Survival or death of neurons during development is mediated by the integration of a diverse array of signal transduction cascades that are controlled by the availability and acquisition of neurotrophic factors and agonists acting at G protein-coupled receptors (GPCRs). The possible involvement of G_i/o proteins in nerve growth factor (NGF)-induced pro-survival Akt signaling in rat pheochromocytoma PC12 was investigated in the present study. Treatment of PC12 cells with NGF increased the Akt phosphorylation level in a time- and dose-dependent manner. The NGF-dependent Akt activation was partially blocked by treatment of PTX or overexpression of regulators of G protein signaling (RGSZI and GAIP), suggesting participation of G_i/o proteins. Additional experimental results showed that NGF cou...[ Read more ]

Survival or death of neurons during development is mediated by the integration of a diverse array of signal transduction cascades that are controlled by the availability and acquisition of neurotrophic factors and agonists acting at G protein-coupled receptors (GPCRs). The possible involvement of G_i/o proteins in nerve growth factor (NGF)-induced pro-survival Akt signaling in rat pheochromocytoma PC12 was investigated in the present study. Treatment of PC12 cells with NGF increased the Akt phosphorylation level in a time- and dose-dependent manner. The NGF-dependent Akt activation was partially blocked by treatment of PTX or overexpression of regulators of G protein signaling (RGSZI and GAIP), suggesting participation of G_i/o proteins. Additional experimental results showed that NGF could utilize G_i1, G_i3, G_oA, and G_oB proteins, but not G_i2, to stimulate Akt. The use of transducin as a Gβγ scavenger further revealed that Gβγ subunits rather than Gα_i/o acted as the signal transducer. The activation profiles of Akt-regulated downstream effectors, like nuclear factor κB (NFκB), Bad, and tuberin were also obtained. NGF-stimulated transcriptional activity of NFκB, inhibition of Bad, and phosphorylation/degradation of tuberin were sensitive to treatments with PTX and a phosphatidylinositol-3-kinase (PI3K) inhibitor, suggesting that NGF may utilize G_i/o pathways to stimulate PI3K/Akt in order to regulate these effectors. The results also support the scenario that tuberin functions as an inhibitory factor on the pro-survival machinery. Removal of tuberin may constitute a mechanism by which trophic factors can promote neuronal cell survival. However, epidermal growth factor (EGF)-induced Akt activation and tuberin phosphorylation were insensitive to PTX treatment, indicating that not every receptor tyrosine kinase is able to utilize G_i/o proteins to carry out its biological effects.

To further confirm and demonstrate the involvement of G_i/o signaling in Akt/tuberin regulation, cells were treated with various specific agonists to the G_i-coupled receptors. The α₂-adrenoceptor, M₄ muscarinic acetylcholine receptor (mAChR), and all three opioid receptor subtypes (δ, κ, μ) are capable of inducing the phosphorylations of Akt and tuberin rapidly in transfected or native cellular models. Furthermore, co-stimulation of M₄ mAChR and NGF receptor resulted in augmentation of Akt activity and enhancement of cell survival. Finally, after understanding the role of G_i/o proteins, the regulatory effects of various members of the G_s, G_q/11, and G_12/13 subfamilies on Akt and tuberin in cultured mammalian cells were examined. In HEK 293 cells transiently expressing constitutively active mutants of Gα₁₁, Gα₁₄, Gα₁₆, Gα₁₂, or Gα₁₃ (Gα₁₁QL, Gα₁₄QL, Gα₁₆QL, Gα₁₂QL, and Gα₁₃QL, respectively), basal and EGF-induced phosphorylation levels of Akt and tuberin were attenuated. In contrast, Akt and tuberin phosphorylations were unaffected by the overexpression of a constitutively active Gα_s mutant (Gα_sQL). The inhibitory mechanism of G_q family members was independent of phospholipase Cβ activation and calcium signaling, while the active Gα₁₂ and Gα₁₃ inhibit Akt signaling via RhoA signaling.

Collectively, this is the first study that demonstrates the involvement of G_i/o proteins in NGF-induced Akt signaling and reveals the functional role of tuberin in neuronal survival. It shows the regulatory effects of NGF, EGF and various G protein signaling pathways on Akt/tuberin and illustrates the synergistic effect between RTK and GPCR on Akt activity.