Integrin activation
David A. Calderwood
Department of Pharmacology, Yale University School of Medicine, Sterling Hall of Medicine, PO Box 208066, New Haven, CT 06520, USA

Journal of Cell Science 117, 657-666 Published by The Company of Biologists 2004 doi:10.1242/jcs.01014

Summary The ability of cells to regulate dynamically their adhesion to one another and to the extracellular matrix (ECM) that surrounds them is essential in multicellular organisms. The integrin family of transmembrane adhesion receptors mediates both cell-cell and cell-ECM adhesion. One important, rapid and reversible mechanism for regulating adhesion is by increasing the affinity of integrin receptors for their extracellular ligands (integrin activation). This is controlled by intracellular signals that, through Introduction Integrins are heterodimeric adhesion receptors formed by the non-covalent association of α and β subunits. Each subunit is a type I transmembrane glycoprotein that has relatively large extracellular domains and, with the exception of the β4 subunit, a short cytoplasmic tail (Hynes, 2002). Integrins are present in all metazoans, and the number of integrins in the genome generally increases with the complexity of the organism (Hynes, 2002; Bokel and Brown, 2002), which is consistent with the central role of integrins in adhesion, migration and tissue organization. Mammals contain 18 α and 8 β subunits that combine to produce at least 24 different heterodimers, each of which can bind to a specific repertoire of cell-surface, ECM or soluble protein ligands. Cell-cell and cell-substratum adhesion is mediated by the binding of integrin extracellular domains to diverse protein ligands; however, cellular control of these adhesive interactions and their translation into dynamic cellular responses, such as cell spreading or migration, requires the integrin cytoplasmic tails. These short tails bind to intracellular ligands that connect the receptors to signalling pathways and cytoskeletal networks (Critchley, 2000; Calderwood et al., 2000; Liu et al., 2000; Brakebusch and Fassler, 2003; Giancotti and Ruoslahti, 1999; Geiger et al., 2001). Hence, by binding both extracellular and intracellular ligands, integrins provide a transmembrane link for the bidirectional transmission of mechanical force and biochemical signals across the plasma membrane. One important mechanism by which cells regulate integrin function is through tight spatial and temporal control of integrin affinity for extracellular ligands. This is achieved by rapid, reversible changes in the conformation of the extracellular domains of the integrin heterodimer, so-called integrin activation (Sims et al., 1991; Woodside et al., 2001). It has recently been noted that researchers in the integrin field use this term differently from those studying most other receptors and that improved clarity of terminology will be required to understand the complexities of integrin regulation (Humphries et al., 2003; Carman and Springer, 2003). The term their action on integrin cytoplasmic domains, induce conformational changes in integrin extracellular domains that result in increased affinity for ligand. Recent studies have shed light on the final intracellular steps in this process and have revealed a vital role for the cytoskeletal protein talin.
Key words: Integrin, Talin, Cytoplasmic tails

activation was initially applied to integrins in early studies of the platelet GPIIb/IIIa complex (integrin αIIbβ3), where an activation-dependent change in the conformation and/or microenvironment of the complex caused activation of the fibrinogen receptor function of GPIIb/IIIa (Coller, 1985; Shattil et al., 1985; Isenberg et al., 1987). In this sense, integrin activation refers to the changes required to enhance ligandbinding activity (the primary effector function of adhesion receptors), whereas activation of signalling receptors generally refers to the changes induced by ligand binding that enhance signal transduction (the primary effector function of signalling receptors). The finding that integrins also play important roles as signalling receptors (Schwartz and Ginsberg, 2002) serves to emphasise the importance of providing clear definitions of terms. Recently, integrin priming was proposed as a term to denote integrin affinity regulation, whereas integrin activation would be defined as the process of ligand-induced propagation of intracellular signals (Humphries et al., 2003). However, priming has also been proposed to define the affinity- or valency-based regulatory events that serve to enhance ligandbinding efficiency (Carman and Springer, 2003). It is therefore