2 MUTATIONS IN A CONSERVED STRUCTURAL MOTIF IN THE INSULIN-RECEPTOR INHIBIT NORMAL FOLDING AND INTRACELLULAR-TRANSPORT OF THE RECEPTOR

Insulin initiates its biological response by binding to the extracellu lar domain of the insulin receptor. The N-terminal half of the alpha-s ubunit contains several repeats of a loosely conserved motif consistin g of a central glycine plus several hydrophobic amino acid residues up stream from the glycine, Hy phi Xaa-Xaa-Hy phi-Xaa-Hy phi Hy phi-Xaa-G ly (where Hy phi represents a hydrophobic amino acid residue). This st ructural motif has been proposed to be important in determining the th ree-dimensional structure of the insulin binding domain. We have ident ified two naturally occurring mutant alleles of the insulin receptor g ene in an insulin-resistant patient, substitution of Ala for Val(28) a nd Arg for Gly(366). The mu- tations alter conserved amino acid residu es in two distinct repeats of the structural motif described above, Wh en mutant cDNAs were expressed in NIH-3T3 cells, both mutations severe ly impaired proteolytic processing of the proreceptor to mature alpha and beta-subunits. Transport of mutant receptors to the plasma membran e was also impaired. However, the minority (<1O%) of receptors that we re eventually transported to the plasma membrane retained the ability to bind insulin with nor- mal affinity and to undergo insulin-stimulat ed phosphorylation. In conclusion, the effects of these naturally occu rring mutations provide experimental support for the importance of the conserved glycine-containing structural motifs described above. By in terrupting these structural motifs, the Ala(28) and Arg(366) mutations prevent normal folding of the insulin receptor alpha-subunit, thereby inhibiting post-translational processing and intracellular transport of the mutant receptors.