Abstract
In a previous analysis, we identified a point mutation that substituted Pro (CCG) for Leu (CTG) at amino acid 87 in the alpha-subunit of the insulin receptor (IR) in a Japanese patient with leprechaunism. In the present study, we transfected either the wild type (Leu-87) or the mutant (Pro-87) IR cDNA into NIH3T3 cells. Pulse-chase in nonreducing conditions revealed that the dimerization of Pro-87 IR was slightly impaired. However, cell surface biotinylation showed that Pro-87 IR was transported to the cell surface. The Pro-87 IR reduced the insulin binding affinity to about 15% of Leu-87 IR, and the dissociation of insulin in Pro-87 IR was more rapid than in Leu-87 IR. The autophosphorylation of Pro-87 IR was less sensitive to insulin than that of Leu-87 IR, suggesting the reduced insulin binding affinity. Site-directed mutagenesis at amino acid 87 was performed to substitute Ile or Ala for Leu. Both mutant IRs were transported to the cell surface and labeled by cell surface biotinylation. The Ile-87 IR enhanced the insulin binding affinity about 4-fold. The insulin binding affinity of Ala-87 IR was reduced by 85% relative to that of Leu-87 IR. In addition, the dissociation of insulin in Ile-87 IR was slower than in Leu-87 IR, but in Ala-87 IR it was more rapid. These results provide the first direct evidence for a critical role of Leu-87 in binding insulin.
Highlights
Phosphorylation of Mutant Insulin Receptor (Pro-87 Insulin receptor (IR)) in Intact Cells — Because one naturally occurring mutation that substitutes Pro for Arg at amino acid 86 constitutively activates autophosphorylation and tyrosine kinase of IR [22, 23], we assessed the autophosphorylationability of the β-subunit of
Insulin binding to the IR is the first essential step for inducing the biological actions of insulin
The investigation of the structure and function of transmembrane receptor glycoproteins is largely dependent on the genetic analysis of naturally occurring mutations and site-directed mutagenesis
Summary
Site-directedmutagenesis at amino acid 87 was performed to substitute Ile or Ala for Leu. Both mutant IRs were transported to the cell surface and labeled by cell surface biotinylation. On cloning human IR cDNA [4, 5], molecular analyses of mutagenesis at this position, substituting Ile or Ala for Leu, patients with genetic forms of severe insulin resistance, includ- and assessed the effects of these substitutions on the insulin ing type A insulin-resistant syndrome, leprechaunism, and binding affinity of NIH3T3 cells expressing these mutant IRs
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