Construction And Optimization Of FRET System For Conformational Changes Detection Of Chemokine Receptor CXCR4

G-protein-coupled receptors(GPCR)are the largest family of membrane protein receptors on the cell surface,they are involved in trans-membrane signaling inside and between cells,have important biological functions and are important drug targets.Although the analysis of the crystal structure of GPCR provides important information for understanding the structure and function of GPCR,but the dynamic conformational change and selective activation mechanism of GPCR activation process are still unclear.The research on this issue will provide a new opportunity for a deep understanding of GPCR transmembrane signal transduction mechanism and selective drug design.The human chemokine receptor CXCR4 belongs to the group A GPCR and plays an important role in embryonic development,cancer development,tumor migration,and is also an important target for HIV-1 into host cells.In this thesis,CXCR4 was used as the research object,and the fluorescence resonance energy transfer(FRET)was used to systematically detect the changes of the receptor's intramolecular conformation during CXCR4 activation under different agonists,thus laying a foundation for a comprehensive understanding of the dynamic process of CXCR4 activation and safer and more efficient drug design.The FRET detection systems have an important effect on the conformational changes detection in the GPCR molecule.We first constructed three different FRET fluorescence systems on the C-terminus and the third intracellular loop of CXCR4: monomeric red fluorescent protein(m Cherry)-enhanced green fluorescent protein(EGFP),enhanced yellow fluorescent protein(EYFP)-enhanced cyan fluorescent protein(ECFP)and tetracysteine-tag(TC-tag)-ECFP.After transient transfected into HEK 293 cells,their detection sensitivity for the conformational changes of CXCR4 in three fluorescent FRET systems were evaluated.It was found that the FRET system composed of EYFP-ECFP had the most significant detection sensitivity for intramolecular conformational changes in CXCR4 under different agonists.On living cells,the value of FRET ratio(FRET ratio)is 1.50-1.70;it is 1.15-1.25 in cell lysate,which is superior to m Cherry-EGFP and TC-ECFP fluorescent FRET system,therefore EYFPECFP fluorescence FRET system is used in subsequent experiments.On this basis,we investigated the effects of different agonist concentrations on the conformational change of CXCR4.It was found that the optimal concentration of AMD3100 and TC14012 on the conformational change of CXCR4 mutant was 10 ?M,and the Change in FRET ratio(Change in FRET ratio)reached 30%.Different concentrations of SDF-1? had little effect on the FRET detection of the conformational change of CXCR4 mutant.The C-terminus of CXCR4 was used as an insertion site,and the N-terminal,different intracellular loop(IC)or extracellular loop(EC)was selected as another insertion site,the FRET conformational changes of CXCR4 with different insertion sites of FRET pairs were explored.The results showed that,the FRET signal of CXCR4 first extracellular loop mutant(YC3)was the most variable after the action of neutral agonist AMD3100,and the Change in FRET ratio was about 35%,the FRET signal of CXCR4 first intracellular loop mutant(YC2)changed the most after the action of complete agonist SDF-1?,and the Change in FRET ratio was about 13%,the FRET signal of CXCR4 mutant YC3 changed the most after the action of inverse agonist TC14012,and the Change in FRET ratio by about 15%.The conformational changes of CXCR4 mutants in surfactant solutions were also explored and compared those on living cells.The results showed that,the change in FRET ratio was small in the surfactant solution,and the molecular conformation change of the CXCR4 mutant was not significant.After binding with different agonists,there is no obvious regularity in the FRET signal change of CXCR4 in the surfactant solution,it may be that the binding activity and conformational change of the receptor and agonist are inhibited in the surfactant solution,therefore,the FRET signal changes are not obvious.Our study provides important information for the use of FRET technology to detect intramolecular conformational changes during GPCR activation,which lays a foundation for further analysis of the molecular mechanism of GPCR selective activation and its conformational changes.