Study On The Mechanism Of GPCR Regulates The Conformation Diversity Of Arrestin And Guides Its Downstream Signal Transduction

G protein-coupled receptors(GPCRs),also known as seven transmembrane receptors,are the largest membrane receptor family known in the human genome.In the human genome,more than 800 genes encode GPCRs,which are responsible for about 80%of transmembrane signal transduction and participate in the regulation of most pathological and physiological processes in the human body.More than 30%of current clinical prescription drugs play their role by targeting GPCRs.GPCRs are located on the plasma membrane and can recognize various extracellular stimuli,including photons,ions,small molecules,peptide and proteins,and generate extracellular signals across the cell membrane to trigger intracellular signals through G proteins and arrestin.In this process,G protein mainly takes effect by regulating the level of the second messenger in the cell.However,arrestin induce desensitization,endocytosis and activation of arrestin mediated signal transduction pathways by recruiting different downstream proteins.However,the human genome encodes only 16 Ga proteins and four arrestin,a number that is far below the number of the many receptors with which they interact.Among them,how each signal sensor(such as arrestin)realizes the transformation of different receptor-mediated signals into downstream effects and performs different biological functions remains unclear.GPCRs are usually phosphorylated by GPCR kinases(GRKs)before recruiting arrestin,which produces different phosphorylation patterns to determine different functions of arrestin.Arrestin mainly bind to GPCRs in two patterns,one is "hanging conformational state" that only binds to the C-terminal encoded by receptor phosphorylation;the other is "snug conformational state" that binds to the seven transmembrane helical cores of receptors and the C-terminal encoded by receptor phosphorylation.Nevertheless,it is still unclear how a single phosphorylation site of GPCR regulate the conformation and function of arrestin in the "hanging conformational state" and how the dynamic conformation of arrestin is distributed regulated by phosphorylated GPCR.In the "snuggly configuration",how GPCR regulates arrestin is still the difficulty and hot spot in this field.Whether the ligand can directly regulate the configuration of the receptor by binding to the receptor,independent of the phosphorylation of the receptor,to realize the specific guidance of the function of arrestin has been a mystery.In this study,in view of the above scientific problems,X-ray crystallography,DeSipher and BRET methods are used to demonstrate the regulation of a single phosphorylation site at the C-terminal tail of GPCR in the suspended configuration on the conformation of the distal functional domain of arrestin,and found important timeorder effects.Meanwhile,we used DeSipher to find that for the same receptor,multiple ligands can interact with the 7 transmembrane core(TM Core)of GPCR and directly regulate the conformation change of arrestin to mediate different downstream functions.We also used the gene codon extension technology to integrate the developed selenium probe into arrestin,and investigated the dynamic conformation change and distribution of arrestin regulated by phosphorylated GPCR.First,we used X-ray crystallography and DeSipher based on gene codon expansion technology to study a single different phosphorylation site at the C-terminus of GPCRs that directly causes a conformational change at the distal end of arrestin.And the selective correlation between the conformational change and its biological function was proved by BRET.It is found that there is an important time order effect during phosphorylation coding.we designed and synthesized phosphopeptides(V2Rpp-1,-3,-4,-6-7)with mutations in different phosphorylation sites at the C-terminus of vasopressin type-2 receptor(V2R)and proved that Fab30 can improve the stability of different phosphorylated peptides and arrestin complex by thermal stability experiments.Therefore,we co-incubated four V2R C-terminal phosphopeptides(V2Rpp-1,-3,-4,-6-7)with arrestin and Fab30 for crystallization.The complex structure of arrestin binding different phosphopeptides with atomic resolution was analyzed by X-ray diffraction.Through structural comparison analysis,we found that the phosphorylation site mutation of the phosphopeptides lost its interaction with the arrestin binding pocket,and resulted in the conformational change of the different distal functional domains of arrestin.More interestingly,we found that the phosphorylation defect of T360 not only disrupted the interaction between pS357 and pT360 with their binding pockets,but also resulted in a significant conformational rearrangement of the mid-segment of the phosphopeptides.pS357 turns outward and lost its interaction with K11,K160,K138 and R165 of arrestin.However,pT359,which is facing outwards,reverses 180 degrees to face inwards,and reforms hydrogen bond network with K11 and R25 of arrestin.This newly discovered binding pocket is named "V3'4'".Compared with inactive arrestin,it was found that the activation of arrestin by peptides with different phosphorylation modes could cause the corresponding changes in the exposure area of distal functional domain.Meanwhile,compared with the structure of arrestin activated by V2Rpp-FP,the V2Rpp-1,-3,-4,-6-7 activated arrestin forms different structural configurations in multiple functional related structural regions.Are these conformational changes associated with different phosphorylation of the receptor that regulates the biological function of arrestin?To further verify the role of GPCRs phosphorylation in the regulation of arrestin configuration,we used gene codon extension technique to label one-dimensional hydrogen spectrum nuclear magnetic probe 4-trimethylsilyl phenylalanine(TMSiPhe)into the interaction region of arrestin with MEK1 and c-Raf-1.The dynamic configuration information of the probe labeled region can be obtained in a short time with low protein concentrations.After identification by mass spectrometry,we successfully inserted the nuclear magnetic probe TMSiPhe at the specific site of arrestin.Meanwhile,we analyzed the complex structure of TMSiPhe and TMSiPheRS by X-ray crystallography.The specific selective recognition mechanism of TMSiPheRS to TMSiPhe was described.The chemical shift of 