (16). Substitution of cysteine with serine residues was performed with QuickChange site-directed mutagenesis method with Pfu DNA polymerase. The resultant G␣ i2 mutants (KpnI site at the 5Ј-end and HindIII site at the 3Ј-end) were inserted into pTZ-18R. In vitro transcription/translation was performed by the method of Shiina et al. (17) using the TNT quick-coupled transcription/translation system. TPCK-treated trypsin (50 g) was added to each sample (2.5 l of sample) in a final volume of 30 l and incubated for 10 min at room temperature (13). After that, the reaction was stopped by the addition of SDS sample buffer.
HPLC and Tandem Mass Spectrometric Sequencing of Limited Tryptic Digests of ⅐OH-treated G␣ i2 -The purified G␣ i2 was treated by 100 M H 2 O 2 in the presence of equimolar amount of FeSO 4 for 10 min at 30°C. The treated G␣ i2 was precipitated by the addition of acetone and dried. The G␣ i2 was digested by TPCK-treated trypsin using the method of McIntire et al. (18). The separation and analysis of the digested G␣ i2 were performed according to the method of McIntire et al. (18). Both matrix-assisted laser desorption ionization-mass spectrometry (Applied Biosystems Voyager-DE) and high performance liquid chromatographyelectrospray ionization tandem mass spectrometry (Finnigan LCQ) were performed on tryptic digests of unmodified and ⅐OH-treated G␣ i2 . G␣ i2 (50 g) was treated with trypsin (1:50 w/w) in ammonium bicarbonate, pH 7.5, overnight at 37°C. Peptides were separated over a Zorbax C 18 column (1 ϫ 150 mm) with a linear gradient from 5% A (H 2 O, 0.05% trifluoroacetic acid) to 70% B (acetonitrile, 0.05% trifluoroacetic acid) over 90 min at a flow of 25 l/min. One set of samples contained 2-mercaptoethanol (M r 78.1) during H 2 O 2 treatment, and a second set of samples did not but were quenched with N-acetylcysteine (M r 163.2).
[ 35 S]GTP␥S Binding Assay-G␣ i2 was first treated by one of ROS, and the reaction was quenched by the addition of 10 mM N-acetylcysteine. UV light was irradiated 1 cm apart from the sample with a handheld UV lamp (wavelength of 365 nm) for 10 min on ice. Then, [ 35 S]GTP␥S binding to G␣ i2 was performed in buffer containing 25 mM Hepes, pH 7.5, 100 mM NaCl, 5 mM MgSO 4 , 1 mM EDTA, 10 nM [ 35 S]GTP␥S, 1 M GDP. The reaction was stopped by dilution with 25 mM Tris, pH 8.0, 100 mM NaCl, 25 mM MgCl 2 , collected on nitrocellulose filter, and the filters were washed three times with same solution. G␣ i2 that bound [ 35 S]GTP␥S was determined with a liquid scintillation counter.
Trypsin Digestion Analysis-The activation of G␣ i2 was assessed by the conformation-dependent difference of digestion pattern of G␣ i2 by TPCK-treated trypsin (13). [ 35 S]Methionine-labeled in vitro translated WT-or Ser-substituted G␣ i2 were treated with H 2 O 2 in the presence or absence of FeSO 4 and quenched by the addition of N-acetylcysteine. H 2 O 2 -treated G␣ i2 was digested for 10 min at room temperature with 50 g/ml TPCK-treated trypsin. Then the radioactive G␣ i2 was resolved by 12% SDS-PAGE and identified by an Image analyzer Fuji-BAS 2000.
PTX-catalyzed ADP-ribosylation-PTX (100 g/ml) was preactivated by incubation with 1 mM ATP, 1 mM dithiothreitol for 30 min at 30°C. The purified G␣ i2 (50ϳ100 ng) was combined with 300 ng of G␤␥ purified from bovine brain and incubated for 10 min at 30°C with 7 g/ml PTX and 0.3 M [ 32 P]NAD in the same solution as GTP␥S binding reaction. The reaction was stopped by the addition of 4ϫ SDS sample loading buffer and subjected to 12% SDS-PAGE.
