Insulin stimulates tyrosine phosphorylation of its receptor beta-subunit in intact rat hepatocytes.
Insulin stimulates tyrosine phosphorylation of its receptor beta-subunit in intact rat hepatocytes.
复制标题
胰岛素刺激完整大鼠肝细胞中其受体β亚基的酪氨酸磷酸化。
DOI：
10.1042/bj2410099
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
E. van Obberghen
中科院分区：
文献类型：
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作者：
R. Ballotti;A. Kowalski;M. White;Y. Le Marchand;E. van Obberghen
我们研究了完整的新鲜分离的大鼠肝细胞中胰岛素受体β亚基的磷酸化，用[32 P]Pi标记。用胰岛素受体抗体或磷酸酪氨酸抗体免疫沉淀经麦胚凝集素层析法部分纯化的胰岛素受体。在不存在胰岛素的情况下孵育的细胞衍生的受体仅含有磷酸丝氨酸。向肝细胞中添加胰岛素导致受体β亚基磷酸化的剂量依赖性增加，在2 nM胰岛素下观察到半最大刺激。用100 nM胰岛素孵育细胞显示，在暴露于激素1分钟内，发生最大受体磷酸化，随后略有下降，然后达到平台。这种胰岛素诱导的受体磷酸化刺激主要是由酪氨酸残基的磷酸化引起的。用抗磷酸酪氨酸抗体和抗受体抗体对受体进行顺序免疫沉淀，并对免疫沉淀的受体进行磷酸氨基酸分析，结果表明，未能进行酪氨酸磷酸化的受体在丝氨酸残基上被磷酸化。在正常细胞中的功能性胰岛素敏感性胰岛素受体激酶的证明强烈支持了这种受体酶活性在介导胰岛素的生物学效应中的作用。
We studied the phosphorylation of the beta subunit of the insulin receptor in intact freshly isolated rat hepatocytes, labelled with [32P]Pi. Insulin receptors partially purified by wheat-germ agglutinin chromatography were immunoprecipitated with either antibodies to insulin receptor or antibodies to phosphotyrosine. Receptors derived from cells incubated in the absence of insulin contained only phosphoserine. Addition of insulin to hepatocytes led to a dose-dependent increase in receptor beta-subunit phosphorylation, with half-maximal stimulation being observed at 2 nM-insulin. Incubation of cells with 100 nM-insulin showed that, within 1 min of exposure to the hormone, maximal receptor phosphorylation occurred, which was followed by a slight decrease and then a plateau. This insulin-induced stimulation of its receptor phosphorylation was largely accounted for by phosphorylation on tyrosine residues. Sequential immunoprecipitation of receptor with anti-phosphotyrosine antibodies and with anti-receptor antibodies, and phosphoamino acid analysis of the immunoprecipitated receptors, revealed that receptors that failed to undergo tyrosine phosphorylation were phosphorylated on serine residues. The demonstration of a functional hormone-sensitive insulin-receptor kinase in normal cells strongly supports a role for this receptor enzymic activity in mediating biological effects of insulin.