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.
Nishida
Motohiro
M
Laboratory of Pharmacology and Toxicology, Physiological Chemistry, and Bioorganic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Schey
Kevin L
KL
Takagahara
Shuichi
S
Kontani
Kenji
K
Katada
Toshiaki
T
Urano
Yasuteru
Y
Nagano
Tetsuo
T
Nagao
Taku
T
Kurose
Hitoshi
H
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D017665:Hydroxyl Radical; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000494:pharmacology*
D017382:Reactive Oxygen Species; D000246:Adenosine Diphosphate Ribose; D017665:Hydroxyl Radical; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020928:Mitogen-Activated Protein Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins