Fig. 5. Mapping the interaction of IIbß3 integrin and DAB2. (A) Schematic representation of the recombinant GST-IIb integrin and ß3 integrin mutant proteins. The shaded box of IIb integrin (aa 171-334) represents the fibrinogen-binding site. The shaded region (aa 692-722) of ß3 integrin represents the transmembrane domain. (B) Amino acids 171-464 of IIb integrin interact with DAB2. Cell lysates (1 mg) of DAB2-transfected K562 cells were collected for GST-pull-down assays using the indicated GST-IIb integrin (constructs a-d) and GST-ß3 (construct e) proteins or control GST proteins (25 µg). The pull-down lysates (upper panel) and the aliquots of the input GST-proteins (lower panel) were subjected to western blot analysis with anti-DAB2 and anti-GST antibodies. The expected band for each purified protein is denoted by asterisk. (C) Schematic representation of the RGD motif and its flanking sequences for human, rat, and mouse DAB2. (D) The DAB2-RGD peptide interferes with DAB2–IIb-integrin interaction. Cell lysates (1 mg) of DAB2-transfected K562 cells were subjected to GST-pull-down analyses with GST-IIb-integrin aa 171-464 (construct d) in the absence or presence of DAB2-RGD and DAB2-RGE peptide (100 µg/ml). The pull-down lysates were subjected to western blot analysis using anti-DAB2 antibody. (E) The resting or TRAP-stimulated (10 µM) platelets (1.5x10⁸) were added into the plates pre-coated with GST, DAB2-PTB or DAB2-D66E (100 µg/ml) for 3 hours at 37°C. Platelet adhesion was then quantified by the Crystal Violet assay. The data represent the mean ± s.d. (n=5). **P<0.001 compared with the TRAP-stimulated platelet adhesion to GST control protein.