essential that investigators operationally define ‘priming’ or ‘activation’ for their specific experimental system. Here, in keeping with general usage and historical precedent, I will apply the term integrin activation to describe the increase in monomeric affinity that is coupled to alterations in integrin conformation. Despite significant recent advances, the exact nature of the conformational changes leading to integrin activation remains controversial (reviewed by Hynes, 2002; Liddington and Ginsberg, 2002; Shimaoka et al., 2002; Xiong et al., 2003a; Humphries et al., 2003). Additional affinity-independent mechanisms, such as integrin clustering, lateral diffusion of receptors, interactions with and reorganization of the cytoskeleton, and changes in integrin expression patterns also contribute to the control of integrin-mediated adhesion (reviewed by Laudanna et al., 2002; van Kooyk and Figdor, 2000; Hogg et al., 2002). Affinity-dependent and -independent

. Crowe et al.658 Journal of Cell Science 117 (5) GFFKR sequence. The interface between the transmembrane and cytoplasmic regions is generally assumed to lie between the conserved W/Y and K residues. β turn-forming.. 2003)... O’Toole et al. 1993.. 2002.. Furthermore. Ylanne et al. 2001). However. The conserved β tail residues involved in talin binding are indicated. elegans βPAT3 (B) were manually aligned. Integrin activation is essential for normal development because it controls cell adhesion. the first. either stimulation with agonists. Deletion of the entire α subunit cytoplasmic tail. 1998). Alignment of integrin cytoplasmic tails.. and human β and Drosophila βPS and C... possibly by disrupting the interaction between integrin α and β tails (Stephens et al. 1994. 1997. cellular control of integrin activation plays important roles in health and disease throughout development and during the course of adult life. and impairs cardiac function (Keller et al. constitutively activates integrins... 1997). 2003). 1. 2001) and the immune response (McDowall et al. 1997. Mutagenesis studies suggest that a salt bridge between residues R995 of αIIb and D723 of β3 stabilizes the association of the membrane-proximal regions and so maintains αIIbβ3 in a low-affinity state (Hughes et al. shown in bold.. Integrin activation is also involved in angiogenesis (Byzova et al. and deregulated integrin activation disrupts embryonic development (Martin-Bermudo et al. 2003). where β1 and β2 integrins become activated (Laudanna et al.. Li et al. 2001). migration and assembly of an extracellular matrix (Wu et al. whereas deletions that retain the GFFKR sequence do not (O’Toole et al. however. 2000. 1997). Erb et al. 1) and play crucial roles in integrin activation... Palecek et al. Laplantine et al. This review will focus on the roles of integrin cytoplasmic tails and the importance of their interactions with talin for regulating integrin activation. Indeed. Likewise. 2003). 1995. in combination with ligand occupancy. the importance of integrin activation is not limited to blood cells. Hogg et al. 1987. Biochemical evidence for a low-affinity interaction between α and β tails also exists (Muir et al. deletion of the membrane-proximal region of the β tail activates integrins. Lu et al. or of only the conserved membrane-proximal A α1 α2 α3A α4 α5 α6A α7 α8 α9 α10 α11 αV αL αM αX αD αIIb αE αPS1 αPAT2 B β1A β2 β3 β5 β6 β7 βPS βPAT3 LALW AILW LLLW YVMW YILY FILW LLLW LALW VLLW FCLW LALW FVMY IVLY AALY AVLY ATLY LAMW VILF KIGFFKRPLKKKMEK KLGFFKRKYEKMTKNPDEIDETTELSS KCGFFKRARTRALYEAKRQKAEMKSQPSETERLTDDY KAGFFKRQYKSILQEENRRDSWSYINSKSNDD KLGFFKRSLPYGTAMEKAQLKPPATSDA KCGFFKRNKKDHYDATYHKAEIHAQPSDKERLTSDA KMGFFKRAKHPEATVPQYHAVKIPREDRQQFKEEKTGTILRNNWGSPRREGPDAHPILAADGHPELGPDGHPGPGTA KCGFFDRARPPQEDMTDREQLTNDKTPEA KMGFFRRRYKEIIEAEKNRKENEDSWDWVQKNQ KLGFFAHKKIPEEEKREEKLEQ KLGFFRSARRRREPGLDPTPKVLE RMGFFKRVRPPQEEQEREQLQPHENGEGNSET KVGFFKRNLKEKMEAGRGVPNGIPAEDSEQLASGQEAGDPGCLKPLHEKDSESGGGKD KLGFFKRQYKDMMSEGGPPGAEPQ KVGFFKRQYKEMMEEANGQIAPENGTQTPSPPSEK KLGFFKRHYKEMLEDKPEDTATFSGDDFSCVAPNVPLS KVGFFKRNRPPLEEDDEEGE KCGFFKRKYQQLNLESIRKAQLKSENLLEEEN YVLW KVGFFKRIRPTDPTLSGNLEKMNEEKPFLAPSKNTHHVF LLLW RCGFFKRNRPPTEHAELRADRQPNAQYADSQSRYTSQDQYNQGRHGQML LLIW LVIW LLIW LAIW LCIW VLAY KLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVV--------NPKYEGK KALIHLSDLREYRRFEKEKLKSQWNND-NPLFKSATTTVM--------NPKFAES KLLITIHDRKEFAKFEEERARAKWDTANNPLYKEATSTFT--------NITYRGT KLLVTIHDRREFAKFQSERSRARYEMASNPLYRKPISTHTVDFTFNKFNKSYNGTVD KLLVSFHDRKEVAKFEAERSKAKWQTGTNPLYRGSTSTFK--------NVTYKHREKQKVDLSTDC RLSVEIYDRREYSRFEKEQQQLNWKQDSNPLYKSAITTTI--------NPRFQEADSPTL LLLW KLLTTIHDRREFARFEKERMNAKWDTGENPIYKQATSTFK--------NPMYAGK LLLW KLLTVLHDRSEYATFNNERLMAKWDTNENPIYKQATTTFK--------NPVYAGKAN Fig. Thus. Integrin cytoplasmic tails The membrane-proximal regions of the short α and β integrin cytoplasmic tails are well conserved (Fig. In the resting state. where activation of platelet integrin αIIbβ3 is a pivotal event in thrombus formation (Shattil et al.. The importance of tight regulation of integrin activation is evident during haemostasis. NPxY motif is shown in blue and the conserved tryptophan is shown in pink.... indeed. Wang et al. and preventing their association activates integrins (Lu et al. The amino acid sequences of human α tails and Drosophila αPS1 and Caenorhabditis elegans αPAT2 (A). 