Real time kinetics of insulin-like growth factor II (IGF-II) interaction with the IGF-II/mannose 6-phosphate receptor - The effects of domain 13 and pH

The interaction of soluble forms of the human cation-independent insulin-li ke growth factor-II/mannose 6-phosphate receptor (IGF-IIR) with IGFs and ma nnosylated ligands was analyzed in real time. IGF-IIR proteins containing d omains 1-15, 10-13, 11-13, or 11-12 were combined with rat CD4 domains 3 an d 4. Following transient expression in 293T cells, secreted protein was imm obilized onto biosensor chips. beta -Glucuronidase and latent transforming growth factor-beta1 bound only to domains 1-15. IGF-II bound to all constru cts except a control, which contained a point mutation in domain 11. The af finity 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 enh ancer of IGF-II affinity by slowing the rate of dissociation, but additiona l enhancement by domains other than 10-13 also occurs. As the receptor func tions to transport ligands from either the trans-Golgi network or extracell ular space to the endosomes, the interaction of IGF-IIR extracellular domai ns with IGF-II was analyzed over a pH range of 5.0-7.4, The constructs beha ved differently in response to pH and in recovery after low pH exposure, su ggesting that pH stability of the extracellular domains depends on domains other than 10-13.