A Fluorescence Cell Biology Approach to Map the Second Integrin-binding Site of Talin to a 130-Amino Acid Sequence within the Rod Domain

Tremuth, Laurent; Kreis, Stephanie; Melchior, Chantal; Hoebeke, Johan; Rondé, Philippe; Plançon, Sébastien; Takeda, Kenneth; Kieffer, Nelly

doi:10.1074/jbc.m400947200

Cited by 62 publications

(97 citation statements)

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“…Mammalian Cell cDNA Expression Constructs-The cDNAs encoding C-terminal DsRed-tagged talin fragments G (aa 1984 -2344), J (aa 1977-2113), B (aa 430 -1076), and Myctagged talin fragment J have been previously described (1). The cDNAs encoding talin residues 1977-2018 (J1), 2010 -2049 (J2), 2042-2080 (J3), and 2072-2113 (J4) were generated using talin fragment G cDNA as a template and were inserted into the mammalian cell expression vector pcDNA4/TO/myc-HIS (Invitrogen), generating recombinant polypeptides with a C-terminal Myc-His tag.…”

Section: Methodsmentioning

confidence: 99%

“…CHO-␣IIb␤3 Cell Culture and Transient Transfection-Chinese hamster ovary cells (CHO) stably expressing recombinant ␣IIb␤3-GFP or wild type ␣IIb␤3 integrin (19) were cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin and transfected as previously described (1). For each cDNA construct, 1.5 ϫ 10 6 adherent cells were transfected with 3-15 g of cDNA using Lipofectamine TM (Invitrogen) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…Data presented here were obtained as part of a doctoral thesis to be submitted to the "Université Libre de Bruxelles", Belgium. 2 To whom correspondence should be addressed: Laboratoire with native platelet integrin ␣IIb␤3 or a recombinant ␤3 integrin subunit cytoplasmic tail, either in vivo, by intracellular fluorescence resonance energy transfer, or in vitro, by surface plasmon resonance and liquid phase pulldown assays (1). We now report the minimal structure of talin IBS2 and provide evidence that a 2-amino acid point mutation (LI/AA) in this minimal structure inactivates the integrin binding capacity of IBS2.…”

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confidence: 99%

“…These include three well characterized vinculin binding sites (VBS1-3) (13), as well as eight additional vinculin binding sequences (12) and a highly conserved actin binding site located at the C-terminal end upstream of a dimerization motif (14 -16). It also contains an integrin binding site (IBS2) (1,17,18) whose functional role is still elusive.…”

mentioning

confidence: 99%

“…Section 1734 solely to indicate this fact. 1 Recipient of a doctoral fellowship from the Ministè re de la Culture, de l'Enseignement Supé rieur et de la Recherche, Luxembourg. Data presented here were obtained as part of a doctoral thesis to be submitted to the "Université Libre de Bruxelles", Belgium.…”

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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

Journal of Biological Chemistry

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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 ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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

Journal of Biological Chemistry

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The Ins and Outs of Integrin Signaling

Banno

Ginsberg

2008

Cell Junctions

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The conserved C-terminal I/LWEQ module targets Talin1 to focal adhesions

Franco

Senetar

Simonson

et al. 2006

Cell Motil. Cytoskeleton

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The cytoskeletal protein Talin1 is a critical link between integrins and the actin cytoskeleton, where it is required for the structural and signaling functions of integrin-containing adhesion complexes. However, the elements in Talin1 that are responsible for localizing it to adhesion complexes are not known. In this report we have used a series of constructs based on the modular structure of Talin1 to determine the structural elements that specify the subcellular localization of Talin1. We show that the conserved actin-binding I/LWEQ module at the C-terminus of Talin1 is necessary and sufficient for targeting to focal adhesion complexes. We also used truncation and site-directed mutagenesis to demonstrate that this novel targeting function correlates with, but is separable from, the actin-binding properties of the Talin1 I/LWEQ module. In addition, we have shown that focal adhesion targeting, unlike actin binding, is not conserved among I/LWEQ module proteins. Finally, we have demonstrated that the subcellular localization of the Talin1 I/LWEQ module is regulated by an intrasteric interaction with an upstream alpha-helix, suggesting that both the actin binding and adhesion-targeting elements are masked in full-length Talin1. Our results define a novel role for the I/LWEQ module as the primary adhesion-complex targeting determinant of Talin1 and suggest that pathways that can relieve inhibition of I/LWEQ module function will be important for regulating the structural and signaling properties of adhesion complexes.

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A Fluorescence Cell Biology Approach to Map the Second Integrin-binding Site of Talin to a 130-Amino Acid Sequence within the Rod Domain

Cited by 62 publications

References 53 publications

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

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

The Ins and Outs of Integrin Signaling

The conserved C-terminal I/LWEQ module targets Talin1 to focal adhesions

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