Integrin alpha(IIb) tail distal of GFFKR participates in inside-out alpha(IIb)beta(3) activation

Abstract

Background: Increases in ligand binding to integrins (activation) play critical roles in platelet and leukocyte function. Integrin activation requires talin and kindlin binding to integrin beta cytoplasmic tails. Research has focused on the conserved GFFKR motif in integrin alpha(IIb) tails, integrin beta cytoplasmic tails and the binding partners of b tails. However, the roles of alpha(IIb) tail distal of GFFKR motif are unexplored. Objective: To investigate the role of alpha(IIb) tail distal of GFFKR in talin-mediated inside-out integrin signaling. Methods: We used model cell systems to examine the role of alpha(IIb) tail distal of GFFKR in bidirectional alpha(IIb)beta(3) signaling and alpha(IIb)beta(3)-talin interactions. Results: Deletion of amino acid residues after the GFFKR motif in alpha(IIb) tail moderately decreased beta 3(D723R)-induced activation, abolished talin-induced alpha(IIb)beta(3) activation in model cells, and inhibited agonist-induced alpha(IIb)beta(3) activation in megakaryocytic cells. Furthermore, residues in alpha(IIb) tail distal of GFFKR did not affect outside-in alpha(IIb)beta(3) signaling or alpha(IIb)beta(3)-talin interaction. Addition of non-homologous or non-specific amino acids to the GFFKR motif restored alpha(IIb)beta(3) activation in model cells and in megakaryocytic cells. Molecular modeling indicates that beta(3)-bound talin sterically clashes with the alpha(IIb) tail in the alpha(IIb)beta(3) complexes, potentially disfavoring the a-b interactions that keep alpha(IIb)beta(3) inactive. Conclusion: The alpha(IIb) tail sequences distal of GFFKR participate in talin-mediated inside-out alpha(IIb)beta(3) activation through its steric clashes with beta(3)-bound talin.