Hedo J A, Kahn C R, Hayashi M, Yamada K M, Kasuga M
J Biol Chem. 1983 Aug 25;258(16):10020-6.
The biosynthesis and carbohydrate processing of the insulin receptor were studied in cultured human lymphocytes by means of metabolic and cell surface labeling, immunoprecipitation with anti-receptor autoantibodies, and analysis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions. In addition to the two major subunits of Mr = 135,000 and Mr = 95,000, two higher molecular weight bands were detected of Mr = 210,000 and Mr = 190,000. The Mr = 210,000 band and the two major subunits were labeled by [3H]mannose, [3H]glucosamine, [3H]galactose, and [3H]fucose, and were bound by immobilized lentil, wheat germ, and ricin I lectins. On the other hand, the Mr = 190,000 band was labeled only by [3H]mannose and [3H]glucosamine and was bound only by lentil lectin. All four components could be labeled with [35S] methionine; however, in contrast with the other three polypeptides, the Mr = 190,000 band was not labeled by cell surface iodination with lactoperoxidase, suggesting that it is not exposed at the outer surface of the plasma membrane. Pulse-chase studies with [3H]mannose showed that the Mr = 190,000 was the earliest labeled component of the receptor; radioactivity in this band reached a maximum 1 h after the pulse, clearly preceded the appearance of the other components, and had a very brief half-life (t1/2 = 2.5 h). The Mr = 210,000, Mr = 135,000, and Mr = 95,000 bands were next in appearance and reached a maximum 6 h in the chase period. Monensin, an ionophore which interferes with maturation of some proteins, blocked both the disappearance of the Mr = 190,000 protein and the appearance of the Mr = 135,000 and Mr = 95,000 subunits. The mannose incorporated in the Mr = 190,000 component was fully sensitive to treatment with endoglycosidase H while that in the Mr = 210,000 band and the two major subunits was only partially sensitive. Tryptic fingerprints of the 125I-labeled Mr = 210,000 band suggested that this component contains peptides of both the Mr = 135,000 and Mr = 95,000 subunits. In conclusion, the Mr = 190,000 component appears to represent the high mannose precursor form of the insulin receptor that undergoes carbohydrate processing and proteolytic cleavage to generate the two major subunits. In addition, the Mr = 210,000 band is probably the fully glycosylated form of the precursor that escapes cleavage and is expressed in the plasma membrane.
采用代谢标记和细胞表面标记、抗受体自身抗体免疫沉淀以及在还原条件下进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶分析的方法，对培养的人淋巴细胞中胰岛素受体的生物合成及碳水化合物加工过程进行了研究。除了分子量分别为135,000和95,000的两个主要亚基外，还检测到分子量为210,000和190,000的两条高分子量条带。分子量为210,000的条带以及两个主要亚基被[³H]甘露糖、[³H]葡糖胺、[³H]半乳糖和[³H]岩藻糖标记，并与固定化的扁豆、麦芽和蓖麻I凝集素结合。另一方面，分子量为190,000的条带仅被[³H]甘露糖和[³H]葡糖胺标记，且仅与扁豆凝集素结合。所有这四种成分都能用[³⁵S]甲硫氨酸标记；然而，与其他三种多肽不同的是，分子量为190,000的条带不能被乳过氧化物酶进行细胞表面碘化标记，这表明它未暴露于质膜的外表面。用[³H]甘露糖进行脉冲追踪研究表明，分子量为190,000的成分是受体中最早被标记的成分；该条带中的放射性在脉冲后1小时达到最大值，明显先于其他成分出现，且半衰期非常短（t₁/₂ = 2.5小时）。分子量为210,000、135,000和95,000的条带随后出现，并在追踪期6小时达到最大值。莫能菌素是一种干扰某些蛋白质成熟的离子载体，它阻断了分子量为190,000蛋白质的消失以及分子量为135,000和95,000亚基的出现。分子量为190,000成分中掺入的甘露糖对内切糖苷酶H处理完全敏感，而分子量为210,000条带和两个主要亚基中的甘露糖仅部分敏感。¹²⁵I标记的分子量为210,000条带的胰蛋白酶指纹图谱表明，该成分包含分子量为135,000和95,000亚基的肽段。总之，分子量为190,000的成分似乎代表胰岛素受体的高甘露糖前体形式，它经过碳水化合物加工和蛋白水解切割以产生两个主要亚基。此外，分子量为210,000的条带可能是前体的完全糖基化形式，它未被切割并在质膜中表达。
