Figure 1
Figure 1. Integrin αIIb and β3 TMD-tail mini-integrins interact with each other via the TMDs. (A) Amino acid sequences of αIIbTM and β3TM used in this study. Sequences in the gray box represent amino acid residues in membrane region. GXXXG motif and GFFKR motif in αIIb are underlined. Point mutations used in this study are indicated with arrows. The previously designated membrane-proximal regions containing hydrophobic stretches in both α and β integrin are boxed.25 (B) Schematic diagram of TMD-tail constructs. For β3TM-TAP and αIIbTM-TAP baits, β3TM and αIIbTM (A) were fused to TAP tag for purification and an N-terminal FLAG tag for detection. Tac-αIIbTM and Tac-β3TM were made by fusion of αIIbTM and β3TM, respectively, with Tac extracellular domain. (C) CHO cells were transiently transfected with αIIbTM-TAP (bait) and Tac-αIIbTM or Tac-β3TM (preys), and cells were lysed and incubated with calmodulin beads to capture the baits. Bound Tac constructs were analyzed by Western blot using anti-Tac antibody (top panels). Expression of Tac preys (middle panel) and captured αIIbTM-TAP (bottom panel) were verified by Western blot using anti-Tac antibody and anti-FLAG antibody, respectively. The arrows indicate mature cell-surface proteins, and the arrowheads incompletely glycosylated intracellular proteins. Open symbols represent Tac-β3TM, and closed symbols represent Tac-αIIbTM. (D) CHO cells were transiently transfected with baits and preys, as indicated, and cell-surface proteins were biotinylated before cell lysis. Ten percent of the lysates were incubated with neutravidin beads to determine the input of biotinylated proteins in the lysates (middle and bottom panels). The remaining lysates were first incubated with calmodulin beads to capture the baits, and the bound proteins were eluted with 10 mM EDTA. The eluates were then incubated with NeutrAvidin beads to capture the biotinylated surface proteins and the presence of Tac preys was analyzed with Western blot using anti-Tac antibody (top panel). (E) αIIbTM-TAP constructs containing deletion of GFFKR motif or mutations of 2 Gly in GXXXG motif to Leu were tested for their binding to Tac-β3 as in (C).

Integrin αIIb and β3 TMD-tail mini-integrins interact with each other via the TMDs. (A) Amino acid sequences of αIIbTM and β3TM used in this study. Sequences in the gray box represent amino acid residues in membrane region. GXXXG motif and GFFKR motif in αIIb are underlined. Point mutations used in this study are indicated with arrows. The previously designated membrane-proximal regions containing hydrophobic stretches in both α and β integrin are boxed.25  (B) Schematic diagram of TMD-tail constructs. For β3TM-TAP and αIIbTM-TAP baits, β3TM and αIIbTM (A) were fused to TAP tag for purification and an N-terminal FLAG tag for detection. Tac-αIIbTM and Tac-β3TM were made by fusion of αIIbTM and β3TM, respectively, with Tac extracellular domain. (C) CHO cells were transiently transfected with αIIbTM-TAP (bait) and Tac-αIIbTM or Tac-β3TM (preys), and cells were lysed and incubated with calmodulin beads to capture the baits. Bound Tac constructs were analyzed by Western blot using anti-Tac antibody (top panels). Expression of Tac preys (middle panel) and captured αIIbTM-TAP (bottom panel) were verified by Western blot using anti-Tac antibody and anti-FLAG antibody, respectively. The arrows indicate mature cell-surface proteins, and the arrowheads incompletely glycosylated intracellular proteins. Open symbols represent Tac-β3TM, and closed symbols represent Tac-αIIbTM. (D) CHO cells were transiently transfected with baits and preys, as indicated, and cell-surface proteins were biotinylated before cell lysis. Ten percent of the lysates were incubated with neutravidin beads to determine the input of biotinylated proteins in the lysates (middle and bottom panels). The remaining lysates were first incubated with calmodulin beads to capture the baits, and the bound proteins were eluted with 10 mM EDTA. The eluates were then incubated with NeutrAvidin beads to capture the biotinylated surface proteins and the presence of Tac preys was analyzed with Western blot using anti-Tac antibody (top panel). (E) αIIbTM-TAP constructs containing deletion of GFFKR motif or mutations of 2 Gly in GXXXG motif to Leu were tested for their binding to Tac-β3 as in (C).

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