IN VITRO TRANSLATION STUDY OF HUMAN INSULIN RECEPTOR FATTY ACYLATION

人胰岛素受体(HIR)介导急性代谢反应和 细胞对胰岛素的生长促进作用。质膜结合 受体是一种糖蛋白，当与胰岛素结合时，它经历一种 分子内事件导致酪氨酸磷酸激酶的激活。这个 HIR的生物合成处理涉及许多步骤，这些步骤修改 产生成熟α-和α-受体的单链新生前受体 细胞表面异四聚体构型中的β-亚基。 这些生物发生的变化是天冬酰胺(N)连接和酯(O)- 连接糖基化、二聚化、蛋白水解性裂解和脂肪 酰化反应。其中一些对受体功能的意义 结构修饰是已知的，即N-连接的糖基化 对于受体向表面膜的移位很重要。 然而，受体功能的其他变化的作用尚不清楚。 启动该项目的目的是最终确定 HIR的脂肪酰化，特别是肉豆蔻化。具体的 这次调查的目的是通过两个部分的努力来提出的 确定脂肪酰化在HIR功能中的作用。第一部分 是一种新的分子生物学方法的应用来确定 前体受体的酰胺连接肉豆蔻化位点(S)。Hir基因的cdna 在体外转录，在兔网织红细胞裂解液中翻译成mRNA [14C]-肉豆蔻酸标记受体前体。 在体外翻译中加入反义寡核苷酸 反应会抑制蛋白质的伸长。互补的寡核苷酸 Hir基因的特定序列将被用来抑制翻译 并产生预定长度的多肽。系统地截断 翻译产物的前受体及其鉴别 具有放射性的肉豆蔻酸和蛋氨酸将被用于检测 肉豆蔻化位点(S)。第二个也是最令人兴奋的部分 项目依赖于脂肪酰化的清晰演示 网站(S)。确定共价连接的功能意义 脂肪酸，一种缺乏这种翻译后修饰的HIR将 及其与胰岛素结合的酪氨酸激酶的性质 活性，以及调节代谢反应的能力将是 检查过了。通过定点突变的方法，获得了hIR基因。 取代的酰化位点(S)产生的脂肪酸缺乏 将构建前受体。这个突变的受体将会是 插入牛乳头瘤病毒载体并在NIH-3T3中表达 细胞。将在这些细胞和 结果将与表达正常野生型细胞的研究进行比较 类型受体。因此，在无细胞系统中的研究 受体前肌氨酸化可能提供了确定其作用的手段 HIR的脂肪酰化。

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.