Shia M A, Rubin J B, Pilch P F
J Biol Chem. 1983 Dec 10;258(23):14450-5.
We determined that the rate of insulin-stimulated autophosphorylation of the insulin receptor is independent of receptor concentration and thus proceeds via an intramolecular process. This result is consistent with the possibility that ligand-dependent autophosphorylation may be a means by which cells can distinguish occupied from unoccupied receptors. We employed dithiothreitol to dissociate tetrameric receptor into alpha beta halves in order to further elucidate the structural requirements for the receptor-mediated kinase activity. Dithiothreitol had a complex biphasic effect on insulin-stimulated receptor kinase activity. Marked stimulation of kinase activity was observed at 1-2 mM dithiothreitol when the receptor was predominantly tetrameric and kinase activity diminished when dimeric alpha beta receptor halves predominate (greater than 2 mM dithiothreitol). N-Ethylmaleimide inhibits insulin-stimulated receptor kinase activity. We suggest that the tetrameric holoreceptor is the most active kinase structure and this structure requires for maximal activity, a reduced sulfhydryl group at or near the active site. We treated receptor preparations with elastase to generate receptor proteolytically "nicked" in the beta subunit. This treatment completely abolishes insulin-dependent autophosphorylation and histone phosphorylation with essentially no effects on insulin binding as determined by affinity labeling of the receptor alpha subunit. We suggest such treatment functionally uncouples insulin binding from insulin-stimulated receptor kinase activity. The possible physiological significance of these findings is discussed.
我们确定胰岛素受体的胰岛素刺激的自身磷酸化速率与受体浓度无关，因此是通过分子内过程进行的。这一结果与配体依赖性自身磷酸化可能是细胞区分被占据受体和未被占据受体的一种方式的可能性相一致。我们使用二硫苏糖醇将四聚体受体解离为αβ亚基对，以进一步阐明受体介导的激酶活性的结构要求。二硫苏糖醇对胰岛素刺激的受体激酶活性有复杂的双相作用。当受体主要为四聚体时，在1-2 mM二硫苏糖醇下观察到激酶活性明显增强，而当二聚体αβ受体亚基对占主导（二硫苏糖醇浓度大于2 mM）时，激酶活性降低。N-乙基马来酰亚胺抑制胰岛素刺激的受体激酶活性。我们认为四聚体全受体是最具活性的激酶结构，并且这种结构对于最大活性而言，在活性位点或其附近需要一个还原的巯基。我们用弹性蛋白酶处理受体制剂，以产生在β亚基上被蛋白酶“切割”的受体。这种处理完全消除了胰岛素依赖性自身磷酸化和组蛋白磷酸化，而通过受体α亚基的亲和标记测定，对胰岛素结合基本上没有影响。我们认为这种处理在功能上将胰岛素结合与胰岛素刺激的受体激酶活性解偶联。讨论了这些发现可能的生理学意义。
