Effect of receptor phosphorylation on the binding between IRS-1 and IGF-1R as revealed by surface plasmon resonance biosensor

A receptor binding assay based on the surface plasmon resonance (SPR) biosensor technique was developed to study the interaction between insulin-like growth factor-1 receptor (IGF-1R) and its intracellular substrate protein insulin receptor substrate-1 (IRS-1). The sensor surface was modified with anti-IGF-1R (α-subunit) monoclonal antibodies for the capturing of the receptor-containing membrane fragments from cell lysates. The IGF-1R was successfully immobilized on the sensor surface with binding capability for its intracellular substrates. SPR measurements showed that the tyrosine phosphorylation of IGF-1R induced by its extracellular ligand insulin-like growth factor-1 caused the receptor to bind with IRS-1 10 times faster than the unactivated receptor. As a result, the affinity constants of IRS-1 to phosphorylated and unphosphorylated IGF-1R were (8.06 ± 5.18) × 10⁹ M^-1 and (9.81 ± 4.61) × 10⁸ M^-1, respectively. © 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.