MEMBRANE PROTEIN SORTING IN POLARIZED CELLS

偏振细胞中的膜蛋白分选

• 批准号: 2749591
• 负责人: IAN S TROWBRIDGE
• 金额: $ 36.66万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-15 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2749591
• 关键词: MDCK cell; affinity chromatography; apical membrane; basolateral membrane; biological signal transduction; cellular polarity; chimeric proteins; electron microscopy; human tissue; immunofluorescence technique; intracellular transport; membrane proteins; molecular cloning; polymerase chain reaction; protein structure function; protein transport; radionuclides; restriction endonucleases; surface plasmon resonance; tissue /cell culture; transferrin receptor; vesicle /vacuole

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

拟议研究的长期目标是了解 信号依赖膜蛋白运输的分子基础 极化细胞这一目标最终将需要界定 所涉及的分选信号的结构特征， 极化细胞中的运输途径，以及 介导极化细胞分选的分子机制。知识 极化细胞膜蛋白运输的分子基础 例如上皮细胞、内皮细胞和神经元， 亲水性大分子跨细胞传递的意义 药物，粘膜表面的抗原呈递，以及恶性肿瘤 一些侵袭性癌的表型。 研究将集中在 突变型和嵌合型人转铁蛋白受体分子的运输 在Madin-Darby犬肾（MDCK）细胞中表达， 逆转录病毒表达系统这项建议的具体目标是：1） 为了识别和表征基底外侧分选信号， 转铁蛋白受体（TR）胞质结构域; 2）为了表征 MDCK细胞中Ii-TR嵌合体的运输和分选信号 3）鉴定人TR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）鉴定蛋白质的显性负突变体 调节基底外侧分选并使用它们来识别分子 机器和所涉及的细胞内分选位点;以及，5） 鉴定与基底外侧分选信号结合的识别蛋白 并表征分子和动力学参数的 表面等离子体共振和亲和层析相互作用 技术.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来分析 极化细胞中TR分子的定量运输。最近 膜调节分子的鉴定进展 哺乳动物细胞和酵母中的蛋白质运输将被利用， 剖析极化细胞中的运输途径。最后，相对较新的 技术将被用来寻找分子， 特征性基底外侧分选信号