Ellis L, Morgan D O, Clauser E, Roth R A, Rutter W J
Hormone Research Institute, University of California, San Francisco 94143.
Mol Endocrinol. 1987 Jan;1(1):15-24. doi: 10.1210/mend-1-1-15.
Insulin stimulates the autophosphorylation of the beta-subunit of the insulin receptor (IR) on tyrosine residues. Mutations which compromise IR autophosphorylation in vivo result in a decrease of the insulin-activated uptake of 2-deoxyglucose. These results are consistent with previous results which implicate IR autophosphorylation in the generation of the insulin response by cells. To further explore the specificity of the IR tyrosine phosphokinase (TPK) domain in IR function, we have altered the human IR (hIR) cDNA to encode truncated insulin-independent TPKs, which are expressed in chinese hamster ovary (CHO) cells as either membrane-anchored or cytosolic proteins. Both mutant hIRs exhibit TPK activity in vitro, although the cytosolic form is approximately 20 times more active. The carbohydrate moiety of the membrane-anchored form is of the high mannose type, consistent with an intracellular localization for this mutant hIR. The two mutant hIRs mediate very different physiological responses in transfected cells: the membrane-anchored, but not the cytosolic, hIR TPK mediates a constitutively elevated (135% the maximum insulin-stimulated response in CHO cells) insulin-independent uptake of 2-deoxyglucose. These results thus suggest that the hIR TPK is in fact specific for this aspect of IR function and, when membrane-associated, can mediate the insulin-independent uptake of 2-deoxyglucose. Neither of these mutant hIRs appears to transform CHO cells.
胰岛素可刺激胰岛素受体（IR）β亚基上酪氨酸残基的自身磷酸化。在体内损害IR自身磷酸化的突变会导致胰岛素激活的2-脱氧葡萄糖摄取减少。这些结果与先前的结果一致，先前的结果表明IR自身磷酸化参与细胞产生胰岛素反应的过程。为了进一步探究IR酪氨酸磷酸激酶（TPK）结构域在IR功能中的特异性，我们对人IR（hIR）cDNA进行了改造，以编码截短的不依赖胰岛素的TPK，其在中华仓鼠卵巢（CHO）细胞中作为膜锚定蛋白或胞质蛋白表达。两种突变型hIR在体外均表现出TPK活性，尽管胞质形式的活性约高20倍。膜锚定形式的碳水化合物部分为高甘露糖型，与该突变型hIR的细胞内定位一致。两种突变型hIR在转染细胞中介导非常不同的生理反应：膜锚定而非胞质的hIR TPK介导2-脱氧葡萄糖的组成性升高摄取（是CHO细胞中最大胰岛素刺激反应的135%），且不依赖胰岛素。因此，这些结果表明hIR TPK实际上对IR功能的这一方面具有特异性，并且当与膜相关时，可以介导2-脱氧葡萄糖的不依赖胰岛素的摄取。这两种突变型hIR似乎都不会使CHO细胞发生转化。
