IN VITRO TRANSLATION STUDY OF HUMAN INSULIN RECEPTOR FATTY ACYLATION

人胰岛素受体脂肪酰化的体外翻译研究

• 批准号: 5211690
• 负责人: Richard Arakaki
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5211690
• 关键词: antisense nucleic acid; cell free system; complementary DNA; enzyme activity; fatty acylation; genetic manipulation; genetic translation; glucose metabolism; human tissue; insulin receptor; phosphorylation; protein biosynthesis; protein structure function; protein tyrosine kinase; site directed mutagenesis

The human insulin receptor (HIR) mediate acute metabolic responses and growth potentiation of a cell to insulin. The plasma membrane-bound receptor is a glycoprotein that upon binding insulin undergoes an intramolecular event resulting in tyrosine phosphokinase activation. The biosynthetic processing of the HIR involves a number of steps that modify the single-chain nascent proreceptor to generate the mature alpha- and beta-subunits in a heterotetrameric configuration on the cell surface. These changes in biogenesis are Asparagine (N)-linked and Ester (O)- linked glycosylation, dimerization, proteolytic cleavage, and fatty acylation. The significance to receptor function of some of these structural modification are known, i.e., N-linked glycosylation is important for translocation of the receptor to the surface membrane. However, the role of the other changes to receptor function are unknown. This project is initiated to ultimately determine the significance of fatty acylation of the HIR, specifically myristylation. The specific aims for this investigation are presented in a two-part effort to ascertain the role of fatty acylation in HIR function. the first part is a novel application of molecular biology methods to determine the site(s) of amide-linked myristylation of the proreceptor. The HIR cDNA is transcribed in vitro and mRNA translated in rabbit reticulocyte lysate to demonstrate [14C]-myristic acid labeling of the receptor precursor. Anti-sense oligodeoxynucleotides added to an in vitro translation reaction inhibits protein elongation. Oligonucleotides complementary to specific sequences of the HIR mRNA will be used to inhibit translation and produce peptides of pre-determined length. Systematic truncation of the proreceptor and differential identification of translated products with radioactive myristic acid and methionine will be used to determine the site(s) of myristylation. The second and most exciting part of the project is dependent on clear demonstration of the fatty acylation site(s). to determine the functional significance of covalently-linked fatty acids, a HIR deficient in this post-translational modification will be produced and its properties of insulin binding, tyrosine kinase activity, and the ability to mediate metabolic responses will be examined. by site-directed mutagenesis method, a HIR cDNA with the acylation site(s) substituted to produce a fatty acid deficient proreceptor will be constructed. This mutant receptor will then be inserted into a Bovine Papilloma Virus vector and expressed in NIH-3T3 cells. Functional studies will be performed in these cells and the results will be compared to studies of cells expressing the normal wild type receptor. Thus, the investigation in a cell-free system of proreceptor myristylation may provide the means to determine the role of fatty acylation of the HIR.

人胰岛素受体（HIR）介导急性代谢反应， 细胞对胰岛素的生长增强作用。 质膜结合型 受体是一种糖蛋白，与胰岛素结合后会发生 分子内事件导致酪氨酸磷酸激酶活化。 的 HIR的生物合成处理涉及许多步骤， 单链新生前受体产生成熟的α-和 在细胞表面上以异四聚体构型的β亚基。 这些生物发生的变化是天冬酰胺（N）-连锁和酯（O）-连锁。 连接的糖基化、二聚化、蛋白水解裂解和脂肪 酰化反应。 其中一些对受体功能的重要性 结构修饰是已知的，即，N-连接的糖基化是 对于受体转运到表面膜很重要。 然而，受体功能的其他变化的作用是未知的。 该项目的启动是为了最终确定 HIR的脂肪酰化，特别是肉豆蔻酰化。 具体 本次调查的目的分为两部分， 确定脂肪酰化在HIR功能中的作用。 第一部分 是一种新的应用分子生物学方法来确定 前受体的酰胺连接肉豆蔻基化位点。 HIR cDNA 在体外转录并在兔网织红细胞裂解物中翻译mRNA 以证明受体前体的[14 C]-肉豆蔻酸标记。 添加到体外翻译中的反义寡脱氧核苷酸 反应抑制蛋白质延伸。 互补寡核苷酸 HIR mRNA的特定序列将用于抑制翻译 并产生预定长度的肽。 系统截断 翻译产物的前受体和差异鉴定 与放射性肉豆蔻酸和蛋氨酸将用于确定 肉豆蔻基化的位点。 第二个也是最令人兴奋的部分 该项目依赖于脂肪酰化的明确证明 研究中心。 为了确定共价连接的 脂肪酸，缺乏这种翻译后修饰的HIR将 胰岛素结合、酪氨酸激酶 活性，以及介导代谢反应的能力将是 考察 通过定点突变的方法， 酰化位点被取代以产生脂肪酸缺陷 将构建前受体。 这种突变的受体将被 插入牛乳头瘤病毒载体并在NIH-3 T3中表达 细胞 将在这些细胞中进行功能研究， 结果将与表达正常野生型 型受体 因此，在无细胞系统中的研究 前受体肉豆蔻酰化可能提供了确定 HIR的脂肪酰化。