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Regulation of integrin activation and trafficking by the β-cytoplasmic tail Hanneke N Monsuur November 2010 - March 2011 Department of Cell Biology, Netherlands Cancer Institute (NKI), Amsterdam Student number: 3052664 Master: Biology of Disease, University of Utrecht Supervisors: Coert Margadant (NKI) Dr. Arnoud Sonnenberg (NKI) Dr. Peter van der Sluijs (UMCU) Index Regulation of integrin activation and trafficking by the β-cytoplasmic tail.......................... 1 Index ................................................................................................................................................................... 2 1 Introduction ................................................................................................................................................. 3 1.1 The integrin family ........................................................................................................................ 3 1.2 Integrin activation and signaling ............................................................................................. 4 1.3 Integrin domains and β-tail sequences ............................................................................... 6 2 Integrin activation .................................................................................................................................. 7 2.1 Inside-out activation ..................................................................................................................... 7 2.1.1 Regulation of inside-out activation by talin and kindlins ...................................... 9 2.1.2 Regulation of inside-out activation by the NPxY motifs and the serine/threonine-rich region...........................................................................................10 2.2 Outside-in signaling.....................................................................................................................12 2.2.1 Focal adhesions ....................................................................................................................13 2.2.2 Fibrillar adhesions...............................................................................................................14 2.2.3 Regulation of outside-in signaling by the NPxY motifs .........................................15 3 Integrin trafficking ...............................................................................................................................17 3.1 Integrin endocytosis ...................................................................................................................17 3.2 Anterograde integrin transport ..............................................................................................20 3.3 Regulation of integrin trafficking by the β-subunit cytoplasmic tails .....................20 3.4 Functional consequences of integrin trafficking ..............................................................21 4 Discussion ................................................................................................................................................23 5 Abbreviations .........................................................................................................................................27 6 Reference list ..........................................................................................................................................28 2 1 Introduction Integrins are heterodimeric transmembrane receptors, which integrate the interior of the cell with the cell environment. Integrins mediate cell adhesion to extracellular matrix (ECM) proteins and to other cells. Integrins are connected to the cytoskeleton, and regulate several signal transduction pathways. In this way, they control many cellular processes, including adhesion, migration, proliferation, differentiation, survival, and gene expression, which is important for development, hemostasis, and immune surveillance, but also in pathological situations as cancer, thrombosis, and inflammation. 