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.
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