Activation of ERK and Akt-Neonatal myocytes were prepared from 0 -1-day-old Sprague-Dawley rats according to the method described by Nishida et al. (13). Neonatal myocytes were plated at 1 ϫ 10 6 cells per each of 6 wells, treated with deferoxamine at the concentration and length indicated in Fig. 7, and then treated with H 2 O 2 . Activation of ERK and Akt was determined by Western blot with anti-phosphospecific ERK or Akt antibodies. The experiments were repeated at least three times and are shown as the mean Ϯ S.E. Statistical analysis was performed with t-test.

RESULTS
Because H 2 O 2 is easily converted to a more reactive species, ⅐OH, by the Fenton reaction in the presence of Fe 2ϩ , we first examined whether H 2 O 2 directly or indirectly acts on G␣ i . The addition of a Fe 2ϩ chelator (diethylenetriaminepentaacetic acid, 1 M) completely inhibited the activation of recombinant G␣ i2 by H 2 O 2 (Fig. 1A). In contrast, treatment with ⅐OH generated from H 2 O 2 in the presence of Fe 2ϩ increased [ 35 S]GTP␥S binding to G␣ i2 in a concentration-dependent manner. This result indicates that H 2 O 2 is converted to ⅐OH before it reacts with G␣ i2 . The enhanced GTP␥S binding to G␣ i by H 2 O 2 in the previous report reflected contaminating Fe 2ϩ in solution that helped generate ⅐OH from H 2 O 2 . Therefore, all buffers used in the subsequent experiments were deionized by passing through a Chelex column. The half-life of ⅐OH is very short (s to ms). Therefore, ⅐OH may further convert to other radical species by reacting with H 2 O or some components in the solution. However, ⅐OH is used in the present report for simplicity. Many reports show that modification of proteins by ROS occurs largely on Cys and Met (19,20). Fig. 1B shows that the activation of G␣ i2 with ⅐OH (1 M) was almost completely reversed by treatment with dithiothreitol. This result suggests that the modification of G␣ i2 by ⅐OH occurs on Cys. Furthermore, pretreatment for 20 min with dimethylthiourea (100 M), an inhibitor of cysteine oxidation, completely inhibited the activation of G␣ i2 by ⅐OH (data not shown). These results indicate that ⅐OH activates G␣ i2 by modifying cysteine residues. When physiological sulfhydryl compound glutathione was used for quenching the reaction, glutathione also supported the ⅐OHinduced activation of G␣ i2 (Fig. 1C). This action of glutathione was specific for the ⅐OH-induced activation, since the activation of G␣ i2 by mastoparan was not affected by the inclusion of glutathione. The extent of activation by ⅐OH in the presence of glutathione is essentially the same as that by ⅐OH in the presence of N-acetylcysteine.
To examine which cysteine residues were modified by ROS, we analyzed ⅐OH-treated G␣ i2 with mass spectrometry (Fig. 2). Mass spectrometric analysis showed that 7 cysteine residues were modified, Cys 66 , Cys 112 , Cys 140 , Cys 255 , Cys 287 , Cys 326 , and Cys 352 , as summarized in Table I and a supplemental  table. The reaction was quenched by the addition of N-acetylcysteine (Table I) or 2-mercaptoethanol (supplemental table), and the molecular weights of these sulfhydryl compounds are 163.2 or 78.1, respectively. Therefore, the molecular weight of ⅐OH-treated and trypsin-digested G␣ i2 was increased by 161.1 or 76.1 when the fragment contained the modified cysteine residue (Table I and the supplemental table). Mass spectrometric analysis of tryptic products yielded 71% coverage of the G␣ i2 sequence, all of which was confirmed by tandem mass spectrometric sequencing. The only peptides not observed were the large amino-terminal tryptic peptide, a large central peptide (residues 212-243), and several very small tryptic peptides. No other significant modifications were observed with the exception of oxidation of some methionine residues to methionine sulfoxide (supplemental table). The extent of methionine oxidation was very low (3-13%) during 300 M H 2 O 2 treatment in the presence of 2-mercaptoethanol and slightly higher (up to 50%) during 1 mM H 2 O 2 treatment in the presence of Nacetylcysteine. It should be noted that the extent of cysteine modification after these treatments was nearly stoichiometric (Table I and the supplemental table). The extents of modification were calculated from areas under the selected  ion plots of modified and unmodified peptides. The alignment of G␣ proteins shows that Cys 255 , Cys 287 , and Cys 352 are conserved in the ␣ i family and that Cys 140 and Cys 326 are found in ␣ i , ␣ q , and ␣ s (Fig. 3). In contrast to these cysteine residues, Cys 66 and Cys 112 exist only in ␣ i .