2000) and tumour cell metastasis (FeldingHabermann et al. β1 and β3 were omitted. The conserved membrane-proximal α subunit GFFKR and β subunit LLxxxHDREE are shown in red. forced association of the cytoplasmic regions of αLβ2 or αMβ2 blocks activation. and during leukocyte trafficking. The high-affinity state generated by deletion of the mechanisms are not mutually exclusive and can act in concert. or introduction of activating membrane-proximal α subunit mutations led to a reduction in FRET.... which is indicative of conformational rearrangements of the cytoplasmic domains. 1995. whereas deletions that are more C-terminal block activation (Hughes et al... 1996).. also stimulates clustering (Isenberg et al. 1996.. 1998. . 1998). 2003). the α and β tails were sufficiently close together to undergo FRET. 1991. Huttenlocher et al. leading to integrin activation. Kim et al. and activating mutations in the α subunit membraneproximal region disrupt the interaction (Vallar et al. fluorescence resonance energy transfer (FRET) analysis of cyan fluorescent protein-fused and yellow fluorescent proteinfused αL and β2 has provided in vivo evidence for α-β tail associations (Kim et al. 1994.. 2002). α6. 1999). integrin activation and clustering may be mechanistically linked such that activation. 1994).. The divergent human β4 and β8 and alternative splice variants of α3. lipid-modified peptides corresponding to the membrane-proximal regions of the αIIb or α2 tails activate αIIbβ3 or α2β1 when introduced into platelets. 2001. Haas and Plow. More recently. Lu and Springer. Ginsberg et al. 2001...

. Thus. Calderwood et al.. either free in solution. 1993. talin fragments perturb the membrane-proximal residues of β3 tails and tethering the αIIb tail adjacent to the β3 tail inhibits this effect (Ulmer et al. probably by interacting with one another to stabilize an inactive conformation. Although the membrane-proximal regions of both β3 and αIIb generally tend to form α helices. and deletion of α subunit residues after GFFKR inhibits cell-type-specific and agonistinduced α2β1-. Li et al.. in intact integrins. 2003). strongly inhibit integrin activation and induce structural changes at the NPxY site and within the membrane-proximal region (Ulmer et al. O’Toole et al. Wang et al.. Thus. In the presence of this sequence.. Furthermore. Li et al.. another study observed two distinct αβ tail complexes. 2001)... the first conserved NPxY motif. like integrin. Thus. However. Talin is a critical integrin-activating protein Recent data indicate that talin. a major cytoskeletal actinbinding protein that binds to integrin‚ tails and colocalizes with activated integrins (Critchley. The only αβ interaction observed using full-length tails was evident from small chemical shifts and was absent in the presence of micelles (Vinogradova et al. 1994).. point mutations can disrupt their conformation and inhibit their interactions with intracellular proteins (Vinogradova et al... Hibbs et al. 1997. with the notable exception of Rap1induced αLβ2-mediated adhesion in BAF/3 cells. prevents formation of the combinatorial epitope (Ginsberg et al. a portion of the αIIb tail immediately C-terminal to the conserved GFFKR may interact with the β3 tail to inhibit formation of an active conformation (Ginsberg et al. 1991b.. Hughes et al. Nonetheless. 2001). limits the relative mobility of the tails and might facilitate complex formation.. 2001. the entire cytoplasmic tails. 1994. P999A mutation within this region alters the conformation of the α tail (Vinogradova et al. Deletion of β tail sequences outside the membrane-proximal region blocks activation. although integrin cytoplasmic tails may be largely unstructured in solution and their modes of interaction remain controversial. However. 2001. 