CELL SURFACE ANTIGENS--TRANSFERRIN RECEPTORS

这个项目的长期目标是了解结构-功能 转铁蛋白受体及其在细胞生长中的作用这个 拟议的工作将有助于我们理解分子基础 受体介导的内吞作用和循环。它还将有助于 转铁蛋白受体作为一种潜在靶点的评价 免疫疗法在癌症治疗中的应用及进展 抗体介导的细胞特异性药物靶向的有效方法。这个 具体目标是：1)描述人类的外部领域 转铁蛋白受体的免疫化学研究及转铁蛋白结合位点的定位 通过对鸡-人杂交受体的结构分析；2)确定 在氨基末端是否发现YXRF内化信号 人转铁蛋白受体的胞浆结构域足以 诱导其他II型膜蛋白的快速内吞；3)表达 并分离出足够量的可溶性人转铁蛋白受体 用于生物物理鉴定的细胞质结构域；4)鉴定 转铁蛋白受体胞质结构域与蛋白质的相互作用 涂层坑的结构组件；5)调查 人转铁蛋白受体胞浆结构域在内体-内体融合中的作用； 和6)表征细胞内的隔室，通过这些隔室 转铁蛋白受体在内吞和循环过程中的交通。 为了实现这些目标，我们将使用广泛的实验方法 源自分子生物学、蛋白质生物化学、细胞生物学和 免疫学。具体地说，我们将利用：1)cDNA的可用性 人、鼠和鸡的转铁蛋白受体；2)逆转录病毒 表达突变型人转铁蛋白受体的表达系统 鸡胚成纤维细胞及其生物学活性分析 细菌中的高产蛋白质表达系统。中国仓鼠卵巢 细胞和空泡病毒感染的昆虫细胞使 完整的转铁蛋白受体的表达，并且独立地， 胞质和外区作为可溶性重组蛋白；4)和 30多种针对外源和外源病毒的单抗 人转铁蛋白受体的胞质结构域。一个重大的新方面 工作的重点是开发必要的材料和方法来确定 胞质和胞外结构域的三维结构 人类转铁蛋白受体。以前的研究将检验效度 在我们最近提出的模型中，第一类转向是结构 高效内吞作用的识别基序。

The long-term goal of this project is to understand the structure-function relationship of the transferrin receptor and its role in cell growth. The proposed work will contribute to our appreciation of the molecular basis of receptor-mediated endocytosis and recycling. It will also contribute to the evaluation of the transferrin receptor as a potential target for immunotherapy in the treatment of cancer and the development of more effective methods of antibody-mediated cell-specific drug targeting. The specific aims are: 1) to characterize the external domain of the human transferrin receptor immunochemically and map the transferrin binding site by structural analysis of chick-human hybrid receptors; 2) to determine whether the YXRF internalization signal found in the amino-terminal cytoplasmic domain of the human transferrin receptor is sufficient to induce rapid endocytosis of other Type II membrane proteins; 3) to express and isolate sufficient soluble human transferrin receptor external and cytoplasmic domains for biophysical characterization; 4) to characterize the interaction of the transferrin receptor cytoplasmic domain with structural components of coated pits; 5) to investigate the role of the human transferrin receptor cytoplasmic domain in endosome-endosome fusions; and 6) to characterize the intracellular compartments through which the transferrin receptor traffics during endocytosis and recycling. To achieve these goals, we will use a broad range of experimental methods derived from molecular biology, protein biochemistry, cell biology, and immunology. Specifically, we will exploit: 1) the availability of cDNAs for the human mouse and chicken transferrin receptors; 2) a retroviral expression system for expressing mutant human transferrin receptors in chick embryo fibroblasts and analysis of their biological activity; 3) high-yield protein expression systems in bacteria. Chinese hamster ovary cells and vaculovirus-infected insect cells that have allowed the expression of the intact transferrin receptor, and independently, the cytoplasmic and external domains as soluble recombinant proteins; 4) and more than 30 monoclonal antibodies against both the external and cytoplasmic domains of the human transferrin receptor. A major new aspect of the work is to develop the materials and methods necessary to determine the 3-dimensional structure of both the cytoplasmic and external domains of the human transferrin receptor. The former studies will test the validity of the model we have recently proposed that a Type I turn is the structural recognition motif for high-efficiency endocytosis.