Replacements of leucine 87 in @entity1 XXXX alter affinity for insulin.

@entity535 @entity1 @entity8277 @entity10117
In a previous analysis, we identified a point mutation that substituted Pro (CCG) for Leu (CTG) at @entity535 87 in the alpha-subunit of the @entity8277 ( @entity8277 ) in a Japanese @entity1 with @entity10117 . 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 @entity535 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.
