REPLACEMENTS OF LEUCINE-87 IN HUMAN INSULIN-RECEPTOR ALTER AFFINITY FOR INSULIN

In a previous analysis, we identified a point mutation that substitute d Pro (<C(C)under bar G>) for Leu (<C(T)under bar G>) at amino acid 87 in the alpha-subunit of the insulin receptor (IR) in a Japanese patie nt 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 o f 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, an d 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 insu lin than that of Leu-87 IR, suggesting the reduced insulin binding aff inity. Site-directed mutagenesis at amino acid 87 was performed to sub stitute Ile or Ala for Leu. Both mutant IRs were transported to the ce ll surface and labeled by cell surface biotinylation. The Ile-87 IR en hanced 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 tha n in Leu-87 IR, but in Ala-87 IR it was more rapid. These results prov ide the first direct evidence for a critical role of Leu-87 in binding insulin.