Talin activates integrins by altering the topology of the ? transmembrane domain.
ABSTRACT: Talin binding to integrin ? tails increases ligand binding affinity (activation). Changes in ? transmembrane domain (TMD) topology that disrupt ?-? TMD interactions are proposed to mediate integrin activation. In this paper, we used membrane-embedded integrin ?3 TMDs bearing environmentally sensitive fluorophores at inner or outer membrane water interfaces to monitor talin-induced ?3 TMD motion in model membranes. Talin binding to the ?3 cytoplasmic domain increased amino acid side chain embedding at the inner and outer borders of the ?3 TMD, indicating altered topology of the ?3 TMD. Talin's capacity to effect this change depended on its ability to bind to both the integrin ? tail and the membrane. Introduction of a flexible hinge at the midpoint of the ?3 TMD decoupled the talin-induced change in intracellular TMD topology from the extracellular side and blocked talin-induced activation of integrin ?IIb?3. Thus, we show that talin binding to the integrin ? TMD alters the topology of the TMD, resulting in integrin activation.
Project description:Integrin activation regulates adhesion, extracellular matrix assembly, and cell migration, thereby playing an indispensable role in development and in many pathological processes. A proline mutation in the central integrin ?3 transmembrane domain (TMD) creates a flexible kink that uncouples the topology of the inner half of the TMD from the outer half. In this study, using leukocyte integrin ?4?7, which enables development of gut-associated lymphoid tissue (GALT), we examined the biological effect of such a proline mutation and report that it impairs agonist-induced talin-mediated activation of integrin ?4?7, thereby inhibiting rolling lymphocyte arrest, a key step in transmigration. Furthermore, the ?4?7(L721P) mutation blocks lymphocyte homing to and development of the GALT. These studies show that impairing the ability of an integrin ? TMD to transmit talin-induced TMD topology inhibits agonist-induced physiological integrin activation and biological function in development.
Project description:Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrin?IIb?3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin ?IIb?3 activation, but it activates ?IIb?3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of ?IIb?3 is tightly regulated by the topology of ?3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin ?3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin ?-? TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.
Project description:Activation of transmembrane receptor integrin by talin is essential for inducing cell adhesion. However, the pathway that recruits talin to the membrane, which critically controls talin's action, remains elusive. Membrane-anchored mammalian small GTPase Rap1 is known to bind talin-F0 domain but the binding was shown to be weak and thus hardly studied. Here we show structurally that talin-F0 binds to human Rap1b like canonical Rap1 effectors despite little sequence homology, and disruption of the binding strongly impairs integrin activation, cell adhesion, and cell spreading. Furthermore, while being weak in conventional binary binding conditions, the Rap1b/talin interaction becomes strong upon attachment of activated Rap1b to vesicular membranes that mimic the agonist-induced microenvironment. These data identify a crucial Rap1-mediated membrane-targeting mechanism for talin to activate integrin. They further broadly caution the analyses of weak protein-protein interactions that may be pivotal for function but neglected in the absence of specific cellular microenvironments.
Project description:Integrin ?IIb?3 is a member of the integrin family of transmembrane proteins present on the plasma membrane of platelets. Integrin ?IIb?3 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 ?- and ?-subunits known as the inner membrane clasp, hydrophobic packing of a few transmembrane residues on the extracellular side between the ?- and ?-subunits that is known as the outer membrane clasp, and the key interaction group of the ?A domain (located on the ?-subunit head domain) with the ?TD (proximal to the plasma membrane on the ?-subunit). However, molecular details of this key interaction group as well as events that lead to detachment of the ?TD and ?A 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 ?IIb?3 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 ?- and ?-subunits. The second part of our study classifies three types of interactions between RGD peptides and the extracellular domains of integrin ?IIb?3. 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 ?A domain and S673/S674 on the ?TD.
