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

Abstract

To examine the effects of naturally occurring inherited mutations on the ability of the integrin α‐subunit, also termed glycoprotein IIb (GPIIb), to bind metal ions, we prepared small synthetic peptides that encompassed individual cation‐binding domains, and recombinant GPIIb polypeptides that encompassed all four Ca²⁺‐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 Ca²⁺‐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 Ca²⁺‐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 Ca²⁺‐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 Ca²⁺‐independent manner.