Structural Determinants of Integrin Binding to the Talin Rod

BSTRACTStudies on the mechanism of integrin inside-out activation have been focused on the role of b-integrin cytoplasmic tails, which are relatively conserved and bear binding sites for the intracellular activators including talin and kindlin. Cytoplasmic tails for a-integrins share a conserved GFFKR motif at the membrane-proximal region and this forms a specific interface with the b-integrin membraneproximal region to keep the integrin inactive. The a-integrin membrane-distal regions, after the GFFKR motif, are diverse both in length and sequence and their roles in integrin activation have not been well-defined. In this study, we report that the a-integrin cytoplasmic membrane-distal region contributes to maintaining integrin in the resting state and to integrin inside-out activation.Complete deletion of the a-integrin membrane-distal region diminished talin-and kindlin-mediated integrin ligand binding and conformational change. A proper length and suitable amino acids in a-integrin membrane-distal region was found to be important for integrin inside-out activation. Our data establish an essential role for the a-integrin cytoplasmic membrane-distal region in integrin activation and provide new insights into how talin and kindlin induce the high-affinity integrin conformation that is required for fully functional integrins.

Section: Icam-1 Binding (Nomalized Mfi)mentioning

confidence: 99%

The dual structural roles of the membrane distal region of α integrin cytoplasmic tail in integrin inside-out activation

Liu¹,

Wang²,

Thinn³

et al. 2015

“…In vitro and in vivo studies have further shown that the binding of talin IBS-1 to the membrane proximal NPxY sequence in the integrin cytoplasmic tail is required for integrin activation (Calderwood et al, 2002;Tadokoro et al, 2003;Wegener et al, 2007;. The crystal structure of the talin IBS-2 domain has also been solved, and residues have been identified in both talin and integrin that mediate their interaction (Cheung et al, 2009;Gingras et al, 2008;Moes et al, 2007;Rodius et al, 2008). Importantly, IBS-2 has been shown to interact with a membrane-proximal part of the integrin cytoplasmic tail, distinct from the NPxY motif; this interaction does not induce inside-out activation (Gingras et al, 2008;Moes et al, 2007;Rodius et al, 2008;Tremuth et al, 2004).…”

Section: Introductionmentioning

confidence: 98%

“…There are conflicting reports as to whether talin IBS-2 also requires the distal part of the integrin cytoplasmic tail (Gingras et al, 2008;Rodius et al, 2008). To elucidate the mechanism that recruits the IBS-2 domain of talin to integrin we used an allele of the PS integrin subunit that lacks the second half of the integrin cytoplasmic tail (supplementary material Figs S1, S3).…”

Section: The Two Ibs Domains Of Talin Are Recruited To Integrins By Dmentioning

confidence: 99%

“…The crystal structure of the talin IBS-2 domain has also been solved, and residues have been identified in both talin and integrin that mediate their interaction (Cheung et al, 2009;Gingras et al, 2008;Moes et al, 2007;Rodius et al, 2008). Importantly, IBS-2 has been shown to interact with a membrane-proximal part of the integrin cytoplasmic tail, distinct from the NPxY motif; this interaction does not induce inside-out activation (Gingras et al, 2008;Moes et al, 2007;Rodius et al, 2008;Tremuth et al, 2004). However, to date the roles of the IBS-2 domain of talin have not yet been explored in the context of a whole developing organism.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

In vivo functional analysis reveals specific roles for the integrin-binding sites of talin

Ellis

Pines

Fairchild

et al. 2011

SummaryAdhesion receptors play diverse roles during animal development and require precise spatiotemporal regulation, which is achieved through the activity of their binding partners. Integrins, adhesion receptors that mediate cell attachment to the extracellular matrix (ECM), connect to the intracellular environment through the cytoplasmic adapter protein talin. Talin has two essential functions: orchestrating the assembly of the intracellular adhesion complex (IAC), which associates with integrin, and regulating the affinity of integrins for the ECM. Talin can bind to integrins through two different integrin-binding sites (IBS-1 and IBS-2, respectively). Here, we have investigated the roles of each in the context of Drosophila development. We find that although IBS-1 and IBS-2 are partially redundant, they each have specialized roles during development: IBS-1 reinforces integrin attachment to the ECM, whereas IBS-2 reinforces the link between integrins and the IAC. Disruption of each IBS has different developmental consequences, illustrating how the functional diversity of integrin-mediated adhesion is achieved.

“…Data from the constitutively activated YA mutant suggest that binding of talin-1 to the NPIY motif is not strictly required for many outside-in integrin-signaling-dependent processes. Since talin-1 can also bind to the membrane-proximal region of b-tails (Gingras et al, 2009;Wegener et al, 2007), we wanted to further investigate the role of talin-1 in cells adhered by the constitutively activated YA mutant integrin. Thus, we assayed the effects of TLN1 siRNA on cell spreading, focal adhesion, stress fibers, and microtubule Journal of Cell Science 123 (8) assembly in the presence or absence of constitutive integrin activation.…”

Section: Talin-1 Is Required Downstream Of Integrin Activation When Cmentioning

confidence: 99%

The NPIY motif in the integrin β1 tail dictates the requirement for talin-1 in outside-in signaling

Nieves

Jones

Ward

et al. 2010

SummaryProtein interactions with the integrin b-subunit cytoplasmic domain (b-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 b-tails (NPIY in b1 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 b-tail downstream of integrin activation, in the context of recombinant integrins containing either the wild type (WT) or the (YA) mutant b1A 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 b1A 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 DGAP , which lacks GAP activity restores spreading in cells adhered by constitutively activated integrins containing the b1A tail, but not by integrins containing the b1D 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 b1A tail. Our studies emphasize the importance of understanding the mechanisms that regulate the differential binding FLNa and talin-1 to the b1 tail downstream of integrin activation in promoting integrin function.