Integrin inactivators: balancing cellular functions in vitro and in vivo

Integrins are ubiquitously expressed transmembrane receptors, involved in both cell–cell (through binding to cadherins and selectins) and cell–ECM (extra cellular matrix; such as binding to fibronectin, collagen and laminin) interactions1. Integrins are heterodimers of α-subunits and β-subunits, and in vertebrates combina-tions of different α-subunits and β-subunits give rise to 24 different integrins with specific binding properties and tissue distributions. Some integrins are essential, as their loss results in early embryonic lethality; by contrast, mice lacking other integrins are nearly normal, with only minor tissue-specific abnormalities.

Upon extracellular stimulation, integrins undergo a conformational change (integrin activation), which allows the recruitment of several cytoplasmic proteins, including activators such as talins and kindlins, to the generally very short intracellular domain of integrins. Ultimately, this results in the formation of a large protein complex2 that interacts with the actin cytoskeleton (BOX 1; for simplicity, only proteins discussed in this Review are mentioned). This complex promotes the recruitment and activation of several protein kinases (such as focal adhesion kinase (FAK) and SRC), leading to the activation of canonical signalling pathways involving ERK, Jun N-terminal kinase (JNK) or RHO-family small GTPases. These signalling events are crucial for several key cellular activities, includ-ing migration, proliferation, survival, gene expression and receptor Tyr kinase signalling2,3. Importantly, owing to their unique ability to link the extracellular environment to the cytoskeleton, integrins are the main transducers of mechanical forces4,5. This force transduction, as well as integrin signalling, is stimulated by clustering of integrin

molecules, which occurs after ligand binding. These inte-grin clusters form different types of adhesion structures that develop over time in a process known as adhesion maturation (reviewed in REF. 6) (BOX 2).

It is thus clear that integrin activation by specific cyto-plasmic proteins is essential for cell adhesion and inte-grin linkage to the actin cytoskeleton, and much of the research has focused on the different integrin activation steps. However, a substantial proportion of cell surface integrins is inactive7. Inactive integrins adopt a closed conformation, in which the recruitment of both extra-cellular ligand and intra cellular activators is repressed (BOX 1). In addition, inactive integrins are uncoupled from the actin cytoskeleton and important scaffolds such as vinculin and the IPP complex (comprising integrin-linked kinase (ILK), PINCH and parvin). The prevail-ing view has been that this inactive conformation is the passively adopted default state of the receptor8, which is shifted towards integrin activation and adhesion matura-tion upon stimulation and subsequent recruitment of acti-vating proteins9. However, the identification of proteins such as ICAP1 (integrin cytoplasmic domain-associated protein 1), filamin and SHARPIN (SHANK-associated RH domain-interacting protein), which are required for integrin inactivation in different cellular contexts, suggests that both active and inactive receptor states are actively regulated10–12. In addition, an impaired ability to activate integrins is associated with many human diseases (for example, bleeding disorders and immune deficiencies), whereas inappropriate integrin activation has been linked to inflammatory disorders and cancer, indicating that the control of integrin activity is of critical importance in vivo.

1Équipe 04 - Chromatine et épigenetique, Institut National de la Santé et de la Recherche Médicale U823, Institut Albert Bonniot, 38042 Grenoble, Cedex 09, France.2VTT Medical Biotechnology and University of Turku, Centre for Biotechnology, FIN-20520 Turku, Finland.*These authors contributed equally to this work.Correspondence to J.I.e-mail: [email protected]:10.1038/nrm3599 Published online 30 May 2013

Integrin inactivators: balancing cellular functions in vitro and in vivoDaniel Bouvard1,*, Jeroen Pouwels2,*, Nicola De Franceschi2 and Johanna Ivaska2

Abstract | Integrins mediate cell–matrix and cell–cell interactions and integrate extracellular cues to the cytoskeleton and cellular signalling pathways. Integrin function on the cell surface is regulated by their activity switching such that intracellular proteins interacting with the integrin cytoplasmic domains increase or decrease integrin–ligand binding affinity. It is widely accepted that integrin activation by specific proteins is essential for cell adhesion and integrin linkage to the actin cytoskeleton. However, there is also increasing evidence that integrin-inactivating proteins are crucial for appropriate integrin function in vitro and in vivo and that the regulation of integrin–ligand interactions is a fine-tuned balancing act between inactivation and activation.

REVIEWSNature Reviews Molecular Cell Biology | AOP, published online 30 May 2013; doi:10.1038/nrm3599

NATURE REVIEWS | MOLECULAR CELL BIOLOGY ADVANCE ONLINE PUBLICATION | 1

© 2013 Macmillan Publishers Limited. All rights reserved

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Nature Reviews | Molecular Cell Biology

α β

α β

Talin FAK

Talin FAK

Talin FAKKindlin

IPPcomplex

Vinculin

Actin

Outside–inactivation

Inside–outactivation

αβ

Inactiveintegrin

Inactivator

Active integrin

Extracellularligand

α β

α β

PtdIns(4,5)P2

This Review focuses on how a balance of active and inactive integrins is dynamically regulated by intra-cellular proteins binding to integrins and discusses the implications of impaired activity control on the molecula r, cellul ar and organismal levels.

Integrins — bidirectional signalling receptors Integrin activation–inactivation cycle. The mechanisms of integrin activation have been reviewed extensively elsewhere9,13. Briefly, for integrins to function correctly, their activity must be strictly controlled, both spatially and temporally. Integrins can be activated by proteins binding their extracellular or their intracellular domain (outside–in and inside–out signalling, respectively) and, therefore, integrins possess the unique ability to signal bidirectionally. As mentioned above, outside–in signalling is triggered by binding of integrins to ECM

molecules, which induces the formation of a protein complex that binds to the cytoskeleton and activates several signalling pathways (BOX 1). Inside–out signallin g is triggered by changes in the intracellular environment that allow binding of integrin-activating proteins like talins and kindlins to the intracellular domain of integ-rin. This extends integrin through the release of the outer membrane clasp and the inner membrane clasp9 and increases integrin affinity for its ligands (BOX 1). An important aspect of integrin function is linked to its ability to cluster into supramolecular structures at the plasma membrane. This clustering controls integ-rin adhesive function (adhesion, migration or matrix deposition) and cell signalling. Integrin clustering is linked to integrin activation and is triggered by multi-valent ECM ligands and integrin connections with the actin network. However, as integrin clustering has been

