An SPR-based screening method for agonist selectivity for insulin signaling pathways based on the binding of phosphotyrosine to its specific binding protein.
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 receptor of a target peptide (Y939) derived from an insulin receptor substrate-1 (IRS-1) and its subsequent binding to another downstream target, the SH2 domain of PI-3 kinase (SH2N), were detected by surface plasmon resonance (SPR) spectrometry. 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 solution 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 < IGF-I < insulin, and neither vanadium ions nor thiazolidine-type medicines for NIDDM, troglitazone and pioglitazone, directly acted on both the kinase reaction of insulin receptor or the binding of pY939 to SH2N. The present 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 phosphorylated tyrosine in IRS-1 and its corresponding SH2 domain-containing protein such as PI-3 kinase, Grb2-Sos, or SHP2.
Yoshida
T
T
Department of Chemistry, School of Science, University of Tokyo, Japan.
Sato
M
M
Ozawa
T
T
Umezawa
Y
Y
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D007328:Insulin / Q000819:agonists* / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism*; D011972:Receptor, Insulin / Q000819:agonists* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D020349:Surface Plasmon Resonance / Q000379:methods
C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; C122033:phospho-Y939 peptide; D019000:Phosphotyrosine; D011972:Receptor, Insulin