Study of low molecular weight effectors on the binding between cell membrane receptor IGF-1R and its substrate protein IRS-1 by SPR biosensor

Abstract

Surface plasmon resonance (SPR) biosensor technique was used to study the effects of Mg 2+, Mn 2+, ATP, genistein, and quercetin on the binding kinetics between insulin-like growth factor-1 receptor (IGF-1R) and its intracellular substrate protein insulin receptor substrate-1 (IRS-1). IGF-1R was captured from cell lysates using anti-IGF-1R (α-subunit) monoclonal antibodies immobilized on biosensor chip surface, which retained the binding capability with its intracellular substrate. With IGF-1 stimulation, the association rate of IRS-1binding to IGF-1R increased and the affinity constant (1.37 × 10 9 M) is about 10 times higher than that without IGF-1 stimulation (1.27 × 10 8 M). The association and dissociation rates of IRS-1 binding to phosphorylated IGF-1R in the presence of either or both of Mg 2+ and Mn 2+, and in the absence or presence of ATP, genistein, and quercetin, were determined from the real-time binding and dissociation curves and the results indicate that ATP, genistein, and quercetin reduced the affinity constants of IRS-1 and IGF-1R by 4.7-, 6.0-, and 6.6-folds, respectively, in the presence of Mg 2+ and Mn 2+. Mn 2+ slightly increased the affinity mainly due to the increased association rate, while Mg 2+ and Mn 2+ together showed no effect on the affinity. In addition, ATP alone did not affect the binding affinity. The results demonstrate that SPR biosensor could be used as a quantitative technique for studying effects of small molecules on the binding kinetics and affinity of receptor–cellular substrate. The method developed in the study may also be used in other biosensor techniques for studying the mechanism of molecule interactions.