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 phosphorylation of pp 185 was impaired in Chinese hamster ovary cells expressing 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 phosphatidylinositol 3-kinase in the anti-phosphotyrosine and anti-IRS-1 immunoprecipitates, the activation of mitogen-activated protein (MAP) kinase, and biological actions were also impaired. In addition, although the deletion of residues 954-965 severely impaired insulin internalization, the deletion of NPXY (957-960), the internalization signal of the low density lipoprotein receptor, did not affect internalization. Moreover, neither the deletions around Tyr⁹⁵³ nor the alterations of the tyrosines (953, 960, or 972) significantly reduced internalization. These data suggest that: 1) Tyr⁹⁶⁰ is important for the recognition of pp185/IRS-1, the association of phosphatidylinositol 3-kinase with pp185/IRS-1, and the activation of MAP kinase; 2) MAP kinase may lie downstream of pp185/IRS-1 in insulin's signal transduction; and 3) the juxtamembrane domain, but not NPXY or individual tyrosines, is important for insulin internalization.