An SPR-Based Screening Method for Agonist Selectivity for Insulin Signaling Pathways Based on the Binding of Phosphotyrosine to Its Specific Binding Protein

摘要

A new screening method was developed that evaluates physiologically relevant chemical selectivity of agonists for insulin-signaling pathways.Phosphorylation(pY939)by an insulin-activated insulin recepor of a target peptide (Y939)derived from an insulin receptor substrate-1 (IRS-1)and its subsequent binding to anoher downstream target,the SH2 domain of PI-3 kinase (SH2N),were detected by surface plasmon resonance (SPR)spectometry.This method is based on competitive binding of SH2N to pY939 either in a solution or on the gold surface of the SPR sensor chip.With increasing the concentration of pY939 in solution by the insulin-induced kinase reaction of insulin receptor,SH2N bound to pY939 in solutin increases and the one on the sensor chip decreases,thereby causing a decrease in the SPR signal.The amount of thus-detected complex pY939-SH2N was found to depend on added insulin concentrations,confirming that the method utilized part of the sequential transduction mechanism of the insulin-signaling pathways.The kinase activity of insulin receptor-agonist complexes increased in the order of IGF-II