Inherited Mutations within the Calcium‐Binding Sites of the Integrin α_IIb Subunit (Platelet Glycoprotein IIb)

Abstract

To examine the effects of naturally occurring inherited mutations on the ability of the integrin alpha-subunit, also termed glycoprotein IIb (GPIIb), to bind metal ions, we prepared small synthetic peptides that encompassed individual cation-binding domains, and recombinant GPIIb poly peptides that encompassed all four Ca(2+)-binding domains, and examined their interactions with divalent cations by means of Tb3+-luminescence spectroscopy. Replacement of the highly conserved Gly418 residue, located within the flanking region of the fourth Ca(2+)-binding domain of GPIIb, with a negatively charged Asp residue resulted in marked reduction in the ability to bind divalent cations. A variant form of GPIIb with a deletion of two amino acids at the -1 and X positions of the fourth Ca(2+)-binding domain of GPIIb also failed to bind metal ions in a normal manner. In contrast, a Glanzmann mutation at the -1 position of the first Ca(2+)-binding domain of GPIIb had no effect on divalent-cation-binding ability with either synthetic peptides or recombinant GPIIb polypeptides. These data support the hypothesis that the highly conserved Gly normally found 7-8 residues N-terminal to integrin metal-binding domains plays a critical role in the maintenance of the conformation or orientation of surrounding EF-hand structures so that they can effectively interact with and bind divalent cations. Furthermore, inherited mutations at or near the divalent-cation-binding domains of GPIIb do not necessarily exert their biochemical effects by disruption of cation binding, but can interfere with integrin biogenesis in a Ca(2+)-independent manner.