Kasuga M, Hedo J A, Yamada K M, Kahn C R
J Biol Chem. 1982 Sep 10;257(17):10392-9.
We have identified the subunits of the insulin receptor using immunoprecipitation by antibodies to the insulin receptor after either biosynthetic or surface labeling of cultured human lymphocytes (IM-9). With this approach, we have found there are two major, Mr = 135,000 (alpha), Mr = 95,000 (beta) and one minor, Mr = 210,000 (gamma) subunit. Peptide mapping clearly demonstrates that the major peptides of the alpha and beta subunits are different, whereas similarities exist in the peptide fragments of the gamma subunit and the alpha and beta subunits after limited proteolysis. The gamma subunit, however, is not simply a disulfide heterodimer of alpha and beta subunits, since this subunit was not reduced by 100 mM dithiothreitol plus 5% 2-mercaptoethanol, or even under more potent denaturing conditions, such as 8 M guanidine-HCL and mercaptoethanol at pH 10.5. In nonreduced gels, free insulin receptor subunits are observed, as well as two higher molecular weight bands of Mr = 520,000 and 350,000. On reduction, the 520,000 band was composed primarily of Mr = 210,000 and 95,000 subunits, whereas the 350,000 band was composed primarily of Mr = 135,000 and 95,000 subunits. These data suggest that the two major subunits of the insulin receptor (alpha and beta) are distinct. In addition, there is a third component of the receptor identifiable of 210,000 which may be a proreceptor or some closely associated effector protein. Furthermore, it appears that in the native state several kinds of disulfide oligomers of these subunits exist. These findings suggest a complex model for insulin receptor synthesis and insertion into the membrane.
我们通过对培养的人淋巴细胞（IM-9）进行生物合成或表面标记后，利用抗胰岛素受体抗体进行免疫沉淀，鉴定出了胰岛素受体的亚基。通过这种方法，我们发现有两个主要亚基，分子量分别为135,000（α）和95,000（β），以及一个次要亚基，分子量为210,000（γ）。肽图谱清楚地表明，α和β亚基的主要肽段不同，而γ亚基的肽片段与α和β亚基在有限蛋白酶解后存在相似性。然而，γ亚基并非简单的α和β亚基的二硫键异二聚体，因为该亚基在100 mM二硫苏糖醇加5% 2-巯基乙醇的条件下不会被还原，甚至在更强的变性条件下，如8 M盐酸胍和pH 10.5的巯基乙醇中也不会被还原。在非还原凝胶中，可以观察到游离的胰岛素受体亚基，以及分子量为520,000和350,000的两条高分子量条带。还原后，520,000条带主要由分子量为210,000和95,000的亚基组成，而350,000条带主要由分子量为135,000和95,000的亚基组成。这些数据表明胰岛素受体的两个主要亚基（α和β）是不同的。此外，受体还有一个分子量为210,000的可识别的第三成分，它可能是前受体或某种紧密相关的效应蛋白。此外，在天然状态下，这些亚基似乎存在几种二硫键寡聚体。这些发现提示了一个关于胰岛素受体合成和插入膜的复杂模型。
