Kindlins, Integrin Activation and the Regulation of Talin Recruitment to αIIbβ3

Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbβ3, full-length talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment

G-protein–gated inwardly rectifying potassium channels regulate ADP-induced cPLA2 activity in platelets through Src family kinases

ADP-induced TXA2 generation requires the costimulation of P2Y1, P2Y12, and the GPIIb/IIIa receptors. Signaling events downstream of the P2Y receptors that contribute to ADP-induced TXA2 generation have not been clearly delineated. In this study, we have investigated the role of G-protein–gated inwardly rectifying potassium channels (GIRKs), a recently identified functional effector for the P2Y12 receptor, in the regulation of ADP-induced TXA2 generation. At 10-μM concentrations, the 2 structurally distinct GIRK channel blockers, SCH23390 and U50488H, caused complete inhibition of ADP-induced cPLA2 phosphorylation and TXA2 generation, without affecting the conversion of AA to TXA2 or ADP-induced primary platelet aggregation in aspirin-treated platelets. In addition, Src family kinase selective inhibitors abolished 2MeSADP-mediated cPLA2 phosphorylation and TXA2 generation. Furthermore, these GIRK channel blockers completely blocked Gi-mediated Src kinase activation, suggesting that GIRK channels are upstream of Src family tyrosine kinase activation. In weaver mouse platelets, which have dysfunctional GIRK2 subunits, ADP-induced TXA2 generation was impaired. However, we did not observe any defect in 2MeSADP-induced platelet functional responses in GIRK2-null mouse platelets, suggesting that functional channels composed of other GIRK subunits contribute to ADP-induced TXA2 generation, via the regulation of the Src and cPLA2 activity

Effect of talin or THD on interaction between kindlins and αIIbβ3.

<p>(<b>A</b>) Schematic illustration of BiFC in CHO cells depicting αIIb-VC, VN-kindlin-2 and β3. When VN-kindlin interacts with αIIb-VCβ3 through the β3 tail, VN and VC should reconstitute Venus, resulting in BiFC. (<b>B</b>) Over-expression of THD or talin does not promote kindlin-2 interaction with αIIbβ3. CHO cells expressing αIIb-VCβ3 or αIIb-VCβ3Δ724 was co-transfected with Tac as a transfection marker and an expression vector for THD, talin or empty vector (Mock), as indicated. After induction of VN-kindlin-2 expression with doxycycline, BiFC was quantified by flow cytometry. BiFC fluorescence was normalized to αIIbβ3 expression and expressed as fold-increase relative to doxycycline-treated, Mock-transfected cells. Data represent means ± SEM of three experiments (asterisk denotes statistically significant difference against mock/induced, P<0.05). (<b>C</b>) Western blots were performed to monitor expression of VN-kindlin-2, talin and THD in cell lysates. (<b>D</b>) Purified kindlin-3 with or without addition of THD was incubated with the recombinant β3 cytoplasmic tail conjugated to neutravidin beads. After washing, proteins bound to the beads were detected on western blots. Band intensities were quantified in LICOR, normalized to kindlin-3 binding in the absence of THD, and presented as a curve. Insert shows a representative western blot of 3 independent experiments. Increasing amount of β3 tail bound THD failed to promote β3–kindlin-3 interaction. Data represent means ± SEM of three experiments. (<b>E</b>) Similar to (D) but talin was used instead of THD. Increasing amounts of β3 tail-bound talin failed to promote β3–kindlin-3 interaction. (asterisks in D and E denotes statistically significant differences compared to kindlin binding in the absence of talin or THD, P<0.10).</p

Effect of kindlin-2 knockdown on talin recruitment to the membrane.

<p>(<b>A</b>) αIIβb3 CHO cells were transduced with lentivirus encoding either control shRNA or <i>kindlin</i>-2 shRNA. Cells were then transfected as indicated with THD, talin, or talin and RIAM1-176CAAX. Intact cells were surface biotinylated in order to isolate membrane-bound proteins. Cells were broken-up by shear and then underwent serial centrifugation to isolate the nuclear/intact cell fraction, cytosolic fraction and crude membranes. The crude membranes were further purified with streptavidin conjugated beads. The streptavidin bound material was isolated as the plasma membrane fraction. The amount of THD and talin in each fraction as well as in whole cell lysate (WCL) was quantified by western blot. Data is expressed as relative protein recovery normalized to the recovery of integrin αIIb subunit in plasma membrane. Data represent means ± SEM of three independent experiments (asterisk, P<0.10 in paired t-test). (<b>B</b>) Representative western blots of the subcellular fractionation experiments showing the WCL and plasma membrane fraction. Western blot of the WCL showed that αIIb expression levels are unchanged. RhoGDI serve both as a loading control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034056#pone.0034056-Choy1" target="_blank">[46]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034056#pone.0034056-Ammoun1" target="_blank">[47]</a> and a cytosolic marker. Western blots of each target proteins and markers were cut and juxtaposed for clarity. The complete blot images with all the subcellular fractions are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034056#pone.0034056.s003" target="_blank">Figure S3</a>.</p

Subcellular localization of BiFC signals.

