The Role Of The Inhibitory Alpha Subunits 1/3 Of G Protein In Brain-Derived Neurotrophic Factor(BDNF) Signal Transduction Pathway

Objective: Guanine nucleotide–binding proteins(G proteins) involve in many signaling transduction pathways that are conservative and important. G proteins have been reported to exert the function mainly by cooperating with G protein coupled receptors(GPCRs). Our previous study has identified a novel role of the inhibitory alpha subunits 1/3 of heterotrimeric guanine nucleotide–binding proteins(Gαi 1/3 proteins) in the signal cascades of growth factor receptor. It has been demonstrated that Gαi 1/3 played a pivotal role in the activation of AKT-mTORC1 signaling induced by Epidermal Growth Factor Receptor(EGFR). Both Gαi 1 and Gαi 3 associated with the Grb2-associated binding protein 1(Gab1) and EGFR, and were required for the phosphorylation of Gab1. Brain Derived Neurotrophic Factor(BDNF) is an important neurotrophin in nervous system. We aimed to figure out whether Gαi 1/3 involve in the signal transduction by BDNF. The underlying signaling mechanism were also tested.Methods:Wide type(WT) and Gαi1/3 double knockout(DKO) mouse embryonic fibroblasts(MEFs), Gαi1 knockout(Gαi1-KO) MEFs, Gαi3 knockout(Gαi3-KO) MEFs, stable transfected cell line selected from WT MEFs with siRNA targeting Gαi1/3(siRNA MEFs) and Gab1 knockout(Gab1-KO) MEFs were used for our cellular models.WT and DKO MEFs were stimulated by BDNF and tested by Western Blot to obtain the difference of the activation level of MAPK/ERK signaling(Erk) and AKT-mTORC1 signaling(Akt,S6,mTOR,GSK3α/3β). WT, Gαi1-KO, Gαi3-KO and DKO MEFs were stimulated by BDNF and tested by Western Blot to obtain the difference of the activation level of MAPK/ERK signaling(Erk) and AKT-mTORC1 signaling(Akt,S6,mTOR,GSK3α/3β). WT, siRNA and DKO MEFs were stimulated by BDNF and tested by Western Blot to obtain the difference of the activation level of MAPK/ERK signaling(Erk) and AKT-mTORC1 signaling(Akt,S6,mTOR,GSK3α/3β). We selected stable transfected cell line from DKO MEFs introduced with Gαi1 or Gαi3(Rescue-1, Rescue-3 MEFs) and detected the difference of the activation level of MAPK/ERK signaling(Erk) and AKT-mTORC1 signaling(Akt,S6,mTOR,GSK3α/3β) between these cells.We injected lentivirus loaded with siRNA to knockdown Gαi1/3 into hippocampus of ICR mice brain, and took behavioral experiment.Result: We found that BDNF-induced MAPK-Erk(Erk phosphorylation), Akt(Ser473 and Thr308) and mTORC1(S6 and mTOR phosphorylation) activation showed severely defects in Gab1-KO MEFs, as compared with WT MEFs. The results showed Gab1 is a vital part in the pathway of BDNF. Gab1 phosphorylation, MAPK-Erk(Erk phosphorylation), Akt(Ser473 and Thr308) and mTORC1(S6, mTOR and GSK3α/3β phosphorylation) activation-induced by BDNF were almost blocked in Gαi1/3-DKO MEFs. While a minor inhibition in these signalings was found between Gαi1/3-siRNA MEFs, or in Gαi1-KO, Gαi3-KO MEFs. There was no significant difference between Gαi2-KO MEFs and WT MEFs in BDNF signalings. Importantly, Gαi1 or Gαi3 construct restored BDNF signaling in DKO MEFs. Therefore, Gαi1 and Gαi3 are essential to the activation of downstream signalings induced by BDNF.Conclusion: We found that Gαi1/3 played a critical role of signal transduction pathway initiated by BDNF. Our research showed a new mechanism that G protein involving in signal transduction. Gαi 1/3 promise to be a new target of BDNF-associated signaling fields.