Box 1 | Integrin activation

Integrins exist in a continuum of affinity states that are related to different conformational states (see the figure). These include at least the following: bent inactive (to which integrin inactivators are bound); extended (primed); and fully activated integrin coupled to both actin and the extracellular matrix (ECM) (extended with high affinity). Inside–out activation is dependent on binding of intracellular proteins to the integrin cytoplasmic domains. This is thought to be a stepwise process, although the exact hierarchy of the proteins involved is still a matter of debate. Talin and focal adhesion kinase (FAK) are among the first proteins to be recruited. They become activated by phosphatidylinositol‑4,5‑bisphosphate (PtdIns(4,5)P

2) binding, which is locally enriched at integrin clustering sites. In its active conformation, talin binds the

β‑integrin cytoplasmic domain via the talin head domain (THD). This disrupts the salt bridge between the integrin subunits, changing the tilt angle of the β‑integrin transmembrane domain114 and thereby releasing the interactions at the interface between the transmembrane domains of the α‑ and β‑subunits, termed outer membrane clasp (OMC) and inner membrane clasp (IMC)9. Kindlins are integrin co‑activators as they also bind to the β‑integrin cytoplasmic domain and enhance talin‑induced integrin activation9. The IPP complex, formed by integrin‑linked kinase (ILK), PINCH and parvin, recruits important focal adhesion proteins such as paxillin and α‑actinin to adhesion sites6. For outside–in activation, ligand binding induces a transition from a ‘closed’ to an ‘open’ conformation of the β‑subunit I domain, commonly referred to as headpiece opening8. These conformational changes have not been linked to any specific change in the transmembrane and intracellular domains115. In the final step of integrin activation, myosin contraction‑dependent physical forces stretch talin (talin provides the link between the immobile integrin‑bound ECM and the actin cytoskeleton) and unmask cryptic vinculin‑binding sites within the talin carboxyl terminus. Vinculin binding strengthens the association between talin and F‑actin, and this tension is thought to be a crucial factor for the full activation of integrins8.

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2 | ADVANCE ONLINE PUBLICATION www.nature.com/reviews/molcellbio


LamellipodiumLamellum

Transition zone

Ruffle

Nucleus

Nascent adhesion

Actomyosin bundle

Actin filament

a

Focal adhesion

Fibrillar adhesion

Focal complex

b

Branched actin

Actomyosin bundle

Integrin inactivator

Integrin activator

Focaladhesion

Focalcomplex

Nascent adhesion

Inactive integrins

Extended integrinsActive integrins

reviewed recently6,14 and the role of integrin inactivators in this process has not been studied, integrin clustering is not discussed further in this Review.

For many years, the main focus in the field has been on integrin activation, and thus integrin-inactivating proteins have been less well-studied. However, several molecules are now established as negative regulators of integrin activity and they fall into two main classes:

those that directly bind integrins and interfere with the recruitment of activators like talin and kindlin (FIG. 1), and those that inhibit integrins through alternative mechanisms. Negative regulators that function through alternative mechanisms include proteins affecting the phosphorylation status and therefore the functionality of integrins or integrin inactivators, and proteins that affect the amount of cell surface integrin through the regulation of integrin trafficking.

Integrin inactivators that directly interact with integrins. The most direct mechanism of integrin inhibition involves the binding of a molecule to the integrin cyto-plasmic domains, which prevents the association of integrin activators. Such molecules support integrin inactivation by interfering with inside–out signalling and therefore switch or maintain integrins inactive. Several inhibitors have been identified, and they bind either the β-integrin cytoplasmic tails15–18 or the con-served region of the α-integrin cytoplasmic tails and thus inhibit talin and kindlin binding10,19,20 (FIG. 1).

One such inhibitor is filamin, a large, rod-shaped protein with an actin-binding domain and 24 immunoglobuli n-like domains that interact with many proteins. Filamin exists in an autoinhibited state until tension-induced binding to actin causes conforma-tional changes that expose several binding sites for inte-grins and other proteins21,22. Filamin inhibits integrins though competition with talin18 by binding the NXXY (Asp-Xaa-Xaa-Tyr; in which Xaa represents any amino acid) motif present in the β-integrin tails (FIG. 1a,e). Interestingly, the inactivation of integrins in talin-depleted cells is fully restored by concomitant inhibi-tion of filamin expression, suggesting that the switching between talin and filamin binding to the β-integrin tail is a crucial determinant of integrin activity23. Filamin has been shown to inhibit cell spreading and cell migra-tion in some cell types16,24 and to stabilize focal adhe-sions in mouse embryonic fibroblasts25. This may be due to reduced integrin activity upon filamin expression. In addition, the balance between the activity of the small GTPases RHOA and RAC is known to be a key determi-nant of focal adhesion stability and cell spreading, such that increased RAC activity favours spreading. Filamin recruits filamin A-associated RHO GTPase-activating protein (GAP) to integrin22, which locally inhibits RAC activation and therefore cell spreading. Filamin also regulates cell migration independently of its direct inter-action with integrins: through suppression of matrix metalloproteinase activity, leading to ECM degradation and inhibition of cell migration26; and by decreasing the activity of the protease calpain, resulting in reduced cleavage of proteins like talin and FAK and thus inhibi-tion of focal adhesion disassembly27 and cell migration. Recent papers have also shown that filamin is an impor-tant mechanotransducer involved in the regulation of actin network stretching22,25. Therefore, filamin is another integrin inhibitor linked to mechanosensing28.

ICAP1 is also an example of an integrin inactivato r. It is a small protein containing a phosphotyrosine-bindin g (PTB) domain16 that interacts with the distal

Box 2 | Integrin-mediated adhesion maturation

At the leading edge of motile cells, a network of branched actin drives lamellipodium protrusion (see the figure, part a (top view of a moving cell) and part b (side view of a cell showing the localization of active and inactive integrins, integrins activators and inhibitors)). In the lamellipodium, which are rich in integrin inactivators such as SHARPIN (SHANK‑associated RH domain‑interacting protein), nascent adhesions are the first adhesion structures to be observed; they contain focal adhesion kinase (FAK) and paxillin but can be formed independently of the integrin activator talin60. Nascent adhesions are highly transient and either disassemble or mature into the larger focal complexes, which reside at the transition zone between the lamellum and lamellipodium. Additional components such as talin, vinculin and α‑actinin are recruited to these structures, which are already linked to the actin cytoskeleton. Adhesion is strengthened during the subsequent maturation of nascent adhesions and focal complexes into focal adhesions, whereby myosin II‑dependent force induces stronger association between integrin and actin. Additional proteins are recruited at this stage, including the cytoskeletal adaptor zyxin and the actin‑regulatory protein VASP (va sodilator‑stimulated phosphoprotein). Finally, focal adhesions can develop into fibrillar adhesions, in which talin has been replaced by tensin and which are involved in matrix remodelling. Adhesion maturation is a very dynamic process, and all adhesions types can be turned over to facilitate cell movement116.

