Precise mapping of an IGF-I-binding site on the IGF-1R

The IGFAR [type 1 IGF (insulin-like growth factor) receptor] is activated upon binding to IGF-I and IGF-II leading to cell growth, survival and migration of both normal and cancerous cells. We have characterized the binding interaction between the IGF-1R and its ligands using two high-affinity mouse anti-IGF-1R mAbs (monoclonal antibodies), 7C2 and 9E11. These mAbs both block IGF-I binding to the 1GF-1R but have no effect on IGF-II binding. Epitope mapping using chimaeras of the IGF-1R and insulin receptor revealed that the mAbs bind to the CR (cysteine-rich) domain of IGF-I R. The epitope was finely mapped using single point mutations in the IGF-1R. Mutation of Phe(241), Phe(251) or Phe(266) completely abolished 7C2 and 9E11 binding. The three-dimensional structure showed that these residues cluster on the surface of the CR-domain. BIAcore analyses revealed that IGF-I and a chimaeric IGF-II with the IGF-I C-domain competed for the binding of both mAbs with the IGF-I R, whereas neither IGF-II nor a chimaeric IGF-II with the IGF-II C-domain affected antibody binding. We therefore conclude the IGF-I C-domain interacts with the CR (cysteine-rich) domain of the receptor at the cluster of residues Phe(241), Phe(251) and Phe(266). These results allow precise orientation of IGF-I within the IGF-I-IGF-IR complex involving the IGF-I C-domain binding to the IGF-1R CR domain. In addition, mAbs 7C2 and 9E11 inhibited both IGF-I- and IGF-II-induced cancer cell proliferation, migration and IGF-1R downregulation, demonstrating that targeting the IGF-1R is an effective strategy for inhibition of cancer cell growth.