MEMBRANE PROTEIN SORTING IN POLARIZED CELLS

拟议研究的长期目标是了解 细胞信号依赖性膜蛋白转运的分子基础 极化细胞。这一目标最终将需要定义 所涉及的分选信号的结构特征、精确映射 极化细胞中的运输路径，以及对 调节极化细胞分选的分子机制。了解以下内容 极化细胞膜蛋白转运的分子基础 如上皮细胞、内皮细胞和神经元，具有潜在的 亲水性大分子跨细胞递送的意义 药物，粘膜表面的抗原提呈，以及恶性 某些浸润性癌的表型。研究的重点将放在 突变和嵌合的人转铁蛋白受体分子的运输 利用一种新的基因在马丁达比犬肾细胞中表达 逆转录病毒表达系统。这项建议的具体目的是：1) 识别和表征中的基侧分类信号(S) 转铁蛋白受体(Tr)胞浆结构域；2)鉴定 Ⅱ-tR嵌合体在MDCK细胞中的转运及其分选信号 3)识别人类树的结构特征 测定其在极化脑毛细血管中的顶端分布 内皮细胞；4)鉴定蛋白质显性-负性突变 规范基边排序并使用它们来识别分子 涉及的机械和胞内分拣地点(S)；以及，5)至 识别与碱侧向分选信号结合的识别蛋白 并对分子和动力学参数进行了表征 表面等离子体共振和亲和层析的相互作用 技巧。这些目标将通过以下组合来实现 分子生物学和电子显微镜方法分析 Tr分子在极化细胞中的定量传输。近期 膜调控分子的鉴定研究进展 哺乳动物细胞和酵母中的蛋白质运输将被利用来 剖析极化细胞中的运输路径。最后，相对较新的 技术将被用来搜索与井相互作用的分子- 特征化的基侧分选信号

The long-term objective of the proposed research is to understand the molecular basis of signal dependent membrane protein trafficking in polarized cells. This objective will ultimately require defining the structural features of the sorting signals involved, precise mapping of trafficking pathways in polarized cells, and characterization of the molecular machinery that mediates polarized cell sorting. Knowledge of the molecular basis of membrane protein trafficking in polarized cells such as epithelial cells, endothelial cells and neurons, has potential implications for the transcellular delivery of hydrophilic macromolecular drugs, antigen presentation at mucosal surfaces, as well as the malignant phenotype of some invasive carcinomas. Studies will focus on the trafficking of mutant and chimeric human transferrin receptor molecules expressed in Madin-Darby canine kidney (MDCK) cells using a novel retroviral expression system. The specific aims of this proposal are: 1) to identify and characterize the basolateral sorting signal(s) in the transferrin receptor (TR) cytoplasmic domain; 2) To characterize the trafficking of Ii-TR chimeras in MDCK cells and the sorting signals involved; 3) To identify the structural features of the human TR that determine its apical distribution in polarized brain capillary endothelial cells; 4) To identify dominant-negative mutants of proteins that regulate basolateral sorting and use them to identify the molecular machinery and the intracellular sorting site(s) involved; and, 5) To identify recognition proteins that bind to basolateral sorting signals and to characterize the molecular and kinetic parameters of the interaction by surface plasmon resonance and affinity chromatography techniques. These goals will be accomplished using a combination of molecular biological and electron microscopic methods to analyze the quantitative trafficking of TR molecules in polarized cells. Recent advances in the identification of molecules that regulate membrane protein trafficking in mammalian cells and yeast will be exploited to dissect trafficking pathways in polarized cells. Finally, relatively new techniques will be used to search for molecules that interact with well- characterized basolateral sorting signals