2002) and. 2000)..... whereas celltype-dependent and energy-dependent activation requires additional. structural studies provide some support for a membrane-proximal interaction between αIIb and β3 tails and for regulation of this by the integrin-activator talin. Vinogradova et al. or the cytoplasmic tails fused to the transmembrane sequences. Ulmer et al.. 1997).. 2000). Weber et al. Two NMR studies detected no interaction between the αIIb and β3 tails (Ulmer et al.. there are significant differences between the various structures obtained. 2003). β3 or β5 tails. 1994. The α tail contributes to the cell-type specificity of integrin activation (O’Toole et al. 2002.. This probably reflects the different constructs and conditions used. 2000. Zent et al. Tohyama et al. 2002). nonetheless. 1994.... Drosophila (Brown et al. Tohyama et al. 2001) and activates αIIbβ3 (Leisner et al... more C-terminal. 1991a. 2002). 2000). α4β1. Isolated tail complexes may be difficult to observe because of the low-affinity interaction between tails (Vallar et al. tethering α and β tails together did not reveal an interaction (Ulmer et al. 1995. 1997. Chen et al. In Drosophila and C. sequences (O’Toole et al. when bound to the phosphotyrosine binding (PTB)-like domain of talin. and specific β tail regions important for activation have been identified (Hibbs et al. and the dynamic flexible nature of the integrin tails. Fenczik et al. Li et al. Mastrangelo et al. whereas one study detected a weak membrane-proximal interaction (Vinogradova et al.and αLβ2-mediated adhesion (Kawaguchi and Hemler. integrin tails might rely on interaction with intracellular factors to stabilize their structure. which raises questions about its biological significance.. Kassner and Hemler. plays a crucial role in integrin activation (Tadokoro et al. 1993. 2001). 2001)... 2002.. is required for normal development in mice (Monkley et al. using truncated tails. talin .. 2002).. 2003). tethered to membranes or embedded in micelles. 2001. Ulmer et al. and αIIbβ3 activation by the β tail-binding protein talin requires an intact NPxY sequence (Calderwood et al. 1999. 2001).. the sequence of these additional residues is unimportant (Kassner et al... 2003). 1994b.. 2003). have shed further light on interactions involving integrin tails (Vinogradova et al. α and β subunit extracellular domains form a complex that. palmitoylated peptides spanning this region of αIIb suppress αIIbβ3 activation in platelets (Ginsberg et al.Integrin activation membrane-proximal region of either the α or β tail is independent of cell type and metabolic energy.. In the case of αIIbβ3. and evidence exists for a turn at the β3 subunit NPLY. 1999).. Weljie et al. the β3 tail forms epitopes present in native inactive αIIbβ3 and in β3 tails containing an inactivating mutation. Talin.. each significantly different from that seen by Vinogradova et al.. but not α tails nor β tails containing membrane-distal inactivating mutations.. Tyrosine to alanine mutations in one of these sites... Ulmer et al. whereas membrane-distal α sequences regulate β tail conformation and association with activator proteins in a cell-type-specific manner. Isolated β1A.. Bodeau et al. 1995.. 1999). 1999). the complex was disrupted by activating membrane-proximal α subunit mutations and by the integrin activator talin. Mutations in the NPxY motif also perturb the binding of numerous cytoskeletal and signalling proteins to integrin β tails (Liu et al. (Weljie et al. 2000. presumably by competing for limiting amounts of intracellular activators (Chen et al. 2001) and a P988A. β1D. 2000. but not non-activating. 2003).. Hughes et al. 2002. 2003). elegans. If an additional 5-7 amino acids is included after the GFFKR sequence then this is prevented. despite the flexibility of the tails. 2001. 2000)... 1995). membrane-distal portions of the β tail might control integrin activation through interactions with regulatory proteins and direct effects on the conformation of membrane-proximal regions. 1994a. inhibit integrin activation in a dose-dependent manner. O’Toole et al. the β3 tail NPLY motif forms a turn and the preceding seven residues form a β strand that augments the β sandwich present in talin (Garcia-Alvarez et al.. Crystallography reveals that. The more distal regions of the β tails regulate activation through interactions with signalling proteins that might disrupt the membrane-proximal interaction. 2002) and Caenorhabditis elegans (Cram et al. 2003. The membrane-proximal regions of the α and β tails thus play crucial roles in integrin activation. fused to soluble coiled-coil regions. Structure of integrin tails Nuclear magnetic resonance (NMR) studies of peptide 659 constructs representing sequences from the membraneproximal regions of the αIIb and β3 tails... Activating. Ulmer et al.

