GpIb binding to 14-3-3ζ inhibited by truncations or deletions in its cytoplasmic domain.

GpIbα mutants were expressed with wild-type GpIbβ and GpIX. The capacity of these mutants to bind to 14-3-3ζ was examined in cell lysates precipitated with a GST-14-3-3ζ fusion protein (right) or with mAb AN51 that recognizes the extracellular domain of GpIbα (left). Truncation of the cytoplasmic domain of GpIbα at residue 542 eliminated binding to the 14-3-3ζ fusion protein and eliminated endogenous 14-3-3ζ coimmunoprecipitating with GpIbα. Deletion of the large cytoskeletal interaction domain(s) between residues 542 and 590 (ABP in Figure 2, which maintains the C-terminal 14-3-3ζ binding site) also eliminated the capacity of recombinant GpIbα to bind to the GST-14-3-3ζ fusion protein and eliminated the interaction of endogenous 14-3-3ζ with recombinant GpIbα in CHO cells. Truncation of GpIbα at amino acid 594 eliminated 14-3-3ζ coimmunoprecipitating with GpIbα. No immunodetectable GpIbα truncated at residue 594 bound GST-14-3-3ζ (IP, immunoprecipitation; n = 3).