Complementation analysis demonstrates that insulin cross-links both α subunits in a truncated insulin receptor dimer

Abstract

The insulin receptor is a homodimer composed of two αβ half receptors. Scanning mutagenesis studies have identified key residues important for insulin binding in the L1 domain (amino acids 1-150) and C-terminal region (amino acids 704-719) of the α subunit. However, it has not been shown whether insulin interacts with these two sites within the same α chain or whether it cross-links a site from each α subunit in the dimer to achieve high affinity binding. Here we have tested the contralateral binding mechanism by analyzing truncated insulin receptor dimers (midi-hIRs) that contain complementary mutations in each α subunit. Midi-hIRs containing Ala 14, Ala64, or Gly714 mutations were fused with Myc or FLAG epitopes at the C terminus and were expressed separately by transient transfection. Immunoblots showed that R14A+FLAG, F64A+FLAG, and F714G+Myc mutant midihIRs were expressed in the medium but insulin binding activity was not detected. However, after co-transfection with R14A+FLAG/F714G+Myc or F64A+FLAG/F714G+Myc, hybrid dimers were obtained with a marked increase in insulin binding activity. Competitive displacement assays revealed that the hybrid mutant receptors bound insulin with the same affinity as wild type and also displayed curvilinear Scatchard plots. In addition, when hybrid mutant midi-hIR was covalently cross-linked with 125I(A14)- insulin and reduced, radiolabeled monomer was immunoprecipitated only with anti-FLAG, demonstrating that insulin was bound asymmetrically. These results demonstrate that a single insulin molecule can contact both α subunits in the insulin receptor dimer during high affinity binding and this property may be an important feature for receptor signaling. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.