INTERACTIONS BETWEEN SH2 DOMAINS AND TYROSINE-PHOSPHORYLATED PLATELET-DERIVED GROWTH-FACTOR BETA-RECEPTOR SEQUENCES - ANALYSIS OF KINETIC-PARAMETERS BY A NOVEL BIOSENSORS-BASED APPROACH

The interaction between SH2 domains and phosphotyrosine-containing seq uences was examined by real-time measurements of kinetic parameters. T he SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as c apturing molecules, immobilized on a biosensor surface. The associatio n and dissociation rate constants for binding to both sites were deter mined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for al l interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinit y to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be gene rally applicable to the study of the specificity inherent in the assem bly of signaling complexes by activated protein-tyrosine kinase recept ors.