Integrin residues L746 and W739. 2002). 2003.. The structure of the β3 tail residues 738-748 (shown as sticks) bound to the talin PTBlike domain (PDB: 1MK7).. Notably.. Both the turn and augmentation L746 Y747 K357 R358 A360 W739 W359 Fig. deletion of the α subunit membraneproximal regions generates integrins that remain activated in the absence of talin or metabolic energy (O’Toole et al. has been shown to reduce the FRET between fluorphore-tagged α and β integrin in living cells (Kim et al. 1999. 1). Calderwood et al. The crystal structure of talin F3-engaging residues 739-750 of the β3 tail reveals that residues 740-742 (DTA) form a β strand that augments the β sheet of F3. 1999) or to indirect conformational changes induced following formation of the complex. Calderwood et al. 2002.. Knezevic et al.. is shown in yellow. its binding to integrin β tails is an essential final step in integrin activation. Binding of the talin PTB domain to the integrin NPxY motif might thus perturb β tail membrane-proximal residues and disrupt an inhibitory α-β tail interaction. only integrin-activating talin fragments affect the membrane-proximal regions of β3... 1999... inhibits activation (Tadokoro et al. 2). Tadokoro et al. RNAi knockdown of talin expression has revealed that talin is essential for β1 and β3 integrin activation in a variety of cell types (Tadokoro et al. F2 is largely α-helical and resembles the acyl-CoA-binding protein. 2003). and residues 744-747 (NPLY) form a reverse turn with Y747 pointing into an acidic and hydrophobic pocket (GarciaAlvarez et al. 2003). Garcia-Alvarez et al. 2003) (Fig. F3 binds with a fourfold higher affinity (Kd = 120 nM) than F2 (Calderwood et al.. 2003). but NMR analysis reveals that NPxY-mediated talin binding induces spectral perturbations in the membrane-proximal residues (Vinogradova et al. 2003). FERM domains have three subdomains – F1. 2001. Y747. Calderwood et al. more recently. when over-expressed. which inhibits talin. 2. Each subunit consists of an N-terminal ~50 kDa globular head and an ~220 kDa C-terminal rod (Rees et al.. F2 and F3 – and often mediate interactions with the cytoplasmic tails of transmembrane proteins (Pearson et al. 2002.. 2000)..660 Journal of Cell Science 117 (5) of the β strand are classical features of PTB domain-ligand interactions (Schlessinger and Lemmon. Tadokoro et al.. expression of talin F3. whereas F3 is a sandwich of two orthogonal antiparallel β-sheets followed by an α-helix (Garcia-Alvarez et al. Ulmer et al. NPxY motifs are highly conserved in integrin β tails (Fig. However... 2002). 2002). however.. whereas mutation of K357 (shown in orange) did not. talin knockdown prevents αIIbβ3 activation following stimulation with physiological agonists. β1D. 2003). 1998. and mutations that disrupt this motif perturb β turn formation... 2003). 1998. talin regulates integrins in several ways (Calderwood and Ginsberg. Pfaff et al. 2002). Pfaff et al.1. 2003). 2003). and the role of defective integrin activation in the phenotypes of talindeficient animals has not been assessed. .. in megakaryocytes (the precursors of platelets). 2002) and. 2003).... 1996. A distinctive feature of the β3-F3 complex is a pocket occupied by the side chain of a conserved β tail tryptophan residue (W739). ezrin. The crystallized talin-β3 complex does not include the membrane-proximal β3 sequence. 1999.... Notably. and tethering the αIIb tail parallel to the β3 tail inhibits this effect (Ulmer et al. β2. Talin binds strongly to β1A. Sampath et al. but not F2 or other high-affinity β tailbinding proteins. 1999. 1998.. radixin. Finally. O’Toole et al. Talin is an antiparallel homodimer of two ~270 kDa subunits (Critchley. The talin-β3 interaction is disrupted and integrin activation inhibited by mutation of either this tryptophan. 2). Calderwood et al. This might be owing to direct interactions with the membraneproximal region of the tail (Patil et al. W359 or A360 (shown in green) inhibit β3 tail binding. or of residues involved in β turn formation. 2003). 2002. talin fragments containing this binding site activate β3 integrins (Calderwood et al. by mutations in either talin or the β3 tail. 1990).. 2003). 1990. This figure was first published as Supporting Online Material at Science online (Tadokoro et al. 1994. as in other FERM domains. Talin mediates cellular-energy-dependent integrin activation by binding to integrin β tails.. moesin) domain (Rees et al.. 2001) and. β3 and β5 integrin tails and weakly to the β7 integrin tail (Horwitz et al. are shown in red. Furthermore. 2003). 1). indicating that talin is required for normal integrin function in vivo.... X-ray crystallography of a talin fragment spanning the F2 and F3 subdomains revealed that. suggesting that the talinβ3 structure represents a general integrin-activation complex. 2001. the talin head domain prevents detection of the membrane-proximal interaction between αIIb and β3 tail peptides by NMR (Vinogradova et al. Calderwood et al. The key sites mediating talin binding are well conserved in integrin β tails (Fig. 2003. or of talin residues that contact the β tail (Garcia-Alvarez et al. 1995). 1986. deficiency generates phenotypes similar to those produced by integrin deficiency... Yan et al. Structure of a β3 tail-talin F3 complex.. The talin head possesses a FERM (4.. 2003) (Fig.. Furthermore. The major integrin-binding site lies within the talin head (Calderwood et al. The requirement for talin cannot be bypassed by physiological agonists or by expression of other putative integrin-activating proteins (Tadokoro et al. Thus. the major integrin-binding and activating fragment of talin lies within the 96-residue F3 subdomain. The F3 fold is very similar to that of PTB domains. and disruption of this interaction. activates αIIbβ3 (Calderwood et al. Patil et al. Thus.. inhibit talin binding and interfere with integrin activation (Ulmer et al. The talin F2 and F3 subdomains bind specifically to integrin β3 tails.. Mutations at talin residues R358. 2003). Ulmer et al. filamin and Syk binding when mutated to alanine.. 2000). Calderwood et al. which often recognize peptide ligands containing β turns formed by NPxY motifs (Schlessinger and Lemmon. which selectively inhibit talin binding and integrin activation when mutated to alanine. 2003). Talin binding also affects the membrane-proximal regions of the β3 tail and this is probably required for transmission of the activation signal (Vinogradova et al.

