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-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. beta-Glucuronidase and latent transforming growth factor-beta1 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.
Linnell
J
J
Department of Zoology, University of Oxford, South Parks Rd., Oxford, OX1 3PS, United Kingdom. jane.linnell@zoo.ox.ac.uk
Groeger
G
G
Hassan
A B
AB
D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D006863:Hydrogen-Ion Concentration; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017527:Receptor, IGF Type 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
D017527:Receptor, IGF Type 2; D011993:Recombinant Fusion Proteins; D007335:Insulin-Like Growth Factor II; D008358:Mannose