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 protein 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. In order to examine molecular determinants of the proline motif:SH3 interaction, an enzyme-linked immunosorbent assay was developed to observe binding of 5-lipoxygenase to SH3 domains of growth factor receptor binding protein 2 (Grb2). The assay makes use of glutathione S-transferase fusion proteins of Grb2 and fragments of Grb2 immobilized onto wells of standard 96-well microtiter plates. Equilibrium binding is monitored colorimetrically and the measured absorbance is proportional to 5-LO concentration. The interactions 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 presence of plant lipoxygenases that lack the proline-rich binding motif. Results suggest that the specificity of the Grb2:5-LO interaction is high.
VanderNoot
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Department of Pharmacology C236, University of Colorado Health Sciences Center, Denver 80262, USA.
Fitzpatrick
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D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry*; D001667:Binding, Competitive; D066246:ErbB Receptors / Q000737:chemistry*; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013053:Spectrophotometry; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D011506:Proteins; D011993:Recombinant Fusion Proteins; D001094:Arachidonate 5-Lipoxygenase; D005982:Glutathione Transferase; D066246:ErbB Receptors