title: Precise mapping of an IGF-I-binding site on the IGF-1R.
abstract: The IGF-1R [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 IGF-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-1R. The epitope was finely mapped using single point mutations in the IGF-1R. Mutation of Phe241, Phe251 or Phe266 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-1R, whereas neither IGF-II nor a chimaeric IGF-I 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 Phe241, Phe251 and Phe266. These results allow precise orientation of IGF-I within the IGF-I-IGF-1R 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 down-regulation, demonstrating that targeting the IGF-1R is an effective strategy for inhibition of cancer cell growth.
authors.affiliation: School of Molecular and Biomedical Science, The University of Adelaide, Adelaide 5005, South Australia, Australia.
authors.forename: Mehrnaz
authors.initials: M
authors.lastname: Keyhanfar
authors.forename: Grant W
authors.initials: GW
authors.lastname: Booker
authors.forename: Jonathan
authors.initials: J
authors.lastname: Whittaker
authors.forename: John C
authors.initials: JC
authors.lastname: Wallace
authors.forename: Briony E
authors.initials: BE
authors.lastname: Forbes
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001943:Breast Neoplasms; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D005260:Female; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000737:chemistry / Q000378:metabolism*; D007668:Kidney; D051379:Mice; D010449:Peptide Mapping; D017354:Point Mutation; D011487:Protein Conformation; D017526:Receptor, IGF Type 1 / Q000737:chemistry / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin