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

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摘要：

The interaction of soluble forms of the human cation-independent 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. β-Glucuronidase and latent transforming growth factor-β1 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 n, 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 -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.

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Animals Cell Line Insulin-Like Growth Factor II Mannose Recombinant Fusion Proteins Receptor, IGF Type 2 Biotinylation Surface Plasmon Resonance Protein Structure, Tertiary Protein Binding