Activation mechanism of G_i and G_o by reactive oxygen species

Reactive oxygen species are proposed to work as intracellular mediators. One of their target proteins is the a subunit of heterotrimeric GTP-binding proteins (Gα_i and Gα_o), leading to activation. H₂O₂ is one of the reactive oxygen species and activates purified Gα_i2. However, the activation requires the presence of Fe²⁺, suggesting that H₂O₂ is converted to more reactive species such as ·OH. The analysis with mass spectrometry shows that seven cysteine residues (Cys⁶⁶, Cys¹¹², Cys¹⁴⁰, Cys²⁵⁵, Cys²⁸⁷, Cys³²⁶, and Cys³⁵²) of Gα_i2 are modified by the treatment with ·OH. Among these cysteine residues, Cys⁶⁶, Cys¹¹², Cys¹⁴⁰, Cys²⁵⁵, and Cys³⁵² are not involved in ·OH-induced activation of Gα_i2. Although the modification of Cys²⁸⁷ but not Cys³²⁶ is required for subunit dissociation, the modification of both Cys²⁸⁷ and Cys³²⁶ is necessary for the activation of Gα_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 Gα_i2 but not Cys²⁸⁷- or Cys³²⁶-substituted mutants are activated by UV light, singlet oxygen, superoxide anion, and nitric oxide, indicating that these oxidative stresses activate Gα_i2 by the mechanism similar to ·OH-induced activation. Because Cys²⁸⁷ exists only in G_i family, this study explains the selective activation of G_i/G_o by oxidative stresses.