Measurement of binding kinetics between PI3-K and phosphorylated IGF-1R using a surface plasmon resonance biosensor

Abstract

Quantitative measurement of biochemical and biophysical parameters of molecular recognition events in signaling pathways is very important for understanding biological function. Binding of insulin-like growth factor-1 receptor (IGF-1R) with IGF-1 activates receptor tyrosine phosphorylation and triggers several signaling pathways including phosphatidylinositol-3 kinase (PI3-K)/AKT and extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) pathways. We have analyzed the interactions between PI3-K and IGF-1R with or without IGF-1 stimulation. The results demonstrate that p85 subunits of PI3-K bind to IGF-1R with IGF-1 stimulation in intact cells. The binding kinetics between PI3-K and IGF-1R with or without IGF-1 stimulation were obtained using surface plasmon resonance biosensor. The affinity constant of the PI3-K to phosphorylated IGF-1R was (2.27 ± 0.12) × 108 M−1, which was about 20 times higher than that of PI3-K to unphosphorylated IGF-1R. Moreover, the kinetic effects of Mg2+, ATP and two kinase inhibitors, genistein and quercetin, on the binding between PI3-K and phosphorylated IGF-1R were studied. The data showed that Mg2+ increased the binding affinity of PI3-K with IGF-1R about 2-fold, while genistein decreased the affinity constant 2.7-fold. On the other hand, ATP and quercetin had no significant effects on the affinity constant, although both ka and kd values were increased or decreased by ATP or quercetin, respectively. This study implicated that the PI3-K binding sites on IGF-IR may be different from its phosphorylation and catalytic sites.