STUDIES IN THE ROLE OF EZRIN IN THE CYTOSKELETAL SYSTEM IN THE PARIETAL CELL

本研究以壁细胞为模型，探讨细胞骨架蛋白在分泌过程中的作用。实际的计划是确定ezrin的功能磷酸化位点，并开发一种模型系统，该系统允许将肽引入细胞，以便直接分析蛋白质的功能。在第一年，克隆了人ezrin的部分序列，用PKA处理，没有观察到磷酸化。然而，纯化的兔ezrin被PKA磷酸化，表明ezrin序列的种属差异。在接下来的一年的研究中，格鲁吉亚大学的Goldenring教授告诉我们，他对兔ezrin进行了测序，没有发现PKA位点，即没有发现物种，因此，我们在实验中发现了许多用于分析信号转导的药物探针，并建议建立透化细胞系统 关于我们 ezrin的PKA位点的差异。与此同时，我们获得了一些数据表明ezrin本身不被PKA磷酸化，而胃粘膜含有一种未鉴定的激酶，该激酶被PKA激活并磷酸化ezrin的C-末端位点。这个方向与一开始的计划不同，但它现在已经成为一个有趣得多的项目。目前，实验正在进行中，以确定这种激酶。对于另一个项目，我们成功地开发了一种新的模型，β-七叶皂苷透化兔胃腺，它允许将肽引入细胞。使用该系统，显示不仅PKA抑制肽而且肌球蛋白轻链激酶抑制肽抑制cAMP刺激的酸分泌反应。我们还可以显示小GTP结合蛋白在分泌过程中应用功能肽的系统中的可能参与。因此，该模型被证明是分析壁细胞囊泡运输中细胞骨架蛋白之间相互作用的有力工具。少

The aim of the present project was to elucidate the role of cytoskeletal proteins in the secretory process using the parietal cell as a model. The actual plans were to determine the functional phosphorylation site of ezrin and to develop a model system which allows an introduction of peptides into the cell in order to make a direct analysis of the function of proteins. In the first year, human ezrin was cloned and the partial sequences were treated with PKA, and no phosphorylation was observed. However, purified rabbit ezrin was phosphorylated by PKA suggesting species difference in the sequence of ezrin. In the separate experiments, we showed that many of pharmacological probes commonly used to analyze signal transduction were useless, and suggested that the establishment of permeabilized cell system was indispensable.During the next year's research, Professor Goldenring in the University of Georgia informed us that he sequenced rabbit ezrin and found no PKA sites, namely, no species … More difference in the PKA site of ezrin. In the meantime, we obtained some data suggesting that ezrin itself is not phosphorylated by PKA, whereas gastric mucosa contains an unidentified kinase which is activated by PKA and phosphorylates C-terminal site of ezrin. This direction is different from the plan at the beginning, but it has now become much more interesting project. At present, experiments are in progress to identify this kinase. For the other project, we successfully developed a new model, the beta-escin permeabilized rabbit gastric gland, which allows the introduction of peptides into the cell. Using this system, it was shown that not only the PKA-inhibitory peptide but also the inhibitory peptide for myosin light chain kinase inhibited cAMP-stimulated acid secretory response. We could also show the possible involvement of small GTP-binding proteins in the secretory process applying functional peptides to the system. This model was thus demonstrated to be a powerful tool to analyze the interaction between cytoskeletal proteins in the parietal cell vesicular transport. Less