Real Time Kinetics of Insulin-like Growth Factor II (IGF-II) Interaction with the IGF-II/Mannose 6-Phosphate Receptor

Abstract

The interaction of soluble forms of the human cationindependent insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-IIR) with IGFs and mannosylated ligands was analyzed in real time. IGF-IIR proteins containing domains 1-15, 10 -13, 11-13, or 11-12 were combined with rat CD4 domains 3 and 4. Following transient expression in 293T cells, secreted protein was immobilized onto biosensor chips. ␤-Glucuronidase and latent transforming growth factor-␤1 bound only to domains 1-15. IGF-II bound to all constructs except a control, which contained a point mutation in domain 11. The affinity of domains 1-15, 10 -13, 11-13, and 11-12 to IGF-II were 14, 120, 100, and 450 nM, respectively. Our data suggest that domain 13 acts as an enhancer of IGF-II affinity by slowing the rate of dissociation, but additional enhancement by domains other than 10 -13 also occurs. As the receptor functions to transport ligands from either the trans-Golgi network or extracellular space to the endosomes, the interaction of IGF-IIR extracellular domains with IGF-II was analyzed over a pH range of 5.0 -7.4. The constructs behaved differently in response to pH and in recovery after low pH exposure, suggesting that pH stability of the extracellular domains depends on domains other than 10 -13.

A Novel Binding Site for the Human Insulin-like Growth Factor-II (IGF-II)/Mannose 6-Phosphate Receptor on IGF-II

“…These values were also similar to previous reports (16,36). All 14 mutants were tested for their ability to bind the IGF2R fragments.…”

Insulin‐like growth factor ligands, receptors, and binding proteins in cancer

“…The main structural domains of IGF-IIR that account for affinity and specificity are domains 11 and 13, with key hydrophobic residues (patch1) on domain 11 accounting for the main contribution of affinity [30][31][32].…”

Functional evaluation of novel soluble insulin-like growth factor (IGF)-II–specific ligand traps based on modified domain 11 of the human IGF2 receptor

“…Native IGF2R is too large and complex to mass produce, and so we have manipulated domain 11 to make a stable, soluble chimeric Fc-tagged protein with high affinity for IGF-II that is similar to the affinity and selectivity of the full-length version of the protein (36,44). When expressed as a fusion protein with human IgG1 Fc, the homodimer that is generated has the potential added advantages of increased valency, stability in vivo and clearance of trap and ligand via Fc(Rn) receptors, and enhanced complement activation (51).…”

“…Of the 15 homologous extracellular domains, only domain 11 directly binds IGF-II with domain 13 important for highaffinity binding (10 À10 mol/L; refs. 35,36). IGF2R loss of heterozygosity and mutations occur frequently in common cancers, such as hepatocellular (70%), breast (40%), and cancers associated with mismatch repair defects, and IGF2R is proposed to be a tumor suppressor gene (37 -40).…”