Anthis NJ, Wegener KL, Ye F, Kim C, Goult BT, Lowe ED et al.. The structure of an integrin/talin complex reveals the basis of inside-out signal transduction. EMBO J 28: 3623-3632

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
The EMBO Journal (Impact Factor: 10.43). 10/2009; 28(22):3623-32. DOI: 10.1038/emboj.2009.287
Source: PubMed


Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that uniquely mediate inside-out signal transduction, whereby adhesion to the extracellular matrix is activated from within the cell by direct binding of talin to the cytoplasmic tail of the beta integrin subunit. Here, we report the first structure of talin bound to an authentic full-length beta integrin tail. Using biophysical and whole cell measurements, we show that a specific ionic interaction between the talin F3 domain and the membrane-proximal helix of the beta tail disrupts an integrin alpha/beta salt bridge that helps maintain the integrin inactive state. Second, we identify a positively charged surface on the talin F2 domain that precisely orients talin to disrupt the heterodimeric integrin transmembrane (TM) complex. These results show key structural features that explain the ability of talin to mediate inside-out TM signalling.

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    • "Talin comprises an N-terminal FERM domain (50 kDa) that binds integrin cytodomains and acidic membrane phospholipids synergistically78. The C-terminal talin rod (220 kDa) contains 13 helical bundles (R1–R13) terminating in a single helix that supports talin dimerisation (Fig 1a)9. "
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    ABSTRACT: The force-dependent interaction between talin and vinculin plays a crucial role in the initiation and growth of focal adhesions. Here we use magnetic tweezers to characterise the mechano-sensitive compact N-terminal region of the talin rod, and show that the three helical bundles R1-R3 in this region unfold in three distinct steps consistent with the domains unfolding independently. Mechanical stretching of talin R1-R3 enhances its binding to vinculin and vinculin binding inhibits talin refolding after force is released. Mutations that stabilize R3 identify it as the initial mechano-sensing domain in talin, unfolding at ∼5 pN, suggesting that 5 pN is the force threshold for vinculin binding and adhesion progression.
    Scientific Reports 04/2014; 4:4610. DOI:10.1038/srep04610 · 5.58 Impact Factor
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    • "The F3 domain itself can bind to the β3 cytoplasmic domain and exert αIIbβ3 activation [5]. Other subdomains also have important roles in the activation [6-8]. The kindlin family members (kindlin-1, -2, and -3), which are focal adhesion proteins, have recently been proven to be critical for integrin activation [9,10]. "
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    ABSTRACT: Integrin-linked kinase (ILK) is an important signaling regulator that assembles into the heteroternary complex with adaptor proteins PINCH and parvin (termed the IPP complex). We recently reported that ILK is important for integrin activation in a Chinese hamster ovary (CHO) cell system. We previously established parental CHO cells expressing a constitutively active chimeric integrin (αIIbα6Bβ3) and mutant CHO cells expressing inactive αIIbα6Bβ3 due to ILK deficiency. In this study, we further investigated the underlying mechanisms for ILK-dependent integrin activation. ILK-deficient mutant cells had trace levels of PINCH and α-parvin, and transfection of ILK cDNA into the mutant cells increased not only ILK but also PINCH and α-parvin, resulting in the restoration of αIIbα6Bβ3 activation. In the parental cells expressing active αIIbα6Bβ3, ILK, PINCH, and α-parvin were co-immunoprecipitated, indicating the formation of the IPP complex. Moreover, short interfering RNA (siRNA) experiments targeting PINCH-1 or both α- and β-parvin mRNA in the parent cells impaired the αIIbα6Bβ3 activation as well as the expression of the other components of the IPP complex. In addition, ILK mutants possessing defects in either PINCH or parvin binding failed to restore αIIbα6Bβ3 activation in the mutant cells. Kindlin-2 siRNA in the parental cells impaired αIIbα6Bβ3 activation without disturbing the expression of ILK. For CHO cells stably expressing wild-type αIIbβ3 that is an inactive form, overexpression of a talin head domain (THD) induced αIIbβ3 activation and the THD-induced αIIbβ3 activation was impaired by ILK siRNA through a significant reduction in the expression of the IPP complex. In contrast, overexpression of all IPP components in the αIIbβ3-expressing CHO cells further augmented THD-induced αIIbβ3 activation, whereas they did not induce αIIbβ3 activation without THD. These data suggest that the IPP complex rather than ILK plays an important role and supports integrin activation probably through stabilization of the active conformation.
    PLoS ONE 12/2013; 8(12):e85498. DOI:10.1371/journal.pone.0085498 · 3.23 Impact Factor
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    • "The αIIbF992A/F993Aβ3 mutant (αIIbFFβ3) has been reported to be constitutively active as a result of the mutations disrupting the association of the membrane-proximal portions of the α and β subunit cytoplasmic domains [31]–[33]. Similar disruption of the α and β subunit cytoplasmic domains is proposed to occur with inside-out activation of the receptor [34]. To assess whether the αIIbFF mutations could rescue the XS-O mutants' ability to bind high Mr ligands, we co-expressed the αIIbFF mutant with normal β3 and the αIIb mutant K321C, F992A, F993A with either the β3E358C or the β3R360C mutant. "
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    ABSTRACT: Structural and functional analyses of integrin αIIbβ3 has implicated swing-out motion of the β3 hybrid domain in αIIbβ3 activation and ligand binding. Using data from targeted molecular dynamics (TMD) simulations, we engineered two disulfide-bonded mutant receptors designed to limit swing-out (XS-O). XS-O mutants cannot bind the high Mr ligand fibrinogen in the presence of an activating mAb or after introducing mutations into the αIIb subunit designed to simulate inside-out signaling. They also have reduced capacity to be "primed" to bind fibrinogen by pretreatment with eptifibatide. They can, however, bind the small RGD venom protein kistrin. Despite their inability to bind soluble fibrinogen, the XS-O mutants can support adhesion to immobilized fibrinogen, although such adhesion does not initiate outside-in signaling leading to normal cytoskeletal reorganization. Collectively, our data further define the biologic role of β3 hybrid domain swing-out in both soluble and immobilized high Mr ligand binding, as well as in priming and outside-in signaling. We also infer that swing-out is likely to be a downstream effect of receptor extension.
    PLoS ONE 12/2013; 8(12):e81609. DOI:10.1371/journal.pone.0081609 · 3.23 Impact Factor
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