REGULATED ENDOCYTIC TRAFFIC IN FIBROBLASTS

描述（申请人摘要）：本项目的长期目标是 描述调节，动态保留的机制， 核内体为了实现这一目标，我们将对中国仓鼠进行表征 卵巢（CHO）细胞中vpTR的内吞行为， 转铁蛋白受体和IRAP（胰岛素调节的氨基肽酶）。我们有 以前证明vpTR是由一个专门的， CHO细胞中胰岛素调节的动态保留机制。具体目标 这个建议的特点是确定动态的图案， 保留在内体系统内，以鉴定相互作用的蛋白质 具有专门的靶向基序，并开发一种分子模型， 内体内的动态保留机制。完成这些 目的将联合收割机与核磁共振结构分析相结合 来建立这些图案的结构功能模型。这些模型将在 通过表征CHO细胞中表达的突变体的运输进行测试。的 结合这些基序的蛋白质将使用各种酵母-二 用于检测蛋白质-蛋白质相互作用的杂交方法。各种完好的 和半完整细胞试验将用于研究分子机制 动态保留。专门和规范的贩运程序是关键 细胞功能和正常的全身生理机能。毫不奇怪， 越来越清楚的是，膜运输中的扰动是一种 人类疾病的常见原因，因此获得一个 在分子水平上理解这些过程。的重要性 沿着生物合成途径的动态保持沿着是很好理解的， 很可能内体内的动态保留同样重要。分析 vpTR在CHO细胞中的运输为我们研究这一点提供了机会。 分子细节的机制。胰岛素调节的动态保持 CHO细胞中的机制可能是一种比 胰岛素调节的脂肪细胞运输。从而 这项建议的研究结果可能会对我们的 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。

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.