Two naturally occurring mutant insulin receptors phosphorylate insulin receptor substrate-1 (IRS-1) but fail to mediate the biological effects of insulin - Evidence that IRS-1 phosphorylation is not sufficient for normal insulin action
Two naturally occurring mutant insulin receptors phosphorylate insulin receptor substrate-1 (IRS-1) but fail to mediate the biological effects of insulin - Evidence that IRS-1 phosphorylation is not sufficient for normal insulin action
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DOI：
10.1074/jbc.271.12.7134
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发表时间：
1996-03-22
影响因子：
4.8
通讯作者：
ORahilly, S
中科院分区：
文献类型：
--
作者：
Krook, A;Moller, DE;ORahilly, S
在显性遗传A型胰岛素抵抗患者中发现的两种天然突变型胰岛素受体Arg-1174 --> Gln和Leu-1178 --> Pro在中国仓鼠卵巢（CHO）细胞中稳定表达时显示出不寻常的信号传导特性。这两种突变体受体在细胞表面上表达，并正常结合胰岛素，但对胰岛素的自磷酸化反应明显受损。此外，在体外酪氨酸激酶活性的突变体受体对人工基板也严重受损。尽管有这些缺陷的激酶活性，抗磷酸酪氨酸免疫印迹的全细胞裂解物和抗磷酸酪氨酸免疫沉淀的P-32标记的细胞显示胰岛素刺激的酪氨酸磷酸化的蛋白质的相似的185 kDa的程度相媲美的CHO细胞表达野生型人胰岛素受体。抗胰岛素受体底物-1（IRS-1）免疫沉淀，然后抗磷酸酪氨酸免疫印迹证实这种酪氨酸磷酸化蛋白质是IRS-1。相比之下，表达在ATP结合位点突变的胰岛素受体（Lys-1030 --> Arg）的CHO细胞没有显示胰岛素刺激的IRS-1的自磷酸化或磷酸化。尽管表现出IRS-1酪氨酸磷酸化的明显正常胰岛素刺激，但表达Arg-1174 --> Gln或Pro-1178 --> Leu受体的细胞在胰岛素刺激糖原合成、胸苷掺入和MAP激酶活化方面表现出明显的损伤。这些突变受体不能正常地向代谢和促有丝分裂反应发出信号，这表明胰岛素刺激的IRS-1酪氨酸磷酸化单独不足以完全介导胰岛素作用。
Two naturally occurring mutant insulin receptors, Arg-1174 --> Gln and Leu-1178 --> Pro, found in patients with dominantly inherited Type A insulin resistance, showed unusual signaling properties when stably expressed in Chinese hamster ovary (CHO) cells. Both mutant receptors were expressed on the cell surface and bound insulin normally, but showed markedly impaired autophosphorylation in response to insulin. In addition, the in vitro tyrosine kinase activity of both mutant receptors toward an artificial substrate was also severely impaired. Despite these defects of kinase activity, antiphosphotyrosine immunoblotting of whole cell lysates and anti-phosphotyrosine immunoprecipitation of P-32-labeled cells showed insulin-stimulated tyrosine phosphorylation of a protein of similar to 185 kDa to an extent comparable to that seen in CHO cells expressing wild-type human insulin receptors. Anti-insulin receptor substrate-1 (IRS-1) immunoprecipitation followed by antiphosphotyrosine immunoblotting confirmed that this tyrosine-phosphorylated protein was IRS-1. In contrast, CHO cells expressing an insulin receptor mutated at the ATP binding site (Lys-1030 --> Arg) showed no insulin-stimulated autophosphorylation or phosphorylation of IRS-1. Despite exhibiting apparently normal insulin stimulation of IRS-1 tyrosine-phosphorylation, cells expressing the Arg-1174 --> Gln or Pro-1178 --> Leu receptors showed marked impairment in insulin stimulation of glycogen synthesis, thymidine incorporation, and activation of MAP kinase. The inability of these mutant receptors to signal normally to metabolic and mitogenic responses suggests that insulin-stimulated tyrosine phosphorylation of IRS-1 alone is insufficient to fully mediate insulin action.