COMPETITIVE-BINDING ASSAY OF SRC HOMOLOGY DOMAIN-3 INTERACTIONS BETWEEN 5-LIPOXYGENASE AND GROWTH-FACTOR RECEPTOR-BINDING PROTEIN-2

c-src homology 3 domains (SH3) modulate the formation of a number of p rotein complexes that are important in cell signaling and cytoskeletal organization. The SH3 domain is recognized by short conserved proline -rich motifs which adopt left-handed polyproline helices on binding. I n order to examine molecular determinants of the proline motif:SH3 int eraction, an enzyme-linked immunosorbent assay was developed to observ e binding of 5-lipoxygenase to SH3 domains of growth factor receptor b inding protein 2 (Grb2), The assay makes use of glutathione S-transfer ase fusion proteins of Grb2 and fragments of Grb2 immobilized onto wel ls of standard 96-well microtiter plates. Equilibrium binding is monit ored colorimetrically and the measured absorbance is proportional to 5 -LO concentration. The interaction is specific for the Grb2 portion of the fusion proteins, and 5-LO binds preferentially to Grb2 fragments containing an SH3 domain. Competitive binding assays with a synthetic peptide which mimicked the proline-rich region of 5-LO yielded results that are consistent with previous estimates. Binding was examined in the presence of a number of peptides containing the consensus sequence -PXXP-, in the presence of enzyme activity mediators and in the prese nce of plant lipoxygenases that lack the proline-rich binding motif. R esults suggest that the specificity of the Grb2:5-LO interaction is hi gh. (C) 1995 Academic Press