<p>αIIb-VCβ3 CHO cells were transduced with <i>kindlin</i>-2 (or control) shRNA lentiviruses also encoding DsRed, and VN-talin was induced by doxycycline. (<b>A</b>) Cells were incubated on fibrinogen-coated plates (100 µg/ml coating concentration) for 45 min, fixed, stained with antibody D57 for αIIbβ3, and examined by deconvolution microscopy (BiFC: Green, αIIbβ3: blue, Transduced: red). The arrows point to transduced cells, and the arrowhead to a non-transduced cell. (<b>B</b>) Spreading of transduced cells was examined and data were expressed as mean cell surface areas measured in total pixels as described in Experimental Procedures. Asterisk denotes statistically significant difference against respective control cells, P<0.01 (<b>C</b>) BiFC and αIIbβ3 fluorescence co-localization in transduced cells was evaluated by deconvolution microscopy as described in Experimental Procedures. Data represent 30–60 cells analyzed for each treatment. (<b>D</b>) Western blots were performed to monitor expression of talin and kindlin-2 in cell lysates. The cell lysates were from both uninfected and virus transduced cells whereas only virus transduced cells were analyzed in (A), (B) and (C).</p

Model of agonist-induced αIIbβ3 activation.

<p>(<b>A</b>) Stimulation of a platelet agonist receptor (e.g., PAR1) by an agonist leads to the activation of Rap1, resulting in targeting of its effector, RIAM, to the plasma membrane. (<b>B</b>) Cell stimulation also releases talin from its auto-inhibitory state, resulting in separation of the THD from the talin rod domain and recruitment of talin to the membrane-bound Rap1/RIAM complex. (<b>C</b>) Membrane-bound talin is recruited to αIIbβ3 by interaction of the THD with membrane-distal residues in the β3 cytoplasmic domain. (<b>D</b>) Further interactions of the THD with membrane-proximal β3 tail residues and membrane phospholipids leads to separation of the αIIb and β3 tail and transmembrane domains, triggering propagated changes in the extracellular domains leading to high-affinity binding of adhesive ligands, such as fibrinogen. While kindlins, like talin, can interact with the β3 cytoplasmic tail, they can also bind to other proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034056#pone.0034056-Moser3" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034056#pone.0034056-Malinin1" target="_blank">[42]</a>, and the molecular basis of their integrin co-activating function remains unclear. This working model is based on published studies summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034056#pone.0034056-Shattil1" target="_blank">[2]</a>.</p

Kindlin-2 requirement for talin-dependent, agonist-induced αIIbβ3 activation in CHO cells.

<p>(<b>A</b>) Agonist-induced PAC-1 binding determined in kindlin-2 knockdown cells. αIIbβ3 CHO cells engineered to conditionally express PAR1 and talin were transduced with lentivirus encoding control (Ctrl) or <i>kindlin-2</i> shRNAs as described in Experimental Procedures. Cells were incubated for 20 min at room temperature with 100 µM SFLLRN (or vehicle), and specific PAC-1 binding was quantified by flow cytometry as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034056#pone.0034056-Watanabe1" target="_blank">[11]</a>. To control for off target effects of knock-down constructs, shRNA-transduced cells were transiently co-transfected with an shRNA-resistant form of Flag-kindlin-2 (or empty vector, Mock) and Tac as a transfection marker. After induction of PAR1 and talin with doxycycline, specific PAC-1 binding was measured and normalized to integrin expression as determined by D57 staining. For clarity, data are expressed as the fold-increase in PAC-1 binding relative to binding observed with doxycycline-induced cells transduced with control shRNA. Data represent means ± SEM of six independent experiments (asterisk, P<0.01). (<b>B</b>) western blots were performed to assess expression of talin and kindlin-2 in lysates of cells studied in panel A. β-actin was monitored as a loading control. In the kindlin-2 rescue experiments, kindlin-2 was assessed both with an antibody to kindlin-2 and an antibody to the Flag epitope. The cell lysates were from both uninfected and virus transduced cells whereas using flow cytometry gating, only virus transduced cells were analyzed in panel A. (<b>C</b>) Kindlin-2 shRNA has no effect on PAC-1 binding induced by THD. αIIbβ3 CHO cells were transduced with lentivirus encoding <i>kindlin</i>-2 (or control) shRNA. Cells were transfected as indicated with THD, empty vector (Mock) and DsRed. PAC-1 binding to transfected cells was quantified by flow cytometry. PAC1 binding was normalized to PAC1 binding when integrins are fully activated by an activating antibody, which also is sensitive to the integrin expression level <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034056#pone.0034056-OToole1" target="_blank">[28]</a>. For clarity, data are expressed as the fold-increase in PAC-1 binding relative to binding observed with THD transfected/control shRNA transduced cells. Data represent means ± SEM of 7 experiments. (Asterisk, P<0.01 against mock transfected/control shRNA transduced cells).</p

Hematopoietic lineage cell–specific protein 1 (HS1) is a functionally important signaling molecule in platelet activation

Collagen activates platelets through an intracellular signaling cascade downstream of glycoprotein VI (GPVI). We have investigated the contribution of hematopoietic lineage cell–specific protein 1 (HS1) downstream of GPVI in platelet activation. Stimulation of GPVI leads to tyrosine phosphorylation of HS1, which is blocked by Src-family kinase inhibitors. Coimmunoprecipitation experiments revealed that HS1 associates with Syk and phosphatidylinositol 3-kinases. HS1-null mice displayed increased bleeding times and increased time to occlusion in the FeCl3 in vivo thrombosis model compared with their wild-type littermates. In addition, aggregation and secretion responses were diminished in HS1-null mouse platelets after stimulation of GPVI and protease-activated receptor 4 (PAR-4) agonists compared with wild-type littermate mouse platelets. Finally, Akt phosphorylation was diminished after GPVI or PAR-4 stimulation in platelets from HS1-null mice compared with their wild-type littermates. These results demonstrate that phosphorylation of the HS1 protein occurs downstream of GPVI stimulation and that HS1 plays a significant functional role in platelet activation downstream of GPVI and PARs