.. 2003). 1997). Talin head has a sixfold higher affinity than intact talin for β3. 1999. 2001. possibly in a Rap1-dependent manner (Fenczik et al. 1997. β3-endonexin binds specifically to β3. 1999)... might also directly activate integrins.... Hamada et al. 2000.. Law et al. and tyrosine phosphorylation of the β tail NPxY motif inhibits talin binding (Tapley et al. Integrin phosphorylation. 1989). 2001). 2003). and one α tail-binding protein. 2002).and C-terminal domains to unmask the integrin-binding site. 2001) and competition between integrin tail-binding proteins (Bouvard et al. 2002). and calpain cleavage increases talin binding to integrins in vitro (Yan et al... Zent et al. Korthauer et al. Turner et al. in the absence of talin. Eigenthaler et al. 1999. The Cterminal domains of CIB bind specifically to the membraneproximal residues of the αIIb tail and a portion of the predicted transmembrane region in a divalent-cation-dependent fashion (Naik et al. such as integrin clustering. the effects of these modifications remain unclear.. calcium.... 2003). However. 2002. 2000. rather than integrin activation. 1999. 1996. Its over-expression does not activate integrins but can reverse suppression of integrin activation mediated by over-expression of free β tails (Fenczik et al.. 2001. Barry et al. Kloeker et al. Sampath et al. 1989).. 2001). 2002). 1997) and GFP-β3-endonexin activates αIIbβ3 in CHO cells (Kashiwagi et al.. 2003). In ERM proteins. 2003).. β3-endonexin (Kashiwagi et al. 1998) and activation of platelets leads to calpain-mediated talin cleavage and αIIbβ3 activation (Hayashi et al... 2001). These effects are consistent with disruption of integrin-talin interactions and are reversed by expression of non-phosphorylatable integrins (Datta et al. Cytohesins-1 and -3 bind β2 integrin tails and have guanine nucleotide exchange factor (GEF) activity for the ARF family of small GTPases (Kolanus et al. CD98hc is unable to reverse integrin suppression mediated by RNAi knockdown of talin (Tadokoro et al. 1997. Sakai et al. hemostasis (Law et al. Naik and Naik. 2003).. 2000). Tails containing nonphosphorylatable NPxF motifs retain talin-binding activity and can be activated by physiological stimuli (Xi et al. Dok-1 and kindlerin (Calderwood et al.. therefore. phosphatidylinositol (4. whereas over-expression of cytohesin-1 or -3 increases adhesion (Kolanus et al...and integrin-binding protein (CIB) (Tsuboi. The side chain of β3 Y747 occupies an uncharged pocket in talin F3. 2003). Beckerle. 2003..5)P2] binding (Martel et al. These putative activators are each selective for only one integrin tail and thus probably represent specialist pathways that collaborate with talin-mediated activation in specific cells. Src-family kinases may also phosphorylate talin (Pasquale et al. 1999. Schoenwaelder . do not activate αIIbβ3.. One report indicates that CIB activates αIIbβ3 by binding to the αIIb tail (Tsuboi. Several potential mechanisms exist. 2000). their effect on integrin activation and details of their in vivo roles remain to be determined.. this activation is very weak (Tadokoro et al. Sakai et al. Kolesnikova et al. interactions between the FERM domain and the C-terminal portion of the molecule also mask ligand-binding sites (Pearson et al. 1999. 2002.. 2001). Numb. 1990). but their relative significance. leads to integrin displacement from focal adhesions (Johansson et al. talin proteolysis (Yan et al. and this might account for any effects of CIB on integrin activation. 