A mechanism of platelet integrin alpha IIb beta 3 outside-in signaling through a novel integrin alpha IIb subunit-filamin-actin linkage

The communication of talin-activated integrin alpha IIb ss 3 with the cytoskeleton (integrin outside-in signaling) is essential for platelet aggregation, wound healing, and hemostasis. Filamin, a large actin crosslinker and integrin binding partner critical for cell spreading and migration, is implicated as a key regulator of integrin outside-in signaling. However, the current dogma is that filamin, which stabilizes inactive alpha IIb ss 3, is displaced from alpha IIb ss 3 by talin to promote the integrin activation (inside-out signaling), and how filamin further functions remains unresolved. Here, we show that while associating with the inactive alpha IIb ss 3, filamin also associates with the talin-bound active alpha IIb ss 3 to mediate platelet spreading. Fluorescence resonance energy transfer-based analysis reveals that while associating with both aIIb and ss 3 cytoplasmic tails (CTs) to maintain the inactive alpha IIb ss 3, filamin is spatiotemporally rearranged to associate with alpha IIb CT alone on activated alpha IIb ss 3. Consistently, confocal cell imaging indicates that integrin alpha CT-linked filamin gradually delocalizes from the ss CT-linked focal adhesion marker-vinculin likely because of the separation of integrin alpha/ss CTs occurring during integrin activation. High-resolution crystal and nuclear magnetic resonance structure determinations unravel that the activated integrin alpha IIb CT binds to filamin via a striking alpha-helix ->ss-strand transition with a strengthened affinity that is dependent on the integrin-activating membrane environment containing enriched phosphatidylinositol 4,5-bisphosphate. These data suggest a novel integrin alpha IIb CT-filamin-actin linkage that promotes integrin outside-in signaling. Consistently, disruption of such linkage impairs the activation state of alpha IIb ss 3, phosphorylation of focal adhesion kinase/proto-oncogene tyrosine kinase Src, and cell migration. Together, our findings advance the fundamental understanding of integrin outside-in signaling with broad implications in blood physiology and pathology.

Automated summary

This study reveals that filamin associates with both inactive and active forms of alpha IIb ss 3, promoting platelet spreading. Furthermore, the study demonstrates that filamin undergoes spatiotemporal rearrangement upon alpha IIb ss 3 activation, binding to the alpha IIb cytoplasmic tail alone. This novel alpha IIb CT-filamin-actin linkage plays a crucial role in integrin outside-in signaling.