α_IIb TMD-tail binds to the β₃ subunit of native integrin α_IIbβ₃. (A) α_IIb^TM-TAP, α_IIb^TMΔGFFKR-TAP, or empty vector was transfected into A5 cells, and after 24 hours, cells were lysed and incubated with calmodulin beads to capture TAP constructs. β₃ was detected by Western blot using anti-β₃ antibody as representative of the bound α_IIbβ₃ (top). Expression level of β₃ in lysates (middle) and captured TAP constructs (bottom) was assessed by Western blot. (B) A5 cells transfected with Tac-α_IIb^TM or Tac-α_IIb were detached and stained with anti-Tac antibody and PAC1. In the merged image, green and red represent Tac and active integrin, respectively. The 2.7-fold digital enlargement of areas in small rectangles is shown in insets. Bar = 5 μm. (C) A5 cells were incubated for 30 minutes at 37°C with TBS (20 mM Tris, pH 8.4, 150 mM NaCl) containing either 1 mM Ca/Mg or 5 mM EDTA, and stained with α_IIbβ₃ complex-specific antibody (D57). D57 binding was measured by flow cytometry and plotted as histogram. (D) A5 cells transfected with TAP constructs were detached, and surface proteins were biotinylated and treated with EDTA to dissociate α_IIbβ₃ complex before the cells were lysed. Five percent of the lysates were incubated with neutravidin beads to determine the input of biotinylated proteins in the lysates (lanes 5-8). The remaining lysates were first incubated with calmodulin beads to capture TAP tag, and the bound proteins were eluted with 10 mM EDTA. The eluates were further incubated with NeutrAvidin beads to capture the biotinylated protein (lanes 1-4). Anti-α_IIb antibody (PMI-1), anti-β₃ antibody (Rb8053), and anti-FLAG antibody (M2) were used for the Western blot, as indicated. Schematic procedure for this experiment is shown in Figure S4. (E) Model of how the α_IIb TMD-tail induced α_IIbβ₃ activation. α_IIb TMD-tail interacts with TMD-tail region of β₃ in the native integrin and competes for the heterodimeric interaction between α_IIb and β₃, resulting in rearrangement of the TMD and activation.