BIOSYNTHESIS AND PROCESSING OF THE IGF-II RECEPTOR

IGF-II 受体的生物合成和加工

• 批准号: 3446013
• 负责人: RICHARD G. MACDONALD
• 金额: $ 5.79万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1987-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3446013
• 关键词: gel electrophoresis; genetic translation; immunologic techniques; insulinlike factor; neoplastic cell; oligosaccharides; peptide hormone biosynthesis; proteolysis; radiotracer; scintillation counter

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.

该项目的长期目标是提高对 质膜生物合成的机制和调控步骤 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研究的模型进行研究。 受体。 IGF-II的生物合成和出现的动力学 将在脉冲追踪研究中使用示踪氨基酸测量受体。 来自质膜的标记受体将被免疫沉淀， 然后通过十二烷基硫酸钠电泳定量。 动力学 新合成的受体在其细胞内途径上的转运 将通过用放射性标记细胞来分析朝向细胞表面的 或密集氨基酸，然后监测标记的 受体在亚细胞膜部分中随时间的变化（例如，微粒体， Golgi）。 IGF-II的翻译后和共翻译蛋白水解修饰 将对受体进行检测。 可能的前导序列 合成的受体将通过受体mRNA的翻译来分析， 体外，免疫沉淀和N-末端残基的分析， 使用丹磺酰氯的翻译产物。 新生IGF-II受体链 连接到tRNA上的蛋白质将被标记，并通过凝胶电泳进行分级， 确定受体是否被翻译为单链。 基化 将在用3H-糖代谢标记后研究受体的活性 使用细胞寡糖侧链加工抑制剂 （莫能菌素、衣霉素）。 抑制剂诱导的掺入变化 标记的糖将在凝胶电泳后测量。 从亚细胞膜部分分离的免疫沉淀受体。 受体寡糖侧链的加工程度 也将在这些实验中通过处理标记的受体来检查 与内切糖苷酶H，其选择性地去除"高甘露糖" 加工不足的N-连接寡糖和内切糖苷酶F， 水解"高甘露糖"和"复杂"（完全加工）侧 店 最后，研究了加工抑制剂对受体的影响。 特性即，125I-IGF-II的结合和新的插入率 受体进入质膜，将进行研究，以确定 受体糖基化的功能意义。