A SINGLE AMINO-ACID SUBSTITUTION FLANKING THE 4TH 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 Gly(418) --> Asp amino acid substitution in GPIIb. Gly(418) is a highly conserved residue that flanks the fourth calcium binding domain of GPIIb. Co-transfection of Asp(418) 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 Asp(418) GPIIb, creating Ala(418) 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.