C-末端碱性氨基酸在α2肾上腺素能受体外输转运过程中的作用机理研究

All G protein-coupled receptors (GPCRs) share a common molecular topology with a hydrophobic core of seven transmembrane-spanning α-helices, three intracellular loops, three extracellular loops, an N-terminus outside the cell, and a C-terminus(CT) inside the cell. The magnitude of cellular response elicited by a given signal is dictated by the level of GPCR expression at the plasma membrane, which is the balance of elaborately regulated endocytic and exocytic trafficking. GPCR export trafficking between the ER, the Golgi and the plasma membrane is directly linked to the pathogenesis of a number of human diseases, including nephrogenic diabetes insipidus (vasopressin V2receptor), retinitis pigmentosa (rhodopsin) and male pseudohermaphroditism (luteinizing hormone receptor). It is well recognized that the CT plays a crucial role in modulating GPCRs export trafficking. However the molecular mechanism remains elusive. In our previous study, by using peptide-conjugated affinity matrix combined with proteomics and glutathione S-transferase-fusion protein pull down assays, we identified tubulin directly interacting with the α2B-AR CT. The interaction domains were mapped to the the basic Arg residues in the α2B-AR CT, particularly Arg437, Arg441 andArg446. More importantly, mutation of these Arg residues to disrupt tubulin interaction markedly inhibited α2B-AR transport to the cell surface and strongly abolished ERK1/2 activation in response to stimulation with UK14304. To further elucidate the molecular mechanism underlying the function of basic residues cluster in α2-AR export transport, we first to confirm if the basic residues in α2A-AR CT could associate with pure tubulin by GST fusion protein pull down assay in vitro, and then to confirm the effect of the mutation on the receptor cell surface expression level. Second, We propose to characterize the interaction between α2-AR CT and tubulin or microtubule in vivo. By using transmission electron microscopy and Confocal laser scanning microscopy we will acquire the ultrastructural visualization of mutant receptor in export trafficking to define the intracellular compartment where the basic residues cluster modulates α2B-AR transport. Finally, as a promising model organism, zebrafish is widely used to study human disease, especially cardiac diseases. Through Morpholino knockdown and TALENT mutant techniques, we aim to explore the functional analysis of α2-AR CT in zebrafish. A molecular understanding of α2-AR export transport could lead to new ways of diagnosing and treating those defects caused by α2-AR CT mutant.

G蛋白偶联受体（GPCRs）外输转运过程受到细胞内多种机制的精细调控，外输转运紊乱与某些遗传性疾病的发生有关，其调控机制相关理论尚在不断完善中。我们前期关于受体羧基末端氨基酸序列的研究发现，α2B型肾上腺素能受体羧基末端的三个精氨酸结构在体外具有确切的与微管蛋白的相互作用，氨基酸突变后受体的细胞膜表面表达水平被显著降低，同时失去与胞外配基结合并激活后续信号通路的功能。然而，氨基酸突变引起受体外输转运过程被阻断的具体机制以及微管蛋白在其中的作用尚未明确。本项目拟在前期研究基础上，采用体外细胞模型和基因敲除斑马鱼模型为研究对象，采用免疫标记和显微成像技术，探讨GPCR羧基末端碱性氨基酸调节受体外输转运过程的分子机制：①明确外输转运过程被阻滞的亚细胞器定位以及可能的相关调节因子；②明确微管蛋白涉及的外输转运机制；③利用斑马鱼模型，进行羧基末端突变可能引起的α2肾上腺素能受体的功能障碍研究。

G蛋白偶联受体（GPCRs）羧基端近膜区富含的碱性氨基酸在受体转运过程中具有重要的调节作用。本项目在前期研究基础上，对于a2B型、a2A型肾上腺素能受体以及AT1R血管紧张素受体C末端的碱性氨基酸进行了突变研究，发现三种受体C末端的碱性氨基酸的排布以及在从ER到Golgi的转运过程中与微管蛋白的作用方式表现出细微的不同。α2B-AR的C末端的5个精基酸437RRxxRRxxxR446中第437，441和446位的精氨酸被突变为谷氨酸后大大降低了与微管蛋白的作用，受体被大量滞留在了内质网，受体完全不能到达细胞表面，而另外两个精氨酸没有此作用。α2A-AR受体C末端的第449，450，453，454，458位碱性氨基酸449RRxxKKxxxRxxRKR463被突变后，受体蛋白失去了与微管蛋白的相互作用，而第461，462，463位的RKR结构对于微管蛋白的结合以及受体的运输没有明显的影响。AT1R受体C末端的四个赖氨酸中第310，311位的赖氨酸突变307KKxKK311可使受体丧失与微管蛋白的相互作用，受体滞留在内质网中而不能运输到细胞表面。数据显示三种受体C末端碱性氨基酸与微管蛋白的作用均与所带正电荷有关，而且作用位点均位于微管蛋白的C端。与其他碱性氨基酸不同的是，AT1R受体C末端第308位的赖氨酸突变为谷氨酸后反而显著增加了细胞表面受体的表达水平，在刺激因子AngII作用条件下的由AT1R受体介导的ERK1/2通路以及RASMCS迁移功能也有所增强。数据显示该作用并不是通过抑制受体降解途径产生的，突变减弱了K308与COPI的直接相互作用，可能通过Rab6依赖的逆向运输途径影响了受体从Golgi到ER的运输，此结果揭示了C末端碱性氨基酸在AT1R受体运输调节中的一种新的作用机制。