INTERACTION OF PHOSPHORYLATED FC-EPSILON-RI-GAMMA IMMUNOGLOBULIN RECEPTOR TYROSINE ACTIVATION MOTIF-BASED PEPTIDES WITH DUAL AND SINGLE SH2DOMAINS OF P72(SYK) - ASSESSMENT OF BINDING PARAMETERS AND REAL, TIMEBINDING-KINETICS

To examine the characteristics of the interaction of the Fc epsilon RI gamma ITAM with the SH2 domains of p72(syk), the binding of an I-125- labeled dual phosphorylated Fc epsilon RI gamma ITEM-based peptide to the p72(syk) SH2 domains was monitored utilizing a novel scintillation proximity based assay. The K-d for this interaction, determined from the saturation binding isotherm, was 1.4 nM. This high affinity bindin g was reflected in the rapid rate of association for the peptide bindi ng to the SH2 domains. Competition studies utilizing a soluble C-termi nal SH2 domain knockout and N-terminal SH2 domain knockouts revealed t hat both domains contribute cooperatively to the high affinity binding . I-125-labeled dual phosphorylated peptide competed with the I-125-la beled peptide for binding to the dual p72(syk) SH2 domains with an IC5 0 value of 4.8 nM. Monophosphorylated 24-mer Fc epsilon RI gamma ITAM peptides, and phosphotyrosine also competed for binding, but with subs tantially higher IC50 values. This, and other data discussed, suggest that high affinity binding requires both tyrosine residues to be phosp horylated and that the preferred binding orientation of the ITAM is su ch that the N-terminal phosphotyrosine occupies the C-terminal SH2 dom ain and the C-terminal phosphotyrosine occupies the N-terminal SH2 dom ain.