Dissociation of the Extracellular α-Tail/β-Tail Interface Interaction Activates αIIbβ3 Integrin.

It is postulated that integrins undergo structural rearrangement from the low affinity bent conformer to the high affinity extended conformer upon activation. In inside-out signaling, the binding of talin to β cytoplasmic tail has been shown to activate integrin by disrupting the endogenous α/β cytoplasmic tail interaction that constrains integrin in low affinity state. How this intracellular event triggers structural rearrangement in the extracellular part of the integrin has not been elucidated. In integrin bent conformer, β-tail not only makes interface with β-head, but also makes large interface with α-tail. We previously reported that the membrane-proximal α-tail/β-tail interface interaction regulates integrin activation, and that stabilization of this interface completely blocks the propagation of inside-out signaling. From these results, we hypothesized that dissociation of the α-tail/β-tail following the dissociation of the α/β cytoplasmic tails triggers disruption of the β-head/β-tail interaction that constrains integrin in the bent state, thus activates integrin in inside-out signaling. Here we report that dissociation of the extracellular α-tail/β-tail actually activates αIIbβ3 integrin. To induce dissociation of the tails, we introduced N-linked glycosylation site (N-X-T/S) in αIIb amino acid residue Met-660 (M660NRT) which is located at the calf-1/EGF-3 interface that compose part of the α-tail/β-tail interface. When expressed in CHO cells, the mutant αIIbβ3 constitutively bound fibrinogen. Introduction of any sequences other than N-X-T/S in this site failed to activate αIIbβ3, suggesting this activation depends on the binding of a bulky N-glycan to the site. This activation was completely abolished when the β-head/β-tail interface was stabilized. Notably, stabilization of the membrane-proximal calf-2/EGF4-βTD interface with a disulfide bridge significantly inhibited activation induced by calf-1/EGF-3 dissociation, while it did not have any effect on fully extended αIIbβ3. These results suggest that disruption of the membrane-proximal α-tail/β-tail interface rather than the dissociation of the membrane-distal calf-1/EGF-3 interface is critical for initiating the structural rearrangement. Next, we examined whether disruption of the β-head/β-tail interface activates αIIbβ3 by introducing N-linked glycosylation site in β3 amino acid residue Gly-382 or in Thr-564 that are located at the β-head/β-tail interface. The resulting G382NLT and T564N mutants constitutively bound fibrinogen. This activation was completely abolished when αIIbβ3 was constrained in the bent conformer. Different from the M660NRT, this activation was not inhibited by stabilizing the calf-2/EGF4-βTD interface. In summary, these results suggest, 1) that disruption of the β-head/β-tail interface is required for the α-tail/β-tail dissociation to activate αIIbβ3, 2) that αIIbβ3 has to be able to extend for the disruption of the β-head/β-tail interface to activate αIIbβ3, 3) dissociation of the α-tail/β-tail is not required for activation, once β-head/β-tail interface is disrupted or αIIbβ3 is fully extended. Taken together, these results are consistent with our hypothesis and indicate that the α-tail/β-tail interface interaction is the key regulator of integrin activation in inside-out signaling.