1.1 The integrin family Each integrin is formed by dimerization of an α- and a β-subunit. Currently 18 α- and eight β-subunits are known in mammals, which form 24 specific integrins (reviewed by Hynes, 2002). The integrin family comprises collagen receptors (β1+α1/2/10/11), laminin receptors (β1+α3/6/7 and α6β4), and RGD receptors (αIIbβ3, α5β1, α8β1, and the αv-containing integrins) (Fig 1). RGD receptors recognize Arg-Gly-Asp (RGD)containing ligands including vitronectin, fibronectin (Fn), the latency-associatedpeptide of transforming growth factor-β1/3 (LAP-TGF-β1/3) and osteopontin. Ligand specificity is defined by the combination of an α- and a β-subunit. Integrin expression and activation, together with the availability of ligand ultimately determines which interactions with ligands occur in vivo (reviewed by van der Flier and Sonnenberg, 2001; Humphries et al., 2006). The integrin family members and their ligands are depicted in table I. Figure 1. The integrin family. 18 α- and 8 β-subunits form 24 integrins, which can be distinguished according to ligand specificity or cell-type-specific expression (from Hynes, 2002). 3 Integrin α1β1 α2β1 α3β1 α4 α5β1 α6 β1 β7 β1 β4 α7β1 α8β1 α9β1 α10β1 α11β1 αV β1 β3 β5 β6 β8 αIIbβ3 αLβ2 αMβ2 αXβ2 αDβ2 αEβ7 Ligand ECM Soluble Col; Col; Ln; Fn; Op Fn; Op Fn; Op Ln Ln Ln Fn; Op; Tn; Vn (Nn; LAP-TGF-β1/3) Op; Tn Col; Ln Col Fn; Op; LAP-TGF-β1/3 Fn; Op; Tsp; Vn; Tn; vWF; Fg; Fb LAP-TGF- β1/3 Fn; Op; Vn; LAP-TGFβ-1/3 Fn; Op; LAP-TGF-β1/3 Fn; LAP-TGF-β1/3 Fn; TSP; Vn vWF; Fg Col (Fn; Vn) Fg, FX; iC3b Fg; iC3b Cell-cell (E-Cadherin) VCAM-1; MAdCAM-1 VCAM-1; MAdCAM-1 VCAM-1 PECAM-1 ICAM ICAM ICAM VCAM-1; ICAM E-Cadherin Table I. The 24 mammalian integrins and their ligands. Col = collagen, E-Cadherin = Epithelial Cadherin, Fb = fibrillin, Fg = fibrinogen, Fn = fibronectin, FX = factor X, ICAM = Inter-Cellular Adhesion Molecule, iC3b = inactive complement factor 3b, LAP-TGF-β = Latency-Associated Peptide of Transforming Growth Factor-β, Ln = laminin, MAdCAM-1 = Mucosal Addressin Cell Adhesion Molecule-1, Nn = nephronectin, Op = osteopontin, PECAM-1 = Platelet Endothelial Cell Adhesion Molecule-1, TSP = thrombospondin, Tn = tenascin, VCAM-1 = Vascular Cell Adhesion Molecule-1, Vn = vitronectin, vWF = von Willebrand factor (from Humphries et al., 2006) 1.2 Integrin activation and signaling Integrin binding to ligand occurs upon integrin activation, which is defined as the conformational change that causes a shift from a low to a high affinity for ligands. Integrin activation can be induced by cytoplasmic events; a process designated ‘insideout’ activation or ‘inside-out’ signaling (reviewed by Ginsberg et al., 1992). After ligand binding, integrins mediate cytoskeletal reorganization and cell spreading over the substratum, and several cellular signal transduction pathways are activated that affect cell motility, proliferation, survival, and gene expression, which is called ‘outside-in’ signaling (Hynes, 1992; Ginsberg et al., 1992). In addition to high affinity, tight adhesion also requires integrin clustering (high avidity). Regulation of integrin activation by ‘inside-out’ signals is particularly important in nonadherent cells such as leukocytes and platelets. Whereas these cells have inactive integrins by default, inside-out activation occurs in response to specific cues, e.g. during hemorrhaging or inflammation. Upon endothelial injury, platelet integrins are activated, after which the platelets adhere to the damaged vascular endothelium and stop the hemorrhage. However, inappropriate integrin activation on platelets and subsequent uncontrolled platelet adhesion results in a pathological situation called thrombosis. Integrins on leukocytes and platelets adhere, in addition to immobilized ECM proteins, to cellular receptors including ICAM (Inter-Cellular Adhesion Molecule), VCAM-1 (Vascular Cell Adhesion Molecule-1) and E-Cadherin, or to soluble ligands like fibrinogen and von Willebrand Factor (vWF). In contrast to non-adherent cells, most adherent cells need to be attached to the ECM at all times in order to maintain tissue integrity, requiring continuously active integrins. Integrins in adherent cells reside in focal adhesions (FAs) or hemidesmosomes (HDs), which are large macro-molecular complexes containing clusters of active integrins connected to the cytoskeleton (reviewed by Zamir and Geiger, 2001; van der Flier and Sonnenberg, 2001; Legate and Fassler, 2009). The interaction with the cytoskeleton increases integrin clustering, and thus adhesion