REGULATED ENDOCYTIC TRAFFIC IN FIBROBLASTS

成纤维细胞内吞交通的调节

• 批准号: 6628582
• 负责人: TIMOTHY E MCGRAW
• 金额: $ 28.82万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-15 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6628582
• 关键词: CHO cells; aminopeptidase; chimeric proteins; cytoplasm; endocytosis; enzyme activity; fibroblasts; glucose transporter; hormone regulation /control mechanism; insulin; nuclear magnetic resonance spectroscopy; site directed mutagenesis; transferrin receptor; yeast two hybrid system

DESCRIPTION (Applicant's abstract): The long-term objective of this project is to characterize the mechanism that mediates regulated, dynamic retention in endosomes. To accomplish this goal, we will characterize in Chinese hamster ovary (CHO) cells the endocytic behavior of vpTR, a chimera between the human transferrin receptor and IRAP (insulinregulated aminopeptidase). We have previously demonstrated that vpTR is trafficked by a specialized, insulin-regulated, dynamic retention mechanism in CHO cells. The specific aims of this proposal are to characterize the motifs that determine dynamic retention within the endosomal system, to identify the proteins that interact with specialized targeting motifs, and to develop a model for the molecular mechanism of dynamic retention within the endosomes. To accomplish these objectives we will combine site-directed mutagenesis and NMR structure analysis to develop structure-function models for the motifs. These models will be tested by characterizing the trafficking of mutants expressed in CHO cells. The proteins that bind these motifs will be identified using various yeast-two hybrid methods for detecting protein-protein interactions. A variety of intact and semi-intact cell assays will be used to investigate the molecular mechanism of dynamic retention. Specialized and regulated trafficking processes are key for cell function and normal whole body physiology. Not surprisingly, it is becoming increasingly clear that perturbations in membrane trafficking are a common cause of disease in humans, and it is therefore important to gain an understanding of these processes at a molecular level. The importance of dynamic retention along the biosynthetic pathway is well appreciated, and it is likely that dynamic retention within endosomes is equally important. Analysis of vpTR trafficking in CHO cells provides us the opportunity to studying this mechanism in molecular detail. The insulin-regulated, dynamic retention mechanism in CHO cells is likely to be a more common mechanism than the specialized insulin-regulated trafficking in fat cells. Consequently the results of the studies in this proposal may have signficant impact on our understanding of membrane trafficking mechanisms in general as well as being instructive for future studies in fat and muscle cells.

描述（申请人的摘要）：该项目的长期目标是 表征介导调节的动态保留的机制 内体。为了实现这一目标，我们将以中国仓鼠为特征 卵巢（CHO）细胞 vpTR 的内吞行为，vpTR 是人类与 转铁蛋白受体和 IRAP（胰岛素调节氨肽酶）。我们有 先前证明 vpTR 是由专门的、 CHO 细胞中胰岛素调节的动态保留机制。具体目标 该提案的目的是描述决定动态的主题 在内体系统内保留，以识别相互作用的蛋白质 具有专门的靶向基序，并开发分子模型 内体内动态保留的机制。为了完成这些 我们将结合定点诱变和 NMR 结构分析的目标 开发主题的结构功能模型。这些模型将 通过表征 CHO 细胞中表达的突变体的运输进行测试。这 结合这些基序的蛋白质将使用各种酵母进行鉴定——两种 检测蛋白质-蛋白质相互作用的混合方法。各种完好无损 和半完整细胞测定将用于研究分子机制 动态保留。专业化且受监管的贩运流程是关键 用于细胞功能和正常的全身生理机能。毫不奇怪，这是 越来越清楚的是，膜运输的扰动是一个 人类疾病的常见原因，因此重要的是获得 在分子水平上理解这些过程。的重要性 沿生物合成途径的动态保留受到广泛赞赏，并且 内体内的动态保留可能同样重要。分析 CHO 细胞中 vpTR 转运的研究为我们提供了研究这一问题的机会 分子细节的机制。胰岛素调节的动态保留 CHO 细胞中的机制可能是比 CHO 细胞中更常见的机制 专门由胰岛素调节的脂肪细胞运输。因此 本提案中的研究结果可能会对我们产生重大影响 对膜运输机制的一般理解以及 对未来脂肪和肌肉细胞的研究具有指导意义。