Site-directed mutagenesis of the juxtamembrane domain of the human insulin receptor

Y Kaburagi,K Momomura,R Yamamoto-Honda,K Tobe,Y Tamori,H Sakura,Y Akanuma,Y Yazaki,T Kadowaki

doi:10.1016/s0021-9258(19)85463-7

Abstract

We have studied the functions of the juxtamembrane domain (941-989) of the human insulin receptor by site-directed mutagenesis. Tyrosine phosphorylation of pp185 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.

Highlights

Domain (941-989) of the human insulin receptor by antibody immunoprecipitates immediately after insulin stimsite-directedmutagenesis.Tyrosinephosphorylation ulation (White et al, 1985; Kadowaki et al, 1987; Momomura of ppl85 was impaired in Chinese hamster ovarycells et al, 1988; Tobe et al, 1990)
Al- including six YMXM and three YXXM motifs, which are though the deletionof residues 964-965 severely im- considered to associate with Src homology (SH)-2domains in paired insulin internalization, the deletion ofNPXY the regulatory 85-kDa subunitof phosphatidylinositol (PI) 3
humaninsulin receptor (HIR) cells, respectively(data not shown).Sincethese receptors exhibited normal tyrosine kinase activity toward an exogenous substrate in vitro, these data suggestedthat TyrW plays an important role in pp185 recognition.To study further the important site for the recognition of pp185, 2 other tyrosine residues in the juxtamembrane domain of the insulin receptor, Tyr9" and Tyr972w, ere changed to Phe, respectively, and the effects of these mutations on the receptor tyrosine kinase activity in intact cells were examined

Summary

RESULTS

Insulin Binding-Following transfection and selection by G418, the cells with high levelsof insulin binding were identified by '261-insulin binding.The clones that bound 33-80% of added '2SI-insulinwere selected for the following analysis (data not shown). MBP Kinase Activity in Total Cell Lysates and Immune was reported previously that PI 3-kinaseactivity in aPY Complexes-To analyze the relationship between the tyrosine immunoprecipitates from the cells expressing the wild-type phosphorylation of pp185/IRS-l and the activation of a kiinsulin receptors was elevated by the treatment with insulin nase cascade involving MAP kinase, we examined insulin-. The kinase activity in the MBP because the reaction mixtures contained EGTA and a aPY immunoprecipitates from insulin-stimulated CHO-HIR peptide kinase inhibitor of CAMP-dependent protein kinase cells was completely inhibited by 1% Nonidet P-40 (data not (Ahn et al, 1990).Fig. 7A shows an increase in kinase activity shown). When the results of both decreased insulin-induced MBP kinase activity compared with CHO-HIR cells These data indicated that insulin-inof the analysis were similar, we denoted the results as derived from duced MAP kinase activationwas severely impaired in CHO-. - - Insulin(l0”M) - + - + - + lmin I ’CHO-KI CHCOH-OH-IRY960F Immunoprecipitates aPY

85 K subunit

DISCUSSION

CHO-V960A