strengthening (Fig. 2; Legate and Fässler, 2009). Integrin connection to the cytoskeleton occurs via proteins that bind the cytoplasmic tail of the -subunit (the -tail). Interestingly, the -subunit targets the integrin into FAs, while the α-subunit seems to inhibit integrin localization into FAs (reviewed by Burridge et al., 1996). HDs mediate stable adhesion of basal epithelial cells to the underlying basement membrane. HDs are formed by binding of the integrin α6β4 and BP180 to laminin-332, which is intracellularly stabilized by connection to the intermediate filament system through BP230 and plectin (reviewed by Margadant et al., 2008). In FAs, integrins connect to actin filaments, and FAs contain many additional proteins that either provide a structural link to the cytoskeleton (structural adaptors), or binding sites for other proteins (scaffold adaptors). In contrast to growth factor receptors, integrins lack intrinsic enzymatic activity. Therefore, they require the binding of signaling proteins to initiate signal transduction pathways (catalytic adaptors), which are also abundant in FAs (Legate and Fässler, 2009). Structural adaptors in FAs include talin and tensin, scaffold adaptors include paxillin and vinculin, and catalytic adaptors are focal adhesion kinase (FAK) and Src. Figure 2. Talin connects integrins to the actin cytoskeleton in FAs. Talin is both a structural- and a scaffold adaptor between the integrin -tail and the actin cytoskeleton. Talin interacts with F-actin either directly or indirectly via vinculin (from Legate and Fässler, 2009). 5 1.3 Integrin domains and β-tail sequences Both the α- and the β-subunit contain an extracellular domain of ~800 amino acids, a transmembrane domain of ~20 amino acids, and a cytoplasmic tail of 13-70 amino acids, with the exception of β4 which has a cytoplasmic tail of 1088 amino acids (reviewed by Moser et al., 2009). The αextracellular domains comprise a βpropeller or head-domain, a thigh-domain, and two calf-domains. About half of the αsubunits contain an additional I-domain. The β-subunits contain a β-I-domain (or βA-domain after von Willebrand A domain), which is analogous to the α-I-domain, a hybrid domain that links the β-I-domain to the PSI (plexin/semaphorin/integrin) domain, four EGF (epidermal growth factor) repeats, and an intracellular tail. Ligands bind either to a metal ion-dependent adhesion site (MIDAS) in both I-domains, or Figure 3. Structural organization of integrins to the MIDAS in the β-I-domain and the (from Moser et al., 2009). propeller domain of the α-subunit, if no α-Idomain is present (Fig 3). Integrin -tails harbor a relatively conserved membrane-proximal (MP) NPxY motif and a membrane-distal (MD) NPxY (in β1) or NxxY motif (in β3, β5, and β6) (Table II). NPxY motifs form β-turns, which are recognized by proteins containing a phosphotyrosinebinding domain (PTB). The NPxY or NxxY motifs serve as docking sites for a multitude of proteins including talin, kindlins, Src, Dok1/2, tensin, filamin, Numb, Disabled (Dab)1, Dab-2, Eps8, and the Ras-related proteins in brain (Rabs). Whereas Dab1/2, 3endonexin, Src, and kindlins bind the MD-NxxY motif, talin, Numb, Dok1/2, and tensin bind the MP-NPxY motif. In this way, these motifs are involved in integrin activation, internalization, and recruitment of integrins to FAs. In addition, the β-tail of most integrins contains a serine/threonine-rich sequence, which has also been implicated in integrin activation. Here, we review how the -tails control integrin functions, with particular emphasis on the regulation of integrin activation and trafficking by the NPxY and NxxY motifs in β1 and β3. β1A: KLLMII HDRREFAKFEKEKMNAKWDTGE NPIYKSAVTTVV β1D: KLLMII HDRREFAKFEKEKMNAKWDTGE NPIYKSPINNFK NPKYEGK NPNYGRKAGL β2: KALIHL SDLREYRRFEKEKLKSQWNND. NPLFKSATTTVM NPKFAES β3: KLLITI HDRKEFAKFEEERARAKWDTAN NPLYKEATSTFT NITYRGT β5: KLLVTI HDRREFAKFQSERSRARYEMAS NPLYRKPISTHTVDFTFNKF NKSYNGTVD β6: KLLVSF HDRKEVAKFEAERSKAKWQTGT NPLYRGSTSTFK NVTYKHREK QKVDLSTDC β7: RLSVEI YDRREYSRFEKEQQQLNWKQDS NPLYKSAITTTI NPRFQEADSPTL β8: RQVILQ WNSNKIKSSSDYRVSASKKDKLILQSVCTRAVTYRREKPEEIKMDISKLNAHETFRCNF Table II. Amino acid sequences of the cytoplasmic tails of all integrin β-subunits apart from β4. The MP-NPxY and MD-NxxY motifs are highlighted in bold, and the serine/threonine-rich region is highlighted in red and bold. 6 2 Integrin activation Integrins on non-adherent cells exist on the plasma membrane in a low-affinity state, an intermediate-affinity state, or a high-affinity state for ligand. The different states are in a dynamic equilibrium, and the conformational change that mediates the transition from the low-affinity state to the intermediate-/high-affinity state is called ‘integrin activation’. Integrin activation can be induced by inside-out signals. After ligand binding, outside-in signals are generated, which regulate a variety of cellular processes. 