Structural Determinants of Integrin Recognition by Talin

García‐Álvarez, Begoña; Pereda, José M. de; Calderwood, David A.; Ulmer, Tobias S.; Critchley, David R.; Campbell, Iain D.; Ginsberg, Mark H.; Liddington, Robert

doi:10.1016/s1097-2765(02)00823-7

Cited by 475 publications

(556 citation statements)

References 43 publications

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“…Thus, we instead examined binding of the main integrin-binding domain of talin, purified talin F2/F3 domains, to the ␤2 integrin peptides. As expected, because the talin-binding site in integrins does not overlap with Thr758, 21,34 phosphorylation of Thr758 did not influence talin binding to the integrin ( Figure 1C). However, the phosphorylation indirectly influences talin binding, as addition of 14-3-3 led to detachment of talin from the phosphorylated integrin peptide ( Figure 1C).…”

Section: Resultssupporting

confidence: 77%

“…However, it appears that 14-3-3 binding to the phosphorylated integrin may lead to detachment of talin from the integrin tail. The talin-binding site in the ␤3 integrin 21 and the 14-3-3-binding In additional allowed regions 5.3 11.9…”

Section: Discussionmentioning

confidence: 99%

“…24 Both of the structures were solved by molecular replacement with Phaser (University of Cambridge, Cambridge, United Kingdom). 25 Protein Data Bank (PDB) entry 1A4O 21,22,26 was used as the search model for 14-3-3 and PDB entry 2BRQ 3 for IgFLNa21. Peptide backbone of the IgFLNa/␤2 structure was built with the program ARP/wARP version 6.1 (European Molecular Biology Laboratory, Hamburg, Germany).…”

Section: Protein Crystallographymentioning

confidence: 99%

“…The sample was further purified by HiTrap Q ion exchange chromatography (Amersham Biosciences) in 20 mM Tris-HCl, pH 8.0, with NaCl gradient elution. Final purification was achieved by gel filtration in a Superdex G75 21 was a generous gift from Dr D. A. Calderwood (Yale University, New Haven, CT). The fusion protein was expressed in E coli BL21 Gold in the presence of 4 mM IPTG.…”

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confidence: 99%

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β2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding

Takala

Nurminen

Nurmi³

et al. 2008

Blood

149

235

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Leukocyte integrins of the ␤2 family are essential for immune cell-cell adhesion. In activated cells, ␤2 integrins are phosphorylated on the cytoplasmic Thr758, leading to 14-3-3 protein recruitment to the ␤2 integrin. The mutation of this phosphorylation site impairs cell adhesion, actin reorganization, and cell spreading. Thr758 is contained in a Thr triplet of ␤2 that also mediates binding to filamin. Here, we investigated the binding of filamin, talin, and 14-3-3 proteins to phosphorylated and unphosphorylated ␤2 integrins by biochemical methods and x-ray crystallography. 14-3-3 proteins bound only to the phosphorylated integrin cytoplasmic peptide, with a high affinity (K d , 261 nM), whereas filamin bound only the unphosphorylated integrin cytoplasmic peptide (K d , 0.5 mM). Phosphorylation did not regulate talin binding to ␤2 directly, but 14-3-3 was able to outcompete talin for the binding to phosphorylated ␤2 integrin. X-ray crystallographic data clearly IntroductionIntegrins are heterodimeric plasma membrane receptors that mediate binding to the extracellular matrix and to ligands present on the surface of other cells. Their function is tightly regulated; they bind ligands only after activation. Modulation of integrin activity occurs through tightly regulated interactions between cytoplasmic molecules and integrin intracellular tails. Factors binding to integrin cytoplasmic domains regulating integrin adhesiveness include the cytoskeletal proteins talin 1,2 and filamin, 3 and the 14-3-3 proteins, which are molecular adaptors that bind to phosphorylated serine or threonine (pSer/ pThr) containing polypeptide sequences. 4 The ␤2 integrins are expressed exclusively on leukocytes and bind ICAM molecules on other leukocytes and endothelial cells after cell activation. 5,6 Talin binds to ␤2 integrins in vitro and in cells and is involved in activating the ␤2 integrins, resulting in binding to ICAMs. 1,4,[7][8][9] The ␤2 integrin polypeptide chain is phosphorylated on the intracellular domain on several residues after cell stimulation with various agents. 10 Thr758 is a physiologically important amino acid residue in the ␤2 cytoplasmic tail, and becomes phosphorylated after T-cell stimulation with T-cell receptor (TCR) antibodies or with phorbol esters. [11][12][13] After its phosphorylation, ␤2 binds to 14-3-3 proteins both in vitro and in cells. 4 Blocking of this interaction with a ␤2 Thr758 to Ala mutation, or by expression of constructs that bind to 14-3-3 proteins and block their interactions with target proteins, leads to abrogation of actin cytoskeleton rearrangements, cell spreading, and adhesion to ICAM ligands. 4 ␤2-Thr758 phosphorylation leads to the activation of the actin cytoskeleton modulators, Rac1/Cdc42, in cells. 13 The region in the ␤2 cytoplasmic tail that binds 14-3-3 proteins has been reported to interact with filamin in other integrins, 14 and for the strong filamin-binder ␤7 integrin, phosphorylation mimicking substitutions of 3 threonine residues (TTT) reduces filamin affinity. 3 Fi...

