Preliminary Study On The Rap1and Ras Signaling Pathways Induced By GABA_B Receptor In Mice Cerebella Granule Neurons

Gamma-aminobutyric acid （GABA） is one of mammals main neurotransmitter in thebrain, regulating many physiological processes. It mediates fast synaptic inhibitionthrough ionotropic GABAAreceptors, as well as slow and prolonged synaptic inhibitionthrough both pre-and post-synaptic metabotropic GABA_Breceptors. GABA_Breceptorsare heterodimeric G-protein coupled receptors that function as heterodimers composed ofGABA_B1and GABA_B2subunits. The GABA_Breceptor is broadly distributed in neuronsand represent promising drug targets for the treatment of epilepsy, pain, drugaddiction,anxiety and depression.In this study, we study the signaling pathway of Rap1activation induced by GABA_Breceptor in cerebellar granule neurons （CGNs）. When we incubated the CGNs withBaclofen which is the agonist of GABA_Breceptor, We found that activation of GABA_Breceptor could induce persistent activation of endogenous Rap1. We also found that theGABA_Breceptor-mediated activation of Rap1is the Ca²⁺-dependent, but the intracellularCa²⁺concentration has not changed. Increased cAMP levels can induce Rap1activation,and this process doesn’t involve PKA activation. But GABA_Breceptor activation does notcause the changes of intracellular cAMP levels in cerebella granule neurons, so cAMP isnot involved GABA_Breceptor-mediated activation of Rap1. In addition, we also foundthat PLCε, which is Rap1GEF, don’t participate in GABA_Breceptor-mediated Rap1activation. In summary, this paper is only a preliminary study on the Rap1activationinduced by the activation of GABA_Breceptor in cerebellar granule neurons. The resultsprovide a certain foundation for the explaination of mechanism of GABA_Breceptormediated Rap1signal transduction pathways，their physiological functions and to explorethe GABA_B receptor targets for the drug screening and development.In order to further study the effects activation of GABA_Breceptor on endogenous Rasprotein, we made an active probe GST-Raf1-RBD, which is a GST fusion protein, by plasmid construction, protein purification, GST-pulldown assay. The protein can be usedas a specific probe to selectively isolate and pull-down RasGTP, which is the active formof Ras, from purified samples or endogenous lysates. Subsequently, the precipitatedRas-GTP is detected by western blot analysis using an Anti-Ras specific polyclonalantibody. The study provide an effective technical method for further study the effects ofactivation of GABA_Breceptor on endogenous Ras protein and other Ras signalingpathways.