Knutson V P, Ronnett G V, Lane M D
J Biol Chem. 1983 Oct 25;258(20):12139-42.
Chronic treatment of 3T3-C2 fibroblasts with insulin causes the slow (t1/2 = 3-4 h) down-regulation of cellular insulin receptor to a new steady state level by accelerating receptor decay (Knutson, V.P., Ronnett, G.V., and Lane, M.D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2822-2826). In the present investigation, the synthesis and turnover of the receptor during the transition to the down-regulated state was examined by the heavy isotope density-shift method. It was observed that within two h after insulin addition, receptor decay increased abruptly for several hours then gradually declined until the "down-regulated" rate was achieved. The abrupt increase in receptor decay induced by insulin was preceded by a more rapid (t1/2 less than or equal to 10 min) translocation of cell surface receptor to an "intracellular" trypsin-resistant compartment. Thus, upon exposure to ligand, insulin receptor rapidly redistributes from the cell surface to an intracellular compartment, without an initial net loss of cellular receptors. The translocation process was rapidly reversed (t1/2 less than or equal to 20 min) upon removal of insulin. With prolonged exposure to insulin, the initial rapid translocation of receptor was followed by a slower inactivation of receptor apparently in the intracellular compartment. Cycloheximide, which lengthens receptor half-life by blocking a step in receptor inactivation, had no effect on receptor internalization. Internalization of insulin receptor and its bound ligand were, however, rapidly (less than 10 min) blocked by phenylarsine oxide. These results support the following sequence of events. Upon exposure to ligand, insulin receptors are translocated from the cell surface to an intracellular site which results in accelerated receptor decay and ultimately to a lower steady state cellular receptor level.
用胰岛素对3T3 - C2成纤维细胞进行长期处理，会通过加速受体降解，使细胞胰岛素受体缓慢下调（半衰期(t_{1/2}=3 - 4)小时）至新的稳态水平（克努森，V.P.，罗内特，G.V.，和莱恩，M.D.（1982年）《美国国家科学院院刊》79，2822 - 2826）。在本研究中，通过重同位素密度转移法检测了受体在向下调状态转变过程中的合成和周转情况。观察到在添加胰岛素后的两小时内，受体降解突然增加数小时，然后逐渐下降，直至达到“下调”速率。胰岛素诱导的受体降解突然增加之前，细胞表面受体向“细胞内”胰蛋白酶抗性区室发生更快的（半衰期(t_{1/2}\leq10)分钟）转位。因此，在暴露于配体时，胰岛素受体迅速从细胞表面重新分布到细胞内区室，而细胞受体最初并无净损失。去除胰岛素后，转位过程迅速逆转（半衰期(t_{1/2}\leq20)分钟）。长时间暴露于胰岛素后，受体最初的快速转位之后，显然是细胞内区室中受体的较慢失活。环己酰亚胺通过阻断受体失活的一个步骤来延长受体半衰期，但对受体内化没有影响。然而，胰岛素受体及其结合配体的内化被苯砷氧化物迅速（小于10分钟）阻断。这些结果支持以下事件顺序。在暴露于配体时，胰岛素受体从细胞表面转位到细胞内位点，这导致受体降解加速，并最终使细胞受体达到较低的稳态水平。
