Obliteration of the binding of full-length FLNa to the GPIb complex in CHO cells by the point mutations in IgFLNa17 and by thiol alkylation. (A) ClustalW alignment31 of domain 17 of FLN isoforms. Residues inferred from the atomic structure to be involved in interactions with GPIbα and tested by mutagenesis are indicated by the gray bars. Potential targets of NEM are indicated by the black bars. (B) Double mutations at G1897D and C1912D of IgFLNa17 abolish IgFLNa17/GPIb binding. Increasing amounts of purified MBP-IgFLNa17 (WT, wild type) and its mutants (G1897D, I1910M, C1912D, and G1897D+C1912D) were incubated with GPIbα556-577–peptide beads, and bound proteins were detected by SDS-PAGE followed by Coomassie blue staining. (C) GFP-FLNa and its mutants were expressed in CHO-GPIb/IX cells and coimmunoprecipitated with the GPIb-IX complex. GFP-FLNa is detected by immunoblot with anti-GFP mAb. The intensity of the band was analyzed by densitometry and the relative intensity graphed. Data are presented as mean ± SD of 3 independent experiments. (D) FLN is expressed in CHO-GPIb/IX cells and coimmunoprecipitated with the GPIb-IX complex. The linkage is uncoupled by treating the cell lysate with cystein-blocking reagent, NEM. Since only anti-FLNb, but neither anti-FLNa nor anti-FLNc antibodies available cross-react with hamster FLN, immunoblot was performed with anti-FLNb mAb (1-11c). IP indicates immunoprecipitation; IB, immunoblot.
