【作者】 黄思罗；

【导师】 刘剑峰；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2009， 博士

【摘要】 C族G蛋白偶联受体(GPCRs)(以下简称C族受体)包括mGluRs、GABA_BR、CaSR、TasterR等成员，在体内广泛分布并具有重要的生理功能，其活性受到精密的调控，并与多种疾病的病理进程密切相关。除了GPCR所共有的参与G蛋白偶联的HD以及膜内的C末端，C族受体还具有膜外巨大的参与配体结合的VFT，以及连接HD与VFT之间的CRD。同时，相对于其他亚家族受配体诱导的不稳定的二聚化，C族受体在体内形成组成性的二聚体。可以想见，结构复杂的C族C族受体具有复杂的激活机制。在大量的研究基础上，简化的基于单体的两步激活模型得到了广泛的承认。配体结合到VFT后导致VFT关闭，进而将激活信号传导到HD，构象发生改变后的HD与G蛋白偶联从而激活受体。但是，对于这一激活过程中的精细机制，还尚不了解。比如VFT是如何将构象变化传导到HD，CRD在其中所扮演的角色，目前还几乎是空白。而更重要的是，C族受体在体内是组成性的二聚体，对于二聚体在激活过程中复杂的构象变化以及由之而产生的各功能域间的相互作用和拓扑学效应，简化的两步激活模型并不能够完全解释。另一方面，功能的重要性和结构上的易操控性，使得C族GPCR成为重要的药物靶点。因此，对于C族GPCR激活机制的精细研究，在理论和应用上都具有重要的意义。本文研究了异源二聚体GABA_BR激活过程中的精细机制。我们根据生物信息学研究推测LB1-LB1结合界面对于受体的结构形成，以及GABA_BR的VFT-VFT相互作用具有重要的意义。由此我们根据构建的三维模型在可能的LB1-LB1结合界面上引入了一系列糖基化位点，进而检测了突变体的功能变化。结果发现，GB2-N114和GB2-N141失活，并丧失了所有二聚化的生理效应；而在同样位点引入的非糖基化突变GB2-Q114和GB2-Q141则无相应改变。这说明引入的糖基化位点所产生的VFT-VFT间的结构阻碍，导致GB1和GB2不能正常二聚化。类似的结果也在GB1中发现，位于LB1-LB1结合界面的GB1-N229和GB1-N251突变具有类似的生理现象。因此，这些结果探明了GABA_BR中VFT-VFT相互作用的精细位点，并对GABA_BR异源二聚化的形成机制提供了新的证据。我们还将糖基化突变引入到GB2的LB2底部，结果发现GB2-N209丧失了活性，但突变体并不影响异源二聚化，据此推测，该位点的失活与糖基化引入的结构阻碍影响了VFT的构象变化以及与HD的偶联有关。本文在前面工作的基础上，进一步在CRD中突变了其中严格保守的9个Cys残基。结果发现，所有的突变体均可以正常上膜、二聚化、结合配体，但其药物反应性质均发生改变，其中mGluR2-C500A产生高组成性活性，mGluR2-C518A激活强度降低，而其余的突变体则完全失活。同时还发现，上述突变体均可以被PAM所激活。这些结果证明了CRD参与了VFT-HD之间的偶联。利用其他手段我们证实了mGluR2-C500A具有完整的活性，并且其组成性活性依赖于CRD间的二硫键的存在，由此推测，其组成性活性可能与VFT和CRD之间的相对位置发生改变有关。为了证实这一点，我们进一步在CRD中进行了双突变体的研究，结果功能实验表明，与预测的一致，mGluR2-C500AC519A恢复了配体诱导的激活性质；而生物化学研究表明，VFT的C234具有与CRD中的C500、C518、C519间形成二硫键的潜力，从而形成不同的构象并呈现不同的生理效应。类似的结果在mGluiR4和mGluR5中也得到了验证。这些结果对我们解析CRD的空间结构，以及理解CRD在二聚体激活过程中所发生的构象变化，具有重要的意义。本文还利用生物信息学的方法研究了C族受体的进化路径。C族受体的VFT与一些细菌中转运小分子的PBPs具有共同的起源，但不同于PBPs，不同受体的VFT的配体结合具有高度的专一性。本文运用一种扩展的系统进化分析法，通过比较C族受体中的五种典型成员的VFT，来研究配体结合的专一性，这种基因／结构复制引起的功能上的趋异变化，是否是由在相关位点上选择参数的变化而导致的。结果显示，从统计上来看，这些不同的成员的VFTMs中的选择参数改变(或改变的进化率)是重要的。而且，通过比较产生在早期脊椎动物C族GPCR基因的进化图谱，我们发现C族受体的VFTMs有很高的位点专一性改变率，直接结果可能是在这些基因中的功能趋异变化，并最终导致了专一性的产生。这些结果有助于探究C族受体的VFT的功能位点，也为我们研究C族受体的进化提供了新的思路。更多还原

【Abstract】 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.更多还原