STRUCTURAL BASIS BY WHICH A RECESSIVE MUTATION IN THE ALPHA-SUBUNIT OF THE INSULIN-RECEPTOR AFFECTS INSULIN BINDING

Recently, a mutation substituting Leu for Ser(323) in the alpha-subuni t of the human insulin receptor has been identified in an insulin-resi stant patient. The Leu(323) mutation leads to a severe impairment in i nsulin binding without significantly altering the processing or cell s urface expression of the receptor. In order to study how alpha beta ha lf-receptors interact to form the insulin-binding site, we cotransfect ed NIH-3T3 cells with two insulin receptor cDNA constructs: a truncate d insulin receptor lacking the C-terminal 43 amino acids (Delta 43) an d the full-length Leu(323) mutant receptor. A clonal cell line from co transfected cells expresses a hybrid receptor consisting of a Leu(323) half-receptor and a Delta 43 half-receptor. We demonstrate that the L eu(323)-Delta 43 hybrid receptor binds insulin with high affinity. Fur thermore, by cross-linking I-125-insulin to immobilized hybrid recepto rs, we show that only the alpha beta(Delta) half of the hybrid recepto r binds insulin. Since the isolated half-insulin receptor has low affi nity for insulin, this suggests that the addition of even a non-bindin g alpha-subunit can result in high affinity binding to the holorecepto r (alpha alpha(mut)beta(Delta)beta). Both beta and beta(Delta)-subunit s of the Leu(323)-Delta 43 hybrid receptor are phosphorylated in vivo and in vitro in an insulin-dependent manner, suggesting an intramolecu lar transphosphorylation mechanism and that the presence of the Leu(32 3) mutant receptor that lacks an intrinsic high affinity binding site does not prevent the associated beta-subunit from functioning either a s a tyrosine kinase or as a phosphate acceptor in the hybrid insulin r eceptor molecule (alpha alpha(mut)beta(Delta)beta). Furthermore, we sh ow that the hybrid receptor can phosphorylate insulin receptor substra te-1 (IRS-1) in response to insulin and can be coimmunoprecipitated to gether with IRS-1 by anti-IRS-1 antibody.