Structural basis for the autoinhibition of talin in regulating integrin activation.
ABSTRACT: Activation of heterodimeric (alpha/beta) integrin transmembrane receptors by the 270 kDa cytoskeletal protein talin is essential for many important cell adhesive and physiological responses. A key step in this process involves interaction of phosphotyrosine-binding (PTB) domain in the N-terminal head of talin (talin-H) with integrin beta membrane-proximal cytoplasmic tails (beta-MP-CTs). Compared to talin-H, intact talin exhibits low potency in inducing integrin activation. Using NMR spectroscopy, we show that the large C-terminal rod domain of talin (talin-R) interacts with talin-H and allosterically restrains talin in a closed conformation. We further demonstrate that talin-R specifically masks a region in talin-PTB where integrin beta-MP-CT binds and competes with it for binding to talin-PTB. The inhibitory interaction is disrupted by a constitutively activating mutation (M319A) or by phosphatidylinositol 4,5-bisphosphate, a known talin activator. These data define a distinct autoinhibition mechanism for talin and suggest how it controls integrin activation and cell adhesion.
Project description:The establishment of a multicellular body plan requires coordinating changes in cell adhesion and the cytoskeleton to ensure proper cell shape and position within a tissue. Cell adhesion to the extracellular matrix (ECM) via integrins plays diverse, essential roles during animal embryogenesis and therefore must be precisely regulated. Talin, a FERM-domain containing protein, forms a direct link between integrin adhesion receptors and the actin cytoskeleton and is an important regulator of integrin function. Similar to other FERM proteins, talin makes an intramolecular interaction that could autoinhibit its activity. However, the functional consequence of such an interaction has not been previously explored in vivo. Here, we demonstrate that targeted disruption of talin autoinhibition gives rise to morphogenetic defects during fly development and specifically that dorsal closure (DC), a process that resembles wound healing, is delayed. Impairment of autoinhibition leads to reduced talin turnover at and increased talin and integrin recruitment to sites of integrin-ECM attachment. Finally, we present evidence that talin autoinhibition is regulated by Rap1-dependent signaling. Based on our data, we propose that talin autoinhibition provides a switch for modulating adhesion turnover and adhesion stability that is essential for morphogenesis.
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:Talin binding to integrin β tails increases ligand binding affinity (activation). Changes in β transmembrane domain (TMD) topology that disrupt α-β TMD interactions are proposed to mediate integrin activation. In this paper, we used membrane-embedded integrin β3 TMDs bearing environmentally sensitive fluorophores at inner or outer membrane water interfaces to monitor talin-induced β3 TMD motion in model membranes. Talin binding to the β3 cytoplasmic domain increased amino acid side chain embedding at the inner and outer borders of the β3 TMD, indicating altered topology of the β3 TMD. Talin's capacity to effect this change depended on its ability to bind to both the integrin β tail and the membrane. Introduction of a flexible hinge at the midpoint of the β3 TMD decoupled the talin-induced change in intracellular TMD topology from the extracellular side and blocked talin-induced activation of integrin αIIbβ3. Thus, we show that talin binding to the integrin β TMD alters the topology of the TMD, resulting in integrin activation.
Project description:Cell migration is a dynamic process that requires temporal and spatial regulation of integrin activation and focal adhesion assembly/disassembly. Talin, an actin and beta-integrin tail-binding protein, is essential for integrin activation and focal adhesion formation. Calpain-mediated cleavage of talin has a key role in focal adhesion turnover; however, the talin head domain, one of the two cleavage products, stimulates integrin activation, localizes to focal adhesions and maintains cell edge protrusions, suggesting that other steps, downstream of talin proteolysis, are required for focal adhesion disassembly. Here we show that talin head binds Smurf1, an E3 ubiquitin ligase involved in cell polarity and migration, more tightly than full-length talin does and that this interaction leads to talin head ubiquitylation and degradation. We found that talin head is a substrate for Cdk5, a cyclin-dependent protein kinase that is essential for cell migration, synaptic transmission and cancer metastasis. Cdk5 phosphorylated talin head at Ser 425, inhibiting its binding to Smurf1, thus preventing talin head ubiquitylation and degradation. Expression of the mutant tal(S425A), which resists Cdk5 phosphorylation thereby increasing its susceptibility to Smurf1-mediated ubiqitylation, resulted in extensive focal adhesion turnover and inhibited cell migration. Thus, talin head produced by calpain-induced cleavage of talin is degraded through Smurf1-mediated ubiquitylation; moreover, phosphorylation by Cdk5 regulates the binding of Smurf1 to talin head, controlling talin head turnover, adhesion stability and ultimately, cell migration.
Project description:Talin plays an important role in regulating integrin-mediated signaling. Talin function is autoinhibited by intramolecular interactions between the integrin-binding F3 domain and the autoinhibitory domain (R9). We determined the crystal structure of a triple-domain fragment, R7R8R9, which contains R9 and the RIAM (Rap1-interacting adaptor molecule) binding domain (R8). The structure reveals a crystallographic contact between R9 and a symmetrically related R8 domain, representing a homodimeric interaction in talin. Strikingly, we demonstrated that the ?5 helix of R9 also interacts with the F3 domain, despite no interdomain contact involving the ?5 helix in the crystal structure of an F2F3:R9 autoinhibitory complex reported previously. Mutations on the ?5 helix significantly diminish the F3:R9 association and lead to elevated talin activity. Our results offer biochemical and functional evidence of the existence of a new talin autoinhibitory configuration, thus providing a more comprehensive understanding of talin autoinhibition, regulation, and quaternary structure assembly.
