【作者】 刘亚；

【导师】 姚雪彪；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2007， 博士

【摘要】 胞吐（Exocytosis）是细胞的一项重要生理活动，它参与了细胞生长与极化、细胞通讯、细胞外被和细胞外基质的形成、受精过程及免疫反应等诸多重要生理过程，因而胞吐调节机制受到人们的关注。对神经递质释放的分子机制的研究表明：SM（Sec1／Munc18）蛋白通过调节可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）复合物的组装达到调节膜融合，进而调节胞吐的作用。胃壁细胞（gastric parietal cell）是一种极化的泌酸上皮细胞，胞质内充满富含H⁺，K⁺-ATPase的管状囊泡。在受到内分泌、旁分泌及神经分泌的刺激时，激活了cAMP-依赖的蛋白激酶级联反应，进而引起管状囊泡之间的融合（同型融合）及管状囊泡向顶膜移动，并与项膜融合（异型融合），使微绒毛向腺腔内急剧伸长，大大增加了顶膜面积，为最大限度地容纳质子泵奠定了基础。同时，原管状囊泡膜上的H⁺，K⁺-ATPase也随之转移到质膜，将H⁺运送至胞外并释放内因子（intrinsic factor）。由于胃壁细胞极化明显并在激活时发生了显著的囊泡转运和膜融合反应，其形态学变化特征有助于生物光子学研究，同时还可以较容易地得到生物化学研究应用所需的大量细胞，因而胃酸分泌过程成为研究cAMP介导的SNARE复合体组装及细胞骨架动力学的极佳模式系统。在调节壁细胞分泌的磷酸化蛋白质组学的研究中，我们发现CDK5和Munc18b是壁细胞内cAMP-依赖的胃酸分泌过程所必需的蛋白。体外的生化实验显示Munc18b N-端156个氨基酸序列可与syntaxin（Stx）的四个同源物Stxs 1-4以相同强度结合，而C-端的54个氨基酸序列特异性地与Stx3结合；特别是当Munc18b的Thr 572被CDK5磷酸化后，Munc18b-Stx3的相互作用大大降低了，但同时却促进了Munc18b-Stx3-SNAP25三元复合物的形成，进而导致了功能性的Munc18b-Stx3-SNAP25-VAMP2膜融合机器的组装；体内实验也证明CDK5是介导的Munc18b磷酸化是SNARE复合物形成所必需的。据此，我们提出了调节性胞吐中一个新的膜融合调节机制：CDK5磷酸化Munc18b调节了SNARE复合物的形成，进而调控了囊泡停泊与融合。Ezrin是连接细胞膜与细胞骨架的ERM（ezrin／radixin／moesin）蛋白家族的一员，它含有两个很重要的结构域N-ERMAD（N-terminal ERM association domains）和C-ERMAD（C-terminal ERM association domains），通常情况下，ezrin的N-ERMAD结构域被自身或其它分子的C-ERMAD结构域所结合而处于失活状态。Ezrin参与了上皮细胞的极化、质膜蛋白的定位及功能的调节，在壁细胞中主要定位于顶膜囊泡，参与了管状囊泡向顶膜的转运，但其机制目前仍不清楚。由于Stx3是一种定位在上皮细胞顶膜的整合膜蛋白，参与了囊泡与顶膜的融合及上皮细胞的极化，因此我们推测Stx3和Ezrin之间可能存在某种相互作用，共同促进了壁细胞激活时管状囊泡向顶膜的转运。我们的体外生化实验证实了Stx3与ezrin存在相互作用，并进一步Map出Stx3的Habc结构域和ezrin的N-端N-ERMAD结构域介导了Stx3-ezrin相互作用。这种相互作用受到了ezrin的磷酸化状态的调节：Stx3特异性地与模拟Ser 66磷酸化的ezrin^S66D相互作用，而不与野生型ezrin^wt结合，提示Ser66磷酸化可能改变了Ezrin分子N端的构象。利用原子力显微镜，我们对Ezrin进行了单分子形态研究，我们的结果揭示：Ser66磷酸化开启了Ezrin分子，使整个分子更为伸展，从而使Stx3-ezrin复合物形成。为此，我们的研究说明了壁细胞酸分泌过程中Ezrin调控管状囊泡定向转运的重要性。更多还原