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DOK1

DOK1


α β1

α β

α β

Filamin

P

P

Active integrin

d

ba

c

e

Inactive integrin

SHARPIN MDGI

Talin

Talin (all β) Talin (all β)

DOK1(β2,β3,β5,β7) SHARPIN (α1,α2,α5)

MDGI (α1,α2,α5,α6,α10,α11,)ICAP1 (β1)

Filamin (β1,β3,β7)

Kindlin (all β)

Talin

ICAP1

ICAP1

KRIT1

Talin

SRC

RSK2

β1 WKLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGKβ3 WKLLITIHDRKEFAKFEEERARAKWDTANNPLYKEATSTFTNITYRGT

α1 LALWKIGFFKRPLKKKMEKα2 AILWKLGFFKRKYEKMTKNPDEIDETTELSSα5 YILYKLGFFKRSLPYGTAMEKAQLKPPATSDA

IMC IMCProximalNPXY

DistalNXXY

α β α β

α β α

α β

β1

NXXY motif of β1 integrin and specifically inhibits β1 integrin activity (ICAP1 does not bind β3 integrin or β5 integrin)15 (FIG. 1b,e). This site is important for kind-lin binding and, in line with the ability of kindlin to activate integrin in a talin-dependent manner29, ICAP1 negatively regulates talin and kindlin binding to β1 inte-grin11,30. ICAP1 colocalizes with β1 integrin in protrud-ing actin-containing ruffles before the formation of focal complexes31 and supports cell migration on fibronectin32. Its functions have been well-studied in osteoblasts owing to the osteopenic phenotype of mice lacking Icap1 (also known as Itgb1bp1)33 (see below), and in vitro Icap1-null osteoblasts display defective adhesion and migration. In addition, ICAP1 is important for mechanotrans-duction28 and regulates the formation of focal adhesions and their maturation into fibrillar adhesions28,30. This is linked to the ability of ICAP1 to bind the β1 integrin tail

and inhibit kindlin 2 (also known as FERMT2) binding, which seems to be a necessary step for the formation of integrin-containing fibrillar adhesions and fibronectin assembly in osteoblasts30. Recently, structural studies have shown that β1 integrin competes with KRIT1 (Krev inter action trapped 1) for binding to ICAP1 (REF. 34) and that KRIT1 promotes β1 integrin activation by relieving ICAP1 inhibition (FIG. 1b).

Docking protein 1 (DOK1) also contains a PTB domain capable of binding integrins16 (FIG. 1c,e) and has been shown to inhibit integrin activity17. DOK1 binds to the same region on the β-integrin tail as talin, and Tyr phosphorylation of this NPXY motif in β1, β3 and β7 integrins by SRC kinases (see below) greatly increases DOK1 binding to the β-integrin cytoplasmic tail. As the affinity of talin for integrins decreases upon Tyr phosphorylation, this phosphorylation event might

Figure 1 | Integrin inactivation. a–d | Schematic of an inactive integrin molecule interacting with the integrin inhibitors filamin, ICAP1 (integrin cytoplasmic domain-associated protein 1), DOK1 (docking protein 1) and SHARPIN (SHANK-associated RH domain-interacting protein) and MDGI (mammary-derived growth inhibitor) (that is, the proteins that bind the integrin cytoplasmic domain and thus inhibit talin and kindlin recruitment to integrins) and the proteins or phosphorylation events that regulate their binding. Phosphorylation of filamin on Ser2152 by epidermal growth factor-activated ribosomal protein S6 kinase 2 (RSK2) promotes binding of filamin to the β-integrin cytoplasmic domain (a). KRIT1 (Krev interaction trapped 1) binds and sequesters ICAP1, preventing ICAP1 binding to the β1 integrin tail (b). Phosphorylation of the Tyr residue in the conserved NPXY motif in the β-integrin cytoplasmic domain by SRC enhances DOK1 binding (c). SHARPIN and MDGI interact with the α‑integrin tail (d). e | Binding sites of integrin inhibitory proteins depicted in panels a–d as well as the integrin-activating proteins talin and kindlin in the α‑integrin or β‑integrin cytoplasmic tails. The integrin subunits that bind to each of the integrin-inhibiting or -activating proteins are indicated in brackets. The residues involved in the formation of the inner membrane clasp (IMC) between the α- and β‑subunits are shown in green, and the proximal NPXY and distal NXXY motifs are shown in blue.

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http://www.ncbi.nlm.nih.gov/gene/16413

provide a switch for integrin inactivation and a transi-tion from talin-dependent to DOK1-dependent integ-rin signalling35. However, many open questions remain regardin g the functional role of DOK1 in the regulation of i ntegrin-dependent processes in cells and in vivo.

SHARPIN is another key regulator of integrin activ-ity10, which is also involved in nuclear factor-κB (NF-κB) signalling as part of the LUBAC (linear ubiquitylation assembly complex)36. SHARPIN is thus far the only ubiquitously expressed integrin inhibitor that binds to the cytoplasmic tail of α-integrins10 (FIG. 1d). SHARPIN preferentially engages non-adherent integrins, and its ability to inhibit integrins is independent of the catalytic activity of the LUBAC10. Specifically, SHARPIN binds to the highly conserved WKXGFFKR sequence of the α-integrin tail (FIG. 1e), suggesting that it could inhibit most, if not all integrins. However, even though its role in the inhibition of β1 integrins has now been established10, whether it also regulates other integrins remains to be investigated. Importantly, the Arg residue at the end of the conserved WKXGFFKR sequence, which forms an integrin-inhibiting salt bridge with the β-integrin tail37, is not required for SHARPIN binding, suggesting that SHARPIN can bind integrins with their salt bridge intact and might actually stabilize the salt bridge. This hypothesis is further supported by the nuclear magnetic resonance (NMR) structure of an in active integrin38, in which the GFFKR sequence is accessible to cytoplas-mic proteins. However, this remains to be investigated. Similarly to ICAP1, SHARPIN co-localizes with in active integrin to ruffles outside of focal adhesions. Even though SHARPIN binds to the α-integrin tail, it inhibits integrin activity through direct competition with talin binding to the β-integrin tail, which is in close proximity to the α-integrin tail10 (J.P., N.D.F. and J.I., unpublished observations) and therefore affects cell adhesion and migration.

Recently, another cytoplasmic protein, MDGI (mammary-derived growth inhibitor), was also shown to bind to the α-subunit (FIG. 1d,e) and inhibit β1-integrin specifically in mammary epithelial cells19. However, the relationship between MDGI- and SHARPIN-mediated integrin inhibition is currently not known19.

