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

Abstract

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...

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

“…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).…”

“…S3). Previous in vitro studies identified two sets of mutations, K2085D and K2089D (KS>DD), and L2094A and I2095A (LI>AA) in vertebrate talin (equivalent to K2094D and S2098D, and L2103A and I2104A in Drosophila talin, respectively) that disrupt the binding of IBS-2 to the integrin cytoplasmic tail (Moes et al, 2007;Rodius et al, 2008). We introduced these mutations into UAS-GFP-IBS2 and found that the mutated IBS-2 domain was not enriched at MTJs (Fig.…”

“…5J). Additionally, the N828A mutant PS integrin was able to recruit, albeit at reduced levels, both talin and paxillin to MTJs and there was no statistically significant change in colocalisation between integrin and talin, integrin and paxillin, or talin and paxillin ( Mutations have been identified in the integrin cytoplasmic tail that interfere with its binding to the IBS-2 domain of talin (Rodius et al, 2008). We generated a PS integrin rescue transgene containing point mutations in the corresponding residues in Drosophila talin, E810Q and E817Q (equivalent to E726Q and E733Q in vertebrate 3 integrin; supplementary material Fig.…”

“…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).…”

“…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.…”