Study On In Vivo Structure Model And Fuction Of GPR17 Dimer

The 800+G-protein coupled receptors(GPCRs)make up a prominent family of membrane receptors.GPCRs are responsible for myriad aspects of cellular function.They also constitute more than 30%of current drug targets.A GPCR is characterized by seven transmembrane helices(TM1 to TM7)and adopts a highly conserved tertiary structure.Experimental evidence has shown that GPCRs form dimers and high-order oligomers.It has been well established that class C GPCRs form dimers and oligomers for their functions.However,the molecular basis and functional relevance of class-A GPCR dimers remain to be established.A class-A GPCR contains a relatively short and often unstructured N-terminal segment,comparing to the class C GPCRs that have a large extracellular and dimeric N-terminal domain.Class-A GPCRs have long been shown to dimerize and oligomerize in the cell membrane.Recombinantly purified,many class A GPCRs have been crystallized as a dimer.To be crystalized,the GPCRs were in vitro reconstituted in the detergent,liquid cubic phase,or nanodisc.As a result,the GPCR dimer structure may or may not manifest the physiological quaternary arrangements of the GPCRs.Multiple experimental methods have been developed and employed to understand how a GPCR dimerizes in the cell membrane.Biochemical methods can only perform simple qualitative studies on GPCR dimers and oligomers.In contrast,the methods based on fluorescence can not only obtain structural information,but also be used for extrapolate of GPCR dimerization in the cell membrane.With two fluorophores introduced,RET techniques can be used to extrapolate of receptor dimerization.Among the RET techniques,FLIM-FRET measures the shortening of the fluorescence lifetime of the fluorescence donor.The fluorescence lifetime of the donor fluorophore is not affected by the protein level of the donor fluorophore.Problems associated with leakage from donor emission,photobleaching during detection,and direct excitation of the acceptor also can be minimized.Therefore,FLIM-FRET provides more accurate distance between fluorophores.GPR17 is a class A GPCR involved in ischemic injuries of the kidney,heart,and brain.UDP-glucose can activate GPR17,leading to the inhibition of adenylyl cyclase and the activation of phospholipase C.Moreover,the treatment of UDP-glucose can lead to the downregulation of the GPR17 signal,which has been attributed to ERK1/2 activation and GPR17 receptor internalization.We found that GPR17 forms dimer and higher oligomers in the cell membrane by using Western blotting and His-tag pull-down analysis.To analyze how GPR17 dimerizes,we performed FLIM-FRET measurement between the two protomers of GPR17 at the cell membrane.The result showed that GPR17 protomers adopt a specific arrangement in the homo-dimer.We converted the FRET efficiencies to FRET distances restraints.Based on the FRET distances,we build the dimeric structure of the GPR17.The dimer interface mainly involves transmembrane helix5(TM5).In particular,a pair of phenylalanines,residues F229 in TM5 is located at the dimer interface,interacting with each other.With introduce alanine or cysteine mutations to F229,the wild-type or mutant receptors have similar protein levels at the cell surface.And the point mutations in F229 can make GPR17 monomeric or dimeric in the cell membrane.Experiment showed that F229A mutants disrupted the dimerization of GPR17.While wildtype and F229C mutants of GPR17 protein could forms dimer.We dissect the respective functions of GPR17 monomer and dimer upon ligand binding.Monomer and dimer of GPR17 have different functions.The administration of 0.5 mM UDP-glucose significantly increased the intracellular Ca2+level for cells transfected with wild-type and cysteine mutant GPR17.Thus,the monomeric mutations of GPR17 impaired the responses of intracellular Ca2+-level and ERK1/2 activation upon agonist binding.By labeling the GPCRs at one of the extracellular loops,we obtained multiple interprotomer distance measurements using fluorescence lifetime imaging microscopy fluorescence resonance energy transfer(FLIM-FRET).The structural model of the dimer manifests the quaternary arrangement of the GPCR in its native membrane environment.We envision the same approach can be used to characterize physiological interactions of other essential membrane proteins.Many GPCRs form homodimer or heterodimer detected by biochemical or fluorescent methods.But their atomic resolution structures have not been reported.Our method can be used to study their homodimer/heterodimer structure and how it dimerizes in the cell membrane.