IMMUNOLOGICAL ANALYSIS OF LDL RECEPTOR DEFECTIVE CELLS

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Understanding in greater molecular detail the mechanism of low density lipoprotein (LDL) receptor-mediated endocytosis and Golgi apparatus structure and function are the long term objectives of this proposal. The molecular mechanisms by which the structure and processing of LDL receptors determine their function will be examined in detail in wild-type and LDL receptor-deficient mutant Chinese hamster ovary (CHO) cells. Anti-LDL receptor antibodies will be used to study 4 groups of genetically distinct CHO mutants, ldlA, ldlB, ldlC and ldlD. The survey and analysis of our large collection of ldlA (structural gene) mutants, with special emphasis on those mutant types which have not been identified in human familial hypercholesterolemics, will help define the relationship of receptor structure and function. Biochemical. ultrastructural, and gene cloning (ldlB only) experiments will be used to deduce the key biochemical defects which cause the pleiotropic Golgi abnormalities in the ldlB and ldlC mutants. Further analysis of the receptor's structure, including detailed carbohydrate analysis, and its abnormally rapid turnover in ldlB-ldlD mutants (ldlD is UDP- Gal/UDP-GAlNAc deficient) will help define the role of glycosylation in determining receptor function. In addition, the analysis of endogenous hamster and transfected human LDL receptor processing in all 4 classes of ldl mutants may help identify important determinants of intracellular protein sorting and new mechanisms of protein degradation. For example, several mutant forms of the human LDL receptor (e.g., an internalization defective receptor) will be transfected into the ldlB-ldlD mutants, and the effects of the ldlB-ldlD mutations on the mutant receptors will be examined. This information will be useful not only because of its direct relevance to LDL receptors and to cholesterol and LDL metabolism (and thus hypercholesterolemia and atherosclerosis), but also because the LDL receptor provides a powerful model for many other surface receptors and membrane glycoproteins. Our unique mutants and the powerful immunochmical tools developed during the provious grant period provide us with the opportunity to answer fundamental questions about mammalian cell biology in general, and LDL receptors and endocytosis in particular.