A Vinculin Binding Domain from the Talin Rod Unfolds to Form a Complex with the Vinculin Head

Talin establishes a major link between integrins and actin filaments and contains two distinct integrin binding sites: one, IBS1, located in the talin head domain and involved in integrin activation and a second, IBS2, that maps to helix 50 of the talin rod domain and is essential for linking integrin ␤ subunits to the cytoskeleton (Moes, M., Rodius, S., Coleman, S. J., Monkley, S. J., Goormaghtigh, E., Tremuth, L., Kox, C., van der Holst, P. P., Critchley, D. R., and Kieffer, N. (2007) J. Biol. Chem. 282, 17280 -17288). Through the combined approach of mutational analysis of the ␤3 integrin cytoplasmic tail and the talin rod IBS2 site, SPR binding studies, as well as site-specific antibody inhibition experiments, we provide evidence that the integrin ␤3-talin rod interaction relies on a helix-helix association between ␣-helix 50 of the talin rod domain and the membrane-proximal ␣-helix of the ␤3 integrin cytoplasmic tail. Moreover, charge complementarity between the highly conserved talin rod IBS2 lysine residues and integrin ␤3 glutamic acid residues is necessary for this interaction. Our results support a model in which talin IBS2 binds to the same face of the ␤3 subunit cytoplasmic helix as the integrin ␣IIb cytoplasmic tail helix, suggesting that IBS2 can only interact with the ␤3 subunit following integrin activation.Integrins are ␣␤ heterodimeric receptors that mediate attachment of cells to the extracellular matrix (ECM) and therefore play important roles in cell adhesion, migration, proliferation, and survival (2, 3). Integrin clustering at sites of cellular attachment to the ECM triggers the assembly of large multifunctional submembrane protein complexes called focal adhesions (FAs), 5 that orchestrate the two-directional processing of stimuli across the cell membrane (4, 5). Both the integrin ␣ and ␤ chains participate in regulating the integrin extracellular ligand binding capacity, while the ␤ chain alone appears to link integrins to the actin cytoskeleton. Among the cytoskeletal proteins that directly interact with the ␤ chain cytoplasmic domain, four proteins: talin, ␣-actinin, filamin, and tensin possess both integrin and actin binding affinities (6, 7). Talin and ␣-actinin also bind to vinculin, an additional major component of FAs with actin binding activity.Talin and ␣-actinin are of particular interest as they have two integrin binding sites as well as multiple vinculin binding sites and display a similar structural organization comprising an extended rod domain composed of ␣-helical bundles. The ␣-actinin central rod domain, which is composed of 4 spectrin repeats of 3 ␣-helices (8, 9), associates with integrin ␤ subunits as well as vinculin through distinct helix-helix interactions (10 -16). Also, the 3 major talin rod-vinculin head (Vh) contacts have a similar architecture based on hydrophobic helixhelix interactions (17)(18)(19). Other FA proteins, such as members of the paxillin supergene family comprising paxillin, Hic-5, leupaxin, and PaxB, rely on an ␣-helical LD motif for their inter...

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confidence: 69%

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confidence: 99%

The Talin Rod IBS2 α-Helix Interacts with the β3 Integrin Cytoplasmic Tail Membrane-proximal Helix by Establishing Charge Complementary Salt Bridges

Rodius

Chaloin

Moes

et al. 2008

“…VBSs are defined by a single amphipathic helix of ϳ25 residues forming six turns, with the hydrophobic residues involved in vinculin binding clustered on one face of the amphipathic helix. Crystallographic and NMR structures have shown that the three major VBSs are contained within helical bundles, with the 6 key hydrophobic residues involved in vinculin binding buried within the hydrophobic core of the ␣-helical bundles (31)(32)(33). Talin binds vinculin with relatively low affinity, confirming that the majority of VBSs in talin are indeed cryptic (34).…”

Section: Discussionmentioning

confidence: 99%

The Integrin Binding Site 2 (IBS2) in the Talin Rod Domain Is Essential for Linking Integrin β Subunits to the Cytoskeleton