1H-NMR shows the dynamic conformational changes of arrestin under the induction of different phosphopeptides.In this study,we found that the S2 conformation state of arrestin2-R307TMSiPhe by V2Rpp-l and V2Rpp-3 stimulated was significantly less than that of V2Rpp-FP,while the phosphorylation defects of V2Rpp-4 and V2Rpp-6-7 did not cause S2 conformation,and all of them were in the S0 conformation.Arrestin2-K357TMSiPhe was all in the M2 conformation under the stimulus of V2Rpp-FP and V2RPP-1.The difference is that arrestin by V2Rpp-3,V2Rpp-4 and V2Rpp-6-7 induced has a new M3 conformation state.The results of NMR analysis showed that the single phosphorylation site defect of GPCR could induce specific dynamic conformational changes in the interaction region of arrestin with MEK1 and c-Raf-1,which could directly affect the configuration of the distal functional domain of arrestin.To reveal whether the conformational diversity of arrestin in different phosphopeptides is related to biological function mediated by arrestin,we conducted related mutations at different phosphorylation sites at the C-terminal of V2R.The interaction of V2R dependent phosphorylation of arrestin with MEK1 and c-Raf-1 was detected by FlAsH-BRET assay.The results showed that the phosphorylation patterns of the receptor caused by different phosphorylation modification sites of V2R Cterminal could affect the recruitment ability of arrestin against MEK1 and c-Raf-1.These results indicate that GPCR phosphorylation causes the configuration difference of activated arrestin,which can lead to different signal transduction of arrestin and regulate the function of arrestin.Different phosphorylation coding patterns of the receptor play an important role in regulating the interaction between arrestin and the downstream proteins c-Raf-1 and MEK1.In order to investigate the regulation of arrestin conformation by the seven transmembrane core regions of the receptor,the newly developed silicon probe TMSiPhe was selectively and specifically inserted into the polar core of arrestin(H295)and the ERK interaction site(R285)using the gene codon extension technique.We successfully inserted the NMR probe TMSiPhe at the specific site of arrestin after mass spectrometry identification.Meanwhile,we analyzed the complex structure of TMSiPhe and TMSiPheRS by X-ray crystallography.The specific selective recognition mechanism of TMSiPheRS to TMSiPhe was revealed.In combination with 950 MHz 1H-NMR,we found that multiple ligands for the same receptor can interact with seven transmembrane core(TM core)of GPCR and directly regulate the conformational changes of arrestin to mediate different downstream functions.We named this innovative technique "DeSipher" and we also applied DeSipher to clathrin that the downstream protein of arrestin,to detect phosphorylated receptors that regulate various conformational changes in arrestin mediated clathrin.In order to obtain the dynamic conformational distribution of arrestin regulated by GPCR,we also developed the selenium probe SeF and applied it to smFRET to detect the dynamic conformational distribution of downstream arrestin regulated by phosphorylated GPCR.First,we developed new FRET fluorescence pairs:Bodipy593FlAsH-EDT2,and then use the gene codon expansion technology to selectively insert the non-natural amino acid SeF into the specific site of arrestin,and label the fluorescent dye Bodipy593 into SeF through substitution reaction.Thus,the site-specific marker of Bodipy593 on arrestin was used as the receptor,and CCPGCC motif was inserted at the specific site of arrestin to label FlAsH-EDT2 as the donor.The purified GPCR expressed in insect cells Sf9 was linked with a synthetic phosphopeptide GGG-V2RppFP through Sortase A to achieve uniform phosphorylation of GPCR.Finally,with the help of TIRF fluorescence microscope,single-molecule F(?)rster resonance energy transfer was used to detect phospho-GPCR to regulate the distribution of different dynamic conformational states of arrestin.This method innovatively uses codon extension technology to integrate selenium probe SeF into specific sites of arrestin to specifically label Bodipy593.Compared with the traditional Cy3-Cy5 labeling method,it avoids all cysteine mutations in arrestin.SeF-Bodipy593 and FlAsH realized the specific labeling of the dye on the protein with shorter linker,and it was easier to accurately detect the subtle conformational changes and state distribution of arrestin before and after activation.In summary,through the above experiments,we analyzed the crystal structure of the complex between V2R C-terminal phosphopeptides and arrestin in four different phosphorylation modes,and found that a single phosphorylation site defect can cause conformational changes in the distal functional domain of arrestin.Combined with DeSipher and BRET and other technical methods,we demonstrated that the deficiency of a single phosphorylation site in GPCR could lead to different conformational changes in the distal functional domains of arrestin,and found its correlation with the biological functions of arrestin.We also revealed the mechanism of phospho-selective regulation by a single phosphorylation site in GPCR on the dynamic conformational diversity and function of arrestin.This single phosphorylation binding pattern is interdependent and tandem in different phosphorylation coding binding pockets,and possess an important sequence principle in the phosphorylation coding process.This is the main mechanism that determines the special conformational changes and functional selectivity of arrestin.This study provides direct evidence to study the phosphoselective mechanism of dynamic conformational diversity and function of arrestin.We used the newly developed DeSipher technique to demonstrate for the first time that ligand regulation of arrestin can be independent of GRK selection.This finding clarifies many years of skepticism in GPCR field that ligands should be effective in manipulating arrestin.It also provides important guidance for the development of biased ligands of arrestin and G protein in the future.Meanwhile,we developed a selenium probe that can specifically label Bodipy593 and applied it to smFRET to detect the dynamic conformational state distribution of arrestin regulated by phosphorylated GPCR,which provided a new method for studying the dynamic conformational change and distribution of downstream proteins regulated by GPCR.