Calcium-dependent properties of CIB binding to the integrin alpha IIb cytoplasmic domain and translocation to the platelet cytoskeleton

The alpha IIb beta 3 integrin receives signals in agonist-activated platelets, resulting in its conversion to an active conformation that binds fibrinogen, thereby mediating platelet aggregation. Fibrinogen binding to alpha IIb beta 3 subsequently induces a cascade of intracellular signalling events. The molecular mechanisms of this bi-directional alpha IIb beta 3-mediated signalling are unknown but may involve the binding of proteins to the integrin cytoplasmic domains. We reported previously the sequence of a novel 22-kDa, EF-hand-containing, protein termed CIB (calcium- and integrin-binding protein) that interacts specifically with the alpha IIb cytoplasmic domain in the yeast-two-hybrid system. Further analysis of numerous tissues and cell lines indicated that CIB mRNA and protein are: widely expressed. In addition, isothermal titration calorimetry indicated that CIB binds to an alpha IIb cytoplasmic-domain peptide in a Ca2+-dependent manner, with moderate affinity (K-d,: 700 nM) and 1:1 stoichiometry. In aggregated platelets, endogenous CIB and alpha IIb beta 3 translocate to the Triton X-100-insoluble cytoskeleton in a parallelmanner; demonstrating that the cellular localization of CIB is regulated, potentially by alpha IIb beta 3. Thus CIB may contribute to integrin-related functions by mechanisms involving Ca2+-modulated binding to the alpha IIb cytoplasmic domain and changes in intracellular distribution.