Divalent cations differentially regulate integrin alphaIIb cytoplasmic tail binding to beta3 and to calcium- and integrin-binding protein.

We have used recombinant or synthetic alphaIIb and beta3 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 KD 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 alphaIIb but not by the beta3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an alphaIIb-specific ligand, bound to the alphaIIb cytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with a KD value of 12 microM. In contrast, in vitro liquid phase binding of CIB to intact alphaIIbbeta3 occurred preferentially with Mn2+-activated alphaIIbbeta3 conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated alphaIIbbeta3, suggesting that Mn2+ activation of intact alphaIIbbeta3 induces the exposure of a CIB-binding site, spontaneously exposed by the free alphaIIb peptide. Since CIB did not stimulate PAC-1 binding to inactive alphaIIbbeta3 nor prevented activated alphaIIbbeta3 occupancy by PAC-1, we conclude that CIB does not regulate alphaIIbbeta3 inside-out signaling, but rather is involved in an alphaIIbbeta3 post-receptor occupancy event.
Mesh Terms:
Antigens, CD, Binding Sites, Blood Platelets, Calcium, Calcium-Binding Proteins, Carrier Proteins, Cations, Divalent, Cytoplasm, Dimerization, Dual Specificity Phosphatase 2, Fibrinogen, Humans, Integrin beta3, Ligands, Manganese, Mutation, Peptide Fragments, Platelet Glycoprotein GPIIb-IIIa Complex, Platelet Membrane Glycoproteins, Protein Binding, Protein Phosphatase 2, Protein Tyrosine Phosphatases, Receptors, Vitronectin, Recombinant Fusion Proteins, Surface Plasmon Resonance
J. Biol. Chem.
Date: Jun. 11, 1999
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