Moes

Rodius

Coleman

et al. 2007

Self Cite

Talin1 is a large cytoskeletal protein that links integrins to actin filaments through two distinct integrin binding sites, one present in the talin head domain (IBS1) necessary for integrin activation and a second (IBS2) that we have previously mapped to talin residues 1984 -2113 (fragment J) of the talin rod domain (1 Tremuth, L., Kreis, S., Melchior, C., Hoebeke, J., Ronde, P., Plancon, S., Takeda, K., and Kieffer, N. (2004) J. Biol. Chem. 279, 22258 -22266), but whose functional role is still elusive. Using a bioinformatics and cell biology approach, we have determined the minimal structure of IBS2 and show that this integrin binding site corresponds to 23 residues located in ␣ helix 50 of the talin rod domain (residues 2077-2099). Alanine mutation of 2 highly conserved residues (L2094A/I2095A) within this ␣ helix, which disrupted the ␣-helical structure of IBS2 as demonstrated by infrared spectroscopy and limited trypsin proteolysis, was sufficient to prevent in vivo talin fragment J targeting to ␣IIb␤3 integrin in focal adhesions and to inhibit in vitro this association as shown by an ␣IIb␤3 pulldown assay. Moreover, expression of a full-length mouse green fluorescent protein-talin LI/AA mutant in mouse talin1 ؊/؊ cells was unable to rescue the inability of these cells to assemble focal adhesions (in contrast to green fluorescent protein-talin wild type) despite the presence of IBS1. Our data provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the cytoskeleton.Talin1 is a multifunctional ϳ270-kDa (2541 amino acids, aa) 3 cytoskeletal protein that functions both as a key structural component of focal adhesions linking integrins to the cytoskeleton and also as a major regulator of integrin-dependent bidirectional signal transduction (2). In vivo, talin is found in equilibrium between a functionally inactive cytoplasmic form and a membrane-bound active form (3, 4). Talin activation is a complex process that is still poorly understood. In some cells, integrin outside-in signaling leads to association of the large isoform of type 1 phosphatidylinositol phosphate kinase with talin. This activates the phosphatidylinositol phosphate kinase, and together they translocate to the plasma membrane (5). Local phosphatidylinositol 4,5-biphosphate production by the phosphatidylinositol phosphate kinase is then thought to activate talin (6), possibly by relieving an autoinhibitory head-tail association unmasking the integrin binding site in the talin head (IBS1). In platelets, talin becomes activated through a protein kinase C ␣-dependent signaling pathway that stimulates activation and translocation of the small GTPase Rap1 to the plasma membrane where it interacts with its effector Riam, leading to the recruitment of talin and the unmasking of its integrin binding site in the head domain (7). Active talin, which can form anti-parallel homodimers (3), finally binds to and activates integrins allowing the exposure of a high affinity binding site for integrin extracellular ligands ...

“…The rod domain of talin comprises 62 amphipathic ␣-helices that are assembled into a series of ␣-helical bundles (33,34). Therefore, to avoid disrupting the tertiary structure of the resulting protein, we introduced stop codons between ␣-helical bundles as designated by arrowheads in Fig.…”

Section: Discrete Region Of Rod Domain Maintains a Cytosolic Pool Ofmentioning

confidence: 99%

“…These lipid-binding sites, which are aligned along one surface of the extended FERM domain, make extensive contacts with the plasma membrane (22) and play a critical role in integrin activation (21,26,31). The rod domain of talin consists of 62 amphipathic ␣-helices that are assembled into a series of ␣-helical bundles (33,34), and autoinhibitory interactions between THD and the rod domain regulate the functions of talin including its interactions with integrins (17,(35)(36)(37)(38)(39)(40).…”

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confidence: 99%

Subcellular Localization of Talin Is Regulated by Inter-domain Interactions

Banno

Goult

Lee

et al. 2012

Self Cite

Background: Talin regulates integrin affinity and nucleates the integrin-cytoskeleton linkage at the plasma membrane. Results: Specific inter-domain interactions between the talin head and two rod regions block interaction with actin or plasma membrane localization. Conclusion: Autoinhibitory interactions between the talin head and rod domains maintain cytosolic talin. Significance: Structurally defined inter-domain interactions regulate talin localization and function.