Phosphorylation regulates integrin inactivation. In many cases, the signals that regulate integrin inside–out acti-vation are not fully understood. However, significant progress has been made in understanding how phos-phorylation events regulate integrin affinity modula-tion. It was initially observed that oncogenic activation of SRC can lead to cell rounding, which is a result of loss of cell adhesion, possibly owing to SRC-mediated inte-grin inhibition. The Tyr residues of both NXXY motifs and a Ser residue in the β-integrin cytoplasmic tail are phosphorylated in an SRC-dependent manner39. This inhibits talin and kindlin binding35,40 and promotes the recruitment of DOK1 to the β-integrin tail35 (FIG. 1c) (see above). In line with this, phosphorylation of these residues inhibits integrin localization to focal adhesions41 and supports the transition of integrins from focal adhe-sions to fibrillar adhesions, which is consistent with the

absence of talin from fibrillar adhesions42 (BOX 2). There are also several examples of how Ser and/or Thr phospho-rylation regulates integrin activity (reviewed in REF. 43). Interestingly, the phosphorylation status of Thr758 of β2 integrin seems to be important for integrin function, as the integrin inhibitor filamin can only bind the non-phosphorylate d integrin, whereas the cytoplasmic scaffold protein and signalling integrator 14-3-3 binds exclusively to the phosphorylated form44. Phosphorylation of Thr758 and subsequent 14-3-3 binding take place after inside–out activation and result in cytoskeletal rearrangements45.

In addition to integrin phosphorylation, phosphoryla-tion of inhibitors regulates their binding to integrins and therefore integrin activation (FIG. 1a). Specifically, filamin is phosphorylated by ribosomal protein S6 kinase 2 (RSK2) in response to adhesion and epidermal growth factor (EGF) stimulation46. RSK2 interacts with β1 inte-grin and β7 integrin via the membrane-proximal NPXY motif and, upon activation through autophosphorylation and phosphorylation by ERK and other kinases (reviewed in REF. 47), phosphorylates filamin, triggers filamin–integrin interaction and integrin inactivation48. Thus, adhesion triggers a filamin phosphorylation-dependent negative feedback loop whereby outside–in signalling induces inhibitory inside–out signalling to facilitate migration and focal adhesion turnover. Moreover, ICAP1 is phosphorylated following integrin-mediated adhe-sion to fibronectin, and this may affect ICAP1-mediated integri n inactivation32.

Several other kinases are also known regulators of cell migration49, and modulating their activity can influ-ence the integrin activity status. In prostate cancer cells, AKT1 and AKT2 inhibit β1 integrin activity through dis-tinct mechanisms. AKT1 signalling inhibits integrins by attenuating crosstalk between the receptors and receptor Tyr kinases50. By contrast, AKT2 inhibits β1 integrins by upregulating the miR-200 family of microRNAs, which regulates integrin activity by controlling the expression of currently unknown targets without affecting total β1 inte-grin levels50. In addition, several other kinases, includ-ing the poorly studied microtubule-associated Ser/Thr kinase 2 (MAST2), were recently identified as important negative regulators of integrin activity in many different cancer cell lines51.

The (in)active life of an integrinEmerging evidence suggests that appropriate control of integrin activity is critically important throughout the life cycle of an integrin heterodimer, immediately following translation of individual subunits all through to receptor degradation in lysosomes (FIG. 2).

Integrin activity regulation in the Golgi and the ER. Integrin activity has mainly been studied at the plasma membrane, where integrins engage extracellular sub-strate molecules to mediate adhesion. However, activity regulation seems to be important starting from when the newly translated receptors are targeted from the endo-plasmic reticulum (ER) via the Golgi to the plasma mem-brane. β1 integrin precursors form a stable pool in the ER52. To exit the ER and to be transported to the plasma

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Actin

4 Focal adhesion

Plasmamembrane

Lysosome

Nucleus

Golgi

ER

FAKFAK? ?

Talin

Integrininactivator

Integrininactivator

Inactive integrinExtended integrin Active integrin

SNX17

3 Nascent adhesion

1

2

5 Early endosome

membrane, α- and β-subunits need to associate to form a stable and mature heterodimer53. The newly synthesized β1 integrin subunit immediately adopts a bent inactive conformation following heterodimerization with the α-subunit. This inactive conformation is maintained dur-ing trafficking of the receptor to the cell surface by bindin g to calcium54. Upon arrival at the plasma membrane, integ-rins are exposed to higher extracellular magnesium levels and can potentially become activated54.

Activity switching at the plasma membrane. Integrins that are inactive or extended (that is, integrins that have high-affinity conformation but are unoccupied by ligand)55 freely diffuse within the plasma membrane56,57. Both inactive integrins and the integrin-inactivating proteins SHARPIN and ICAP1 accumulate in actin-positive ruffles that are not bound to the ECM. SHARPIN and ICAP1 colocalize with integrin until their recruitment to focal adhesions10,31, suggesting that these inhibitors might main-tain integrins uncoupled from actin. This might facilitate integrin movement to the leading edge of the cell, where activation and subsequently adhesion to the substrate take place to sustain directional migration.

As discussed above, once the integrin binds either extracellular ligands or activating proteins like talin, it becomes activated (BOX 1) and immobilized (full immo-bilization requires both talin and ligand binding)57 and initiates signalling. However, the actual sequence of inte-grin activation events, and the definition of upstream and downstream effectors in forming adhesion sites, may not be as straightforward as previously thought. The initial cell adhesion is talin independent58, and it is not known whether as-yet-unidentified integrin activators are involved in this step. Another possibility is that the dis-sociation of inactivating proteins (SHARPIN and ICAP1 are not detected in focal adhesions) allows the integrin to adopt an extended conformation and then undergo ligand-induced outside–in activation, as bent integrins can bind their ligands59. Interestingly, FAK was recently shown to be recruited to adhesion sites before talin and to be required for talin recruitment to nascent adhesions, although focal adhesion formation and paxillin recruit-ment are FAK independent60. This suggests that a crucial point in early integrin activation in adherent cells could be the release of integrin inhibitors and FAK recruitment rather than talin binding to the receptor, which would

Figure 2 | The life of an integrin. Following dimerization and calcium binding, a bent inactive α–β-integrin heterodimer exits the endoplasmic reticulum (ER) and traffics through the Golgi to the plasma membrane (1). Subpopulations of integrins in different activation states coexist at the plasma membrane, depending on the availability of extracellular ligand and whether they are bound by activators or inactivators (2). Focal adhesion kinase (FAK) is localized to nascent adhesions before talin, suggesting that integrin inhibitors may still be present in nascent adhesions (indicated by a question mark) (3). Nascent adhesions mature into focal adhesions (4). Although both ligand-bound and ligand-free integrins can be endocytosed, recycling is faster for inactive integrin. Sortin nexin 17 (SNX17) is important for controlling the balance between recycling and lysosomal degradation of integrins (5).

