Two 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 extracellular domain of the insulin receptor. The N-terminal half of the α-subunit contains several repeats of a loosely conserved motif consisting of a central glycine plus several hydrophobic amino acid residues upstream from the glycine, Hyφ-Xaa-Xaa-Hyφ-Xaa-Hyφ-Hyφ-Xaa-Gly (where Hyφ represents a hydrophobic amino acid residue). This structural motif has been proposed to be important in determining the three-dimensional structure of the insulin binding domain. We have identified two naturally occurring mutant alleles of the insulin receptor gene in an insulin-resistant patient, substitution of Ala for Val²⁸ and Arg for Gly³⁶⁶. The mutations alter conserved amino acid residues in two distinct repeats of the structural motif described above. When mutant cDNAs were expressed in NIH-3T3 cells, both mutations severely impaired proteolytic processing of the proreceptor to mature α- and β-subunits. Transport of mutant receptors to the plasma membrane was also impaired. However, the minority (<10%) of receptors that were eventually transported to the plasma membrane retained the ability to bind insulin with normal affinity and to undergo insulin-stimulated phosphorylation. In conclusion, the effects of these naturally occurring mutations provide experimental support for the importance of the conserved glycine-containing structural motifs described above. By interrupting these structural motifs, the Ala²⁸ and Arg³⁶⁶ mutations prevent normal folding of the insulin receptor α-subunit, thereby inhibiting post-translational processing and intracellular transport of the mutant receptors.