To determine which cysteine is responsible for the activation of G␣ i2 , each Cys was individually mutated to Ser. Cysteine at position 352 was excluded from mutational analysis, since H 2 O 2 still activated G␣ i2 subjected to PTX-catalyzed ADP-ribosylation (13). Using in vitro translated WT and mutated forms of G␣ i2 , the effect of the substitution on ⅐OHinduced activation was assessed by a change in the conformation-dependent trypsin digestion pattern. Mutants of Cys 287 or Cys 326 , but not mutants of other cysteine residues, showed resistance to ⅐OH-induced activation (Fig. 4, A and  B). This result demonstrates that Cys 287 and Cys 326 are possible candidates for ⅐OH-induced modification and consequent activation of G␣ i2 . To correlate the modification of Cys 287 and Cys 326 with the functional activation of G␣ i2 , we expressed and purified Cys to Ser mutants of G␣ i2 (C287Sand C326S-G␣ i2 ). The C255S-G␣ i2 mutant was also purified to demonstrate the functional integrity of the mutant after the substitution of Cys to Ser. Cys 255 was chosen by the fact that Cys 255 was also modified and located at the closest position of two cysteine residues. Table II shows that K d values of these mutants were essentially the same as the value of recombinant WT-G␣ i2 . These K d values were nearly the same as the K d value of G␣ i purified from rabbit liver membranes (21). Therefore, cysteine modification does not induce conformational changes in the guanine nucleotide binding pocket of G␣ i2 . The basal GTP␥S binding activities of these mutants were similar to that of WT-G␣ i2 (about 0.3-0.4 mol/mol of G␣ i2 ). As expected, exposure of ⅐OH increased the binding of [ 35 S]GTP␥S to WT-G␣ i2 and C255S-G␣ i2 mutants but did not increase the binding of [ 35 S]GTP␥S to C287S-and C326S-G␣ i2 mutants (Fig. 5). This result clearly shows that the target amino acids of ⅐OH are cysteine residues at position 287 and 326, and the modification of these cysteine residues activates G␣ i2 .
Our previous report indicated that the dissociation of G␤␥ from G i and G o by ROS is critical for generation of downstream signaling (13). We next examined which cysteine is involved in the dissociation of G i and G o into their respective subunits. Treatment of heterotrimeric G o with ⅐OH decreased PTX-catalyzed ADP-ribosylation in a dose-dependent manner, indicating the ⅐OH-induced subunit dissociation of G o (13). The subunit dissociation induced by ⅐OH was also observed in recombinant WT-G␣ i2 in the presence of G␤␥ (Fig. 6). However, the ⅐OH-induced subunit dissociation was almost completely inhibited in C287S-G␣ i2 but not C326S-G␣ i2 mutants. These results suggest that oxidation of Cys 287 by ⅐OH is only required for the subunit dissociation of G i , and oxidation of Cys 326 does not contribute to the FIG. 3. Alignment of sequences of various G␣ subunits. Cysteine residues of ␣ i , indicated by the box, were modified by ⅐OH treatment. The sequences of ␣ i2 , ␣ oA , and ␣ s were derived from rat (35), and the sequences of ␣ q and ␣ 12 were from mouse (36,37). subunit dissociation. However, both modifications of Cys 287 and Cys 326 are necessary for the ⅐OH-induced activation of G␣ i2 . We previously reported that exposure of H 2 O 2 induces liberation of G␤␥ from G i , leading to the activation of ERK and Akt in rat neonatal cardiomyocytes (13). However, we have demonstrated in the present report that the species acting on G␣ i was ⅐OH but not H 2 O 2 itself. This suggests that intracellular Fe 2ϩ is involved in conversion of H 2 O 2 to ⅐OH for the generation of downstream signaling. Therefore, we pretreated rat neonatal myocytes with a cell-permeable Fe 2ϩ chelator deferoxamine and determined the effect on ERK and Akt activation. The treatment with deferoxamine significantly decreased H 2 O 2 (1 mM)-induced activation of ERK and Akt in a concentration-dependent manner (Fig. 7). The maximum extent of inhibition is about 40%, consistent with the value reported in the previous study that sequestration of G␤␥ by expressing the carboxylterminal region of G protein-coupled receptor kinase 2 inhibited H 2 O 2 -induced activation of ERK and Akt. These results clearly show that ⅐OH is generated from H 2 O 2 in cells and liberates G␤␥, leading to the activation of ERK and Akt in rat neonatal cardiomyocytes.
