Requirement Of Gab1-G?i1/3 For GDNF-induced Signaling

Purpose.To understand the potential role and underlying mechanisms of G?i 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;? G?i1 or G?i3 was exogenously expressed in Gail/3 DKO MEFs;? WT-G?i3 was exogenouly expressed in WT MEFs;? Dominant negative G?i1/3 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 G?i 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 G?i1/3 DKO MEFs;? In MEFs,G?i1 or G?i3 single knockout inhibits GDNF-induced downstream signaling activation;? In MEFs,combined-knockdown of G?i1 and G?i3 by targeted shRNAs largely inhibited GDNF-induced downstream signaling activation;? Exogenous expression of G?i1 or G?i3 restores GDNF-induced downstream signaling activation in MEFs;? Forced-overexpression of G?i3 facilitated GDNF-induced downstream signaling activation in MEFs;? Forced-overexpression of dominant negative G?i1/3 inhibited GDNF-induced signaling transduction;? Following GDNF stimulation,G?i1/3 physically associated with GFR?;? Gab1 KO almost completely blocked GDNF-induced downstream signaling activation.Conclusion.G?i-Gab1 signaling complex,as the key adaptor protein for GDNF-GFR,mediates downstream signaling transduction.