2001). Shock et al. However.. This effect is independent of its role in amino acid transport and is dependent on its binding to the suppressive β tail (Fenczik et al. Thus.. tails through both membrane-proximal and -distal motifs (Shattil et al. 1995. 2003). Those 661 tested to date.... Suga et al. However..g. and this lack of positively charged residues is consistent with recognition of non-phosphorylated tyrosine and disruption of integrin binding by phosphorylation (Garcia-Alvarez et al.. The role of other PTB-like domains during integrin activation requires further study. along with the observation that many PTB domains bind integrin β tails (Calderwood et al. 1996. 1999. Clustering cell-surface CD98 activates pathways of integrin signalling and stimulates cell adhesion. 1997... may therefore be an important negative regulator of integrin activation. 1997).. 2001). cytohesin-1 over-expression induces appearance of an activation epitope on αLβ2 but binding of soluble ligand is unaffected (Geiger et al. 2000. Byzova and Plow. the importance of the talin PTB domain-NPxY interaction in integrin activation. However. 1998).. Kaapa et al. 2002. 3). 2003) (Fig. Although PTB domains were initially characterized as domains that bind to phosphorylated tyrosines.. cytohesins might increase cell adhesion through affinityindependent processes. their binding is often independent of phosphotyrosine (Schlessinger and Lemmon.... 2002). β3-endonexin may cooperate with talin during αIIbβ3 activation in platelets. Vallar et al.. 1996). 1999) and transformation (Datta et al. 1989. 2000). Ogasawara et al.. serine or threonine phosphorylation of talin (e. Haataja et al. The protease calpain provides an in vivo mechanism for the separation of talin N.. including integrin phosphorylation (Tapley et al... 1994) and is important in cell migration (Sakai et al. 1997) and cytohesin (Kolanus et al. Tyrosine phosphorylation of the NPxY motifs in integrin β tails by Src-family kinases reduces cell adhesion (Datta et al... CD98 is important for integrin function but is probably not directly involved in integrin activation.. 1998). Calpain is implicated in αVβ3 activation on vascular cells (Byzova et al. Control of talin-integrin interactions Integrin activation is dynamically regulated and a major new challenge will be to understand how cellular signalling pathways control binding of talin to integrin tails and so integrin activation. Finally.. Therefore.5)bisphosphate [PtdIns(4. 1986). Antisense cytohesin-1 oligonucleotides reduce β2 integrin-mediated cell adhesion (Hmama et al. 2002.. 2000. but not β1 or β2. others implicate CIB in post-receptor occupancy events not activation (Vallar et al.. Korthauer et al. The type II transmembrane protein CD98 heavy chain (CD98hc) – an amino acid transporter – is another class of β tail-binding protein implicated in integrin activation (Fenczik et al. however. which suggests that the β3 tail-binding site is masked in intact talin (Yan et al.. despite numerous reports of tyrosine.. Rintoul et al. 2000). suggests that other integrin-binding PTB or FERM domain-containing proteins could activate integrins.Integrin activation Other regulators of integrin activation At least two other β tail-binding proteins. 2001). and (as discussed below) some PTB domain proteins might antagonize talin binding to integrins and so inhibit activation.. Zent et al.. by Src-family kinases or other kinases.