【Abstract】 Exocytosis is essential for many physiological activities including fertilization, immune response, cell signaling, cellular growth and polarization, the formation of extracellular matrix. However, the molecular mechisms underlying regulated exocytosis have remained elusive despite great progresses made over the last two decades.Gastric parietal cells are polarized epithelial cells in which hydrochloric acid secretion is triggered by paracrine, endocrine, and neurocrine pathways. Stimulation of acid secretion typically involves an initial elevation of intracellular calcium and cAMP followed by activation of a cAMP-dependent protein kinase cascade, which triggers the translocation and insertion of the proton pump enzyme, H, K-ATPase, into apical plasma membranes of parietal cells and results in an activation of the pump for acid secretion into the glandular lumen. After secretory stimuli are withdrawn, the H⁺, K⁺-ATPase is withdrawn back into the cytoplasmic compartment. The stimulation-mediated membrane transformation formulates the membrane recycling hypothesis of HC1 secretion. Therefore, acid secretion in parietal cells provides a perfect model system to study cAMP-regulated vesicular trafficking and its relationship to proton pump dynamics.Membrane-associated helical proteins known as soluble N-ethyl maleimide sensitive factor attachment protein receptors （SNAREs） are crucial for vesicle fusion. In the brain, fusion of synaptic vesicles with the plasma membrane requires three SNARE proteins: syntaxin 1, SNAP25 （synaptosomal-associated protein 25 kDa） and synaptobrevin. The three SNARE proteins form a four-helical bundle—the SNARE complex that mediates membrane fusion. Our recent studies demonstrate the functional significance of membrane trafficking and fusion machinery components such as syntaxin 3, VAMP2, and SNAP25, in the parietal cell activation. In addition, we show the dynamic stimulation-associated redistribution of VAMP2 from H⁺, K⁺-ATPase-rich tubulovesicles to co-localize with SNAP-25 on the apical plasma membrane. Despite our demonstration of the functional importance of syntaxin 3 in parietal cell activation, it is still unclear how syntaxin 3 is involved in the tubulovesicular membrane dynamics triggered by histamine stimulation.In the present study, we have employed 2-dimention-electrophoresis and mass spectrometry to screen for phosphor-proteins required for live parietal cell activation, we identified the requirements of CDK5 and Munc18b in gastric acid secretion in parietal cells. Our pull-down assay revealed that Munc18b contains two binding sites for Stx3 which are localized to the N-terminal 156 amino acids and the most C-termianl 54 amino acids, respectively. The C-terminal Munc18 only bound Stx3 while the N-terminal Muncl8b bound to all syntaxin isoforms tested （stx1, 2, 3, and 4） equally. Further experiments in vitro and in vivo suggested the phoshorylation of Munc18b Thr 572 by CDK5 reduced its affinity for Stx3, however enhanced the complex of Munc18b with SNARE. Our studies reveal a novel regulatory mechanism underlying by which CDK5-mediated phosphorylation of Munc18b operates secretory vesicle docking and fusion in regulated exocytosis.Ezrin is a member of the ERM （ezrin/radixi/moesin） family which mediates the dynamic association between plasma membrane and actin cytoskeleton. In gastric parietal cell, it mainly localizes to the apical membrane, regulates the cell polarization and the localization of membrane proteins, furthermore, it plays an important role in the tubulovesicular membrane dynamics triggered by histamine stimulation. Since stx3 is an integral membrane protein involved in the epithelial cell polarization and mediates the fusion between tubulovesicle and apical membrane in parietal cells, then we presume that Stx3 may interact wih ezrin and this interaction may play an important role during the translocation of H⁺, K⁺-ATPase from plasma to the apical membrane in parietal cells.Our experiments in vitro verified our guess, and further assays revealed that Stx3-ezrin interaction was regulated by the phosphorylation of ezrin: when hatched Stx3 with various mutants of ezrin (ezrin^wt, ezrin^S66D, ezrin^T567D), it specifically interacted with ezrin^66D at physiological pH. Deletion mutants of Stx3 and ezrin were used to map the precise regions of Stx3-ezrin physical contacts. Our biochemical characterization demonstrated that the Habc domain of Stx3 and N-ERMAD domain of ezrin, suggesting that Ser66 phosphorylation-induced conformation change enables ezrin-Stx3 physical contact. To validate this hypothesis, we conducted atomic force microscopic analysis of recombinant ezrin proteins. Our analysis demonstrated Ser66 phosphorylation unfolds ezrin molecule to allow its N-terminus to bind Stx3. Taken together, our studies demonstrate an essential role of ezrin in the spatial control of polarized epithelial secretion.更多还原