Understanding in greater molecular detail the key role played by receptor-mediated endocytosis in a variety of cellular processes such as lipid metabolism and its derangements (e.g., familial hypercholesterolemia) is the long-term objective of the proposed research. Receptor-mediated endocytosis is a process by which cells bind and internalize transport proteins and peptide hormones. To understand in greater detail the mechanism by which animal cells internalize the cholesterol transport protein low density lipoprotein (LDL), we have isolated dozens of clones of mutant Chinese hamster ovary (CHO) cells that cannot take up LDL normally. The mutant clones represent at least three different complementation groups, including mutants in the gene for the LDL receptor. The goal of this proposal is the isolation of antibodies which react with the LDL receptor of CHO cells and the utilization of these antibodies to examine the structure of the LDL receptor in normal and mutant cells. We will initially use immunoblotting and immunoprecipitation procedures to characterize the receptors. We anticipate characterizing several classes of mutants, including mutants in the gene for the receptor and mutants in genes which affect the structure of the receptor. These classes would include those with receptors which can bind LDL but cannot be delivered properly to the cell surface and with receptors which are delivered to the cell surface but cannot bind LDL, as well as those with incompletely processed receptors. These classes should lead to greater insights into the relationship between the structure and the function of the LDL receptor, and should therefore contribute to our basic understanding of cellular lipoprotein metabolism.
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