The three-lobed FERM domain within the talin head is indicated.. PIPKIγ-90 and integrin β tails compete for overlapping binding sites on the talin F3 subdomain (Barsukov et al. the integrin cytoplasmic domainassociated protein-1α (ICAP-1α). binds to integrin β1A through a PTB domain-NPxY interaction (Chang et al. Bouvard and Block. 1998. ICAP-1α inhibits integrin activation.. 2003)... which suggests that PIPKIγ-90 may positively regulate integrin activation. Calpain also cleaves integrin β tails (Pfaff et al.. Furthermore.5)P2-producing enzyme: phosphatidylinositol phosphate kinase type Iγ-90 (PIPKIγ-90) (Ling et al.g. 3. 1993. a kinase implicated in the control of β1 activation.5)P2 Inactive talin F1 F3 F2 ECM B Extracellular Intracellular Src P PIPKIγ PTB Fig. so inhibiting integrin activation. (A) Stimulation of talin binding. PIPKIγ-90). 2002.. PIPKIγ-90 might inhibit integrin activation by displacing talin from β tails. under some conditions. Therefore. 2003) and so.. Talin binding to integrin β tails induces conformational changes in the extracellular domain. It remains to be determined whether. the role of integrin tyrosine phosphorylation in determining PTB- . Talin interacts with the first β tail NPxY motif... Calpain cleavage or PtdIns(4. 2002). (B) Inhibition of talin binding. Hence. Potential mechanisms regulating talinmediated integrin activation. Two hypothetical models of inactive talin are shown. ICAP-1α binds to the second NPxY motif in β1 and it is likely that tyrosine phosphorylation at this site inhibits binding (Chang et al. calpain might induce integrin activation by cleaving talin and subsequently downregulating activation by cleaving the β tail. calpain also plays roles downstream of integrin activation (Fox et al.662 Journal of Cell Science 117 (5) A ECM ECM Extracellular α β Intracellular Calpain PtdIns(4.. 2000). phosphorylates ICAP-1α and this phosphorylation appears to regulate integrin-mediated processes (Bouvard et al. dynamic interplay between the stimulatory and inhibitory pathways might determine the integrin activation state. However. Binding of PtdIns(4. 2002). albeit more slowly than talin (Xi et al. Notably. and differential phosphorylation of the β tail tyrosines may therefore provide a means to control ICAP-1α or talin binding independently. increasing their affinity for ligands (the nature of the conformational changes remains controversial and the model shown represents only one of several possibilities). talin binds to and activates one splice variant of the PtdIns(4. 2003). Mechanisms that regulate talin binding may therefore control integrin activation. raising the possibility that they might compete with talin. hence. 2000).. or other talin-binding proteins (e..g. 2002) and inhibits β1A-talin association (Bouvard et al. In addition to the talin F3 subdomain. et al.. and competition with other β tail-binding proteins (e. 2003). 1999). calcium/calmodulin-dependent protein kinase II. potentially activating integrins. It will be important to determine the roles of ICAP-1α and the other integrin-binding PTB or FERM domains as positive or negative regulators of integrin activation. may prevent integrin-talin interactions. talin can stimulate PtdIns(4.5)P2 binding unmasks the binding site. One such protein. 2001).5)P2 to talin induces a conformational change that unmasks the tail-binding site within the talin FERM domain and enhances its association with integrin β1 tails (Martel et al. and it will be important to determine whether talin cleavage is sufficient to activate integrins. 1998).5)P2 production that in turn enhances talinintegrin interactions. However. where regions of the rod mask the β tailbinding site in the F3 subdomain. Di Paolo et al.. The putative salt bridge stabilizing the interaction between membrane-proximal regions of the α and β tails in the inactive conformation is illustrated as a black bar. Src-mediated tyrosine phosphorylation (P) of integrin NPxY motifs. PTB domain proteins). Schoenwaelder et al. by preventing talin binding to integrin β1 tails. However. many other PTB domains can bind integrin NPxY motifs (Calderwood et al. suggesting that calpain could regulate talinmediated αIIbβ3 activation.

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R. talin. and include pistoning. Mol. Many cell signalling pathways regulate integrin activation. their membrane-proximal regions and of talin-integrin β tail interactions is now well established. Biophys. However. Acad. D... N.. This work is supported by a grant from the American Heart Association. Calderwood. 282-287.. Weiss.. H. 2003) suggests that talin might be the final step between these pathways and the integrin cytoplasmic tails. R. J. M. Res. C.. The integrin-actin connection. C. P. Xiong et al. Bhanji. 274. J. von Heijne. White. N. Li et al.. Armulik.. crystal structures of the αvβ3 extracellular domains (Xiong et al. Determination of the border between the transmembrane and cytoplasmic domains of human integrin subunits. Cell Biol.. W. L. 31202-31209 Beckerle. A. (2002). Floyd. S..g. R. additional structures of active and inactive integrins along with integrinligand co-crystals will be required to elucidate fully these conformational changes. C. 1. PIP kinase type 1γ and β1-integrin cytoplasmic domain bind to the same region in the talin FERM domain. responding to.. 2002). whereas enhancing separation promotes activation (Takagi et al. and LaFlamme.. (1998). Hynes. G. The pathways that regulate integrin activation and the mechanisms by which they act (e. 278. 2002). Cell 6. Dev. J.. F. Cell Biol. The inability of R-Ras to activate integrins in talindeficient cells (Tadokoro et al. Bokel. E. and . (1998). (2003). but conformational changes in the membrane-proximal extracellular domains have been documented.. J. Shock. L. Beglova et al. I. Chem. L.. G.. Bouvard. 278. Pampori. 1994. Cysteinerich module structure reveals a fulcrum for integrin rearrangement upon activation. Chem.. H. Retta. 2002.. Liddington and Ginsberg. Boudignon-Proudhon.. Several non-mutually exclusive models have been proposed to explain transmission of the activation signal (Williams et al. Vignoud. (2003).. E. T. Cell Regul. Abed. C. cell spreading. 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