An SPR-based screening method for agonist selectivity tar insulin signaling pathways based on the binding of phosphotyrosine to its specific binding protein

A new screening method was developed that evaluates physiologically relevan t chemical selectivity of agonists for insulin-signaling pathways, Phosphor ylation (pY939)by an insulin-activated insulin receptor of a target peptide (Y939) derived from an insulin receptor substrate-1 (IRS-1) and its subseq uent binding to another downstream target, the SH2 domain of PI-3 kinase (S H2N), were detected by surface plasmon resonance (SPR) spectrometry,This me thod is based on competitive binding of SH2N to pY939 either in a solution or on the gold suyface of the SPR sensor chip. With increasing the concentr ation of pY939 in solution by the insulin-induced kinase reaction of insuli n receptor, SH2N bound to pY939 in solution increases and the one on the se nsor chip decreases, thereby causing a decrease in the SPR signal. The amou nt of thus-detected complex pY939-SN2N 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 activ ity of insulin receptor-agonist complexes increased in the order of IGF-II < IGF-I < insulin, and neither vanadium ions nor thiazolidine-type medicine s for NIDDM, troglitazone and pioglitazone, directly acted on both the kina se reaction of insulin receptor or the binding of pY939 to SH2N, The presea l approach will thus become a general method for screening agonists for one specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling , which is regulated by specific protein-protein interaction between a phos phorylated tyrosine in IRS-1 and its corresponding SH2 domain-containing pr otein such as PI-3 kinase, Grb2-Sos, or SHP2.