Project description:The activation of heterodimeric (?/?) integrin transmembrane receptors by cytosolic protein talin is crucial for regulating diverse cell-adhesion-dependent processes, including blood coagulation, tissue remodeling, and cancer metastasis. This process is triggered by the coincident binding of N-terminal FERM (four-point-one-protein/ezrin/radixin/moesin) domain of talin (talin-FERM) to the inner membrane surface and integrin ? cytoplasmic tail, but how these binding events are spatiotemporally regulated remains obscure. Here we report the crystal structure of a dormant talin, revealing how a C-terminal talin rod segment (talin-RS) self-masks a key integrin-binding site on talin-FERM via a large interface. Unexpectedly, the structure also reveals a distinct negatively charged surface on talin-RS that electrostatically hinders the talin-FERM binding to the membrane. Such a dual inhibitory topology for talin is consistent with the biochemical and functional data, but differs significantly from a previous model. We show that upon enrichment with phosphotidylinositol-4,5-bisphosphate (PIP2) - a known talin activator, membrane strongly attracts a positively charged surface on talin-FERM and simultaneously repels the negatively charged surface on talin-RS. Such an electrostatic "pull-push" process promotes the relief of the dual inhibition of talin-FERM, which differs from the classic "steric clash" model for conventional PIP2-induced FERM domain activation. These data therefore unravel a new type of membrane-dependent FERM domain regulation and illustrate how it mediates the talin on/off switches to regulate integrin transmembrane signaling and cell adhesion.
Project description:Integrin αIIbβ3 is a predominant type of integrin abundantly expressed on the surface of platelets and its activation regulates the process of thrombosis. Talin and kindlin are cytoplasmic proteins that bind to integrin and modulate its affinity for extracellular ligands. Although the molecular details of talin-mediated integrin activation are known, the mechanism of kindlin involvement in this process remains elusive. Here, we demonstrate that the interplay between talin and kindlin promotes integrin activation. Our all-atomic molecular dynamics simulations on complete transmembrane and cytoplasmic domains of integrin αIIbβ3, talin1 F2/F3 subdomains, and the kindlin2 FERM domain in an explicit lipid-water environment over a microsecond timescale unraveled the role of kindlin as an enhancer of the talin interaction with the membrane proximal region of β-integrin. The cooperation of kindlin with talin results in a complete disruption of salt bridges between R995 on αIIb and D723/E726 on β3. Furthermore, kindlin modifies the molecular mechanisms of inside-out activation by decreasing the crossing angle between transmembrane helices of integrin αIIbβ3, which eventually results in parallelization of integrin dimer. In addition, our control simulation featuring integrin in complex with kindlin reveals that kindlin binding is not sufficient for unclasping the inner-membrane and outer-membrane interactions of integrin dimer, thus ruling out the possibility of solitary action of kindlin in integrin activation.
Project description:Side chains of Lys/Arg near transmembrane domain (TMD) membrane-water interfaces can 'snorkel', placing their positive charge near negatively charged phospholipid head groups; however, snorkelling's functional effects are obscure. Integrin ? TMDs have such conserved basic amino acids. Here we use NMR spectroscopy to show that integrin ?(3)(Lys?716) helps determine ?(3) TMD topography. The ?(??b)?(3) TMD structure indicates that precise ?(3) TMD crossing angles enable the assembly of outer and inner membrane 'clasps' that hold the ?? TMD together to limit transmembrane signalling. Mutation of ?(3)(Lys?716) caused dissociation of ?(??b)?(3) TMDs and integrin activation. To confirm that altered topography of ?(3)(Lys?716) mutants activated ?(??b)?(3), we used directed evolution of ?(3)(K716A) to identify substitutions restoring default state. Introduction of Pro(711) at the midpoint of ?(3) TMD (A711P) increased ?(??b)?(3) TMD association and inactivated integrin ?(??b)?(3)(A711P,K716A). ?(3)(Pro?711) introduced a TMD kink of 30?±?1° precisely at the border of the outer and inner membrane clasps, thereby decoupling the tilt between these segments. Thus, widely occurring snorkelling residues in TMDs can help maintain TMD topography and membrane-embedding, thereby regulating transmembrane signalling.
