Va. Vandernoot et Fa. Fitzpatrick, COMPETITIVE-BINDING ASSAY OF SRC HOMOLOGY DOMAIN-3 INTERACTIONS BETWEEN 5-LIPOXYGENASE AND GROWTH-FACTOR RECEPTOR-BINDING PROTEIN-2, Analytical biochemistry, 230(1), 1995, pp. 108-114
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