CONSTRUCTION OF AN SH2 DOMAIN-BINDING SITE WITH MIXED SPECIFICITY

SH2 domains bind to specific phosphotyrosine-containing sites in a fas hion dictated by the amino acids flanking the phosphotyrosine. Attenti on has focused on the role of the three COOH-terminal positions (+1 to +3) in generating specificity. Autophosphorylation of Tyr(1021) in th e tail of the beta-receptor for platelet-derived growth factor creates a specific binding site for the COOH-terminal SH2 domain of phospholi pase C (PLC)-gamma 1. We show that the residues 4 and 5 amino acids CO OH-terminal to Tyr(1021) (+4 Leu and +5 Pro) are required for efficien t PLC-yl binding, and that their replacement with the corresponding re sidues from a phosphatidylinositol 3'-kinase binding site abrogates st able association with PLC-gamma 1. In contrast, replacement of the +3 Pro with Met produces a Tyr(1021) Site with mixed specificity that bin ds both PLC-gamma 1 and phosphatidylinositol 3'-kinase. This motif is rendered specific for phosphatidylinositol 3'-kinase by further substi tution of the +4 Leu. These results indicate that the +4 and +5 residu es are important for the selective binding of specific SH2 domains. Th is study suggests that phosphotyrosine sites can be tailored to bind o ne or more SH2 domains with high affinity, depending on the combinatio n of residues in the +1 to +5 positions.