The Fine Research Of The Activation Mechanism Of GPCR Family C

mGluRs,GABA_BR,CasR,TasterR are also members of family C GPCRs,whichhave wide distribution in vivo and important physiological function.The activity of thesereceptors are precisely regulated and are involved in numerous types of nociception,cognitive impairment,epilepsy,spasticity and drug addiction.All class-C GPCRs but theorphan receptors,possess a large extracellular domain (ECD)composed of two mainregions:a Venus Flytrap module (VFTM).This domain is connected to a heptahelicaldomain (HD)typical of all GPCRs via a cysteine-rich region (except for the GABABreceptor subunits).In contrast to unstable dimerization of other families,class-C GPCRspresent as constitutive dimers in vivo.This complex architecture raises a number of important questions.Based on numerousstudies,simple two-step activation model has been widely accepted.Agonist bindingwithin the large ECD triggers the necessary change of conformation,or stabilize a specificconformation,of the heptahelical domain leading to G-protein activation.However,howligands acting within the heptahelical domain can change the properties of these complexmacromolecules and the role of CRD in this process are less well known.Moreover,thetwo-step activation model is still too simple to precisely explain the complexconformation change and interaction between each functional domain of class-C familyGPCR dimmers during activation.On the other hand,class-C GPCRs is crucial drug targetbecause of their important physiological function and structure that is easy to manipulative.Therefore,it is significant to explore the accurate activation mechanism both on theoryand application.Here we study on the accurate activation mechanism of GABABR heterodimer.Baseon Bioinformatic prediction,we speculate that LB1-LB 1 interface play an important roleon structure formation and inaction on VFT domain between two subunits.we haveidentified the VFT dimerization interface and used a glycan wedge scanning approach toanalyze its functional relevance.Our data demonstrate that lack of activity of theGB2-N114 or-N141 mutants was likely due to the presence of the N-glycan on the VFT,while analagous mutants GB2-Q114 and GB2-Q141 which cannot be glycosylated,do notlost activity,so direct interaction between the VFTs of the GABAB subunits is requiredfor cell surface targeting and agonist activation of the receptor.Similar results are foundon GB1-N229 and GB1-N251 mutants.Hereby,we provide direct evidence that a changein the dimerization interface takes place during agonist activation of the receptor. Moreover,we introduction of N-glycans at the bottom of VFT,the GB2-N210 mutant lostactivity but can form heterodimer,so we speculate that such site mutation just blockcouple between VFT and HD.How the CRD servers as a mediator in signal transduction from VFT to HD in vivo?We first replaced the 9 conserved cysteine by alanine in mGluR2,and find the receptorsdisplay different pharmacology phenotype,the most remarkable is the high constitutiveactivity of C500A.Based on a series of biochemistry and functional assay results,we haveproved the spontaneous formed disulfide bridge between two new'free'C519 in eachmonomer accounts for the constitutive activity of C500A.Substitution of cysteine for theresidue which close to C519 and in the putative CRD-CRD interface to form analogousinter-monomer bridge also produces constitutive activity.The similar results are also gainin mGluR4,mGluR5 and CaSR.Taking into account the bioinformatics results for thesereceptors,we demonstrate that the activation involves the horizontal nearness and thetangent rotation of two CRD in each monomer,thereby provides a simple explanation to along-standing question of how agonist binding to VFT induces allosteric conformationchange conduct to effector domain.And our study is also important for medical researchand drug screening.Several independent homologous modeling of family C GPCRs have shown that theVFTM has a structurally overall similarity with bacterial PBPs,suggesting their commonorigin via internal domain duplication.Unlike the bacterial PBPs binding extensiveligands,most members of family C GPCRs mainly expressing in the central nervesystem bind only one kind of natural ligand,which imply that the VFTMs of family CGPCRs have undergone loss of some functions such as more ligand-binding abilities andsimultaneously gained of unknown pivotal functions.we perform an extensivelyphylogenetic analysis for subfamily members of family C GPCRs to inspect thehypothesis that the altered selective constraints at specific sites occurred in the VFTMs.Here,we report that the functional divergence occurs after three continuous geneduplication events as a result of site-specific altered selective constraint in family CGPCRs evolution.Our study provide a new insight for understanding the specification ofligand-binding properties and refine the activation or modulation mechanism of thefamily C GPCRs.