Project description:Integrin activation is essential for dynamically linking the extracellular environment and cytoskeletal/signaling networks. Activation is controlled by integrins' short cytoplasmic tails (CTs). It is widely accepted that the head domain of talin (talin-H) can mediate integrin activation by binding to two sites in integrin beta's CT; in integrin beta(3) this is an NPLY(747) motif and the membrane-proximal region. Here, we show that the C-terminal region of integrin beta(3) CT, composed of a conserved TS(752)T region and NITY(759) motif, supports integrin activation by binding to a cytosolic binding partner, kindlin-2, a widely distributed PTB domain protein. Co-transfection of kindlin-2 with talin-H results in a synergistic enhancement of integrin alpha(IIb)beta(3) activation. Furthermore, siRNA knockdown of endogenous kindlin-2 impairs talin-induced alpha(IIb)beta(3) activation in transfected CHO cells and blunts alpha(v)beta(3)-mediated adhesion and migration of endothelial cells. Our results thus identify kindlin-2 as a novel regulator of integrin activation; it functions as a coactivator.
Project description:Focal adhesions (FAs) are protein machineries essential for cell adhesion, migration, and differentiation. Talin is an integrin-activating and tension-sensing FA component directly connecting integrins in the plasma membrane with the actomyosin cytoskeleton. To understand how talin function is regulated, we determined a cryoelectron microscopy (cryo-EM) structure of full-length talin1 revealing a two-way mode of autoinhibition. The actin-binding rod domains fold into a 15-nm globular arrangement that is interlocked by the integrin-binding FERM head. In turn, the rod domains R9 and R12 shield access of the FERM domain to integrin and the phospholipid PIP2 at the membrane. This mechanism likely ensures synchronous inhibition of integrin, membrane, and cytoskeleton binding. We also demonstrate that compacted talin1 reversibly unfolds to an ?60-nm string-like conformation, revealing interaction sites for vinculin and actin. Our data explain how fast switching between active and inactive conformations of talin could regulate FA turnover, a process critical for cell adhesion and signaling.
Project description:Protein interactions with the integrin beta-subunit cytoplasmic domain (beta-tail) are essential for adhesion-dependent processes, including cell spreading and the connection of integrins with actin filaments at adhesion sites. Talin-1 binds to the conserved membrane-proximal NPxY motif of beta-tails (NPIY in beta1 integrin) promoting the inside-out activation of integrins and providing a linkage between integrins and the actin cytoskeleton. Here, we characterize the role of interactions between talin-1 and beta-tail downstream of integrin activation, in the context of recombinant integrins containing either the wild type (WT) or the (YA) mutant beta1A tail, with a tyrosine to alanine substitution in the NPIY motif. In addition to inhibiting integrin activation, the YA mutation suppresses cell spreading, integrin signaling, focal adhesion and stress-fiber formation, as well as microtubule assembly. Constitutive activation of the mutant integrin restores these integrin-dependent processes, bringing into question the importance of the NPIY motif downstream of integrin activation. Depletion of talin-1 using TLN1 siRNA demonstrated that talin-1 is required for cell spreading, focal adhesion and stress-fiber formation, as well as microtubule assembly, even when cells are adhered by constitutively activated WT integrins. Depletion of talin-1 does not inhibit these processes when cells are adhered by constitutively activated mutant integrins, suggesting that the binding of an inhibitory protein to the NPIY motif negatively regulates integrin function when talin-1 is depleted. We identified filamin A (FLNa) as this inhibitory protein; it binds to the beta1A tail in an NPIY-dependent manner and inhibition of FLNa expression in talin-1-depleted cells restores integrin function when cells are adhered by constitutively activated WT integrins. FLNa binds FilGAP, which is a negative regulator of Rac activation. Expression of the dominant inhibitory mutant, FilGAP(DeltaGAP), which lacks GAP activity restores spreading in cells adhered by constitutively activated integrins containing the beta1A tail, but not by integrins containing the beta1D tail, which is known to bind poorly to FLNa. Together, these results suggest that the binding of talin-1 to the NPIY motif is required downstream of integrin activation to promote cell spreading by preventing the inappropriate recruitment of FLNa and FilGAP to the beta1A tail. Our studies emphasize the importance of understanding the mechanisms that regulate the differential binding FLNa and talin-1 to the beta1 tail downstream of integrin activation in promoting integrin function.
Project description:The cytoskeletal, actin-binding protein talin has been previously implicated in phagocytosis in Dictyostelium discoideum and mammalian phagocytes. However, its mechanism of action during internalization is not understood. Our data confirm that endogenous talin can occasionally be found at phagosomes forming around IgG- and C3bi-opsonized red blood cells in macrophages. Remarkably, talin knockdown specifically abrogates uptake through complement receptor 3 (CR3, CD11b/CD18, alpha(M)beta(2) integrin) and not through the Fc gamma receptor. We show that talin physically interacts with CR3/alpha(M)beta(2) and that this interaction involves the talin head domain and residues W747 and F754 in the beta(2) integrin cytoplasmic domain. The CR3/alpha(M)beta(2)-talin head interaction controls not only talin recruitment to forming phagosomes but also CR3/alpha(M)beta(2) binding activity, both in macrophages and transfected fibroblasts. However, the talin head domain alone cannot support phagocytosis. Our results establish for the first time at least two distinct roles for talin during CR3/alpha(M)beta(2)-mediated phagocytosis, most noticeably activation of the CR3/alpha(M)beta(2) receptor and phagocytic uptake.
Project description:The activation of heterodimeric (α/β) 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 β 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.