Study On The Modulatory Mechanism Of GABA_B Receptors By Lipid Rafts And Development Of Activity-based Fluorescent Probe Targeting To GABA_B Receptors

Lipid raft domains have attracted much recent attention as platforms for plasma membrane signalling complexes. In particular, evidence is emerging that shows them to be key regulators of G protein coupled receptor function. Previous researches had shown that the G protein coupledγ-aminobutyric acid receptor B (GABA_B receptor) co-isolates with lipid raft domains from rat brain cerebellum, so exploring the effect that rafts play on GABA_B receptor may shed new light on understanding regulation mechanism of GABA_B receptor.In the present study, single particle tracking approach was applied to follow surface diffusion of GABA_B receptors in real time when expressed ectopically in HEK293 cells. We found that the surface mobility of GABA_B receptors depends on the GABA_B: sunbunit. Furthermore, sucrose density gradients display that GABA_B receptors which diffused slowly presented in raft fractions, while GABA_B1-ASA which diffused freely did not localized in rafts selectively. Therefore, location in rafts is critical for restricting the lateral diffusion of GABA_B receptors.However, the association between GABA_B receptors and rafts was not permanent. Treatment of tranfected HEK293 cells with agonist GABA significantly increased its diffusion coefficience, similar to the action of MCD (Methyl-β-cyclodextrin). In parallel with change on lateral mobility, confocal microscope imaging and sucrose density gradients both indicated the colocalization of GABA_B receptors with rafts decreased significantly upon activation in both heterogenous cells and neurons. Activation induced translocation of GABA_B receptor was convictively proved in this research.The implication of the translocation of GABA_B receptors to nonraft domain is highlighted by attenuated G protein-dependent signaling, such as agonist-induced ERK phosphorylation and IP₃ acccumulation mediated by chimeric G_qi9, with the decrease in location within rafts. Moreover, lipid rafts facilitated the spatial organization of signal compotents involved in GABA_B-mediated network by including or excluding them to variable extents, so that the transduction was switched on or off dynamicly.Thus, a new perspective on surface behavior and signal transduction of GABA_B receptors modulated by rafts were proposed in the present study, so the intrinsic location property of the GABA_B receptors deserves more attentions on it.In order to label the surface GABA_B receptors on living cells, a trimodular activity-based fluorescent probe was designed, synthesized and characterized based on the structure of CGP64213, an antagonist of GABA_B receptor. This probe can be applied to photoaffinity label the GABA_B receptor transiently expressed in CHO (Chinese hamster ovary) cells. Moreover, it exhibits specific binding activity at the ligand-binding pocket of GABA_B1 subunits and high specificity of photoaffinity labeling, which makes the probe valuable for studying the localization and function of GABA_B receptors on living cells.