The goals of the proposal are to use the human transferrin receptor as a model system to elucidate the proteins involved in the uptake of receptors into cells. Receptor-mediated endocytosis is a fundamental process in eukaryotic cells which allows for the specific internalization of proteins. The transferrin receptor is an excellent model system for the study of this phenomenon. Transferrin is the major iron transport protein in the blood of vertebrates and some invertebrates. All proliferating cells require a net uptake of iron into the cell for incorporation into cytoplasmic and mitochondrial proteins and therefore have measurable numbers of transferrin receptors. The first specific aim of this proposal is to test the hypothesis that receptors interact with the same set of proteins associated with clathrin- coated pits. DNA coding for full length transferrin receptor will be stably overexpressed to levels which will saturate the endocytic path way of this receptor. The effect of overexpression of the transferrin receptor on the internalization of other ligands will be measured to determine whether they compete for the same proteins in the endocytic path way. The second specific aim is to determine the portions of the transferrin receptor sufficient to interact with the endocytic apparatus. Initially, five different constructs of the transferrin receptor ranging in size from the cytoplasmic domain to the cytoplasmic through transmembrane and part of the extracellular domain will be generated by site-directed mutagenesis. Cell lines expressing these constructs will be selected. The efficiency of internalization of the constructs and/or the ability to inhibit the endocytosis of the native transferrin receptor will be measured. The third specific aim is to identify the proteins that interact with the transferrin receptor. In vitro competition assays using portions of the cytoplasmic domain of the transferrin receptor will be used to determine if the cytoplasmic portion of the receptor reacts directly with a clathrin-associated protein, AP-2, implicated in receptor interactions, or with proteins within the plasma membrane. Cleavable crosslinking agents will be used to identify transferrin receptor-binding proteins. Affinity chromatography and gel filtration will be per-formed to measure both high and low affinity interactions of the cytoplasmic domain of the transferrin receptor with other proteins. Affinity chromatography will be used to isolate TR binding proteins.
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