Calcium Integrin Binding Protein Associates with Integrins α_Vβ₃ and α_IIbβ₃ Independent of β₃ Activation Motifs

The Calcium Integrin Binding protein (CIB) has been identified as interacting specifically with the cytoplasmic tail of the integrin α_IIb domain to induce receptor activation and integrin α_IIbβ₃ mediated cell adhesion to extracellular proteins. In K562 cells stably expressing mutated integrin α_Vβ₃, or chimeric α_Vβ₃ carrying α_IIb cytoplasmic tail, we report that the interaction of CIB with β₃ integrins is not α_IIbβ₃ specific but binds α_IIb as well as α_V cytoplasmic tail domains. A double mutation of two proline residues to alanine residues in the α_IIb cytoplasmic domain, previously shown to disturb its conformation, inhibits chimeric α_V/α_IIbβ₃-CIB interaction. This demonstrates that α_IIb cytoplasmic domain loop-like conformation is required for interaction with CIB. Moreover, mutations of β₃ cytoplasmic domain residues Tyr-747 and/or Tyr-759 to phenylalanine residues (Y747F, Y759F, and Y747,759F) as well as residues Ser-752 to proline or alanine (S752P and S752A), do not affect the α_IIbβ₃ or α_Vβ₃ interaction with CIB. Since tyrosine residues Tyr-747 and/or Tyr-759 are the sites of tyrosine phosphorylation of β₃ subunit, these results suggest that the β₃ integrin-CIB interaction occurs through a β₃-phosphorylation independent mechanism. Likewise, ablation of conformation-dependent affinity change in β₃ Ser752Pro mutation had no affect on CIB-β₃ interaction. In summary, our results demonstrate that the α_IIb-subunit integrin and CIB interaction is non-exclusive and requires the loop-like α_IIb-cytoplasmic domain conformation. An interaction of CIB with α_V-containing integrins provides an additional role for this molecule in keeping with its expression outside of platelets.