A potential role for α-actinin in inside-out αIIbß3 signaling.

ABSTRACT

Many different biochemical signaling pathways regulate integrin activation through the integrin cytoplasmic tail. Here, we describe a new role for α-actinin in inside-out integrin activation. In resting human platelets, α-actinin was associated with αIIbß3, whereas inside-out signaling (αIIbß3 activation signals) from protease-activated receptors (PARs) dephosphorylated and dissociated α-actinin from αIIbß3. We evaluated the time-dependent changes of the αIIbß3 activation state by measuring PAC-1 binding velocity. The initial velocity analysis clearly showed that PAR1-activating peptide stimulation induced only transient αIIbß3 activation, whereas PAR4-activating peptide induced long-lasting αIIbß3 activation. When αIIbß3 activation signaling dwindled, α-actinin became rephosphorylated and reassociated with αIIbß3. Compared with control platelets, the dissociation of α-actinin from αIIbß3 was only transient in PAR4-stimulated P2Y(12)-deficient platelets in which the sustained αIIbß3 activation was markedly impaired. Overexpression of wild-type α-actinin, but not the mutant Y12F α-actinin, increased its binding to αIIbß3 and inhibited PAR1-induced initial αIIbß3 activation in the human megakaryoblastic cell line, CMK. In contrast, knockdown of α-actinin augmented PAR-induced αIIbß3 activation in CMK. These observations suggest that α-actinin might play a potential role in setting integrins to a default low-affinity ligand-binding state in resting platelets and regulating αIIbß3 activation by inside-out signaling.