The Physiological Function And Mechanism Of GABA_B Receptor Mediated Rap1Signaling

GABA_Breceptor, belonging to GPCR class C family, mediates slow inhibitoryneurotransmission in the central nervous system. GABA_Breceptor is functional as ahetero-dimer composed by GABA_B1and GABA_B2subunits. GPCR-mediated signalingstrength is critically dependent on the availability of the receptor at the cell surface.Desensitization of GABA_Breceptors is distinct from the classical mechanism ofG-protein-coupled receptors, undergo rapid constitutive endocytosis and recycling,activation of GABA_Breceptors accelerates the rates of their endocytosis and recycling. Itwas concluded that GABA_Breceptors are very stable at the cell surface. The molecularmechanism regulates the availability of GABA_Breceptor at the cell surface remainselusive. It is still not known whether GABA_Breceptor interacting proteins may regulatecell surface availability of the receptor upon agonist exposure. Indeed, The C-terminaldomains of both subunits of GABA_Breceptor contain a coiled-coil regions, which areinvolved in receptor assembling, cell surface trafficking and constitutes a binding domainfor GABA_Breceptor interacting proteins. Here, we demonstrated for the first time thatagonist-stimulated GABA_Breceptor interacts with the activated form of Rap1, Rap1GTP,in mouse cerebellar granule neurons. Furthermore, we found that selective activation ofGABA_Breceptor induced persistent activation of Rap1. GABA_Breceptor-induced Rap1activation is via Gi/o-protein and Rap1GAPII. We then identified that the region withinGABA_B1C-terminal to interact with Rap1GTP is RVHLLYK. By using synthesizedpeptides which can impair the interaction between Rap1GTP and GABA_B1, wedemonstrated that pretreatment the cells with this synthesized peptide decrease GABA_B1expressing at the cell surface which in turn inhibit the GABA_Breceptor downstreamsignaling, thus suggesting the important role of Rap1for controlling the availability ofGABA_Breceptor at the cell surface. Rap1was activated by GABA_Breceptors selectiveagonist baclofen stimulation via Gi/odependence and Rap1GAPII. The stability of GABA_Breceptors at the cell surface was controled by Rap1signaling via the interaction betweenGB1subunit and Rap1GTP. We also identified the region within GB1C-terminal and Rap1GTP required for its interaction. Once impairing this interaction, the cell surfaceavailability of GABA_Breceptor decreases. Our data also suggest that GABA_Breceptor-induced Rap1GTP may provide a feedback regulation for its signaling strength.