Harmon J T, Hedo J A, Kahn C R
J Biol Chem. 1983 Jun 10;258(11):6875-81.
Using the technique of radiation inactivation we have previously shown that the insulin receptor behaves as if it is composed of at least two functional components: a binding component (Mr approximately equal to 100,000) and an affinity regulatory component (Mr approximately equal to 300,000). The interaction between the affinity regulator and binding component results in a decrease in the affinity of the receptor for insulin. To examine in more detail the interaction between this "affinity regulator" and the binding component we have studied the insulin receptor by radiation inactivation under conditions which alter receptor concentration or receptor affinity. Liver membranes of ob/ob mice exhibit a decrease in insulin binding when compared to their lean litter mates which is due to a decrease in receptor concentration. When studied by radiation inactivation, however, there was no detectable change in the interaction or size of the two receptor components. By contrast, under circumstances in which the affinity of the receptor was increased (treatment with high salt, high pH, 1 mM dithiothreitol, 1-5 micrograms/ml of trypsin), the interaction between the regulatory and binding components was either decreased or absent, i.e. there was no increase in binding with irradiation. Conversely, conditions which produce a decrease in receptor affinity resulted in an increase in the interaction between the regulatory and binding components. The changes in receptor affinity and interactions of the two components produced by either high salt or pH were reversible. Partial purification of the solubilized receptor on lectin affinity columns resulted in the apparent removal of the affinity regulator, i.e. receptor affinity was increased. In this state, radiation inactivation studies revealed a monoexponential decay indicating no interaction between binding and regulatory components. Taken together, these results suggest that the affinity regulator is a membrane protein which is both trypsin-sensitive and has disulfide bond(s) essential for its function. The interaction between the affinity regulator and binding component is not via a covalent bond and the two components appear to be separated by lectin chromatography. The interaction between these components appears to be altered in most states associated with altered receptor affinity.
我们之前利用辐射失活技术表明，胰岛素受体的行为表现就好像它至少由两个功能成分组成：一个结合成分（分子量约为100,000）和一个亲和力调节成分（分子量约为300,000）。亲和力调节因子与结合成分之间的相互作用导致受体对胰岛素的亲和力降低。为了更详细地研究这种“亲和力调节因子”与结合成分之间的相互作用，我们在改变受体浓度或受体亲和力的条件下，通过辐射失活研究了胰岛素受体。与瘦的同窝小鼠相比，ob/ob小鼠的肝细胞膜胰岛素结合能力下降，这是由于受体浓度降低所致。然而，通过辐射失活研究发现，两种受体成分的相互作用或大小没有可检测到的变化。相比之下，在受体亲和力增加的情况下（用高盐、高pH、1 mM二硫苏糖醇、1 - 5微克/毫升胰蛋白酶处理），调节成分与结合成分之间的相互作用要么减弱要么不存在，即辐射后结合没有增加。相反，导致受体亲和力降低的条件会导致调节成分与结合成分之间的相互作用增加。高盐或pH引起的受体亲和力变化以及两种成分的相互作用变化是可逆的。在凝集素亲和柱上对溶解的受体进行部分纯化导致亲和力调节因子明显去除，即受体亲和力增加。在这种状态下，辐射失活研究显示单指数衰减，表明结合成分与调节成分之间没有相互作用。综上所述，这些结果表明，亲和力调节因子是一种膜蛋白，它对胰蛋白酶敏感且具有对其功能至关重要的二硫键。亲和力调节因子与结合成分之间的相互作用不是通过共价键，并且这两种成分似乎可以通过凝集素色谱法分离。在大多数与受体亲和力改变相关的状态下，这些成分之间的相互作用似乎会发生改变。