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be necessary later on to allow adhesion site maturation through connection to the actin network.

Nascent adhesions subsequently disassemble or mature into focal complexes and focal adhesions in an actin-dependent manner61 (BOX 2). β3 integrins enter focal adhesions unbound to talin, which is recruited to these sites directly from the cytoplasm. Thus, talin-medi-ated integrin immobilization can be specifically restricted to focal adhesions57. Simultaneous binding of talin to the β-integrin cytoplasmic tail and actin results in physi-cal forces that are transduced through myosin II. These forces relieve talin autoinhibition, thereby exposing sev-eral cryptic vinculin-binding sites in talin and resulting in stronger actin binding and increased stability of the focal adhesion62. These so-called ‘catch bonds’, which derive their name from the fact that the bond between the focal adhesion and the actin cytoskeleton becomes stronger with increasing tension63,64, are thought to be important for the formation of stable focal adhesions and for firm adhesion of the cell to the extracellular ligand64. New data, however, show that β3 integrins within focal adhesions undergo repeated immobilization and diffusion cycles and are therefore constantly switching between active and inactive conformations57. Thus, the duration of an active integrin–ECM bond is much shorter than the relatively long half-life of detectable mature focal adhesions.

One important step in focal adhesion formation is the synthesis of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) by type I phosphatidylinositol phos phate kinase isoform-γ 661 (PIPKIγ661), which recruits and activates FAK, talin and vinculin (reviewed in REF. 65). Therefore, integrin activation is intimately coupled to the generation of PtdIns(4,5)P2.

Together, these data suggest that the balance between inactive and active integrins is a two-step transition (BOX 1) whereby the inactive integrin first switches to an extended but non-actin bound state regulated by local-ized generation of PtdIns(4,5)P2 and the recruitment of FAK and possibly other proteins57,60. These integrins may still be associated with integrin inhibitors that com-pete with talin for binding, but further work is needed to confirm this. Subsequently, upon ligand binding the extended integrin interacts with talin to support full inte-grin activation and promote binding to actin to transduce mechanical forces58,63.

Integrin activation, endocytosis and degradation. Endocytosis facilitates integrin turnover from the plasma membrane, and integrin activity has recently been linked to the regulation of integrin trafficking. Several reports have suggested that endocytosed integrins can be in an active state as they can bind ECM substrate frag-ments7,66,67. Active and inactive integrins are trafficked with different kinetics such that the inactive receptors rapidly recycle to the plasma membrane to form new adhesion sites, whereas active receptors are recycled more slowly7. Furthermore, endocytosis of constitutively in active integrins (those carrying a mutation in the mem-brane-proxima l, talin-binding NPXY motif) seems to be reduced, further linking the integrin activity status to integrin trafficking68. The membrane distal NXXY motif

(which binds the integrin activator kindlin), but not the membrane proximal NPXY, in β1 integrin has an essen-tial role in the regulation of integrin recycling68–70. At the moment, the exact sequence of events is not known, but current data are in line with a model whereby kindlin binding at the plasma membrane controls integrin activ-ity, but upon endocytosis of the active receptor kindlin dissociates from the β1 integrin tail, and sortin nexin 17 (SNX17) binds the NXXY motif in early endosomes to promote integrin recycling and prevent degradation69,70. It is likely that integrins are inactivated before recycling, but this remains to be demonstrated.

Integrins are relatively long-lived proteins, and their degradation is strongly linked to recycling rates. Several studies have shown that non-recycled integrins are targeted for lysosomal degradation68–71. Lysosomal tar-geting, however, does not necessarily result in integrin degradation, as late-endosomal integrins can be retro-gradely targeted and subsequently recycled back to the plasma membrane67. This recycling depends on CLIC3 (chloride intracellular channel protein 3) and facilitates cell invasion by releasing the cell rear, which facilitates cell motility. Surprisingly, in these cells, data obtained with a constitutively active integrin mutant suggest that endocytosed active integrins are targeted to the rear of the cell for recycling, whereas wild-type integrins are mostly recycled to the cell front67, further highlighting the importance of the integrin activity status for integrin trafficking.

Maintaining the balanceAbove we discussed in vitro data revealing the impor-tance of regulating integrin activity levels. This is further emphasized in vivo: mice and flies lacking integrin regu-lators, or carrying mutant integrins with altered activit y levels, show numerous defects, emphasizing that the integri n activity status needs to be tightly controlled.

Mice with integrin loss‑of‑function mutations. The importance of integrin signalling, in particular for devel-opment and tissue homeostasis, has become evident from studies of integrin-knockout mice. To provide a basis for our discussion about the phenotypes that are induced by genetic ablation of integrin-activating and -inactivating proteins, we briefly describe the phenotypes resulting from loss of different integrin subunits in mice. Defects incurred by integrin loss fall into three major classes: embryonic lethality, perinatal lethality and either no obvious phenotype or a very mild defect (TABLE 1; supple mentary information S1 (table)). Interestingly, the first phenotypic group encompasses fibronectin- and vitronecti n-binding integrins such as α4 and α5 integ-rins, which corresponds well with the essential role of these receptors in ECM organization and signalling. The second group includes integrins that are more restricted in their tissue expression (such as α6β4 integrin) or that recognize non-ubiquitously expressed molecules (for example, laminins or collagens), indicating a role for these receptors in tissue function, maintenance or cohesion. The third group includes the four collagen-binding α1, α2, α10 and α11 integrins. Because they have

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overlapping ligand specificity and are fairly ubiquitously expressed, redundancy is a likely explanation for the mild phenotype s observed in vivo.

Loss of integrin activators. Full activation of integrins requires the direct binding of actin-linking activators to their cytoplasmic domain in vitro. Thus, it is expected that the loss of integrin-activating proteins causes inte-grin inactivation and results in phenotypes closely resembling the loss of integrins (TABLE 2; supplementary information S2 (table)).

