Divalent cations differentially regulate integrin αIIb cytoplasmic tail binding to β3 and to calcium- and integrin-binding protein

We have used recombinant or synthetic alpha(IIb) and beta(3) integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. alpha(.)beta heterodimerization occurred in a 1:1 stoichiometry with a weak K-D in the micromolar range. Divalent cations were not required for this association but stabilized the alpha(.)beta complex by decreasing the dissociation rate. alpha(.)beta complexation was impaired by the R995A substitution or the KVGFFKR deletion in alpha(IIb) but not by the beta(3) S752P mutation. Recombinant calcium-and integrin-binding protein (CIB), an alpha(IIb)-specific ligand, bound to the alpha(IIb) cytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with a K-D value of 12 mu M. In contrast, in vitro liquid phase binding of CIB to intact alpha(IIb)beta(3) occurred preferentially with Mn2+-activated alpha(IIb)beta(3) conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated alpha(IIb)beta(3), suggesting that Mn2+ activation of intact alpha(IIb)beta(3) induces the exposure of a GIB-binding site, spontaneously exposed by the free alpha(IIb) peptide. Since CIB did not stimulate PAC-1 binding to inactive alpha(IIb)beta(3) nor prevented activated alpha(IIb)beta(3) occupancy by PAC-1, we conclude that CIB does not regulate alpha(IIb)beta(3) inside-out signaling, but rather is involved in an alpha(IIb)beta(3) post-receptor occupancy event.