FUNCTIONAL-PROPERTIES OF 2 NATURALLY-OCCURRING ISOFORMS OF THE HUMAN INSULIN-RECEPTOR IN CHINESE-HAMSTER OVARY CELLS
FUNCTIONAL-PROPERTIES OF 2 NATURALLY-OCCURRING ISOFORMS OF THE HUMAN INSULIN-RECEPTOR IN CHINESE-HAMSTER OVARY CELLS
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DOI：
10.1210/endo-129-4-2058
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发表时间：
1991-10-01
期刊：
影响因子：
4.8
通讯作者：
MOLLER, DE
中科院分区：
文献类型：
--
作者：
YAMAGUCHI, Y;FLIER, JS;MOLLER, DE
我们和其他人之前已经证明，人胰岛素受体信使 RNA (mRNA) 被选择性剪接，从而包含 (Ex11+) 或排除 (Ex11-) 受体基因外显子 11 编码的 36 核苷酸序列。 虽然 Ex11- 和 Ex11+ 胰岛素受体（其羧基末端 α 亚基是否存在 12 个氨基酸的差异）已被证明在独立过表达和研究时具有胰岛素受体的功能，但两种异构体之间存在细微功能差异的可能性受到的关注有限。 鉴于两种 mRNA 转录物的相对丰度以组织特异性方式受到高度调节，两种受体变体的功能特性的差异可能导致已知存在的胰岛素受体功能和胰岛素作用的组织特异性差异。 为了解决这个假设，我们将编码两种受体亚型的 cDNA 转染到中国仓鼠卵巢 (CHO) 细胞中，并制备了几种表达大量 Ex11- 或 Ex11+ 受体的稳定 CHO 细胞系。 所表达的胰岛素受体的几个功能特性与以下结果平行比较：1)胰岛素与表达Ex11-亚型的细胞的稳态结合表现出更高的(几乎等于2倍)亲和力； 2)使用两种不同的方法，注意到受体介导的胰岛素内化的显着差异，使得Ex11-亚型表现出更高的内化速率（几乎等于速率常数K(e)增加25%)； 3) 部分纯化的 Ex11- 和 Ex11+ 受体在受体自身磷酸化和对外源底物（聚 Glu-Tyr，4:1）的激酶活性方面表现出相似的最大和胰岛素剂量反应特征； 4)表达的Ex11-和Ex11+受体与胰岛素的代谢（葡萄糖掺入糖原）和促有丝分裂（胸苷掺入DNA）作用的能力没有明显不同。 因此，当在 CHO 细胞中表达时，胰岛素受体的两种选择性剪接亚型在胰岛素结合亲和力和配体刺激的内化动力学方面具有细微的差异，预计这会以组织特异性方式影响体内胰岛素受体表达和信号传导的模式。
We and others have previously demonstrated that the human insulin receptor messenger RNA (mRNA) is alternatively spliced such that the 36-nucleotide sequence encoded by exon 11 of the receptor gene is included (Ex11+) or excluded (Ex11-). Although both Ex11- and Ex11+ insulin receptors which differ in the presence or absence of 12 amino acids in the carboxy-terminal alpha-subunit have been demonstrated to function as insulin receptors when independently overexpressed and studied, the possibility that subtle functional differences between the two isoforms exists has received limited attention. Given that the relative abundance of the two mRNA transcripts is highly regulated in a tissue-specific manner, differences in the functional properties of the two receptor variants might contribute to tissue-specific differences in insulin receptor function and insulin action that are known to exist. To address this hypothesis, we transfected cDNAs encoding the two receptor isoforms into Chinese hamster ovary (CHO) cells and prepared several stable CHO cell lines expressing high numbers of Ex11- or Ex11+ receptors. Several functional properties of the expressed insulin receptors were compared in parallel with the following results: 1) steady state binding of insulin to cells expressing the Ex11- isoform exhibited higher (almost-equal-to 2-fold) affinity; 2) using two different methods, a significant difference in receptor-mediated insulin internalization was noted such that the Ex11- isoform displayed a higher (almost-equal-to 25% increase in the rate constant, K(e)) rate of internalization; 3) partially purified Ex11- and Ex11+ receptors displayed similar maximal and insulin dose-response characteristics for receptor autophosphorylation and kinase activity toward an exogenous substrate (poly Glu-Tyr, 4:1); 4) the ability of expressed Ex11- and Ex11+ receptors to couple to a metabolic (glucose incorporation into glycogen) and mitogenic (thymidine incorporation into DNA) action of insulin was not discernibly different. Thus, when expressed in CHO cells, the two alternatively spliced isoforms of the insulin receptor have subtle differences in insulin binding affinity and the kinetics of ligand-stimulated internalization that would be expected to influence the pattern of insulin receptor expression and signaling in vivo in a tissue-specific manner.