Reconstitution of Functional Integrin αIIbβ3 and Its Activation in Plasma Membrane-Mimetic Lipid Environments.
Ontology highlight
ABSTRACT:
SUBMITTER: Janke U
PROVIDER: S-EPMC8304682 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Increased ligand binding to cellular integrins ("activation") plays important roles in processes such as development, cell migration, extracellular matrix assembly, tumor metastasis, hemostasis, and thrombosis. Integrin activation encompasses both increased integrin monomer affinity and increased receptor clustering and depends on integrin-talin interactions. Loss of kindlins results in reduced activation of integrins. Kindlins might promote talin binding to integrins through a cooperative mechanism; however, kindlins do not increase talin association with integrins. Here, we report that, unlike talin head domain (THD), kindlin-3 has little effect on the affinity of purified monomeric αIIbβ3, and it does not enhance activation by THD. Furthermore, studies with ligands of varying valency show that kindlins primarily increase cellular αIIbβ3 avidity rather than monomer affinity. In platelets or nucleated cells, loss of kindlins markedly reduces αIIbβ3 binding to multivalent but not monovalent ligands. Finally, silencing of kindlins reduces the clustering of ligand-occupied αIIbβ3 as revealed by total internal reflection fluorescence and electron microscopy. Thus, in contrast to talins, kindlins have little primary effect on integrin αIIbβ3 affinity for monovalent ligands and increase multivalent ligand binding by promoting the clustering of talin-activated integrins.
| S-EPMC3912999 | biostudies-literature
Project description:In the present work, the factors that determine EPR line shapes from spin labels at the protein-hydrocarbon interface of a β-barrel membrane protein are examined. The EPR spectra from hydrocarbon facing sites in the outer membrane protein A (OmpA) are highly dependent upon the detergent or lipid into which OmpA is reconstituted. In general, line shapes at these sites are correlated with the solvent accessibility in the supporting amphiphile. A notable exception is CHAPS, which yields rigid limit EPR line shapes for labels at every position along a transmembrane β-strand in OmpA. EPR line shapes from the surface of OmpA are not strongly influenced by steric interference with neighboring side chains, but are modulated by solutes that should interact with hydrophobic surfaces. These results suggest that differences in EPR spectra in different supporting environments are not the result of differences in protein dynamics but are a result of different configurations or rotameric states that are assumed by the label. This conclusion is supported by distance measurements across the OmpA β-barrel, which indicate that labels yielding more motionally restricted line shapes interact more closely with the protein surface. These results have implications for the use of spin-label-derived distance constraints in protein structure determination and demonstrate that spin labels on membrane proteins provide a highly sensitive probe for the environment surrounding a membrane protein.
| S-EPMC3237940 | biostudies-literature
Project description:The number of membrane protein structures in the Protein Data Bank is becoming significant and growing. Here, the transmembrane domain structures of the helical membrane proteins are evaluated to assess the influences of the membrane mimetic environments. Toward this goal, many of the biophysical properties of membranes are discussed and contrasted with those of the membrane mimetics commonly used for structure determination. Although the mimetic environments can perturb the protein structures to an extent that potentially gives rise to misinterpretation of functional mechanisms, there are also many structures that have a native-like appearance. From this assessment, an initial set of guidelines is proposed for distinguishing native-like from nonnative-like membrane protein structures. With experimental techniques for validation and computational methods for refinement and quality assessment and enhancement, there are good prospects for achieving native-like structures for these very important proteins.
| S-EPMC3731949 | biostudies-literature
Project description:Kindlins play an important role in supporting integrin activation by cooperating with talin; however, the mechanistic details remain unclear. Here, we show that kindlins interacted directly with paxillin and that this interaction could support integrin αIIbβ3 activation. An exposed loop in the N-terminal F0 subdomain of kindlins was involved in mediating the interaction. Disruption of kindlin binding to paxillin by structure-based mutations significantly impaired the function of kindlins in supporting integrin αIIbβ3 activation. Both kindlin and talin were required for paxillin to enhance integrin activation. Interestingly, a direct interaction between paxillin and the talin head domain was also detectable. Mechanistically, paxillin, together with kindlin, was able to promote the binding of the talin head domain to integrin, suggesting that paxillin complexes with kindlin and talin to strengthen integrin activation. Specifically, we observed that crosstalk between kindlin-3 and the paxillin family in mouse platelets was involved in supporting integrin αIIbβ3 activation and in vivo platelet thrombus formation. Taken together, our findings uncover a novel mechanism by which kindlin supports integrin αIIbβ3 activation, which might be beneficial for developing safer anti-thrombotic therapies.
| S-EPMC6040092 | biostudies-literature
Project description: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.
| S-EPMC3311585 | biostudies-literature
Project description:Integrin αIIbβ3 is a member of the integrin family of transmembrane proteins present on the plasma membrane of platelets. Integrin αIIbβ3 is widely known to regulate the process of thrombosis via activation at its cytoplasmic side by talin and interaction with the soluble fibrinogen. It is also reported that three groups of interactions restrain integrin family members in the inactive state, including a set of salt bridges on the cytoplasmic side of the transmembrane domain of the integrin α- and β-subunits known as the inner membrane clasp, hydrophobic packing of a few transmembrane residues on the extracellular side between the α- and β-subunits that is known as the outer membrane clasp, and the key interaction group of the βA domain (located on the β-subunit head domain) with the βTD (proximal to the plasma membrane on the β-subunit). However, molecular details of this key interaction group as well as events that lead to detachment of the βTD and βA domains have remained ambiguous. In this study, we use molecular dynamics models to take a comprehensive outside-in and inside-out approach at exploring how integrin αIIbβ3 is activated. First, we show that talin's interaction with the membrane-proximal and membrane-distal regions of integrin cytoplasmic-transmembrane domains significantly loosens the inner membrane clasp. Talin also interacts with an additional salt bridge (R734-E1006), which facilitates integrin activation through the separation of the integrin's α- and β-subunits. The second part of our study classifies three types of interactions between RGD peptides and the extracellular domains of integrin αIIbβ3. Finally, we show that the interaction of the Arg of the RGD sequence may activate integrin via disrupting the key interaction group between K350 on the βA domain and S673/S674 on the βTD.