2.1 Inside-out activation Integrin activation by inside-out mechanisms has been studied mostly in circulating leukocytes and platelets, which should not adhere under normal physiological circumstances, and therefore have inactive integrins by default. The main integrin on platelets is αIIbβ3, and a wealth of evidence indicates that αIIbβ3 is regulated tightly by conformational changes induced by ‘inside-out’ signals. αIIbβ3 is considered an archetype for all integrins, and it is often assumed that the mechanisms of activation of other integrins are similar to those of αIIbβ3. Whereas a bent form with a closed head is thought to represent the low-affinity state, an extended form with a closed head represents the intermediate state, and the extended form with an open head represents activated- and ligand-bound integrins (Fig. 4; Xiong et al., 2001; Nishida et al., 2006; Takagi et al., 2002). Stabilization of the low-affinity conformation is thought to occur by interactions between the α- and β-subunit (sometimes referred to as a ‘clasp’), particularly a salt bridge between R995 in αIIb and D723 in β3 (Vinogradova). The disruption of this salt bridge leads to separation of the cytoplasmic tails or the ‘unclasping’ of the αIIb and the β3 subunits, and is thus important in the transition of the low-/intermediate- to the high-affinity state (Nishida et al., 2006; Takagi et al., 2002). Figure 4. Structural rearrangements occurring in integrin activation. The α-headpiece domains are displayed in red, the α-tailpiece in pink, the β-headpiece domains are displayed in blue, and the βtailpiece in cyan. The transmembrane and cytoplasmic domains are represented in black (adapted from Takagi et al., 2002). Inside-out activation of αIIbβ3 is initiated by ligands for G-protein coupled receptors (GPCRs) including thrombin, sphingosine-1-phosphate, lysophosphatidic acid (LPA), 7 and phorbol myristate acetate. Ligand binding to a GPCR leads to an increase of intracellular Ca2+ and formation of diacylglycerol (DAG). This results in activation of protein kinase C (PKC) and a Rap guanine nucleotide exchanger (Rap-GEF), which then cooperate to activate Rap1. Activated Rap1 interacts with RIAM, after which talin associates with RIAM to form a complex inducing integrin activation (Fig 5; Han et al., 2006). Figure 5. Inside-out integrin activation. Ligand binding to a GPCR results in increased intracellular Ca2+ and DAG. PKC and a Rap-GEF are then activated, which induce Rap1 activation. RIAM associates with talin upon interaction with Rap1, resulting in integrin activation due to the unmasking of an integrin-binding site in talin (from Han et al, 2006). Inside-out activation of IIb3 and other integrins on non-adherent cells is of vital importance, as illustrated by the human disorders Glanzmann thrombasthenia (GT) and leukocyte adhesion deficiency syndrome-I and -III (LAD-I,-III). GT is a rare bleeding disorder caused by reduced binding of αIIbβ3 to fibrinogen or vWF, which leads to defective platelet adhesion and aggregation during a bleeding. The patients suffer from mild bruising to severe hemorrhages. GT is caused by mutations in either the α- or βsubunit of αIIbβ3. Many different mutations have been found across the entire sequence of the α- or the β-subunit, leading to quantitative or qualitative defects (Nurden et al., 2006). Whereas many mutations lead to reduced surface expression of αIIbβ3, probably by compromising the transport of integrins to the platelet surface, other mutations affect the ability to bind to ligands. Some mutations affect surface expression, as well as activation and subsequent outside-in signaling (e.g. the S752P mutation in the β3subunit; Chen et al., 1994). LAD-I results from defects in functions