Kishimoto M, Hashiramoto M, Yonezawa K, Shii K, Kazumi T, Kasuga M
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
J Biol Chem. 1994 Apr 15;269(15):11349-55.
We studied a patient with severe insulin resistance and a remarkable decrease in the in vivo autophosphorylation of the insulin receptor. Using a polymerase chain reaction-single strand conformation polymorphism method and direct sequencing, we identified a heterozygous mutation substituting Gln for Arg1131 in the putative "catalytic loop" of the tyrosine kinase domain of the insulin receptor gene. The Gln1131 mutant receptor was expressed by transfection in Chinese hamster ovary cells and compared with cells expressing the wild-type insulin receptor. Both mutant and wild-type receptors were expressed on the cell surface and displayed similar insulin-binding affinity. The Gln1131 mutation impaired the activity of the receptor tyrosine kinase and inhibited the ability of insulin to phosphorylate the endogenous substrate insulin receptor substrate-I. In addition, the Gln1131 mutant receptor exhibited diminished tyrosine-phosphorylated phosphatidylinositol 3-kinase and myelin basic protein kinase activities compared with the wild-type cells. It also demonstrated a defective mediation of the insulin signal stimulating 2-deoxy-D-glucose transport and thymidine incorporation, resistance to endocytosis, and insulin-induced down-regulation. Unlike a previously described mutation in the putative catalytic loop of the receptor that substituted Glu for Ala1135, the Gln1131 mutation retained proteolytic cleavage of the proreceptor into separate subunits. Our results demonstrate that a naturally occurring mutation (R1131Q) in the putative catalytic loop of the insulin receptor results in severe impairment of the tyrosine kinase function in our patient. In addition, our results indicate that Arg1131 is important for receptor-mediated insulin action in vivo and suggest that the amino acids constituting the catalytic loop of protein kinases may possess different modes in order to retain kinase function.
我们研究了一名严重胰岛素抵抗且胰岛素受体体内自磷酸化显著降低的患者。通过聚合酶链反应 - 单链构象多态性方法和直接测序，我们在胰岛素受体基因酪氨酸激酶结构域的假定“催化环”中鉴定出一个杂合突变，该突变用谷氨酰胺（Gln）替代了精氨酸（Arg）1131。通过转染在中国仓鼠卵巢细胞中表达谷氨酰胺1131突变体受体，并与表达野生型胰岛素受体的细胞进行比较。突变体和野生型受体均在细胞表面表达，且显示出相似的胰岛素结合亲和力。谷氨酰胺1131突变损害了受体酪氨酸激酶的活性，并抑制了胰岛素磷酸化内源性底物胰岛素受体底物 - I的能力。此外，与野生型细胞相比，谷氨酰胺1131突变体受体的酪氨酸磷酸化磷脂酰肌醇3 - 激酶和髓鞘碱性蛋白激酶活性降低。它还表现出在介导胰岛素信号刺激2 - 脱氧 - D - 葡萄糖转运和胸苷掺入、抵抗内吞作用以及胰岛素诱导的下调方面存在缺陷。与先前描述的在受体假定催化环中用谷氨酸（Glu）替代丙氨酸（Ala）1135的突变不同，谷氨酰胺1131突变保留了前体受体蛋白水解切割成单独亚基的过程。我们的结果表明，胰岛素受体假定催化环中的一个自然发生的突变（R1131Q）导致了我们患者酪氨酸激酶功能的严重受损。此外，我们的结果表明精氨酸1131对体内受体介导的胰岛素作用很重要，并提示构成蛋白激酶催化环的氨基酸可能具有不同模式以保留激酶功能。
