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

Surface plasmon resonance (SPR) biosensor technique was used to study the effects of Mg²⁺, Mn²⁺, 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 × 10⁹ M) is about 10 times higher than that without IGF-1 stimulation (1.27 × 10⁸ M). The association and dissociation rates of IRS-1 binding to phosphorylated IGF-1R in the presence of either or both of Mg²⁺ and Mn²⁺, 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 Mg²⁺ and Mn²⁺. Mn²⁺ slightly increased the affinity mainly due to the increased association rate, while Mg²⁺ and Mn²⁺ 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. © 2007 Elsevier B.V. All rights reserved.