CELL SURFACE ANTIGENS--TRANSFERRIN RECEPTORS

本项目的长期目标是了解结构-功能 转铁蛋白受体的关系及其在细胞生长中的作用。 的 拟议的工作将有助于我们了解的分子基础， 受体介导的内吞作用和再循环。 它还将协助 转铁蛋白受体作为潜在靶点的评价 免疫疗法在治疗癌症和发展更多 抗体介导的细胞特异性药物靶向的有效方法。 的 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和地图转铁蛋白结合位点 通过鸡-人杂交受体的结构分析; 2）确定 在氨基末端发现的YXRF内化信号 人转铁蛋白受体的胞质结构域足以 诱导其他II型膜蛋白快速内吞; 3）表达 并分离足够的可溶性人转铁蛋白受体， 用于生物物理表征的胞质结构域; 4）表征 转铁蛋白受体胞质结构域与 涂层坑的结构成分; 5）调查的作用， 内体-内体融合中人转铁蛋白受体胞质结构域; 和6）表征细胞内区室， 转铁蛋白受体在内吞作用和再循环期间运输。 为了实现这些目标，我们将使用广泛的实验方法 衍生自分子生物学、蛋白质生物化学、细胞生物学， 免疫学 具体来说，我们将利用：1）cDNA的可用性 人、小鼠和鸡转铁蛋白受体; 2）逆转录病毒 用于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 细菌中的高产蛋白表达系统。 中国仓鼠卵巢 细胞和空泡病毒感染的昆虫细胞， 完整转铁蛋白受体的表达，并且独立地， 作为可溶性重组蛋白的胞质和外部结构域; 4）和 超过30种单克隆抗体， 人转铁蛋白受体的胞质结构域。 一个主要的新方面 的工作是开发必要的材料和方法，以确定 细胞质和外部结构域的三维结构 人类转铁蛋白受体 以往的研究将检验其有效性 我们最近提出的模型，即I型转弯是结构性的 用于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.