There are two genes that encode talin, Tln1 and Tln2, with the former being more widely expressed than the latter72. Consistent with this, deletion of Tln1 leads to an embryonic arrest at the peri-implantation stage73, albeit slightly later than deletion of the gene coding for β1 integrin, possible owing to compensa-tion by Tln2, as observed in fibroblasts58 (however, this remains speculative as the germline deletion of talin 1 and talin 2 has not yet been reported). In line with the high expression of talin 2 in muscles, talin 2-mutant mice develop a mild myopathy74. Similarly to what is

Table 1 | Phenotypes of integrin-knockout mice

Integrin subunit Mouse phenotype Effects of loss Link to human disease

α1 Viable, fertile Defects in collagen synthesis, tumour angiogenesis, small kidneys, skin hardening

Unknown

α2 Viable, fertile Defects in platelet adhesion, mammary ductal branching and kidney development

Unknown

α3 Perinatal lethal Defects in kidney and lung development, skin blistering Interstitial lung disease, nephrotic syndrome, epidermolysis bullosa

α4 Embryonic lethal Defects in placenta formation, cardiac development and haematopoietic cell maintenance and homing

Unknown

α5 Embryonic lethal Defects in neural crest cell survival and mesoderm formation Unknown

α6 Perinatal lethal Defects in brain cortex organization, skin blistering Junctional epidermolysis bullosa with pyloric stenosis

α7 Perinatal lethal Defects in vascular development and placenta formation, muscular dystrophy

Muscular dystrophy

α8 Perinatal lethal Kidney defects, deafness Unknown

α9 Perinatal lethal Defects in lymphatic valve formation and in the development of the lymphatic system, congenital chylothorax*

Congenital chylothorax

α10 Viable, fertile Dwarfism, mild chondrodysplasia Unknown

α11 Viable, fertile Dwarfism, increase mortality, defective incisor eruption Unknown

αv Embryonic or perinatal lethal

Defects in placenta formation and in CNS and GI blood cell development, cleft palate

Unknown

αx Viable, fertile T cell defects, increased susceptibility to bacterial infections Systemic lupus erythematosus

αL Viable, fertile Impaired leucocyte recruitment and tumour rejection, defects in osteoclast development

Psoriasis

αD Viable, fertile Increased susceptibility to bacterial infections Unknown

αM Viable, fertile Impaired phagocytosis and PMN apoptosis, obesity, defective mast cell development

Systemic lupus erythematosus

αE Viable, fertile Reduced lymphocyte numbers Unknown

αΙIb Viable, fertile Platelet abnormalities Glanzmann’s thrombasthenia, thrombocytopenia

β1 Embryonic lethal Inner cell mass deterioration Unknown

β2 Viable, fertile Impaired leucocyte migration, skin infections, osteoporosis LADI

β3 Viable, fertile Platelet abnormalities, bone defects Glanzmann’s thrombasthenia, thrombocytopenia

β4 Perinatal lethal Skin blistering Epidermolysis bullosa

β5 Viable, fertile No apparent phenotype Unknown

β6 Viable, fertile TGFβ activation defect, juvenile baldness, asthma, periodontal disease

Asthma

β7 Viable, fertile Immunological defects Inflammatory bowel disease

β8 Embryonic or perinatal lethal

Defects in placenta and in CNS and GI blood cell development, cleft palate

Unknown

CNS, central nervous system; GI, gastrointestinal tract; LADI, leucocyte adhesion deficiency type I; PMN, polymorphonuclear neutrophil; TGFβ, transforming growth factor-β. A fully referenced version of this table is available in supplementary information S1 (table).*Lymphatic fluid accumulation in the pleural cavity.

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observed in talin-deficient mice, loss of the ubiquitously expressed kindlin 2 is embryonic lethal (as is loss of β1 integrin or talin 1)75, whereas loss of the two genes encoding k indlin 1 and kindlin 3 (Fermt1 and Ferm3, respectively) results in tissue-specific defects as these kindlins have a more restricted expression pattern76–78. Furthermore, phenotypic analyses of tissue-specific deletions of talin 1 or a null-mutation in the gene encoding kindlin 3 support the idea that in leukocytes, talin 1 has a key role during integrin pre-activatio n, whereas both talin 1 and kindlin 3 seem to be required for full activation79. Except for early development, when the germline-deletio n phenotypes of talin 1 and kindli n 2 resemble that of β1 integrin, loss of those two activators leads to a stronger phenotype than the loss of individual integrin chains (TABLES 1,2), confirming that the two activators can regulate the activity of many differen t integrin heterodimers.

Importantly, loss of talin in Drosophila melanogaster phenocopies the deletion of integrin βPS (the fly ortho-logue of β-integrin)80, indicating that the functions of integrin activators are conserved and are crucial for development.

Loss of integrin inactivators. Recently, the importance of integrin-inactivating proteins has been validated in vivo (TABLE 2; supplementary information S2 (table)). The fact that loss of integrin-inactivating proteins manifests in complex in vivo phenotypes demonstrates that integ-rins are not passively returning to their inactive state in the absence of activating proteins but require activity-limitin g proteins for a correct balance of the integrin activity status. As discussed above, in vitro findings sug-gest that filamins compete directly with talin for binding to β-integrins and thereby act as negative functional reg-ulators12,81. Mice lacking specific filamins display cardiac and platelet (filamin A), skeletal (filamin B) and muscu-lar defects (filamin C), which is in line with the expres-sion patterns of these proteins. Interestingly, filamin B-deficient mice display an osteopenic phenotyp e, simila r to ICAP1- and SHARPIN-deficient mice82.

Loss of function of ICAP1 leads to a more or less penetrant perinatal lethal phenotype depending on the genetic background, and surviving mutant mice suffer from several defects, such as premature death and defective osteogenesis33. Interestingly, isolated ICAP1-deficient osteoblasts have increased integrin

Table 2 | Phenotypes of mice lacking an integrin activator or inactivator

Protein* Integrin domain affected ‡ Phenotype Integrin activity Link to human diseases

Inactivator

ICAP1 (LOF) Second NXXY motif Perinatal lethal (partial), reduced fertility, osteogenesis defects, ataxia

Increased Unknown

SHARPIN (LOF) Salt bridge, α-subunit Reduced fertility, osteogenesis defects, premature death, proliferative dermatitis

Increased Unknown

Filamin A (LOF) Between the two NXXY motifs Embryonic lethal at embryonic day 14.5

Increased Periventricular heterotopia

Filamin B (LOF) Between the two NXXY motifs Perinatal lethal, skeletal defects Increased Spondylocarpotarsal synostosis syndrome

Filamin C (LOF) Between the two NXXY motifs Perinatal lethal, muscular defects Increased Myopathy

Activator

Talin 1 (LOF) First NXXY motif and IMC (salt bridge) Embryonic lethal at embryonic day 7.5

Reduced Talin 1 overexpressed in cancer

Talin 2 (LOF) First NXXY motif and IMC (salt bridge) Myopathy Unknown Unknown

Talin 1 and talin 2 (muscle-specific LOF)

