α-Thrombin-mediated Phosphatidylinositol 3-Kinase Activation through Release of Gβγ Dimers from Gαq and Gαi2

Abstract

Chinese hamster embryonic fibroblasts (IIC9 cells) express the G␣ subunits G␣ s , G␣ i2 , G␣ i3 , G␣ o , G␣ q/11 , and G␣ 13 . Consistent with reports in other cell types, ␣-thrombin stimulates a subset of the expressed G proteins in IIC9 cells, namely G i2 , G 13 ␣-Thrombin is a potent mitogen for Chinese hamster embryonic fibroblasts (IIC9) (1). The ␣-thrombin receptor is a member of the 7-membrane spanning family of G-protein-coupled receptors (GPCRs) 1 known as the proteinase-activated receptors (2).Upon activation, GPCRs catalyze the exchange of GTP for GDP on the G␣ subunit of specific G protein trimers, promoting the dissociation of G␣-GTP from the G␤␥ subunits, which remain tightly associated (3). Both the G␣-GTP and the G␤␥ dimers stimulate effectors including phospholipase C (4), Ras (5), and PI 3-kinases (6, 7). Twenty distinct G␣ subunits have been cloned that can be divided into four families based on sequence homology: G s , G i , G q , and G 12 (8). Members of the G i family can be identified by their sensitivity to ADP-ribosylation by pertussis toxin (PTX), which uncouples the G protein from the receptor (9). In addition to the 20 G␣ subunits, 5 G␤ subunits and 12 G␥ subunits have been identified (10). The downstream events associated with a particular GPCR depend on the heterotrimeric G proteins that associate with the receptor. Several G protein-coupled receptors, including ␣-thrombin, are known to activate multiple G proteins and these G proteins can activate different signaling pathways (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). IIC9 cells express G s , G i2 , G i3 , G o , G q/11 , and G 13 (21). Pertussis toxin-sensitive G proteins are involved in ␣-thrombin-induced Ras activation (22), and G q family members mediate the activation of PLC-␤1 (4) and protein kinase C (22).We have previously found that in IIC9 cells ␣-thrombin induces PI 3-kinase and Akt activities that are dependent on 24). At present the G␣ subunits that mediate activation of PI 3-kinase and Akt by ␣-thrombin are unknown. This article identifies the specific G␣ subunits by using transient expression of specific G␣ peptides, which block coupling of GPCRs with selective G proteins, as well as dominant negative xanthine nucleotide-binding G␣ mutants. We show that ␣-thrombin stimulates G q , G 13 , and G i2 in IIC9 cells. Interestingly, PI 3-kinase and Akt stimulation are sensitive to uncoupling of both G q and G i2 . Efforts to delineate the individual role of G q and G i2 in this common pathway show that Ras activation and the translocation of ␤-arrestin1 to membranes are mediated by G␤␥ dimers released from G␣ i2 and G␣ q , respectively. These data indicate that ␣-thrombin induces PI 3-kinase and Akt stimulation via the G␤␥ dimers from G␣ q acting cooperatively with the G␤␥ dimers from G␣ i2 , and that PI 3-kinase activation is a point of convergence in the action of the two G proteins. MATERIALS AND METHODSCell Culture and Reagents-IIC9 cells, a clonal population of Chinese hamster embryo fibroblasts were maintained in Dulbe...

β-Arrestins and Cell Signaling

“…The function of β-arrestin1-mediated Akt activation is still unclear, as its inhibition does not affect cell cycle progression (81)(82)(83).…”

Disruption of Type 5 Adenylyl Cyclase Enhances Desensitization of Cyclic Adenosine Monophosphate Signal and Increases Akt Signal With Chronic Catecholamine Stress

“…8 Long-term isoproterenol increased the activation of Akt, as demonstrated by an increased amount of phospho-Akt (Ser-473) in both WT and AC5KO, presumably through the release of Gs␣ from ␤-AR on activation with isoproterenol. 19 We initially thought that the degree of Akt activation was similar between AC5KO and WT hearts because the amount of released Gs␣ was similar as well; however, the magnitude of Akt activation was much greater in AC5KO ( Figure 5A and 5D). Phosphorylation of GSK3␣/␤ (Ser-21/Ser-9), a target molecule of Akt-mediated phosphorylation, was increased after long-term isoproterenol in both WT and AC5KO ( Figure 5B and 5D).…”

Rig-I−/− mice develop colitis associated with downregulation of Gαi2

“…Heterotrimeric G proteins consist of α, β and γ subunits. Gαi2 is one of the Gα subunits, which has a role in a large variety of cellular and metabolic processes [19][20][21][22][23]. Furthermore, a decreased number of Peyer's patches was also observed in Gαi2 -/-mice [16,17,19].…”