Project description:Integrins function as bi-directional signaling transducers that regulate cell-cell and cell-matrix signals across the membrane. A key modulator of integrin activation is talin, a large cytoskeletal protein that exists in an autoinhibited state in quiescent cells. Talin is a large 235-kDa protein composed of an N-terminal 45-kDa FERM (4.1, ezrin-, radixin-, and moesin-related protein) domain, also known as the talin head domain, and a series of helical bundles known as the rod domain. The talin head domain consists of four distinct lobes designated as F0-F3. Integrin binding and activation are mediated through the F3 region, a critically regulated domain in talin. Regulation of the F3 lobe is accomplished through autoinhibition via anti-parallel dimerization. In the anti-parallel dimerization model, the rod domain region of one talin molecule binds to the F3 lobe on an adjacent talin molecule, thus achieving the state of autoinhibition. Platelet functionality requires integrin activation for adherence and thrombus formation, and thus regulation of talin presents a critical node where pharmacological intervention is possible. A major mechanism of integrin activation in platelets is through heterotrimeric G protein signaling regulating hemostasis and thrombosis. Here, we provide evidence that switch region 2 (SR2) of the ubiquitously expressed G protein (G?13) directly interacts with talin, relieves its state of autoinhibition, and triggers integrin activation. Biochemical analysis of G?13 shows SR2 binds directly to the F3 lobe of talin's head domain and competes with the rod domain for binding. Intramolecular FRET analysis shows G?13 can relieve autoinhibition in a cellular milieu. Finally, a myristoylated SR2 peptide shows demonstrable decrease in thrombosis in vivo Altogether, we present a mechanistic basis for the regulation of talin through G?13.
Project description:Cell-matrix adhesion is essential for building animals, promoting tissue cohesion, and enabling cells to migrate and resist mechanical force. Talin is an intracellular protein that is critical for linking integrin extracellular-matrix receptors to the actin cytoskeleton. A key question raised by structure-function studies is whether talin, which is critical for all integrin-mediated adhesion, acts in the same way in every context. We show that distinct combinations of talin domains are required for each of three different integrin functions during Drosophila development. The partial function of some mutant talins requires vinculin, indicating that recruitment of vinculin allows talin to duplicate its own activities. The different requirements are best explained by alternative mechanisms of talin function, with talin using one or both of its integrin-binding sites. We confirmed these alternatives by showing that the proximity between the second integrin-binding site and integrins differs, suggesting that talin adopts different orientations relative to integrins. Finally, we show that vinculin and actomyosin activity help change talin's orientation. These findings demonstrate that the mechanism of talin function differs in each developmental context examined. The different arrangements of the talin molecule relative to integrins suggest that talin is able to sense different force vectors, either parallel or perpendicular to the membrane. This provides a paradigm for proteins whose apparent uniform function is in fact achieved by a variety of distinct mechanisms involving different molecular architectures.
Project description:Recruitment and tethering of talin to the plasma membrane initiate the process of integrin activation. Multiple factors including the Rap1 proteins, RIAM (also known as APBB1IP) and PIP2 bind talin proteins and have been proposed to regulate these processes, but not systematically analyzed. By expressing specific talin mutants into talin-null fibroblasts, we show that binding of the talin F0 domain to Rap1 synergizes with membrane lipid binding of the talin F2 domain during talin membrane targeting and integrin activation, whereas the interaction of the talin rod with RIAM was dispensable. We also characterized a second Rap1-binding site within the talin F1 domain by detailed NMR analysis. Interestingly, while talin F1 exhibited significantly weaker Rap1-binding affinity than talin F0, expression of a talin F1 Rap1-binding mutant inhibited cell adhesion, spreading, talin recruitment and integrin activation similarly to the talin F0 Rap1-binding mutant. Moreover, the defects became significantly stronger when both Rap1-binding sites were mutated. In conclusion, our data suggest a model in which cooperative binding of Rap1 to the talin F0 and F1 domains synergizes with membrane PIP2 binding to spatiotemporally position and activate talins to regulate integrin activity.