Regulatory Role of the Extracellular α-Tail/β-Tail Interaction in αIIbβ3 Integrin Activation
Authors: Kamata Tetsuji , Handa Makoto , Kawai Yohko , Ikeda Yasuo , Aiso Sadakazu 
Published: October 2019

Abstract
Abstract 3199
The platelet αIIbβ3 integrin changes its conformation to increase the affinity for ligands upon platelet activation. It has been shown that binding of the intracellular proteins to the cytoplasmic tails activate αIIbβ3 by disrupting the endogenous interaction between the two cytoplasmic tails in inside-out signaling. However, the mechanism how separation of the cytoplasmic tails initiate structural rearrangement of the extracellular domains has not been investigated. In αIIbβ3 crystal structure, the α-extracellular tail consisting of thigh, calf-1, and calf-2 domains makes large interface with the β-extracellular tail consisting of EGF-2, -3, -4, and βT domains. We have previously shown that clasp formation between the two extracellular tails completely abrogated αIIbβ3 activation induced by the cytoplasmic tail truncation. This suggests that separation of the two extracellular tails is essential for activation induced by inside-out signaling. To examine whether this is the case, N-glycan binding sites consisting of the N-X-T/S motif were introduced in αIIb amino acid residues located in the thigh/EGF-2, calf-1/EGF-3, or calf-2/βT interfaces. Attachment of a bulky N-glycan to these sites would prevent endogenous interface formation, thus forces the tails to separate from each other. When expressed in CHO cells, Q513NKT mutation in the thigh, M660NRT mutation in the calf-1, V760NVT mutation in the calf-2 domain all induced robust fibrinogen binding without any activators. Attachment of N-glycan was confirmed by SDS-PAGE analysis on the αIIb fragments carrying these mutations. This activation was inhibited by stabilizing the β-head/β-tail interface or by limiting the αIIb movement with artificially introduced disulfides, both of which prevents integrin from assuming the extended conformer. Prevention of the hybrid domain swing-out also completely inhibited activation. These results suggest that separation of the tails activate αIIbβ3 by inducing integrin extension. To confirm if endogenous α-tail/β-tail interaction indeed keeps integrin in low affinity state, αIIb amino acid residues that participate in the interface formation were mutated and the effect on fibrinogen binding was examined. Among the mutants, the K514A in the thigh, R671A in the calf-1, and R751A in the calf-2 domains induced only weak activation. However, combining these mutations resulted in robust activation. This activation was completely inhibited by the C-terminal clasp formation. Taken together, these results suggest that the α-tail/β-tail interface is kept by a group of key interdomain interaction that individually is not strong enough to do so. In conclusion, the α-tail/β-tail interface interaction regulates integrin activation by keeping integrin in low affinity state. Disruption of the interaction induces separation of the extracellular tails and activates integrin by initiating the structural rearrangement from the low affinity bent conformer to the high affinity extended conformer.

Disclosures:
No relevant conflicts of interest to declare.