Integrin activation, the rapid conversion of integrin adhesion receptors from low to high affinity, occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin ␤ subunits. Talin binding to integrin ␤ tails provides one key activation signal, but additional factors are likely to cooperate with talin to regulate integrin activation. The integrin ␤ tailbinding proteins kindlin-2 and kindlin-3 were recently identified as integrin co-activators. Here we report an analysis of kindlin-1 and kindlin-2 interactions with ␤1 and ␤3 integrin tails and describe the effect of kindlin expression on integrin activation. We demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin ␤ tails in pulldown binding assays. or stably expressed ␣IIb␤3 integrins. This inhibition is not dependent on direct kindlin-integrin interactions because mutant kindlins exhibiting impaired integrin binding activity effectively inhibit integrin activation. Consistent with previous reports, we find that when co-expressed with the talin head, kindlin-1 or -2 can activate ␣IIb␤3. This effect is dependent on an intact integrin-binding site in kindlin. Notably however, even when co-expressed with activating levels of talin head, neither kindlin-1 or -2 can cooperate with talin to activate ␤1 integrins; instead they strongly inhibit talin-mediated activation. We suggest that kindlins are adaptor proteins that regulate integrin activation, that kindlin expression levels determine their effects, and that kindlins may exert integrin-specific effects.

Talin is a 270-kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for β-integrin tails, F0 and F1 are also required for activation of β1-integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated activation of β1-integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins.

The activation of integrin adhesion receptors from low to high affinity in response to intracellular cues controls cell adhesion and signaling. Binding of the cytoskeletal protein talin to the ␤3 integrin cytoplasmic tail is required for ␤3 activation, and the integrin-binding PTB-like F3 domain of talin is sufficient to activate ␤3 integrins. Here we report that, whereas the conserved talin-integrin interaction is also required for ␤1 activation, and talin F3 binds ␤1 and ␤3 integrins with comparable affinity, expression of the talin F3 domain is not sufficient to activate ␤1 integrins. ␤1 integrin activation could, however, be detected following expression of larger talin fragments that included the N-terminal and F1 domains, and mutagenesis indicates that these domains cooperate with talin F3 to mediate ␤1 activation. This effect is not due to increased affinity for the integrin ␤ tail and we hypothesize that the N-terminal domains function by targeting or orienting talin in such a way as to optimize the interaction with the integrin tail. Analysis of ␤3 integrin activation indicates that inclusion of the N-terminal and F1 domains also enhances F3-mediated ␤3 activation. Our results therefore reveal a role for the N-terminal and F1 domains of talin during integrin activation and highlight differences in talin-mediated activation of ␤1 and ␤3 integrins.Integrins are a family of ␣␤ heterodimeric transmembrane receptors that mediate cell adhesion to extracellular matrix, cell surface, or soluble protein ligands and modulate a variety of intracellular signaling cascades. Cells regulate integrin function through tight temporal and spatial control of integrin affinity for extracellular ligands. This is achieved by rapid, reversible changes in the conformation of the integrin extracellular domains; integrin activation (1-3). Activation of the platelet integrin ␣IIb␤3 is a pivotal event in thrombus formation (4), and ␣IIb␤3 has served as a prototype in studies on integrin activation. However, activation of other integrins, including the widely expressed ␤1 family, is essential for normal development because it controls cell adhesion, migration, and assembly of an extracellular matrix (5-10), and deregulated ␤1 integrin activation contributes to neoplasia (11) and impairs cardiac function (12) and the immune response (13).A large body of evidence points to regulation of integrin activation through interactions of the ␤ subunit tail (3, 14), although ␣ tail-binding proteins also have a role (15, 16). Using ␣IIb␤3 as a model system we and others have shown that binding of talin to the ␤3 cytoplasmic tail is necessary and sufficient for integrin activation (17-23). Talin, a cytoskeletal actin-binding protein, consists of an N-terminal ϳ50-kDa globular head and an ϳ220-kDa C-terminal rod (3,24,25). The talin head is composed of an N-terminal 85-amino acid region followed by a FERM (4.1, ezrin, radixin, moesin) domain and a 33-amino acid stretch (18, 24 -26). FERM domains are made up of three subdomains, F1, F2, and F3 (27...

The integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and integrin activation requires binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the β-integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for talin-induced integrin activation. FERM domains are typically composed of F1, F2, and F3 domains, but the talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains. Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support talin-induced αIIbβ3 integrin activation and for the localization of kindlin-1 to focal adhesions.