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Section: Resultssupporting

confidence: 77%

Section: Discussionmentioning

confidence: 99%

Section: Protein Crystallographymentioning

confidence: 99%

mentioning

confidence: 99%

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β2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding

Takala

Nurminen

Nurmi³

et al. 2008

Blood

149

235

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“…In recent years, structural and biochemical studies indicate that the FERM head region of talin binds to a highly conserved structural element formed by the NPxY motifs in the integrin β cytoplasmic domains [38]. X-ray crystallographic studies of the talin F3 region of the FERM domain engaging residues 739-750 of the β3 tail, reveals classic features of PTB domain-ligand interactions that directly interact and stabilize the NPxY motif [39]. Binding of talin leads to disruption of auto-inhibitory interactions between the α and β chains, and long-range allosteric changes in the trans-membrane and extracellular domain resulting in enhanced affinity for VTN [6,40].…”

Section: Discussionmentioning

confidence: 99%

A NPxY-independent β5 integrin activation signal regulates phagocytosis of apoptotic cells

Singh

D’Mello

Henegouwen

et al. 2007

Biochemical and Biophysical Research Communications

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Integrin receptors are heterodimeric trans-membrane receptors with critical functions in cell adhesion and migration, cell cycle progression, differentiation, apoptosis, and phagocytosis of apoptotic cells. Integrins are activated by intracellular signaling that alter the binding affinity for extracellular ligands, so-called inside to outside signaling. A common element for integrin activation involves binding of the cytoskeletal protein talin, via its FERM domain, to a highly conserved NPxY motif in the beta chain cytoplasmic tails, which is involved in long-range conformation changes to the extracellular domain that impinges on ligand affinity. When the human beta-5 (β5) integrin cDNA was expressed in αv positive, β5 and β3 negative hamster CS-1 cells, it promoted NPxY-dependent adhesion to VTN-coated surfaces, phosphorylation of FAK, and concomitantly, β5 integrin-EGFP protein was recruited into talin and paxillin-containing focal adhesions. Expression of a NPxY destabilizing β5 mutant (Y750A) abrogated adhesion and β5-Y750A-EGFP was excluded from focal adhesions at the tips of stress fibers. Surprisingly, expression of β5 Y750A integrin had a potent gainof-function effect on apoptotic cell phagocytosis, and further, a β5-Y750A EGFP fusion integrin readily bound MFG-E8-coated 10 um diameter microspheres developed as apoptotic cell mimetics. The critical sequences in β5 integrin were mapped to a YEMAS motif just proximal to the NPxY motif. Our studies suggest that the phagocytic function of β5 integrin is regulated by an unconventional NPxY-talin independent activation signal and argue for the existence of molecular switches in the β5 cytoplasmic tail for adhesion and phagocytosis.

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Integrins: Signalling and Disease

Danen

2013

Encyclopedia of Life Sciences

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Cell‐surface adhesion molecules of the integrin family are not a passive glue, but rather are dynamic molecules that mediate the transfer of information across the membrane in both directions. Integrin‐mediated adhesion can be regulated in response to signals by clustering and conformational changes triggered at their cytoplasmic tails. Vice versa, integrins probe chemical and physical aspects of the extracellular environment and, in concert with growth factor receptors, activate signalling pathways in response. Integrin signalling controls cell survival, cell cycle progression, and differentiation, and the dynamic regulation of integrin‐mediated adhesion structures is critical for many forms of cell migration. Lastly, integrins contribute to the pathogenesis of a diverse array of acquired and hereditary diseases, and hence represent major targets for therapeutic intervention. Key Concepts: Integrins are heterodimeric transmembrane receptors that mediate cell adhesion to extracellular matrix proteins and other cells. Integrins cluster in cell–matrix or cell–cell adhesions where they assemble large multiprotein complexes including actin‐binding proteins. Integrin‐containing adhesions contain enzymes such as FAK, Src, Rac and many others as well as their upstream activators and downstream effectors allowing local activation of cellular signalling cascades. Through a series of conformational changes in mechanoresponsive proteins, integrin‐containing adhesions provide mechanical coupling between the environment and the cytoskeletal network. Signalling through various growth factor receptors is amplified in response to integrin‐mediated adhesion. The ability of integrins to modulate cellular shape and cytoarchitecture in accordance with environmental stiffness as well as their role in a variety of biochemical signal transduction cascades underlies their critical function in cell proliferation, survival, differentiation and motility. Defective integrin function underlies several human diseases. Integrins represent potential drug targets and therapeutic strategies based on interference with integrin‐mediated adhesion have entered clinical trials.

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Structural Determinants of Integrin Recognition by Talin

Cited by 475 publications

References 43 publications

β2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding

β2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding

A NPxY-independent β5 integrin activation signal regulates phagocytosis of apoptotic cells

Integrins: Signalling and Disease

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