of β2 integrins on leukocytes due to mutations in the gene encoding the β2-subunit. As a consequence, LAD-I patients suffer from recurrent bacterial infections and impaired wound healing. LAD-III is caused by defects in the functions of β1, β2, and β3 integrins on leukocytes and platelets, leading to LAD-I-like infections and additional GT-like bleeding abnormalities. Although agonist-induced activation of αIIbβ3 has been studied mostly in adherent Chinese Hamster ovary (CHO) cells, which express talin and PKC at levels comparable to those in platelets, it is unclear whether the same inside-out mechanisms exist to activate endogenous integrins in adherent cells, and even whether the same conformational states exist for integrins in adherent cells. A recent study suggests that α5β1 in adherent cells does adopt either the bent, inactive state or the extended state with separated legs, and that the conversion from the former to the latter occurs in FAs (Askari et al., 2010). Integrin activation in adherent cells is probably induced by cytoskeletal tension generated by myosin-II, in response to ECM stiffness. Depending on the degree of tension, binding of α5β1 to Fn can occur in two ways. Under low tension, 51 forms ‘relaxed’ bonds solely to the RGD site of Fn, whereas under high tension, 51 binds both the RGD and additional sites on Fn, leading to stronger, ‘tensioned’ bonds (Friedland et al., 2009). 8 2.1.1 Regulation of inside-out activation by talin and kindlins Inside-out activation of integrins occurs by talin binding to the MP-NPxY motif. Talin consists of an N-terminal head domain and a C-terminal rod or tail. The head is constituted by a FERM (4.1, ezrin, radixin, moesin) domain, of which the F3 domain harbors the PTB-site (Fig 6; Moser et al., 2009). The PTB-site recognizes the MP-NPxY motif in β1, β2, β3, β5, and β7 (Pfaff et al., 1998, Calderwood et al., 2003). Subsequently, the talin-head binds additional membrane-proximal residues. Talin exists in the cytoplasm in an auto-inhibitory conformation, in which the C-terminal domain interacts with the PTB-domain. Talin recruitment to the membrane and disruption of the autoinhibitory conformation are mediated by phosphatidylinositol-4,5-biphosphate (PI4,5P2), generated by phosphatidylinositol phosphate kinase (PIPK1; Martel et al., 2001, Goksoy et al., 2008). Interestingly, talin can in turn bind to and activate PIPK, generating a positive feedback loop. Moreover, PI4,5P2 is important for the recruitment of additional proteins important for FA assembly (Ling et al., 2003). Figure 6. Structural organization of talin and kindlin (from Moser et al., 2009). Talin binding disrupts the ‘clasp’ between the α- and the β-tail, and requires the D723 residue in β3 (Vinogradova et al., 2002; Anthis et al., 2009). Both the binding of talin and the disruption of the salt bridge are required for activation of αIIbβ3 (Anthis et al., 2009; Tadokoro et al., 2003; Wegener et al., 2007; Lu et al., 2001; Takagi et al., 2001). The salt bridge appears to have no apparent function in β1-integrins under normal physiological conditions in vivo, as mice carrying a D759A substitution in β1, which impairs formation of the salt bridge, had no obvious defects (Czuchra et al., 2006). Although binding of the talin-head alone is sufficient for integrin activation, the talinrod is required to connect integrins to the actin cytoskeleton, and thus to target integrins to FAs (Calderwood et al., 2003; Tanentzapf and Brown, 2006; Bouaouina et al., 2008; Zhang et al., 2008). The importance of talin for integrin function is underlined by in vivo studies; disruption of the talin gene in mice leads to death during gastrulation, due to a failure of integrin-mediated cell migration (Monkley et al., 2000). In addition, platelet-restricted deletion of talin in mice impairs integrin α2β1-mediated adhesion to collagen and αIIbβ3-mediated adhesion to Fn, and causes severe hemostatic defects (Nieswandt et al., 2007; Petrich et al., 2007). It should be noted that in Drosophila melanogaster, loss of talin expression results in wing blistering, much like the integrinknockout phenotype, but integrin binding to ECM is not impaired (Brown et al., 2002). A similar phenotype is observed in Drosophila mutants expressing talin-head alone (Tanentzapf and Brown, 2006). These observations suggest that talin-requirement for inside-out activation in adherent cells can be bypassed. Indeed, integrin activation can also be induced by ligand from the outside (Du et al., 1991). However, integrin-ligand binding crucially depends on cytoskeletal association, as in the absence of the talin-rod, loss of integrin function is observed. 