BIOSYNTHESIS AND PROCESSING OF THE IGF-II RECEPTOR

这个项目的长期目标是提高对 质膜生物合成的机制及调控步骤 多肽激素受体。胰岛素样生长因子II(IGF-II) H-35肝癌细胞中的受体将作为这些的模型进行研究 感受器。胰岛素样生长因子-II的生物合成和出现动力学 受体将在脉冲追逐研究中使用示踪氨基酸进行测量。 来自质膜的标记受体将被免疫沉淀，并且 用十二烷基硫酸钠电泳法进行定量。动力学 新合成受体在其细胞内途径上的转运 通过用放射性标记细胞来分析朝向细胞表面 或稠密的氨基酸，然后监测标记的外观 亚细胞膜部分中的受体随时间的变化(例如，微粒体， 高尔基体)。IGF-II的翻译后和共翻译蛋白水解性修饰 将对受体进行检查。最初可能的前导序列 合成的受体将通过受体mRNA的翻译进行分析 牛血清白蛋白N端残基的体外免疫沉淀及分析 翻译产品使用丹磺酰氯。新生的IGF-II受体链 贴在tRNA上，通过凝胶电泳法进行标记和分级以 确定受体是否被翻译成单链。糖基化 将在用~3H-糖进行代谢标记后对该受体进行研究 利用细胞低聚糖侧链加工的抑制剂 (莫能菌素、衣霉素)。抑制物诱导的参入改变 标记的糖将在凝胶电泳后被测量 从亚细胞膜部分分离的免疫沉淀受体。 受体低聚糖侧链的加工程度 也将在这些实验中通过处理标记的受体进行检查 带有内切糖苷酶H，选择性地去除“高甘露糖” 未加工的N-连接低聚糖和内切糖苷酶F，它 同时对“高甘露糖”和“复杂”(完全加工)端进行水解 锁链。最后，加工抑制剂对受体的影响 性质，即125I-IGF-II的结合和新的插入速率 受体进入质膜，将被研究以确定 受体糖基化的功能意义。

The long-range goal of this project is to improve understanding of the mechanism and regulatory steps governing biosynthesis of plasma membrane polypeptide hormone receptors. The insulin-like growth factor II (IGF-II) receptor in H-35 hepatoma cells will be studied as a model for these receptors. The kinetics of biosynthesis and appearance of the IGF-II receptor will be measured in pulse-chase studies using tracer amino acids. Labeled receptors from the plasma membrane will be immunoprecipitated, and then quantitated by sodium dodecyl sulfate electrophoresis. The kinetics of transit of newly-synthesized receptor on its intracellular pathway toward the cell surface will be analyzed by labeling cells with radioactive or dense amino acids and then monitoring the appearance of labeled receptors with time in subcellular membrane fractions (e.g., microsomes, Golgi). Post-and co-translational proteolytic modifications of the IGF-II receptor will be examined. Possible leader sequences on the initially synthesized receptor will be analyzed by translation of receptor mRNA in vitro, immunoprecipitation and analysis of the N-terminal residues of the translation products using dansyl chloride. Nascent IGF-II receptor chains attached to tRNA will be labeled and fractionated by gel electrophoresis to determine if the receptor is translated as a single chain. Glycosylation of the receptor will be studied after metabolic labeling with 3H-sugars using inhibitors of cellular oligosaccharide side-chain processing (monensin, tunicamycin). Inhibitor-induced changes in incorporation of the labeled sugars will be measured after gel electrophoresis of the immunoprecipitated receptors isolated from subcellular membrane fractions. The extent of processing of the oligosaccharide side chains of the receptor will also be examined in these experiments by treating the labeled receptor with endoglycosidase H, which selectively remove "high mannose" underprocessed N-linked oligosaccharides, and endoglycosidase F, which hydrolyzes both "high mannose" and "complex" (fully processed) side chains. Finally, the effects of the processing inhibitors on receptor properties i.e., binding of 125I-IGF-II and rate of insertion of new receptor into the plasma membrane, will be studied to determine the functional significance of receptor glycosylation.