IMMUNOLOGICAL ANALYSIS OF LDL RECEPTOR DEFECTIVE CELLS

在更大的分子细节的理解低的机制， 密度脂蛋白（LDL）受体介导的内吞作用， 高尔基体的结构和功能是长期的 这一提案的目标。 的分子机制 LDL受体的结构和加工决定了它们 将在野生型和LDL中详细检查功能 受体缺陷型突变中国仓鼠卵巢（CHO）细胞。 抗LDL受体抗体将用于研究4组 遗传上不同的CHO突变体，ldlA、ldlB、ldlC和ldlD。 本文对我国大量收集的低密度脂蛋白A（结构型）进行了调查和分析， 基因）突变体，特别强调那些突变类型， 在人类家族性高胆固醇血症中尚未发现， 将有助于确定受体结构和 功能 生物化学的。超微结构和基因克隆（ldlB 只有）实验将被用来推断关键的生化 缺陷，导致多效性高尔基体异常， ldlB和ldlC突变体。 对受体的进一步分析 结构，包括详细的碳水化合物分析， ldlB-ldlD突变体（ldlD是UDP- Gal/UDP-GAlNAc缺陷）将有助于确定 糖基化决定受体功能。 此外该 内源性仓鼠和转染的人LDL的分析 在所有4类LDL突变体中的受体加工可能有助于 鉴定细胞内蛋白质分选重要决定因素 和蛋白质降解的新机制。 比如说， 人低密度脂蛋白受体的几种突变形式（例如，一个 内化缺陷型受体）将被转染到 ldlB-ldlD突变体，以及ldlB-ldlD突变对 将检查突变受体。 这些信息将是有用的，不仅因为它的直接 与LDL受体、胆固醇和LDL的相关性 代谢（以及因此高胆固醇血症和动脉粥样硬化）， 还因为LDL受体为 许多其他表面受体和膜糖蛋白。 我们 独特的突变体和强大的免疫化学工具开发 在临时拨款期间， 来回答哺乳动物细胞生物学的基本问题， 一般，LDL受体和内吞作用，特别是。

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.