Calcium Integrin Binding Protein Associates with Integrins αVβ3 and αIIbβ3 Independent of β3 Activation Motifs.

Abstract

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.