We next examined whether other ROS or ultraviolet (UV) irradiation can activate G i and G o . G protein activation was assessed by PTX-catalyzed ADP-ribosylation of G o purified from bovine brain and [ 35 S]GTP␥S binding of recombinant G␣ i2 . The ADP-ribosylation of G o was decreased by the treatment with KO 2 (a generator of superoxide anion, O 2 . ), EP-1 (a generator of singlet oxygen, 1 O 2 , NOC13 (a generator of nitric oxide (NO)), and UV light (wavelength 365 nm) (Fig. 8A). In contrast, treatment with H 2 O 2 (in the absence of Fe 2ϩ ) has no effect on PTX-catalyzed ADP-ribosylation. Higher concentrations of EP-1 (200 M), KO 2 (100 M), or NOC13 (300 M) than H 2 O 2 were required for the activation of G i and G o . GTP␥S binding to WT-G␣ i2 was also increased by these ROS and UV light treatments (Fig. 8B). Because UV light can generate ROS from O 2 dissolved in the solution, these results indicate that free radicals are the likely species involved in the modification of G i and G o , and the requirement of higher concentration to activate G i and G o reflects differential reactivity of various ROS. To examine whether these treatments modify the same cysteine residues as ⅐OH treatment, the activation was determined with G␣ i2 mutants. K d values of GTP␥S binding of these mutants were not different from the value of WT-G␣ i purified   5. Effect of cysteine mutation on ⅐OH-induced activation of G␣ i2 . Shown is GTP␥S binding to WT-G␣ i2 , Cys 255 -substituted (C255S-G␣ i2 ), Cys 287 -substituted (C287S-G␣ i2 ), and Cys 326 -substituted G␣ i2 (Cys326S-G␣ i2 ) by ⅐OH. Each G␣ i2 was treated with 1 M ⅐OH for 10 min at 30°C and quenched by the addition of 10 mM N-acetylcysteine. Then, GTP␥S binding activities were determined as described under "Experimental Procedures." FIG. 6. Effect of cysteine substitution on the decrease of ADPribosylation by ⅐OH. Serine-substituted mutants of G␣ i2 (100 ng) were first treated with ⅐OH (1 M, 10 min, 30°C), and ADP-ribosylation of these mutants was determined in the presence of G␤␥ (300 ng). from liver membranes (Table II and Ref. 21), and GTP␥S binding to these mutants was increased to a similar extent to recombinant G␣ i2 by mastoparan stimulation (data not shown).
