The Molecular Mechanism Of GABA_ B Receptor-mediated Neuroprotection And The Research Of Dynamic Interaction Between GABA_B Receptor And G Protein

GABA_B receptor is the metabotropic recetor of ?-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the central nevous system (CNS). It is widely expressed presynaptically and postsynaptically. Meanwhile, GABA_B receptor belongs to G protein coupled receptor family (GPCR) group C and couples with Gi/o protein, mediating slow and prelonged synaptic activity. Hypoactivity or hyperactivity of GABA_B receptor would reslut in various nevous system disorders, such as epilepsy, spasticity, anxiety, stress, depression, addiction, pains and memory dysfunction. Therefore, it is an important drug target.The activation of GABA_B receptor is reported to promote neuronal survival under ischemia and metabolic stress. However, the molecular mechanism by which the GABA_B receptor mediates neuroprotection remains to be elucidated. In the first study of our work, we confirmed that the activation of GABA_B receptor protected cerebellar granule cells (CGNs) from potassium-induced apoptosis. Furthermore, we found that the actvation of GABA_B receptor increased PI3K/Akt signal pathway activity and this pathway played an important role in the antiapoptosis function of GABA_B receptor. Interestingly, we found the GABA_B receptor activation increased insulin-like growth factor 1 receptor (IGF-1R) phosphorylation. When we used the inhibitor of IGF-1R to treat the neurons or knockdowned the expression of IGF-1R in both CGNs and transfected MEF cells, the agonist of GABA_B receptor, baclofen-stimulated Akt phosphorylation was reduced significantly, which suggested the activation of GABA_B receptor transactivated IGF-1R and then induced Akt pathway. Meanwhile, using co-immunoprecipitation and ligand competition assay, we showed that there was an interaction between GABA_B receptor and IGF-1R while the transactivation of IGF-1R by GABA_B receptor was ligand-independent. These results showed a new function for GABA_B receptor and further highlighted the importance of functional cross-talk networks between GPCRs and receptor tyrosine kinases (RTKs). Meanwhile, our results revealed GABA_B receptor as a potential drug target for the treatment of neurodegenerative disorders.The new approaches for protein-protein interaction brought challenge to traditional GPCR-G protein collision-interaction model. The research on GPCR group A members found that the receptor and G protein could form stable complex and G protein wouldn't disassociate during the receptor activation. However, there is no result about GPCR group C members. In the secondary part of our work, we used BRET and TR-FRET to study the dynamic change between GABA_B receptor and G protein. We showed that G protein pre-coupled with GABA_B receptor and both the interaction between GABA_B receptor and G protein and G(?) and G(?)(?) were decreased. These results might provide a new insight for drug screening cell model of GABA_B receptor.