Wardzala L J, Simpson I A, Rechler M M, Cushman S W
J Biol Chem. 1984 Jul 10;259(13):8378-83.
Previous studies have proposed that insulin increases the binding of insulin-like growth factor II (IGF-II) in isolated rat adipose cells at 24 degrees C by increasing receptor affinity (Ka). This study re-examines these observations under conditions in which receptor-ligand internalization is blocked by 1 mM KCN. In the absence of KCN, adipose cells bind 0.71 amol of IGF-II/cell with low apparent affinity (0.030 nM-1), of which greater than 75% is not accessible to trypsin. In contrast, in the presence of KCN, IGF-II binding is decreased by 95% and its apparent affinity increased to 0.21 nM-1. Moreover, greater than 60% of the bound IGF-II now is sensitive to trypsin. In either the absence or presence of KCN, approximately 20% of the cell's total IGF-II receptors are present in the plasma membranes and approximately 80% in the low density microsomes. Insulin induces a 5-fold increase in cell surface IGF-II receptors without a change in affinity when IGF-II binding is measured in the presence of KCN. Similarly, insulin increases IGF-II receptor concentration in the plasma membranes and concomitantly decreases that in the low density microsomes. Receptor affinity in these two subcellular membrane fractions is not affected by incubation of intact cells with either insulin or KCN and is similar to that observed in intact cells in the presence of KCN. Addition of KCN prior to insulin abolishes all of these effects of insulin. These data suggest that (a) the effects of KCN reflect a selective blockade of endocytosis; (b) in the absence of KCN, IGF-II binds to receptors of constant affinity that cycle between the plasma membrane and an intracellular pool resulting in an accumulation of intracellular IGF-II; (c) insulin induces an increase in IGF-II binding by causing a steady state redistribution of receptors from this intracellular pool to the plasma membrane; and (d) this redistribution in the intact cell can only be detected using Scatchard analysis when recycling of the receptors is prevented by KCN.
以往研究表明，在24℃条件下，胰岛素可通过增加受体亲和力（Ka）来提高离体大鼠脂肪细胞中胰岛素样生长因子II（IGF-II）的结合量。本研究在1 mM KCN阻断受体-配体内化的条件下重新审视了这些观察结果。在无KCN时，脂肪细胞以低表观亲和力（0.030 nM-1）结合0.71 amol的IGF-II/细胞，其中超过75%的结合物对胰蛋白酶不敏感。相反，在有KCN存在时，IGF-II结合量减少95%，其表观亲和力增加至0.21 nM-1。此外，现在超过60%的结合IGF-II对胰蛋白酶敏感。无论有无KCN，细胞总IGF-II受体中约20%存在于质膜中，约80%存在于低密度微粒体中。当在有KCN存在的情况下测量IGF-II结合时，胰岛素可使细胞表面IGF-II受体增加5倍，而亲和力不变。同样，胰岛素可增加质膜中IGF-II受体浓度，同时降低低密度微粒体中的受体浓度。完整细胞与胰岛素或KCN孵育后，这两个亚细胞膜组分中的受体亲和力不受影响，且与在有KCN存在的完整细胞中观察到的亲和力相似。在胰岛素之前添加KCN可消除胰岛素的所有这些作用。这些数据表明：（a）KCN的作用反映了对胞吞作用的选择性阻断；（b）在无KCN时，IGF-II与具有恒定亲和力的受体结合，这些受体在质膜和细胞内池之间循环，导致细胞内IGF-II积累；（c）胰岛素通过使受体从该细胞内池稳定地重新分布到质膜，从而诱导IGF-II结合增加；（d）只有当KCN阻止受体循环时，使用Scatchard分析才能检测到完整细胞中的这种重新分布。
