Backer J M, Kahn C R, Cahill D A, Ullrich A, White M F
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02215.
J Biol Chem. 1990 Sep 25;265(27):16450-4.
The juxtamembrane region of the insulin receptor (IR) beta-subunit contains an unphosphorylated tyrosyl residue (Tyr960) that is essential for insulin-stimulated tyrosyl phosphorylation of some endogenous substrates and certain biological responses (White, M.F., Livingston, J.N., Backer, J.M., Lauris, V., Dull, T.J., Ullrich, A., and Kahn, C.R. (1988) Cell 54, 641-649). Tyrosyl residues in the juxtamembrane region of some plasma membrane receptors have been shown to be required for their internalization. In addition, a juxtamembrane tyrosine in the context of the sequence NPXY [corrected] is required for the coated pit-mediated internalization of the low density lipoprotein receptor. To examine the role of the juxtamembrane region of the insulin receptor during receptor-mediated endocytosis, we have studied the internalization of insulin by Chinese hamster ovary (CHO) cells expressing two mutant receptors: IRF960, in which Tyr960 has been substituted with phenylalanine, and IR delta 960, in which 12 amino acids (Ala954-Asp965), including the putative consensus sequence NPXY [corrected], were deleted. Although the in vivo autophosphorylation of IRF960 and IR delta 960 was similar to wild type, neither mutant could phosphorylate the endogenous substrate pp185. CHO/IRF960 cells internalized insulin normally whereas the intracellular accumulation of insulin by CHO/IR delta 960 cells was 20-30% of wild-type. However, insulin internalization in the CHO/IR delta 960 cells was consistently more rapid than that occurring in CHO cells expressing kinase-deficient receptors (CHO/IRA1018). The degradation of insulin was equally impaired in CHO/IR delta 960 and CHO/IRA1018 cells. These data show that the juxtamembrane region of the insulin receptor contains residues essential for insulin-stimulated internalization and suggest that the sequence NPXY [corrected] may play a general role in directing the internalization of cell surface receptors.
胰岛素受体（IR）β亚基的近膜区域含有一个未磷酸化的酪氨酰残基（Tyr960），它对于胰岛素刺激某些内源性底物的酪氨酰磷酸化以及某些生物学反应至关重要（怀特，M.F.，利文斯顿，J.N.，巴克尔，J.M.，劳里斯，V.，杜尔，T.J.，乌尔里希，A.，以及卡恩，C.R.（1988年）《细胞》54卷，641 - 649页）。一些质膜受体近膜区域的酪氨酰残基已被证明是其内化所必需的。此外，低密度脂蛋白受体通过有被小窝介导的内化需要在序列NPXY[已修正]背景下的一个近膜酪氨酸。为了研究胰岛素受体近膜区域在受体介导的内吞作用中的作用，我们研究了表达两种突变受体的中国仓鼠卵巢（CHO）细胞对胰岛素的内化：IRF960，其中Tyr960被苯丙氨酸取代；以及IR delta 960，其中包括假定的共有序列NPXY[已修正]在内的12个氨基酸（Ala954 - Asp965）被删除。尽管IRF960和IR delta 960在体内的自身磷酸化与野生型相似，但两种突变体都不能使内源性底物pp185磷酸化。CHO/IRF960细胞正常内化胰岛素，而CHO/IR delta 960细胞中胰岛素的细胞内积累量是野生型的20 - 30%。然而，CHO/IR delta 960细胞中胰岛素的内化始终比表达激酶缺陷受体的CHO细胞（CHO/IRA1018）中发生的内化更快。胰岛素的降解在CHO/IR delta 960和CHO/IRA1018细胞中同样受损。这些数据表明胰岛素受体的近膜区域含有胰岛素刺激内化所必需的残基，并表明序列NPXY[已修正]可能在指导细胞表面受体的内化中起普遍作用。
