The Study Of The Homologous Dimerization Of A G Protein-coupled Receptor-GPR17

G Protein-Coupled Receptor (GPCRs) is a large family of membrane proteins coupled with G protein. They all contain seven transmembrane a helices, and the C-terminal and the intracellular bop of the fifth and sixth transmembrane helices have G protein binding sites. GPCRs interact with the corresponding ligand in the surrounding environment of cells, and activate a series of intracellular signaling pathways through various ways, and finally participate in regulation of the cell. GPCRs are closely associated with many diseases, the particularly important is that about 50% of clinical drugs currently treat GPCRs as the target. Therefore, it is very important to study the structure, the conformational change and the mechanism of GPCRs.The mechanism of GPCRs includes G protein activation, receptor internalization and dimerization generation. Activation of G protein signaling pathway is classic as relatively mature research methods, a lot of progresses and cognitions in internalization mechanisms and biological significance have well investigated. However, due to shortage of research methods for GPCRs dimerization, it remains controversial and unclear whether dimerization happens and how it happens. It was thought that GPCRs dimerization would not occur, and the so-called dimerization was just "illusion" generated by overexpression in the cell by the receptors during investigation. Also, some people consider that GPCRs would form homo-or hetero-dimerization, and, even affect the function of receptors. In recent years, more and more studies have showed the presence of GPCRs dimerization.The methods of studying GPCRs dimerization include biochemical methods, such as co-immunoprecipitation, chemical cross-linking combined with Western blot, fluorescence methods, including single-molecule fluorescence, fluorescence resonance energy transfer, and X-ray crystal diffraction technology. Among them, the fluorescence lifetime imaging-fluorescence resonance energy transfer (FLIM-FRET) is a new type of fluorescence imaging technology based on the lifetime change of the donor to study protein-protein interaction.FLIM-FRET is not affected by the intensity of exciting light, the concentration of the fluorophore, photobleaching and other factors, compared with the conventional FRET technology, it provides a great advantage.G protein-coupled receptor 17 (GPR17) is a P2 Y-like receptor, mainly distributed in the brain, kidneys, heart, vascular endothelium and other organs vulnerable to ischemia reperfusion injury. GPR17 participates in various pathophysiobgical processes, such as brain injury, spinal cord injury, oligodendrocyte cell development and maturation, so GPR17 has huge potential to become a drug target of the treatment for relevant disease. In recent years, people have carried out a series of studies on the physiology and pathology of GPR17. Thereinto, as to the structure of GPR17, there is no relevant study on whether there are homodimers or heterodimers in GPR17.Therefore, in this thesis, we adopt FLIM-FRET technology to study whether there is the homodimer in GPR17, and analyzes its conformation of homodimerization.Here, we use split GFP labeling strategy, which was developed recently in our laboratory, to achieve the labeling of GPR17 in cell at different sites, getting five pairs of fluorescent donor and acceptor. Through cellular free calcium imaging experiment, we studied the effect of the labeling on GPR17 function; using FLIM-FRET, we measured the FRET efficiency of five pairs of fluorescent donor and acceptor and calculated the distance between each fluorescent donor and acceptor. In the dimerization conformation calculation, we initially used I-TASSER software to predict the monomer structure of GPR17 monomer, and then based the distance restraints derived from the FRET data to build the homodimer structure of GPR17. In the PDB database, we found 64 GPCRs homodimer structures, and by comparison, we found that 12 structures of GPCRs have similar interface with the GPR17 homodimer we got. In the subsequent studies, we will promote or suppress homodimerization of GPR17 through mutating the amino acid near the dimerization interface, and then study the biological significance of GPR17 dimerization.For fluorescence labeling methodology, this thesis not only develops the fluorescent protein multisite-label strategy for GPR17 in cell, but also develops other two fluorescent methods, which bases on the combination of lanthanides and organic ligands, and the hydrophilicity of Rhodamine 101 inner salt(Rh101). Among them, the tpy-Tb3+and tpy-Eu3+have following features:larger stoke displacement which can effectively avoid self-quenching and the interference caused by excitation, longer fluorescence lifetime, and smaller steric hindrance etc. The advantages of R101 include higher quantum yield, stronger combination with the protein (nanomole degree), and better compatibility for monitoring in vivo due to the spectrum in long wavelength region.In summary, based on fluorescent protein site-label strategy, this thesis indicates that GPR17 can form homodimer using FLIM-FRET and get the homodimer model. In addition, this thesis developes some methods for site-specific labeling of proteins, which will offer much more information in biological issues.