Thus, these mutants retain a conformation necessary for GTP␥S binding in the resting and activated forms. The activation of G␣ i2 by these ROS was inhibited in C326S-and C287S-G␣ i2 mutants, indicating that same cysteine residues are modified irrespective of species of generating ROS (Fig. 8B). Therefore, these ROS and UV treatments modify and activate G␣ i2 with a mechanism similar to the activation mechanism by ⅐OH treatment. DISCUSSION The present study demonstrated that ROS activated G i and G o by modification of specific cysteine residues. The activation of G i and G o by ROS consists of two steps. The first step is the modification of Cys 287 , leading to subunit dissociation. The second step is the modification of Cys 326 , resulting in the enhanced GTP␥S binding that reflects the increased rate of GDP dissociation. However, Cys 326 may be modified by ROS before subunit dissociation. Then modification of Cys 287 simultaneously induces dissociation and activation. Cys 287 and Cys 326 are located near the guanine nucleotide binding motif (N 270 KXD 273 ), Cys 287 at the ␣G/␣4 loop and Cys 326 at the ␤6/␣5 loop (22,23). Iiri et al. (24) report that the naturally occurring mutation of Ala 366 , equivalent to Ala 327 of G␣ i2 , to Ser of G␣ s results in the increased dissociation rate of GDP and shows instability against heat (24). Thomas et al. (25) report that the mutation of Cys 325 of rat G␣ o equivalent to Cys 326 of G␣ i2 decreased the affinity for GDP. Marin et al. (26) demonstrate that the mutation of three amino acids located in ␣5 helix of the transducin ␣ subunit (Thr 325 , Val 328 , and Phe 332 ) greatly increased the basal guanine nucleotide exchange rates. Therefore, it is possible that the modification of Cys 326 of G␣ i2 by ⅐OH in the presence of sulfhydryl compounds increases the dissociation rate of GDP. In contrast to the role of Cys 326 , the role of Cys 287 in activation is not apparent, since the ␣G/␣4 loop that contains Cys 287 does not directly interact with G␤␥ and the nucleotide binding pocket (22). The analysis of crystal structure of ⅐OH-treated G␣ i2 may be necessary for determining the detailed structural changes upon modification. However, it is expected that structural alteration of G␣ i2 modified by ROS is subtle. Posner et al. (27) report that substitution of Ala 326 of G␣ i1 with a serine residue results in the mutant that shows the increased GDP dissociation in the absence of structural alteration. The position of Ala 326 of G␣ i1 is next to Cys 325 of G␣ i1 that corresponds to Cys 326 of G␣ i2 . However, it will be interesting to determine the structure of Cys 287 -modified G␣ i2 . It may provide structural information about subunit dissociation of G protein.
Because ROS activates G␣ i2 , it is interesting to compare the mechanism of ROS-mediated G protein activation with the mechanism of receptor-mediated G protein activation. There are at least two hypotheses of G protein activation mechanism (26,28). One is proposed by Bourne and co-workers (28). They proposed that the intracellular loops of the receptor move G␤␥ away from G␣ like a lever, resulting in the relief of the constraints on switch I and switch II. This relief allows G␣ to dissociate GDP and facilitate the binding of GTP. The other hypothesis is proposed by Sakmar and co-workers (26). They demonstrate that several mutations of the ␣5 helix of transducin drastically increase GDP dissociation without altering nucleotide binding properties. ␣5 helix locates between the carboxyl-terminal tail and the ␤6/␣5 loop, and ␤6/␣5 loop directly contacts the nucleotide binding pocket. Therefore, ␣5 helix interacting with the receptor distantly affects the ␤6/␣5 loop to lead enhanced GDP dissociation. Our result showed that subunit dissociation by modification of Cys 287 is not enough for full activation of G protein, and the modification of Cys 326 locating near ␤6/␣5 is also not enough for activation of G protein. The   FIG. 7. Effect of deferoxamine on H 2 O 2 -induced ERK and Akt activation in rat neonatal myocytes. Neonatal myocytes prepared from 0 -1-day-old rats were treated with 30 -300 M deferoxamine (DFX) for 30 min at 37°C, and then cells were stimulated with 1 mM H 2 O 2 for 10 min at 37°C. Cell lysates were prepared, and activation of ERK and Akt was determined by Western blot with anti-phospho-specific ERK (P-ERK) or Akt antibodies (P-Akt). *, p Ͻ0.05 versus H 2 O 2 (1 mM). , and the reaction was stopped by the addition of 10 mM N-acetylcysteine. Then, PTX-catalyzed ADP-ribosylation was determined as described in the legend of Fig. 6. B, effect of substitution of Cys to Ser mutation on GTP␥S binding. After WT-G␣ i2 , C287S-G␣ i2 , or C326S-G␣ i2 was treated with one of NOC13, EP-1, KO 2 , or UV light with the indicated concentration, the reaction was quenched by the addition of 10 mM N-acetylcysteine. Then GTP␥S binding was determined as described under "Experimental Procedures." The results are shown as the percent increase, with basal GTP␥S binding set as 100%. full activation of G protein requires modification of two amino acids (Cys 287 and Cys 326 ) that are involved in subunit dissociation or release of GDP. This result is consistent with the finding that receptor stimulation dissociates G protein into G␣ and ␤␥ subunits and also increases the GDP dissociation by interacting with the ␣5 helix.
