Effect of replacing residues Val298 and Ile374 with different amino acids on the maturation and processing of pro-αIIb to mature αIIb.

Cells were cotransfected with cDNA constructs expressing control or mutant αIIb and control β3 subunits for 36 hours. Whole cell lysates were prepared and immunoprecipitated with antibodies to αIIb (B1B5 and M-148). Immunoblots were performed with another antibody to αIIb (PMI-1). Bands representing pro-αIIb and mature αIIb are shown by arrows. (A) Schematic model of a calcium-binding loop showing residues numbered −3 to −1 and 1-12. The amino acid sequences of the second and third calcium-binding domains of αIIb are shown. The Val298 and Ile374 residues are underlined and the amino acids in the putative N-linked glycosylation site created by the Ile374Thr mutation are bracketed. The amino acid substitutions for residues Val298, Ile374, and Asn372 are shown in the boxes. (B) Immunoblot of cells expressing amino acid substitutions for residue Val298. (C) Immunoblot of cells expressing amino acid substitutions for residue Ile374. (D) Immunoblot of cells expressing amino acid substitutions for Ile374 and Asn372.