First NXXY motif and IMC (salt bridge) Severe myopathy No effect Unknown

Talin 1Leu325Arg IMC Conditional expression of talin 1Leu325Arg in platelets leads to clot retraction and integrin activation defects

Reduced Unknown

Kindlin 1 (LOF) Second NXXY motif and Ser/Thr stretch Perinatal lethal, skin blistering Reduced Kindler syndrome

Kindlin 2 (LOF) Second NXXY motif and Ser/Thr stretch Embryonic lethal at embryonic day 6.5

Reduced Kindlin 2 overexpressed in cancer

Kindlin 3 (LOF) Second NXXY motif and Ser/Thr stretch Immunological and haematopoietic defects

Reduced LADIII

ICAP1, integrin cytoplasmic domain-associated protein 1; IMC, inner membrane clasp; LADII, leucocyte adhesion deficiency type II; LOF, loss-of-function; SHARPIN, SHANK-associated RH domain interacting protein. *The effect of the mutation is shown in brackets. ‡The interaction site to which the integrin activator and inactivator have been shown to bind. A fully referenced version of this table can be found in Supplementary information S2 (table).

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activation but reduced proliferation and differentia-tion capabilities. Similarly, loss of filamin A in a KRAS-induced lung tumour model leads to reduced cell proliferation and consequent decreased tumour burden83. Therefore, unexpectedly, it seems that, at least in some contexts, integrins need to be inactivated to enable the proper activation of signalling pathways. This concept of integrin cycling (newly activated integrins emerging from a pool of inactive integrins) was first described in endothelial cells that required the constant formation of new adhesions via the recruitment of newly engaged integrin s in order to fully activate the JNK pathway84.

In the case of SHARPIN, spontaneous mutant mice lacking this integrin inactivator have been reported. These mice share some phenotypic traits with ICAP1-deficient mice such as premature death and osteo genesis defects. However, the SHARPIN-deficient mice also develop a strong proliferative dermatitis85, pos-sibly due to the fact that SHARPIN binds many different α-subunits and hence could potentially affect not only β1-pairing integrins but also other integrins in the epi-dermis such as αvβ5, αvβ8 or α6β4 integrins86.

Taken together, these findings highlight that main-taining or promoting integrin inactivation has a key role in optimal tissue function. However, integrin-inactivating proteins also have integrin-independent roles. For exam-ple, SHARPIN has a key role in apoptosis signalling and NF-κB activation, and this function has been linked to the inflammatory phenotype of the mutant mice87. Thus, an important question is which phenotypic abnormalities are related to increased integrin activity in vivo.

In vivo integrin activity mutants. The importance of regulating the integrin activity status has also been revealed by studies in which crucial residues in integrins have been mutated and found to influence their activity and conformational switching (Supplementary informa-tion S3 (table)). These studies stress the importance of carefully balancing the integrin activity status in vivo. Key pioneering work on the in vivo effects of many of these mutations was done in D. melanogaster, and based on the phenotypes it is obvious that mutations that lock integ-rin in an active state are deleterious for D. melanogaster development88.

In mammals, β1 integrins can be alternatively spliced, and the variants display specific functions. For instance, the muscle-specific β1D integrin strongly connects to the contractile machinery to support muscular contraction. This strong connection might be due to increased talin binding and therefore increased activity of β1D integrin compared with the ubiquitously expressed β1A isoform. Not surprisingly, ubiquitous expression of β1D integrin (which is normally specifically expressed in muscle) and not of the β1A isoform is embryonic lethal, partly caused by the disrupted migration of neuroepithelial cells89.

Recently, the in vivo significance of three cytoplas-mic regions of the β-subunit that are important for activity regulation have been analysed in mice: the salt bridge (which stabilizes the bent inactive integrins and is disru pted upon talin binding), the NPXY motif (which recruits talin) and the NXXY motif and a stretch of

Ser/Thr residues in between the two NXXY motifs (the latter two of which recruit talin or kindlin and ICAP1). Although in vitro studies have highlighted the impor-tance of the salt bridge in the regulation of integrin affinity, mouse studies suggested that this region has a more modest contribution in β1 integrin regulation90. Specifically, targeted mutation disrupting the salt bridge in β1 integrin did not interfere with mouse development or tissue function. Whether this region is truly involved in integrin activity regulation in vivo is still an open ques-tion, but mutations within the GFFKR region of α4 inte-grin lead to an increase in its function91. Further work is needed to reconcile this discrepancy, but one hypoth-esis is that the inner membrane clasp is only marginally affected in these mice owing to additional interactions that stabilize this region and allow it to remain intact in the salt-bridge mutants. Conversely, additional effectors, such as ICAP1 and SHARPIN, might still inhibit the activity of integrin mutants with a disrupted salt bridge. Indeed, ICAP1 could still restrict kindlin recruitment to this mutant and therefore inhibit full integrin activation30.

In line with the loss of talin and kindlin binding and thus loss of integrin activation, substitution of the Tyr to an Ala residue in the two NXXY motifs of both β1 and β3 subunits in mice results in a similar lethal phenotype as loss of integrin function90,92,93. Thus, integrin activation is critically important in vivo. To determine whether the phosphorylation of the Tyr residues of both NXXY motifs regulates integrin activity, as suggested by in vitro experi-ments (see above), these residues have been replaced in mice by a more conserved non-phosphorylatable resi-due (Phe). Interestingly, this mutation affects β3 integrin function, resulting in clot retraction94 but not in any overt phenotypes in β1 integrin-mutant mice90,92. This strongly suggests that β1 and β3 integrins are distinctly regulated and have tissue-specific functions despite sharing most of their cytoplasmic interactors.

Recently, attention has focused on the region between the two NXXY motifs. Mice in which the two Thr resi-dues in this region were substituted with Ala had a major developmental defect, with strong impairment of β1 integrin function70. These residues were found to be important for kindlin and SNX17 binding, thereby affect-ing both integrin affinity and integrin trafficking70. This region corresponds to a well-characterized mutation in the β3 integrin chain, found in patients with the blood co agulation disorder Glanzmann’s thrombasthenia, who lack β3 integrin function95. In mice, mutation of these resi-dues in β1 integrin produces a very similar phenotype as the loss of β1 integrin or the loss of the integrin activator kindlin 2. This suggests that this region plays a major part in integrin activation by recruiting kindlin, which is nega-tively regulate d by proteins such as ICAP1 or SHARPIN.

Integrin activity regulation in human diseasesLoss of integrins or defects in the regulation of integrin affinity strongly affect tissue formation or function in vivo in mice (see above). Importantly, mutations affecting the integrin activation status in humans have an impact on normal physiology, and dysregulation of integrin affinity is linked to a number of pathological states.

