Congenital insulin resistance associated with a conformational alteration in a conserved beta-sheet in the insulin receptor L1 domain

The hormone binding site of members of the insulin receptor family is conta ined within a highly conserved extracellular region of the receptor. Recent crystallization of the N-terminal region of the binding site revealed two large domains (L1, L2), each organized as a single-stranded right-handed be ta-helix, connected by a rod-shaped cysteine-rich domain. Here, we analyze two new naturally occurring mutations in a single p-sheet within L1, D59G a nd L62P, that we previously identified in a young woman with classic congen ital insulin resistance (type A). Substitution of D59G, a beta-sheet connec ting loop residue, caused decreased hormone binding but did not disrupt ove rall folding, assembly, or movement to the cell surface. In contrast, repla cement of the adjacent residue L62P, which is located within the beta-sheet , and positioned in a hormone binding surface, completely disrupted intrace llular folding, oligomerization, and trafficking and resulted in aberrant p roteolytic degradation. Immunohistochemistry in combination with biosynthet ic studies showed that misfolded receptors were retained in an incorrect ce llular location and that they colocalized with the resident endoplasmic ret iculum chaperone calnexin, This study, together with other mutagenesis data , shows that formation of beta-sheet elements within the L1 beta-helix are critical for the folding of the entire extracellular domain of the receptor and that the hormone contact site is composed in part by residues in this domain.