It is unknown whether G␣ i2 modified by ROS is functionally active. The sites modified by ROS are assigned to one of effector interaction sites (29,30). Therefore, the modification of G␣ i2 by ROS may impair the functional interaction with effector molecules such as adenylyl cyclase. It also remains to be determined what modification occurs in intact cells. Lee et al. (8) report that the treatment of cells with H 2 O 2 decreased the phosphatase activity of PTP-1B by modifying the cysteine residue located at the catalytic site (8). However, mass spectrometric analysis of PTP-1B prepared from H 2 O 2 -treated cells revealed that cysteine is glutathiolated instead of hydroxylated (9). Therefore, it is possible that cysteine residues in G␣ i and G␣ o are modified by glutathione in a similar manner as PTP-1B. This raises the possibility that G i and G o are reversibly oxidized and reduced in cells like PTP-1B. However, it remains to be determined whether Cys 287 and Cys 326 of G␣ i2 are similarly modified by H 2 O 2 treatment in cells.
Cysteine and methionine residues are reported to be especially sensitive to oxidation by various ROS (19,20). Shakertype K ϩ channels contain the oxidation-sensitive methionine residue that localizes at the amino-terminal domain (31). Oxidation of methionine to methionine sulfoxide by ROS slowed the inactivation kinetics of the channel. This observation was confirmed by the evidence that the expression of methionine sulfoxide reductase abolishes the slow inactivation kinetics of the channel (31). Ryanodine receptor contains free reduced cysteine residues in which NO nitrosylates one of the cysteine residues (32). When these cysteine residues are oxidized, ryanodine receptor is no longer modified by NO and loses the enhanced Ca 2ϩ -releasing activity from the endoplasmic reticulum. Therefore, Ca 2ϩ -releasing activity of ryanodine receptor is reversibly affected by cysteine oxidation and reduction. The present study showed that the cysteine residue of G␣ i2 is only amino acid modified by ROS. Mass spectroscopic analysis did not detect any modification of the methionine residue. Since N-acetylcysteine was added to quench the reaction with ROS, methionine sulfoxide may return to methionine. However, the result of mutational analysis is consistent with the idea that the cysteine residue of G␣ i2 is the target amino acid for ROS. We consistently observed a slightly lower activation by ⅐OH treatment than mastoparan treatment. This may be explained by the fact that ⅐OH treatment induces unstability of G␣ i2 , since the modification by ⅐OH and subsequent binding of sulfhydryl compound increases the GTP␥S binding (i.e. the increased dissociation of pre-bound GDP), and nucleotide-free G␣ i is unstable. It may induce denaturation of G␣ i2 .
López-Barneo et al. (33) mention that ion channels are acutely modified by ROS, and enzymes and transcription factors are modified by chronic exposure to reduced or oxidized conditions. Intracellular signaling proteins such as NADPH oxidase (34), ryanodine receptor (32), and K ϩ channel (31) can work as redox sensors (33). Our results add G i and G o to the growing members of the redox-sensing protein family. These proteins, including G i and G o , may be activated at different lengths and levels of the oxidized state in cells, and cells generate signals dependent on the severity of the intracellular oxidized state. Consequently, cells can adjust to the environmental changes.
The present study reveals a novel mechanism of G i activation by ROS. The activation of G i requires oxidation of two cysteine residues (Cys 287 and Cys 326 ). Each cysteine residue contributes to different processes of G protein activation; oxidation of Cys 287 of G␣ i2 leads to dissociation into G␣ i2 and G␤␥, whereas oxidation of Cys 326 in addition to Cys 287 leads to the activation of G␣ i .