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 a subunit of heterotrimeric GTP-binding proteins (Galpha(i) and Galpha(o)), leading to activation. H2O2 is one of the reactive oxygen species and activates purified Galpha(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 (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 Gai2 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 Gai2 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.