Abstract
The Calcium Integrin Binding protein (CIB) has been identified as interacting specifically with the cytoplasmic tail of the integrin αIIb domain to induce receptor activation and integrin αIIbβ3 mediated cell adhesion to extracellular proteins. In K562 cells stably expressing mutated integrin αVβ3, or chimeric αVβ3 carrying αIIb cytoplasmic tail, we report that the interaction of CIB with β3 integrins is not αIIbβ3 specific but binds αIIb as well as αV cytoplasmic tail domains. A double mutation of two proline residues to alanine residues in the αIIb cytoplasmic domain, previously shown to disturb its conformation, inhibits chimeric αV/αIIbβ3-CIB interaction. This demonstrates that αIIb 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 αIIbβ3 or αVβ3 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 αIIb-subunit integrin and CIB interaction is non-exclusive and requires the loop-like αIIb-cytoplasmic domain conformation. An interaction of CIB with αV-containing integrins provides an additional role for this molecule in keeping with its expression outside of platelets.
Highlights
The Calcium Integrin Binding (CIB) protein is a 22-kDa intracellular member of the family of regulatory calciumbinding proteins that includes calmodulin, calcineurin B and recoverin
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-Calcium Integrin Binding protein (CIB) interaction occurs through a β3-phosphorylation independent mechanism
It has been shown that the association of CIB with integrin αIIbβ3 during outside-in signaling is required for platelet spreading on fibrinogen [6] through regulation of focal adhesion kinase (FAK) activation [7]
Summary
The Calcium Integrin Binding (CIB) protein is a 22-kDa intracellular member of the family of regulatory calciumbinding proteins that includes calmodulin, calcineurin B and recoverin. In the present study using K562 cells expressing mutated integrins, we report that, in addition to the N-terminal membrane proximal GFFKR motif, the acidic C-terminal of the αIIb subunit cytoplasmic domain as well as the αIIb “closed” conformation are important for the αIIbCIB interaction. Mutations of tyrosine or serine residues on the β3 subunit cytoplasmic domain, previously shown to prevent β3 tyrosine phosphorylation or receptor activation, failed to prevent interaction with CIB Together these data indicate that in K562 cells, in addition to the N-terminal GFFKR motif, the binding of CIB to the cytoplasmic domain is αIIb cytoplasmic domain conformation dependent and is not αIIbβ specific, providing a basis for understanding CIB-integrin interaction in vivo and providing an additional role for CIB in non-platelet cell types
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