9 Although one study suggests that the MP-NPxY in β3 is not essential for talin binding and that the actual binding site for talin is located more proximal to the membrane, most studies find that the NPxY motif is necessary for talin binding, and that talin binding is abolished by Y-A substitution in the MP-NPxY motif (Patil et al., 1999; Calderwood et al., 2002; Calderwood et al., 2003; García-Alvarez et al., 2003; Bouaouina et al., 2008). Downstream of the MP-NPxY, a W-A substitution also impairs talin binding (Bouaouina et al., 2008). Interestingly, talin binding was not prevented upon a Y-S substitution in the MP-NPxY of β1, which is probably because this mutation still allows the formation of a β-turn (Vignoud et al., 1997). Other proteins that have been implicated in integrin activation are kindlin-1, -2 and -3. Kindlins contain a C-terminal FERM domain, which is highly homologous to the FERM of talin, but binds the MD-NxxY motif in β-tails (Meves et al., 2009). Kindlin-1 is expressed in epithelial cells, kindlin-2 is ubiquitously expressed, and kindlin-3 is primarily present in hematopoietic cells (Ussar et al., 2006). Mutations in kindlin-1 lead to Kindler Syndrome (KS), which is a rare autosomal, recessive genodermatosis. Hallmarks of KS are skin blistering, photosensitivity, poikiloderma, abnormal pigmentation and thinning of the skin (Siegel et al., 2003). Histologically, KS is characterized by duplication of the basement membrane and microblistering at the dermal-epidermal junction. The clinical and histological features of KS resemble the defects caused by loss of β1 integrins from the epidermis, in particular of α3β1 (Margadant et al., 2009; Raghavan et al., 2000; Brakebusch et al., 2000). Moreover, mutations in kindlin-3 have been disclosed in LADIII patients, illustrating the importance of kindlin-3 in regulating integrin functions in leukocytes and platelets. Genetic disruption of kindlin-3 recapitulates the LAD- and GTlike symptoms in mice, whereas the disruption of kindlin-1 partially resembles KS (Moser et al., 2008; Ussar et al., 2008). Kindlin-2 knockout mice die during development due to defective adhesion of the endoderm and epiblast to the basement membrane (Montanez et al., 2008). Thus, it is clear that the kindlins are crucial for integrin functions, however, their precise role is unclear and controversial. A number of studies suggest that talin and kindlin cooperate to regulate inside-out integrin activation (Moser et al., 2009). In many cell lines, expression of talin alone is sufficient to induce integrin activation, but activation is enhanced when kindlin is co-expressed, whereas kindlin alone only very weakly activates integrins (Ma et al., 2008; Bledzka et al., 2010; Ye et al., 2010; Montanez et al., 2008). An intriguing possibility is that the F3 domain of kindlin binds directly to the talin rod, thereby releasing the auto-inhibitory conformation and thus activating talin. In contrast, other data suggest that expression of kindlin-1 or -2 alone inhibits the activation of both αIIbβ3 and α5β1, whereas coexpression with talin results in activation of αIIbβ3 but inhibition of α5β1, suggesting that kindlins may exert integrin-specific effects (Harburger et al., 2009). In summary, whereas talin seems absolutely required for integrin activation, the effects of kindlins on integrin functions are less clear. It is suggested that kindlins can indirectly favor talin binding over the inhibitory binding of filamin, that kindlins stabilize integrins in FAs, or that kindlins function as adaptors for other proteins (Ye et al., 2010; Harburger et al., 2009). 