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Development Of Chemical Probe Labeling Technology To Explore The Mechanism Of ?-arrestin1 And GPCR

Posted on:2021-02-24Degree:MasterType:Thesis
Country:ChinaCandidate:C Y YangFull Text:PDF
GTID:2370330605468781Subject:Biochemistry and Molecular Biology
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G-protein-coupled receptors(GPCRs)are the largest receptor family in human genome,which are widely distributed in various tissues and organs,and participate in the regulation of almost all life activities.GPCRs mainly mediate intracellular signal transduction through G protein and arrestins.?-arrestins play an important role in the desensitization,internalization,desensitization and recycling of receptors and G protein independent signal transduction.In the first part of this paper,we used TMSiPhe to study the conformational change of ?-arrestin1.In the early study in our laboratory,Yang Fan et al.inserted F2Y into seven phosphate group binding regions(named site 1-7)on ?-arrestinl by genetic codon expansion technology,and found different GPCR phosphorylation patterns on C-tail can induce the differential response of ?-arrestinl phosphate binding pocket by 19F NMR experiment.It can regulate the specific conformational change of ?-arrestin1,and then affect the interaction between ?-arrestinl and downstream protein.However,how does a specific phosphate binding site on?-arrestinl affect the activated configuration of ?-arrestinl and regulate the downstream signal transduction of P-arrestinl?Based on the work published on nature by Arun K.Shukla et al.in 2013,we synthesized a peptide V2Rpp-I whose V2R pT347 position is dephosphorized,in order to eliminate the direct interaction with site 1.Characterized by X-ray crystal,1H NMR and BRET techniques,we analyzed the crystal structure of ?-arrestinl-V2Rpp-l-Fab30 complex,and found that?-arrestinl-R307 TMSiPhe,when added with V2Rpp-WT and V2Rpp-1 respectively,appeared double activated conformation S1/S2.V2Rpp-1 regulated the reduction of the proportion of S2 activated conformation produced by ?-arrestinl,which may reduce the interaction between ?-arrestinl and c-Rafl.The results show that the phosphorylation of V2R T347 can affect the recruitment ability of ?-arrestinl to c-Rafl by regulating the distribution ratio of S1/S2 of the two activated conformations of ?-arrestin1.In the second part of the thesis,we hope to characterize the conformational change of GPCR by DFBQ.Adenosine receptors(AR)are a family of purinergic receptors that can bind to endogenous adenosine and regulate vasodilation and inflammation.AR are involved in the regulation of sleep,angiogenesis,immunity and other physiological processes.Due to its wide application in disease treatment,the conformational changes of A2AR combined with agonists or antagonists have been a scientific hot spot for a long time.At present,the main methods to study the mechanism and conformational state of A2AR are X-ray crystal diffraction,NMR and Cryo-SEM.We hope to develop a cheap,simple and real-time monitoring technology to study the dynamic conformational change of A2AR.Based on the protein bioelectronic interface published on Science by David E.Benson et al.in 2001,we first proposed the concept of GPCR electrochemical sensor,a sensor uses intuitive electrochemical signals to characterize the conformational changes of GPCR after binding with ligands.Here,the idea is to construct SH3 domain fusion protein at the N-terminal of the receptor to direct fixation of A2AR on MWCNTs,and then label the quinone electrochemical probe DFBQ in the region where the conformation of A2AR changes greatly after binding with the ligand.Besides,we have to design the orientation of the probe to face the electrode surface.In theory,the conformation of A2AR changes after ligand binding would lead to the varied relative displacement between DFBQ and the electrode surface,which would give out the fluctuation of electrochemical signal.Currently,we have successfully expressed and purified A2A-WT/V229C receptor in yeast cells,and carried out the preliminary experiment of DFBQ labeling on cysteine and ?-arrestinl.The results showed that DFBQ probe was efficient in labeling protein.The following research plans include DFBQ probe labeling,A2AR electrode coupling and electrical signal analysis under different ligand stimulation.It will establish the relationship among ligand efficiency,GPCR conformation change degree and electrical signal response,to build the foundation for the application of electrochemical probe in GPCR conformation research and drug screening.
Keywords/Search Tags:GPCRs, ?-arrestin1, X-ray crystallography, NMR, Electrochemical Biosensor
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