Oka Y, Rozek L M, Czech M P
J Biol Chem. 1985 Aug 5;260(16):9435-42.
The photoactive insulin-like growth factor (IGF)-II analogue 4-azidobenzoyl-125I-IGF-II was synthesized and used to label specifically and covalently the Mr = 250,000 Type II IGF receptor. When rat adipocytes are irradiated after a 10-min incubation with 4-azidobenzoyl-125I-IGF-II at 10 degrees C and immediately homogenized, most of the labeled IGF-II receptors are associated with the plasma membrane fraction, indicating that receptors accessible to the labeling reagent at low temperature are on the cell surface. However, when the photolabeled cells are incubated at 37 degrees C for various times before homogenization, labeled IGF-II receptors are rapidly internalized with a half-time of 3.5 min as evidenced by a loss from the plasma membrane fraction and a concomitant appearance in the low density microsome fraction. The low density microsomes were previously shown to contain intracellular membranes (Oka, Y., and Czech, M.P. (1984) J. Biol. Chem. 259, 8125-8133). The steady state level of cell surface IGF-II receptors in the presence or absence of IGF-II, measured by the binding of anti-IGF-II receptor antibody to cells, remains constant under these conditions, demonstrating that IGF-II receptors rapidly recycle back to the cell surface at the same rate as receptor internalization. Using the above methodology, it is shown that acute insulin action: 1) increases the steady state number of cell surface IGF-II receptors; 2) increases the number of ligand-bound IGF-II receptors that are internalized per unit of time, as evidenced by a large increase in the photolabeling of intracellular membrane IGF-II receptors when cells are incubated at 37 degrees C with insulin and 4-azidobenzoyl-125I-IGF-II prior to photoactivation; and 3) increases the rate of cellular 125I-IGF-II degradation by a process that is blocked by anti-IGF-II receptor antibody. The results indicate that the action of insulin to elevate the steady state number of cell surface IGF-II receptors leads to an increased internalization flux of IGF-II-bound receptors, mediating increased IGF-II uptake and degradation.
合成了光活性胰岛素样生长因子（IGF）-II类似物4-叠氮苯甲酰基-125I-IGF-II，并用于特异性共价标记分子量为250,000的II型IGF受体。当大鼠脂肪细胞在10℃下与4-叠氮苯甲酰基-125I-IGF-II孵育10分钟后照射并立即匀浆时，大部分标记的IGF-II受体与质膜部分相关联，这表明在低温下可被标记试剂作用的受体位于细胞表面。然而，当光标记的细胞在匀浆前于37℃孵育不同时间时，标记的IGF-II受体迅速内化，半衰期为3.5分钟，这可通过质膜部分标记物的减少以及低密度微粒体部分中相应标记物的出现得到证明。先前已表明低密度微粒体含有细胞内膜（冈，Y.，和捷克，M.P.（1984年）《生物化学杂志》259，8125 - 8133）。在存在或不存在IGF-II的情况下，通过抗IGF-II受体抗体与细胞的结合来测量细胞表面IGF-II受体的稳态水平，在这些条件下保持恒定，这表明IGF-II受体以与受体内化相同的速率迅速循环回到细胞表面。使用上述方法表明，急性胰岛素作用：1）增加细胞表面IGF-II受体的稳态数量；2）增加单位时间内内化的配体结合型IGF-II受体的数量，这可通过在光激活前将细胞与胰岛素和4-叠氮苯甲酰基-125I-IGF-II在37℃孵育时细胞内膜IGF-II受体的光标记大幅增加得到证明；3）通过一个被抗IGF-II受体抗体阻断的过程增加细胞内125I-IGF-II的降解速率。结果表明胰岛素提高细胞表面IGF-II受体稳态数量的作用导致IGF-II结合型受体的内化通量增加，介导了IGF-II摄取和降解的增加。
