Tollefsen S E, Stoszek R M, Thompson K
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri.
Biochemistry. 1991 Jan 8;30(1):48-54. doi: 10.1021/bi00215a008.
We have recently found that association of the two alpha beta dimers of the insulin-like growth factor I (IGF I) receptor is required for formation of a high-affinity binding site for IGF I [Tollefsen, S. E., & Thompson, K. (1988) J. Biol. Chem. 263, 16267-16273]. To determine the structural requirements for IGF I activated kinase activity, we have examined the effect of dissociation of the two alpha beta dimers of the IGF I receptor on beta subunit autophosphorylation. The alpha beta dimers formed after treatment with 2 mM dithiothreitol (DTT) at pH 8.75 for 5 min were separated from IGF I receptor remaining as tetramers after DTT treatment by fast protein liquid chromatography on a Superose 6 gel filtration column. Purification of the alpha beta dimers was confirmed by Western blot analysis using 125I-labeled alpha IR-3, a monoclonal antibody to the IGF I receptor. Autophosphorylation of the IGF I receptor (alpha beta)2 tetramer, treated without DTT or remaining after DTT treatment, is stimulated 1.6-2.9-fold by IGF I. In contrast, autophosphorylation of the alpha beta dimers incubated in the presence or absence of IGF I (100 ng/mL) does not occur. Both IGF I receptor dimers and tetramers exhibit similar kinase activities using the synthetic substrate Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, indicating that the failure to detect autophosphorylation of the IGF I receptor dimers does not result from inactivation of the kinase by DTT treatment. We conclude that autophosphorylation of the IGF I receptor depends upon the interaction of the two alpha beta dimers.
我们最近发现，胰岛素样生长因子I（IGF I）受体的两个αβ二聚体的缔合对于形成IGF I的高亲和力结合位点是必需的[Tollefsen, S. E., & Thompson, K. (1988) J. Biol. Chem. 263, 16267 - 16273]。为了确定IGF I激活激酶活性的结构要求，我们研究了IGF I受体的两个αβ二聚体解离对β亚基自身磷酸化的影响。在pH 8.75下用2 mM二硫苏糖醇（DTT）处理5分钟后形成的αβ二聚体，通过在Superose 6凝胶过滤柱上的快速蛋白质液相色谱，与DTT处理后仍作为四聚体存在的IGF I受体分离。使用125I标记的αIR - 3（一种针对IGF I受体的单克隆抗体）进行蛋白质印迹分析，证实了αβ二聚体的纯化。未经DTT处理或DTT处理后剩余的IGF I受体（αβ）2四聚体的自身磷酸化被IGF I刺激1.6 - 2.9倍。相反，在有或没有IGF I（100 ng/mL）存在的情况下孵育的αβ二聚体均未发生自身磷酸化。使用合成底物Arg - Arg - Leu - Ile - Glu - Asp - Ala - Glu - Tyr - Ala - Ala - Arg - Gly时，IGF I受体二聚体和四聚体均表现出相似的激酶活性，这表明未能检测到IGF I受体二聚体的自身磷酸化并非由DTT处理导致激酶失活引起。我们得出结论，IGF I受体的自身磷酸化取决于两个αβ二聚体的相互作用。
