The Mechanism And Function Of GABA_B Receptor Mediated Signaling Via Different Subunits Of Gi/O Protein

GABAB receptor is the metabotropic recetor of y-aminobutyric acid (GABA), which is the main inhibitory neurotransmitter in the central nevous system (CNS). It belongs to class C G protein coupled receptors(GPCR). GABAB receptors have been shown to play a key role in regulating synaptic transmission and membrane excitability in the brain. The dysregulation of GABAB receptor may lead to multiple diseases, such as epilepsy, depression, spastic, anxiety, drug addiction, and cognitive disorder.Recent studies indicate that GABAB receptors also play an important role in fragile X syndrome. Agonists of GABAB receptor show a significant effect both in animal models and clinical trials. However, most of the previous studies have been focused on abnormal behaviors in FMRP knockout mouse model, and fewer studies have been reported for molecular mechanism signaling pathways between GABAB receptors and FMRP. In the first part, we demonstrate that activation of GABAB receptor up-regulates FMRP expression in cultured cerebellar granule neurons(CGNs) through the CREB phosphorylation. In a series of studies to determine the mechanisms, we found that GPy subunit of Gi/o, PLCβ, Ca2+and FAK were involved in GABAB receptor mediated ERK1/2, Akt and CREB phosphorylation. By using specific IGF-1R inhibitor AG1024or siRNA, GABAB receptor mediated ERK1/2, Akt and CREB phosphorylation were significantly blocked. Interestingly, by using different PKC inhibitor or siRNA, we found that CREB phosphorylation was also blocked while they have no effects on IGF-1R, Akt and ERK1/2phosphorylation. These results suggest both PKC dependent pathway and IGF-1R tranactivation pathway were involved in GABAB receptor mediated CREB activation. Indeed, both PKC pathway and IGF-1R transactivation pathway depend on FAK activation. In addition, GABAB receptor mediated up-regulation of FMRP expression was blocked by inhibitors of FAK, PKC or IGF-1R. In conclusion, our study has provided the first evidence that GABAB receptor activation can induce CREB phosphorylation which in turn up-regulate FMRP expression through both IGF-1R transactivation pathway and PKC dependent pathway, which may help us to understand the pathogenesis of fragile X syndrome. In order to avoid their overstimulation and to terminate their agonist-induced signaling, most GPCR undergoes desensitization and subsequent internalization upon agonist stimulation. However, the internalization of GABAB receptor remains controversy. Initial studies found that GABAB receptors are very stable in the cell surface, ligand stimulation can not enhance their internalization. While more and more studies have found that GABAB receptors undergo constitutive internalization and this process is accelerated by the baclofen. However, the mechanism is poorly understood.Our group found that GABAB receptor activation can induce sustained activation of small G protein Rapl through Ga subunit but not Gβγ subunit of Gi/o protein, the active form of Rap1GTP recruits to the membrane and interacts directly with C terminal of GABAB1subunit. In the second part, our further studies showed that GABAB receptor activation promotes the phosphorylation of serine residue in Rap1GAPII. Phosphorylation of Rap1GAPII is associated with inhibition of GAP activity, which results in increasing Rapl activity. By using biotinylation assay, we found that the Peptide derived form GB1C-terminal which is responsible for interaction with Rap1GTP increased the accumulation of internalized GABAB receptor in CGNs. This was confirmed by using immunofluorescence-based assay in HEK293cells transfected with GB1wild-type and GB1ΔS953. To further analysis the fate of internalized GABAB receptor, we next detect the colocalization of internalized GABAB receptor with different endosome markers. Our results shown that disrupt the interaction between GABAB receptor and Rapl decreased the co-localization ration of internalized receptor with early endosomes marker EEA-1, while increased the co-localization ration with lysosome marker LAMP-1. These results demonstrate for the first time that a small GTPase Rap1can accelerate the internalized receptor recycling to the cell surface, which may provide new view to understand the mechanism of internalizaton.