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

A receptor binding assay based on the surface plasmon resonance (SPR) biose nsor technique was developed to study the interaction between insulin-like growth factor-1 receptor (IGF-1R) and its intracellular substrate protein i nsulin receptor substrate-1 (IRS-1). The sensor surface was modified with a nti-IGF-1R (alpha -subunit) monoclonal antibodies for the capturing of the receptor-containing membrane fragments from cell lysates. The IGF-1R was su ccessfully immobilized on the sensor surface with binding capability for it s intracellular substrates. SPR measurements showed that the tyrosine phosp horylation of IGF-1R induced by its extracellular ligand insulin-like growt h 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 phosp horylated and unphosphorylated IGF-IR were (8.06 +/- 5.18) X 10(9) M-1 and (9.81 +/- 4.61) X 10(8) M-1, respectively. (C) 2001 Published by Elsevier S cience B.V. on behalf of the Federation of European Biochemical Societies.