| S-EPMC3785890 | biostudies-literature
Project description:Anchorage dependence of cell growth, which is mediated by multiple integrin-regulated signaling pathways, is a key defense against cancer metastasis. Detachment of cells from the extracellular matrix triggers caveolin-1-dependent internalization of lipid raft components, which mediates suppression of Rho GTPases, Erk, and phosphatidylinositol 3-kinase in suspended cells. Elevation of cyclic adenosine monophosphate (cAMP) following cell detachment is also implicated in termination of growth signaling in suspended cells. Studies of integrins and lipid rafts, however, examined mainly ganglioside GM1 and glycosylphosphatidylinositol-linked proteins as lipid raft markers. In this study, we examine a wider range of lipid raft components. Whereas many raft components internalized with GM1 following cell detachment, flotillin2, connexin43, and G?(s) remained in the plasma membrane. Loss of cell adhesion caused movement of many components from the lipid raft to the nonraft fractions on sucrose gradients, although flotillin2, connexin43, and H-Ras were resistant. G?(s) lost its raft association, concomitant with cAMP production. Modification of the lipid tail of G?(s) to increase its association with ordered domains blocked the detachment-induced increase in cAMP. These data define the effects of that integrin-mediated adhesion on the localization and behavior of a variety of lipid raft components and reveal the mechanism of the previously described elevation of cAMP after cell detachment.
| S-EPMC3172269 | biostudies-literature
Project description:αIIbβ3 activation in platelets is followed by activation of the tyrosine kinase c-Src associated with the carboxyl terminus of the β3 cytosolic tail. Exogenous peptides designed to interact with the αIIb transmembrane (TM) domain activate single αIIbβ3 molecules in platelets by binding to the αIIb TM domain and causing separation of the αIIbβ3 TM domain heterodimer. Here we asked whether directly activating single αIIbβ3 molecules in platelets using the designed peptide anti-αIIb TM also initiates αIIbβ3-mediated outside-in signaling by causing activation of β3-associated c-Src. Anti-αIIb TM caused activation of β3-associated c-Src and the kinase Syk, but not the kinase FAK, under conditions that precluded extracellular ligand binding to αIIbβ3. c-Src and Syk are activated by trans-autophosphorylation, suggesting that activation of individual αIIbβ3 molecules can initiate αIIbβ3 clustering in the absence of ligand binding. Consistent with this possibility, incubating platelets with anti-αIIb TM resulted in the redistribution of αIIbβ3 from a homogenous ring located at the periphery of discoid platelets into nodular densities consistent with clustered αIIbβ3. Thus, these studies indicate that not only is resting αIIbβ3 poised to undergo a conformational change that exposes its ligand-binding site, but it is poised to rapidly assemble into intracellular signal-generating complexes as well.
| S-EPMC4882439 | biostudies-literature
Project description:This study demonstrates that two orthogonal events regulate integrin αIIbβ3's interactions with fibrinogen, its primary physiological ligand: (1) conformational changes at the αIIb-β3 interface and (2) flexibility in the carboxy terminus of fibrinogen's γ-module. The first postulate was tested by capturing αIIbβ3 on a biosensor and measuring binding by surface plasmon resonance. Binding of fibrinogen to eptifibatide-primed αIIbβ3 was characterized by a k(on) of ~2 × 10(4) L mol(-1) s(-1) and a k(off) of ~8 × 10(-5) s(-1) at 37 °C. In contrast, even at 150 nM fibrinogen, no binding was detected with resting αIIbβ3. Eptifibatide competitively inhibited fibrinogen's interactions with primed αIIbβ3 (K(i) ~0.4 nM), while a synthetic γ-module peptide (HHLGGAKQAGDV) was only weakly inhibitory (K(i) > 10 μM). The second postulate was tested by measuring αIIbβ3's interactions with recombinant fibrinogen, both normal (rFgn) and a deletion mutant lacking the γ-chain AGDV sites (rFgn γΔ408-411). Normal rFgn bound rapidly, tightly, and specifically to primed αIIbβ3; no interaction was detected with rFgn γΔ408-411. Equilibrium and transition-state thermodynamic data indicated that binding of fibrinogen to primed αIIbβ3, while enthalpy-favorable, must overcome an entropy-dominated activation energy barrier. The hypothesis that fibrinogen binding is enthalpy-driven fits with structural data showing that its γ-C peptide and eptifibatide exhibit comparable electrostatic contacts with αIIbβ3's ectodomain. The concept that fibrinogen's αIIbβ3 targeting sequence is intrinsically disordered may explain the entropy penalty that limits its binding rate. In the hemostatic milieu, platelet-platelet interactions may be localized to vascular injury sites because integrins must be activated before they can bind their most abundant ligand.
| S-EPMC3210020 | biostudies-literature
Project description:Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbβ3 rather than α5β1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair.
| S-EPMC8916723 | biostudies-literature