Integrin β3 Membrane-Proximal and -Distal Residues Cooperatively Regulate Talin-Mediated αIIbβ3 Activation.

Integrin αIIbβ3 exists in a low affinity state in resting platelets and requires activation for high affinity binding with soluble ligands. Activation of αIIbβ3 is tightly linked to structural rearrangements of the αIIbβ3 molecule that is initiated from the cytoplasmic tails of the αIIb and β3 subunits. The β3 membrane-distal region has been shown to interact with many signaling and cytoskeletal molecules, and considered as a trigger point of integrin activation. The interaction of the β3 tail with a cytoplasmic protein, talin, largely contributes to integrin activation. In view of the link between integrin activation and allosteric structural rearrangements of integrins, one would expect that structural changes in the β3 membrane-distal region containing binding sites for intracellular proteins would be relayed to the membrane-proximal region, leading to αIIbβ3 activation. However, there has been no evidence that structural rearrangement of the β3 membrane-distal region is directly linked to integrin activation. No activating mutation has so far been reported in the β3 membrane-distal region despite numerous reports of loss-of-function mutants in this region. In this context, a previously reported αIIbβ3 mutant in which the β3 tail was replaced by the β1 tail was noteworthy. This chimeric integrin, αIIbβ3/β1, was constitutively active. Because the β1 and β3 subunits have relatively high sequence homology in their membrane-proximal regions, we reasoned that the residues differing between the β1 and β3 membrane-distal regions may be responsible for αIIbβ3 activation. To identify such residues, we produced 13 αIIbβ3 mutants in which the individual or group residues in the β3 tail were substituted with the corresponding β1 tail residues. The αIIbβ3 mutants were expressed on the surface of CHO cells by cotransfection of mutant β3 and wild-type αIIb cDNAs, and were tested for binding of fibrinogen and PAC1, a ligand-mimetic anti-αIIbβ3 antibody. Among them, only β3I719M and E749S mutants bound significant PAC1 and fibrinogen binding without any stimulation and the RGDS peptide abolished binding of these ligands, indicating a constitutively active state. The similar effect was observed with I719A and E749A mutants. Moreover, the I719M/E749S double mutant showed more PAC1 binding than the single mutants, reaching the same ligand binding activity as αIIbβ3/β1. These β3 mutations also induced αVβ3 activation. Conversely, substitution of M719 or S749 in the β1 tail with the corresponding β3 tail residue (M719I or S749E) inhibited αIIbβ3/β1 activation, and the M719I/S749E double mutant inhibited ligand binding to a level comparable with that of the wild-type αIIbβ3. Knock down of talin by short hairpin RNA inhibited the I719M- and E749S-induced αIIbβ3 activation, indicating talin-mediated activation of mutant integrins. Since I719 is located at the β3 membrane-proximal region, it is likely that the I719 mutation disrupts the well-known membrane-proximal clasp to maintain integrins at a low affinity state. On the other hand, E749 is located at the β3 membrane-distal region. This result provides experimental evidence that structural perturbation of the β3 membrane-distal region is linked to integrin activation. Moreover, our result showed that the mutational effects of the membrane-proximal I719 and the membrane-distal E749 residues were additive and talin-dependent, suggesting that the β3 membrane-proximal and –distal regions cooperatively regulate talin-mediated αIIbβ3 activation. This finding is consistent with a recent model of talin-induced αIIbβ3 activation in which talin cooperatively interacts with the β3 membrane-proximal and distal regions.