Adam Denley

Structure and Functional Analysis of the Igf-II/Igf2R Interaction

Embryonic development and normal growth require exquisite control of insulin‐like growth factors (IGFs). In mammals the extracellular region of the cation‐independent mannose‐6‐phosphate receptor has gained an IGF‐II‐binding function and is termed type II IGF receptor (IGF2R). IGF2R sequesters IGF‐II; imbalances occur in cancers and IGF2R is implicated in tumour suppression. We report crystal structures of IGF2R domains 11–12, 11–12–13–14 and domains 11–12–13/IGF‐II complex. A distinctive juxtaposition of these domains provides the IGF‐II‐binding unit, with domain 11 directly interacting with IGF‐II and domain 13 modulating binding site flexibility. Our complex shows that Phe19 and Leu53 of IGF‐II lock into a hydrophobic pocket unique to domain 11 of mammalian IGF2Rs. Mutagenesis analyses confirm this IGF‐II ‘binding‐hotspot’, revealing that IGF‐binding proteins and IGF2R have converged on the same high‐affinity site.

Computational model for the IGF-II/IGF2r complex that is predictive of mutational and surface plasmon resonance data.

Insulin‐like growth factors (IGFs) are key regulators of cell proliferation, differentiation, and transformation, and are thus pivotal in cancer, especially breast, prostate, and colon neoplasm. Their potent mitogenic and anti‐apoptotic actions depend primarily on their availability to bind to the signaling IGF cell surface receptors. One mechanism by which IGF‐II availability is thought to be modulated is by binding to the nonsignaling IGF‐II receptor (IGF2R). This binding is essentially mediated by domain 11 in the multidomain IGF2R extracellular region. The crystal structure of domain 11 of the human IGF‐II receptor (IGF2R‐d11) has identified a putative IGF‐II binding site, and a nuclear magnetic resonance (NMR) solution structure for the IGF‐II ligand has also been characterized. These structures have now been used to model in silico the protein–protein interaction between IGF‐II and IGF2R‐d11 using the program 3D‐Dock. Because the IGF‐II data comprise an ensemble of 20 structures, all of which satisfy the NMR constraints, the docking procedure was applied to each member of the ensemble. Only those models in which residue Ile1572 of IGF2R‐d11, known to be essential for the binding of IGF‐II, was at the interface were considered further. These plausible complexes were then critically assessed using an array of analysis techniques including consideration of additional mutagenesis data. One model was strongly supported by these analyses and is discussed here in detail. Furthermore, we demonstrate in vitro experimental support for this model by studying the binding of chimeras of IGF‐I and IGF‐II to IGF2R fragments. Proteins 2006.