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Blood, Vol. 113, Issue 19, 4747-4753, May 7, 2009
Interactions of platelet integrin  IIb and β3 transmembrane domains in mammalian cell membranes and their role in integrin activation
Blood Kim et al.
113: 4747
Supplemental materials for: Kim et al
Files in this Data Supplement:
- Figure S1. Upper band of Tac-b3™ is glycosylated and plasma membrane-localized (JPG, 64.7 KB)
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(A) αIIb™-TAP and Tac-β3™ or Tac-β3 (containing cytoplasmic tail of β3 (K716-T762) and extracellular and TMD of Tac) were co-transfected into CHO cells, and their interaction was tested as in Fig. 1C. (B) αIIb™-TAP and Tac-β3™ were co-transfected into CHO cells. One day after transfection, cells were lysed and incubated with calmodulin beads to capture αIIb™-TAP. The beads were treated with an enzyme that cleaves N-linked oligosaccharides, PNGase F, as indicated. The beads bound proteins were analyzed by western blot using anti Tac antibody (upper panel) and anti flag antibody (bottom). Arrow and arrow head indicate glycosylated and non-glycosylated form of Tac-αIIb™, respectively. (C) αIIb™-TAP and Tac-β3™ were co-transfected into CHO cells. One day after transfection, cell surface proteins were biotinylated as indicated before the cells were lysed and incubated with calmodulin beads. The bound proteins were eluted with 10 mM EDTA, and the eluates were further incubated with neutravidin beads to capture biotinylated proteins. The bound proteins were analyzed by western blot using anti Tac antibody (upper panel) and anti flag antibody (bottom). Arrow and arrow head indicate Tac-αIIb™ in the cell surface and in intracellular membranes, respectively.
- Figure S2 (JPG, 92.7 KB)
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(A) Distribution of β3 backbone HN-N chemical shift changes upon αIIb-β3 heterodimerization Somewhat increased 15N chemical shift changes are observed at the intracellular membrane border, but, not surprisingly, the magnitudes of HN-N shift changes correlate only weakly with residues that are expected in the heterodimerization interface (e.g., Gly708). The magnitude of HN-N shift changes does not correlate directly with the magnitude of the underlying structural change. Rather, effects from neighboring residues, slight changes in helical hydrogen bonding, and the orientation of aromatic rings can cause large indirect effects, resulting in shift changes for virtually all transmembrane residues. (B) Effect of the Arg995Ala substitution on αIIb chemical shifts. Significant backbone HN-N chemical shifts differences from the side chain substitution were obtained exclusively at the site of mutation, including the preceding and following residues. This verifies that the fold of the transmembrane segment of the αIIb(Arg995Ala) peptide is indistinguishable from wild-type αIIb, confirming that the loss of the heterodimeric set of β3 resonances upon αIIb(Arg995Ala) substitution results from the loss of αIIb(Arg995)-β3(Asp723) electrostatic interactions. The transmembrane helix (Ile966-Lys989) borders and the Lys994-Arg995 transition are marked in all panels by blue lines.
- Figure S3. Tac-αIIb™ does not activate α5 β1 integrin (JPG, 47.1 KB)
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Affinity state of α5 β1 integrin in CHO cells transfected with Tac-αIIb™ or Tac-βIIb were measured by incubating with fibronectin (FN9-11). Cells were also stained in the presence of 10 mM EDTA to determine the non-specific binding of FN9-11, and in the presence of activating antibody, 9EG7. The cells were analyzed with flow cytometry to generate density plot.
- Figure S4. Schematic of the procedure for the experiment in Fig. 5D (JPG, 92 KB)
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