Macaulay S L, Polites M, Frenkel M J, Hewish D R, Ward C W
CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia.
Biochem J. 1995 Mar 15;306 ( Pt 3)(Pt 3):811-20. doi: 10.1042/bj3060811.
Native human insulin receptor (hIR) has been reported to contain only one free thiol group proposed to lie near the ATP-binding. domain of its beta-subunit [Finn, Ridge and Hofmann (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 419-423]. The present study investigated the role of the six cytoplasmic cysteines of the beta-subunit of the hIR using a mutagenic approach in which insulin receptors, mutated at each cytoplasmic cysteine (to alanine) in turn, were transfected into Chinese hamster ovary (CHO) cells. Cell lines expressing hIR mutation at high level were obtained which, by both flow-cytometric analysis towards an hIR-specific monoclonal antibody (83-7) and insulin-binding analysis, were similar to the well-characterized CHOT cell line which overexpresses native hIR. The ED50 and Kd values of the mutant receptors were the same as those of the wild-type hIR. Each of the mutant receptors signalled insulin action to stimulate receptor autophosphorylation and kinase activity as well as glucose utilization to levels appropriate for the receptor level expressed. In contrast, insulin-stimulated thymidine uptake and glucose-transport responses of two of the six mutant cell lines, those expressing Cys981Ala and Cys1245Ala, were impaired compared with that of the native hIR-expressing cell line, CHOT. The beta-subunits of each of the hIR cytoplasmic cysteine mutant cell lines could be alkylated specifically with N-[3H]ethylmaleimide. The kinase activity of each receptor was inhibited by N-ethylmaleimide and stimulated by iodoacetamide, indicating that none of the cytoplasmic cysteines alone contributes the single free thiol group to the hIR structure. We conclude that the cytoplasmic cysteines of the hIR have a predominantly passive role in hIR activity although Cys-981 and Cys-1245 do affect mitogenic and glucose-transport responses of the receptor. Our findings indicate that the stoicheiometry of a single free thiol group/mol of insulin-binding activity noted in previous studies is either spread fractionally over a number of the cytoplasmic cysteines or is one of the four cysteines in the ectodomain of the hIR beta-subunit. Alternatively, the mutagenesis performed in the present study may enable differential exposure of a second titratable cysteine in wild-type and mutant receptors.
据报道，天然人胰岛素受体（hIR）仅含有一个游离巯基，该巯基被认为位于其β亚基的ATP结合结构域附近[芬恩、里奇和霍夫曼（1990年）《美国国家科学院院刊》87卷，419 - 423页]。本研究采用诱变方法研究了hIRβ亚基的六个胞质半胱氨酸的作用，即将胰岛素受体依次在每个胞质半胱氨酸处突变为丙氨酸后转染到中国仓鼠卵巢（CHO）细胞中。获得了高水平表达hIR突变体的细胞系，通过针对hIR特异性单克隆抗体（83 - 7）的流式细胞术分析和胰岛素结合分析，这些细胞系与已充分表征的过表达天然hIR 的CHOT细胞系相似。突变受体的ED50和Kd值与野生型hIR相同。每个突变受体都能传递胰岛素作用信号，刺激受体自身磷酸化和激酶活性以及葡萄糖利用，使其达到与所表达受体水平相适应的水平。相比之下，六个突变细胞系中的两个，即表达Cys981Ala和Cys1245Ala的细胞系，其胰岛素刺激的胸苷摄取和葡萄糖转运反应与表达天然hIR的细胞系CHOT相比有所受损。每个hIR胞质半胱氨酸突变细胞系的β亚基都可以用N - [³H]乙基马来酰亚胺进行特异性烷基化。每个受体的激酶活性都受到N - 乙基马来酰亚胺的抑制并被碘乙酰胺刺激，这表明单独的任何一个胞质半胱氨酸都不会为hIR结构贡献单一的游离巯基。我们得出结论，hIR的胞质半胱氨酸在hIR活性中主要起被动作用，尽管Cys - 981和Cys - 1245确实会影响受体的促有丝分裂和葡萄糖转运反应。我们的研究结果表明，先前研究中所指出的每摩尔胰岛素结合活性中单个游离巯基的化学计量比要么分散在多个胞质半胱氨酸上，要么是hIRβ亚基胞外结构域中的四个半胱氨酸之一。或者，本研究中进行的诱变可能会使野生型和突变型受体中第二个可滴定半胱氨酸的暴露情况有所不同。
