Requirement Of Gab1-Gαi1/3 For GDNF-induced Signaling

Purpose.To understand the potential role and underlying mechanisms of Gαi and Gab1 in GDNF-induced signalingMethods.■ WT,Gai1 and Gai3(Gai1/3)double knockout(DKO)mouse embryonic fibroblasts(MEFs),Gai1 or Gai3 single-knockout(SKO)MEFs,Gai2 KO MEFs were cultured;■ shRNA strategy was applied to selectively knockdown Gai1 and Gai3 in MEFs;■ Gαi1 or Gαi3 was exogenously expressed in Gail/3 DKO MEFs;■ WT-Gαi3 was exogenouly expressed in WT MEFs;■ Dominant negative Gαi1/3 were introduced to WT MEFs;■ Gab1 KO MEFs were also utilized;■ Above cells were treated with various concentrations of GDNF,Western Blotting assay,and co-immunoprecipitation(Co-IP)assay were applied to test GDNF receptor,Gab1,and Gαi expression and association,as well as downstream PI3K-Akt-mTOR、Erk-MAPK signaling activation.Results■ GDNF-induced downstream signaling activation was almost completely blocked in Gαi1/3 DKO MEFs;■ In MEFs,Gαi1 or Gαi3 single knockout inhibits GDNF-induced downstream signaling activation;■ In MEFs,combined-knockdown of Gαi1 and Gαi3 by targeted shRNAs largely inhibited GDNF-induced downstream signaling activation;■ Exogenous expression of Gαi1 or Gαi3 restores GDNF-induced downstream signaling activation in MEFs;■ Forced-overexpression of Gαi3 facilitated GDNF-induced downstream signaling activation in MEFs;■ Forced-overexpression of dominant negative Gαi1/3 inhibited GDNF-induced signaling transduction;■ Following GDNF stimulation,Gαi1/3 physically associated with GFRα;■ Gab1 KO almost completely blocked GDNF-induced downstream signaling activation.Conclusion.Gαi-Gab1 signaling complex,as the key adaptor protein for GDNF-GFR,mediates downstream signaling transduction.