A single amino acid substitution flanking the fourth calcium binding domain of alpha IIb prevents maturation of the alpha IIb beta 3 integrin complex.

To define specific structural domains involved in the biosynthesis and processing of integrin subunits, we examined the biosynthesis of normal and mutated forms of the platelet-specific integrin alpha IIb beta 3. Platelet mRNA was isolated from a Glanzmann thrombasthenic patient who failed to express significant levels of the glycoprotein (GP) IIb-IIIa complex on the platelet surface. Sequence analysis of polymerase chain reaction-amplified platelet GPIIb mRNA revealed a Gly418-->Asp amino acid substitution in GPIIb. Gly418 is a highly conserved residue that flanks the fourth calcium binding domain of GPIIb. Cotransfection of Asp418 GPIIb and GPIIIa plasmid constructs into COS-7 cells resulted in the accumulation of a pre-GPIIb-IIIa complex that failed to reach the cell surface, in effect recreating the thrombasthenic phenotype. Pulse-chase and endoglycosidase studies demonstrated that the biosynthetic blockade occurred in a pre-Golgi compartment. Removal of the negatively charged carboxyl group of Asp418 GPIIb, creating Ala418 GPIIb, rescued intracellular transport and surface expression of the integrin complex. Mutagenesis of a homologous Gly within the integrin alpha subunit alpha v also resulted in the failure to express alpha v beta 3 on the cell surface. These results suggest that the presence of a small, uncharged amino acid 6-8 residues amino-terminal to the calcium coordination complex is crucial for the proper folding and maturation of integrin complexes.