Activation Mechanism of Gi and Go by Reactive Oxygen Species

Abstract

Reactive oxygen species are proposed to work as intracellular mediators. One of their target proteins is the ␣ subunit of heterotrimeric GTP-binding proteins ( (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 2 -treated 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. . generator (KO 2 ) and PMSF were purchased from Sigma, and a singlet oxygen generator EXPERIMENTAL PROCEDURES Materials-[

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal

“…Those proteins are included here as the same critical cysteine(s) can be modified by either ROS or RNS and very likely by HNE as well when the cysteine is in the thiolate form. Among the signaling proteins (and their critical cysteines) that have been well characterized are the PTPs, PTP1B (cys 215 ) (26,27,(34)(35)(36), low molecular weight PTP (cys12,17) (37-39) and Srchomology 2 domain-containing PTP (SHP-2) (40), the small G protein, Ras (cys 118 ) (41)(42)(43)(44), the large G proteins, G i (cys 267 ) and G o (cys 326 ) (45) and the lipid phosphatase, phosphatase and tensin homolog deleted on chromosome 10 (PTEN). In addition, as mentioned above, in its reduced form Trx binds and inhibits ASK1 and possibly other signaling proteins as well (46)(47)(48)(49)(50)(51)(52)(53).…”

Involvement of Both Gq/11 and Gs Proteins in Gonadotropin-releasing Hormone Receptor-mediated Signaling in LβT2 Cells

“…Trypsin Sensitivity Assay for G Protein Activation-The trypsin sensitivity assay was performed as described on membranes prepared from L␤T2 cells (32). The cells were rinsed twice with ice-cold PBS and scraped in ice-cold lysis buffer containing 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 10 g/ml benzamidine, 10 g/ml soybean trypsin inhibitor (type II-S), and 5 g/ml leupeptin.…”

Human Neuroglobin Functions as an Oxidative Stress-responsive Sensor for Neuroprotection

“…G␣ i/o proteins are targets of ROS, which directly activate G␣ i/o without receptor activation by modification of specific cysteine residues that exist only in G␣ i/o but not in other G␣ families, leading to the selective activation of G␣ i/o under conditions of oxidative stress (34,35). ROS, such as hydroxyl radical, were generated in the presence of hydrogen peroxide and Fe 2ϩ .…”

“…Because G␣ i/o proteins have been reported to be targets of reactive oxygen species (ROS) (34,35), we investigated whether ferric HNgb interacts with the G␣ i/o that is modified by ROS, thereby acting as a GDI for modified G␣ i/o . Moreover, we substituted the distal His residue with Val to create an HNgb mutant (H64V HNgb) that cannot form a bis-His conformation and investigated the significance of the structural changes of HNgb induced by oxidative stress.…”