INSULIN RECEPTOR SIGNALLING

胰岛素受体信号传导

基本信息

批准号：
3246324
负责人：
IRA D. GOLDFINE
金额：
$ 14.99万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-06-01 至 1996-03-31
项目状态：
已结题

项目摘要

The insulin receptor is a alpha2beta2 tetrameric protein having an extracellular alpha subunit with an insulin binding domain and a transmembrane beta subunit with an intracellular tyrosine kinase domain. We propose to study these functional domains of the insulin receptor, and potential mechanisms of insulin receptor transmembrane signalling. We will investigate: 1) the location of the insulin binding site in the alpha subunit; 2) the role of tyrosine kinase activity in the beta subunit; and 3) post receptor signalling mechanisms via serine kinase activation. First we will continue our studies of the insulin binding site in the high cysteine domain of the receptor alpha subunit. By employing "site specific" mutagenesis and a panel of insulin analogs we will test our recent hypotheses that sequence 240-250 is an insulin binding site. Next we will "domain swap" regions of the insulin receptor alpha subunit with analogous regions of the IGF-I receptor alpha subunit. Also we will map monoclonal antibody binding sites on the alpha subunit. Second we will investigate which effects of insulin require tyrosine kinase activation by studying insulin receptor beta subunit mutants. We have preliminary data in rat HTC cells that a triple tyrosine autophosphorylation mutant (tyrosines 1158, 1162, 1163) does not undergo receptor autophosphorylation and has no detectable tyrosine kinase activity, but signals regulation of amino acid transport. We plan to express this mutant in mouse 3T3 cells where we can measure a spectrum of biological functions. We will also study the effects of insulin and ATP on receptor beta subunit conformation under conditions where receptor kinase is not activated. We will also continue our studies with insulin receptor monoclonal antibodies that are either insulin agonists or antagonists. Fab fragments will be prepared to test the hypothesis that bivalency and receptor aggregation are important for antibody (and perhaps insulin) action. Third we will investigate intermediate serine/threonine kinase(s) involved in insulin action. As a model we will investigate the kinase cascade leading to the phosphorylation of ribosome protein S6. Intermediate kinases to be investigated include: insulin receptor associated serine kinase (IRSK), multifunctional protein kinase (Fa), and microtubule associated protein kinase (MAP kinase). These studies should provide important information therefore as to the various mechanisms leading to insulin action.

胰岛素受体是一种α2β2四聚体蛋白，具有细胞外α亚基具有胰岛素结合结构域和具有细胞内酪氨酸激酶结构域的跨膜β亚基。我们建议研究胰岛素受体的这些功能域，并且胰岛素受体跨膜信号传导的潜在机制。我们将研究：1) α 中胰岛素结合位点的位置亚基； 2）β亚基酪氨酸激酶活性的作用；和 3）通过丝氨酸激酶激活的后受体信号传导机制。第一的我们将继续研究高胰岛素结合位点受体α亚基的半胱氨酸结构域。通过使用“网站特定的”诱变和一组胰岛素类似物，我们将测试我们的最近假设序列 240-250 是胰岛素结合位点。下一个我们将胰岛素受体α亚基的区域“结构域交换” IGF-I 受体α亚基的类似区域。我们还将绘制地图 α 亚基上的单克隆抗体结合位点。其次我们会研究胰岛素的哪些作用需要酪氨酸激酶激活研究胰岛素受体β亚基突变体。我们有初步数据在大鼠 HTC 细胞中，三重酪氨酸自磷酸化突变体（酪氨酸 1158、1162、1163）不发生受体自身磷酸化没有可检测到的酪氨酸激酶活性，但信号调节氨基酸运输。我们计划在小鼠 3T3 细胞中表达该突变体我们可以测量一系列生物功能。我们也会研究胰岛素和 ATP 对受体 β 亚基构象的影响在受体激酶未激活的条件下。我们也会继续我们的胰岛素受体单克隆抗体研究胰岛素激动剂或拮抗剂。 Fab 片段将准备好检验二价和受体聚集很重要的假设用于抗体（也许还有胰岛素）的作用。第三，我们将进行调查参与胰岛素作用的中间丝氨酸/苏氨酸激酶。作为一个模型中我们将研究导致磷酸化的激酶级联核糖体蛋白S6。要研究的中间激酶包括：胰岛素受体相关丝氨酸激酶（IRSK），多功能蛋白激酶 (Fa) 和微管相关蛋白激酶 (MAP 激酶)。这些因此，研究应该提供关于各种不同情况的重要信息。导致胰岛素作用的机制。