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Genetic mutations in integrins have been associated with several pathologies (TABLE 1). In line with their role in early embryonic development, mutations were rarely found in the genes encoding β1, β8, α4, α5 or αv integ-rins. However, mutations in integrins or integrin regu-lators with more tissue-specific expression have been associated with Glanzmann’s thrombasthenia (caused by mutations in β3 integrin), the immunodeficiency disor-der LAD (leucocyte adhesion deficiency) type I and III (caused by mutations in β2 integrin and kindlin 3, respectively) and skin diseases (caused by mutations in kindlin 1, α2, α6 and β4 integrin) (TABLES 1,2).

For integrin inhibitors, disease-related muta-tions have been reported for filamins96,97. In addition, loss-of-function mutations in KRIT1 are detected in 40% of patients with autosomal-dominant cerebral cavern-ous malformation (CCM; associated with vessel malfor-mation in the brain). Because KRIT1 binds directly to ICAP1 and antagonizes its ability to inhibit integrins34, these mutations could be linked to altered integrin activ-ity regulation. However, as filamins and KRIT1 have additional functions98,99, it remains to be determined whether these diseases are caused by inappropriate integrin activation. Interestingly, activating mutations in integrins have been reported in more restricted cell types such as haematopoietic cells or platelets and are involved in several pathologies100–102. Thus, with the identification of new integrin activity-regulating mech-anisms and the explosion of genomic sequencing data, it is likely that our understanding of the role of integ-rin activity regulation in human diseases will increase significantl y in the future.

Integrins are known to be crucially involved in tumour progression in several different carcinomas. For instance, loss of β1 integrin expression blocks tumour progression in different tumour models103,104, and increased expression of β1 or α5 integrins is asso-ciated with poor prognosis in cancer105. However, at present the link between integrin activity regulation and cancer is not fully elucidated. Recent studies have linked increased expression of the integrin-activating proteins talin, kindlin and CD9 to cancer progression and metastasis106–108. In addition, expression of a mutant, constitutively active form of β1 integrin was shown to drive metastasis in experimental models109. Conversely, deletion of the gene encoding filamin A (which would be expected to increase integrin activity) diminished tumour burden in a lung tumour83, and upregulation of SHARPIN expression has been seen in solid tumours, especially ovarian and liver carcinoma110. Therefore, it is evident that the link between integrin activity and cancer is not always predictable, and the role of integrin inactivators in cance r is complex and context dependent.

Concluding remarksThe canonical models of integrin activation postulate that the inactive integrin conformation is adopted by the receptor in the absence of activating proteins9 and that inside–out activation through talin binding dic-tates the transition from the inactive state to the active one. Therefore, the inactive integrin conformation was

considered to be the default state of the receptor8,111. Although this is indeed the case in in vitro model sys-tems112, the identification of integrin-inhibiting proteins suggests that in cells and tissues integrins need to be actively maintained inactive and that the activity state of integrins is carefully balanced by inactivators and activa-tors. The existence of mechanisms allowing the regulated recruitment of activators and competing inactivators to integrins implies that, in adherent cells, integrin activity regulation needs to be fine-tuned by the cytoskeleton and appropriate scaffolding molecules to facilitate cell adaptation to different ECMs and signalling conditions in different tissues.

For non-adherent cells, like circulating blood cells, it is imperative that their integrins remain in an inac-tive state. These integrins are rapidly activated only upon extracellular stimulation, resulting in cell adhe-sion. In this Review, however, we have mainly focused on integrin inactivation in adherent cells because inte-grin inhibitors have not been extensively studied in non-adherent cells. Although there is some indication that SHARPIN has a role in leukocyte function (J.P., N.D.F, P. Rantakari, M. Karikoski, E. Mattila, C. Potter, J.P. Sundberg, N. Hogg, C. Gahmberg, M. Salmi and J.I, unpublished observations), future studies will need to address whether the same regulatory pathways govern integrin activity in adherent and non-adherent cells.

As currently subcellular regulation of integrin activity in response to extracellular cues is poorly understood, unravelling how these activators and inactivators are regulated and how they function in different conditions, especially in vivo, remains a challenge for the future. Although some progress has been made, one rather under-developed field is the post-translational modification s on integrin activators and especially inhibitors.

One area of intense research and debate is the link between the integrin activation status and integrin endo-cytosis and degradation. Several recent papers have indi-cated that active integrins can be endocytosed and are either recycled or targeted for degradation. However, it has also been reported that active integrins are not detected in endosomes in primary fibroblasts70, raising the possibility that endocytosis of active integrins is lim-ited to cancer cells and fibroblasts66,71. A large study com-paring different cell types, including primary cells and cancer cells with different genetic backgrounds, would shed light on this. Furthermore, if active integrins are truly endocytosed, the possibility that these endocytosed active integrins signal via an ‘inside–in’ mechanism from the endosomes cannot be excluded. Another open ques-tion is the relationship between integrin activity regula-tors and endosomal proteins in integrin binding. RAB GTPases affect integrin endocytosis through direct bind-ing to the cytoplasmic tails of β-integrin (RAB25)113 and α-integrin (RAB21)66 and therefore could compete with talin or SHARPIN binding to the receptor. For RAB25, the exact β-integrin-binding site at the cytoplasmic tail remains to be identified, but the RAB21- and SHARPIN-binding sites overlap considerably. Thus, integrin endocytosis as RAB21-bound cargo might require the dissociation of SHARPIN and this would provide

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selectivity for endocytosis of active integrins. Similarly, SNX17 binds to β1 integrin at the same site as kindlins, providing another possible connectio n between integrin activity and traffic70.

There are several examples of impaired integrin activation leading to human pathological conditions such as bleeding disorders. However, a link for integ-rin-inactivatin g proteins has not been well-established.

It will therefore be important to determine whether inte-grin inactivators have a role in human diseases, and, if so, what their role is. The recent exciting findings on integrin inactivators have caused a paradigm shift in the field of integrin activation regulation. Future studies will deepen our knowledge on how cells balance integ-rin activity with spatio-temporal resolution and how this has an effect on development and disease.

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AcknowledgementsJ.I. has been supported by an European Research Council (ERC) starting grant, and funding from the Academy of Finland, the Sigrid Juselius foundation and the Finnish Cancer Organizations. J.P has been supported by the Cancer Society Finland and the Instrumentarium Foundation. D.B. is funded by INCa (Institut National du Cancer) grants.

Competing interests statementThe authors declare no competing financial interests.

FURTHER INFORMATIONJohanna Ivaska’s homepage: http://www.btk.fi/research/research-groups/ivaska

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Integrin inactivators: balancing cellular functions in vitro and in vivo

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