SITE-DIRECTED MUTAGENESIS OF THE JUXTAMEMBRANE DOMAIN OF THE HUMAN INSULIN-RECEPTOR

We have studied the functions of the juxtamembrane domain (941-989) of the human insulin receptor by site-directed mutagenesis. Tyrosine pho sphorylation of pp185 was impaired in Chinese hamster ovary cells expr essing the receptors with the alteration of Tyr960, but not of Tyr953 or Tyr972, to Phe (CHO-Y960F cells) as compared with cells expressing the normal receptors. In CHO-Y960F cells, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), the activation of phosphatidylin ositol 3-kinase in the anti-phosphotyrosine and anti-IRS-1 immunopreci pitates, the activation of mitogen-activated protein (MAP) kinase, and biological actions were also impaired. In addition, although the dele tion of residues 954-965 severely impaired insulin internalization, th e deletion of NPXY (957-960), the internalization signal of the low de nsity lipoprotein receptor, did not affect internalization. Moreover, neither the deletions around Tyr953 nor the alterations of the tyrosin es (953, 960, or 972) significantly reduced internalization. These dat a suggest that: 1) Tyr960 is important for the recognition of pp185/IR S-1, the association of phosphatidylinositol 3-kinase with pp185/IRS-1 , and the activation of MAP kinase; 2) MAP kinase may lie downstream o f pp185/IRS-1 in insulin's signal transduction; and 3) the juxtamembra ne domain, but not NPXY or individual tyrosines, is important for insu lin internalization.