A mechanism of platelet integrin aIIbb3 outside-in signaling through a novel integrin aIIb subunit-filamin-actin linkage

Abstract

The communication of talin-activated integrin aIIbb3 with cytoskeleton (integrin outside-in signaling) is essential for platelet aggregation, wound healing, and hemostasis. Filamin, a large actin cross-linker 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 aIIbb3, is displaced from aIIbb3 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 aIIbb3, filamin also associates with the talin-bound active aIIbb3 to mediate platelet spreading. FRET-based analysis reveals that while associating with both aIIb and b3 cytoplasmic tails (CTs) to maintain the inactive aIIbb3, filamin is spatiotemporally re-arranged to associate with aIIb CT alone on activated aIIbb3. Consistently, confocal cell imaging indicates that integrin a CT-linked filamin gradually delocalizes from b CT-linked focal adhesion marker - vinculin likely due to the separation of integrin a/b CTs occurring during integrin activation. High-resolution crystal and NMR structure determinations unravel that the activated integrin aIIb CT binds to filamin via a striking a-helix→b-strand transition with strengthened affinity that is dependent on the integrin-activating membrane environment containing enriched phosphatidylinositol 4,5-bisphosphate. These data suggest a novel integrin aIIb CT-filamin-actin linkage that promotes integrin outside-in signaling. Consistently, disruption of such linkage impairs the activation state of aIIbb3, phosphorylation of FAK/Src kinases, and cell migration. Together, our findings advance the fundamental understanding of integrin outside-in signaling with broad implications in blood physiology and pathology.

Interaction of Filamin C With Actin Is Essential for Cardiac Development and Function

“…However, considering the crucial roles of both FLNC and ITGB1 in myocardial integrity, 26 the exclusive involvement of the C-terminal R20 domain of FLNC in interacting with β1 integrin, 26 and that the FLNC F93A/L98E mutations do not impair its ability to interact with ITGB1, it is plausible that FLNC plays a critical role in integrin-mediated outside-in signaling events following integrin activation as indicated in a previous study on FLNA. 37 We also demonstrated that loss of FLNC-actinbinding activity in embryonic cardiomyocytes led to smaller embryonic body size, abnormal cardiac morphology, impaired cardiomyocyte proliferation, loss of ventricular wall integrity, and eventual embryonic lethality. Furthermore, our data demonstrated comparable embryonic body sizes cardiomyocyte proliferation between gMut and control embryos at E8.5, a developmental period when the loss of ventricular wall integrity are not observed in gMut embryos.…”

“…However, considering the crucial roles of both FLNC and ITGB1 in myocardial integrity, 26 the exclusive involvement of the C-terminal R20 domain of FLNC in interacting with β1 integrin, 26 and that the FLNC F93A/L98E mutations do not impair its ability to interact with ITGB1, it is plausible that FLNC plays a critical role in integrin-mediated outside-in signaling events following integrin activation as indicated in a previous study on FLNA. 37…”

Fine-Tuning Levels of Filamins A and B as a Specific Mechanism Sustaining Th2 Lymphocyte Functions

“…Furthermore, we observed decreased in vivo recruitment of Th2 lymphocytes in inflamed lungs of mice submitted to airway inflammation or asthma. These results together with the fact that FLNa bridges the actin cytoskeleton to integrins thereby maintaining them in an inactive state 20 and that FLNa associates with active integrins to inhibit cell migration 24 , raise the possibility that the low levels of FLNa and FLNb driven by ASB2α in Th2 lymphocyte favor αV3 integrin-dependent cell migration to optimize their effector functions. Since compensation by FLNb in the absence of FLNa has been observed 27,43 , functions of FLNa in T lymphocytes may have been missed or underestimated in assays using FLNa knockout/knockdown cells 44 .…”

“…Second, FLNa can form a ternary complex engaging the cytoplasmic tails of both integrin αIIb and 3, thereby stabilizing the inner-membrane clasp and competing with talin recruitment to the  subunit cytoplasmic tail by binding both the C-terminal and membrane-proximal regions of the 3 tail 23 . In addition, FLNa can also associate with the αIIb cytoplasmic tail of active αIIb3 integrin to inhibit cell migration likely because of excessively enhanced integrin outside-in signaling 24 . We previously showed that ASB2α-mediated degradation of FLNa and FLNb regulates actin cytoskeleton remodeling and cell motility in different cell types 14,[25][26][27][28][29] .…”

The Potential of Extracellular Matrix- and Integrin Adhesion Complex-Related Molecules for Prostate Cancer Biomarker Discovery

“…Among them, the consensus adhesome has been recognized, which consists of the sixty most common fibronectin-initiated adhesome proteins [12]. Currently, we are witnessing an increasing interest in the role of integrins that encouraged the quest for IACs' composition (e.g., [13][14][15][16]) and the hierarchy among its components (e.g., [17][18][19]).…”