Background: Kindlins cooperate with talin to activate integrins. Results: A polylysine motif within a loop in the F1 domain of kindlin binds membranes and is required for integrin activation. Conclusion:The membrane-binding site in kindlin F1 is distinct from that in talin and is essential to activate integrins Significance: Understanding the molecular basis of integrin activation requires detailed information on kindlin interactions.

Integrin adhesion receptors are essential for development and functioning of multi-cellular animals. Integrins mediate cell adhesion to the extracellular matrix and to counter-receptors on adjacent cells and the ability of integrins to bind extracellular ligands is regulated in response to intracellular signals that act on the short cytoplasmic tails of integrin subunits. Integrin activation, the rapid conversion of integrin receptors from low to high affinity, requires binding of talin to integrin β tails and, once bound, talin provides a connection from activated integrins to the actin cytoskeleton. A wide range of experimental approaches have contributed to the current understanding of the importance of talin in integrin signaling. Here we describe two methods that have been central to our investigations of talin; a biochemical assay that has allowed characterization of interactions between integrin cytoplasmic tails and talin and a fluorescent activated cell sorting procedure to assess integrin activation in cultured cells expressing talin domains, mutants, dominant negative constructs, or shRNA.

We previously demonstrated that the TNF-α-induced inside-out signaling leading to β2 integrin activation is redox regulated. To identify kinases involved in this pathway, the effects of kinase inhibitors on the expression of β2 integrin activation neoepitope (clone 24) were investigated. We show that both p38 MAPK (inhibited by SB203580) and Src kinases (inhibited by PP2) are involved in β2 integrin activation by TNF and oxidants in human neutrophils. Src kinases appeared constitutively active in resting neutrophils and not further activated by TNF or oxidants in nonadherent conditions. However, PP2 blocked both TNF-induced expression of the 24 epitope and cell adhesion promoted by the integrin activating anti-CD18 KIM185 mAb, showing that both the inside-out and the outside-in signaling involve Src kinases. p38 MAPK was activated by TNF and oxidants in nonadherent conditions i.e., with 10 mM EDTA. This activation in EDTA resulted in CD11b, CD35 and CD66 up-regulation and in an oxidative response, all blocked by SB203580 and PP2. p38 MAPK was not activated upon direct integrin activation by KIM185 mAb. Thus, p38 activation allows the study to distinguish the initial transduction pathway leading to β2 integrin activation from the signaling resulting from integrin engagement. Finally, p38 MAPK activation by TNF was blocked by diphenylene iodonium, an inhibitor of flavoprotein oxidoreductase, and by the free radical scavenger N-acetylcystein. Taken together, these results demonstrate, for the first time, that constitutively activated Src tyrosine kinases and a redox-regulated activation of p38 MAPK are involved in TNF inside-out signaling leading to β2 integrin activation.

Although leukosialin (CD43) membrane expression decreases during neutrophil apoptosis, the CD43 molecule, unexpectedly, is neither proteolyzed nor internalized. We thus wondered whether it could be shed on bleb-derived membrane vesicles. Membrane blebbing is a transient event, hardly appreciated during the asynchronous, spontaneous apoptosis of neutrophils. Cell pre-synchronization at 15°C made it possible to observe numerous blebbing neutrophils for a short 1-h period at 37°C. CD43 down-regulation co-occurred with the blebbing stage and phosphatidylserine externalization, shortly after mitochondria depolarization and before nuclear condensation. Blebs detaching from the cell body were observed by time lapse fluorescence microscopy, and the release of bleb-derived vesicles was followed by flow cytometry. Phosphatidylserine externalization required caspases and protein kinase C (PKC) but not the myosin light chain kinase (MLCK). By contrast, bleb formation and release was caspase-and PKCindependent but required an active MLCK, whereas CD43 down-regulation involved caspases but neither PKC nor MLCK. Furthermore, CD43 appeared mostly excluded from membrane blebs by electron microscopy. Thus, CD43 down-regulation does not result from the release of bleb-derived vesicles. Ultracentrifugation of apoptotic cell supernatants made it possible to recover <1 m microparticles, which contained the entire CD43 molecule. These microparticles expressed neutrophil membrane markers such as CD11b, CD66b, and CD63, together with CD43. In conclusion, we show that the three early membrane events of apoptosis, namely blebbing, phosphatidylserine externalization, and CD43 down-regulation, result from different signaling pathways and can occur independently from one another. CD43 down-regulation results from the shedding of microparticles released during apoptosis but unrelated to the blebbing.