2.1.2 Regulation of inside-out activation by the NPxY motifs and the serine/threonine-rich region It is controversial whether tyrosine–phoshorylation of the NPxY motifs is required for inside-out activation. The role of the NPxY motifs in integrin activation has been 10 WT: KLLMII HDRREFAKFEKEKMNAKWDTGE D759A: HARREFAKFEKEKMNAKWDTGE Y783F: Y783A: Y795F: Y795A: YY783,795FF: YY783,795AA: β1A β1A β1A β1A β1A β1A β1A β1A β3 β3 β3 β3 WT: KLLITI HDRKEFAKFEEERARAKWDTAN Y747A: Y759A: YY747,759FF: MP-NPxY NPIYKSAVTTVV MD-NxxY NPKYEGK NPIF NPIA NPIY NPIY NPIF NPIA NPKY NPKY NPKF NPKA NPKF NPKA MP-NPxY NPLYKEATSTFT NPLA NPLY NPLF MD-NxxY NITYRGT NITY NITA NITF Table III. Amino acid sequences of β1A and β3 cytoplasmic tails. The positions of Y-F and Y-A mutations are indicated. The NPxY and NxxY motifs are highlighted in bold, and the serine/threonine-rich region is highlighted in red and bold. addressed using either chimeric integrins overexpressed in CHO cells, or mutated β1tails expressed in β1-deficient cells (Table III). Results obtained in CHO cells suggest that structural integrity, rather than the phosphorylation status of the MP-NPxY motif is required for integrin activation, because conservative Y-F substitutions in the MP-NPxY of β1 and β3 do not affect integrin activation or cell adhesion, whereas more disruptive Y-A substitutions do (O’Toole et al., 1995; Romzek et al., 1998). Y-A substitutions in the MD-NPxY motifs also slightly reduced integrin activation (O’Toole et al., 1995). Also in fibroblasts, a Y-F substitution in the MD-NPxY motif did not affect adhesion, although there was a loss of directed migration (Sakai et al., 1998). In contrast, Y-F substitutions in both NPxY motifs impaired adhesion of T-lymphoma cells (Stroeken et al., 2000). This probably reflects differences in the regulation of integrin activation between adherent and non-adherent cells. However, mice carrying Y-F substitutions in both NPxY motifs (YY/FF) do not display any defects (Czuchra et al., 2006). In contrast, YY/AA mice are not viable, and keratinocyte-restricted YY/AA substitutions cause epidermal defects similar to the loss of β1 expression in the epidermis (Czuchra et al., 2006). The complete loss of β1 function upon YY/AA mutations in mice was confirmed in another study, while YY/FF substitutions had no effects (Chen et al., 2006). Thus, most in vitro and in vivo evidence supports the concept that structural integrity of the NPxY motifs is required for integrin activation, but tyrosine-phosphorylation is not. Instead, it has even been suggested that phosphorylation may act as a negative regulator of integrin activation, either by reducing the affinity of talin, or by increasing the affinity of other PTB-domaincontaining proteins such as Dok1, which then compete with talin for the binding site (Czuchra et al., 2006; Oxley et al., 2007; McCleverty et al., 2007; Wegener et al., 2007). In this way, phosphorylation may serve as a molecular switch. Dok1 binds β3 but not to β1–tails (Calderwood et al., 2003). The differential effects of Dok1 and talin in integrin activation are probably explained by differences in the structural properties of their PTB-domains (Wegener et al., 2007). Tyrosine-phosphorylation of the NPxY motifs in 1 can occur by Src, and reduces adhesion to Fn and laminin (Sakai et al, 2001). Y-A substitutions in the NPxY motifs impair the binding of a number of proteins including Numb, Dab1, Dab2, EPS8, tensin, Dok1 and talin (Calderwood et al., 2003). Although the NPxY motifs are highly conserved among β1A, β2, β3, β5 and β7, there is a 11 difference in which interactions occur, since not all proteins were able to bind to all βtails. Additional residues (-5 relative to the N, and +2 relative to the Y in the MP-NPxY motif) influence the specificity of the interactions, for example both tensin and talin require W776 downstream of the MP-NPxY motif (McCleverty et al., 2007; GarcíaAlvarez et al., 2003). Whereas talin initially binds the MP-NPxY motif, additional binding to W776 is thought to result in the actual separation of the α- and the β-tail, following a two-step activation pathway (García-Alvarez et al., 2003). The serine/threonine region in between the two NPxY motifs is also considered important for the binding of regulatory proteins and integrin activation. The region is phosphorylated in β1, β2, β3 and β7 after agonist stimulation (Hilden et al., 2003; Valmu and Gahmberg, 1995; Willigen et al., 1996; Nilsson et al., 2006). Phosphorylation can occur by PKC and seems to regulate the exposure of the ligand-binding sites in αIIbβ3 (Willigen et al., 1996). Conservative and non-conservative mutations of the serines or threonines in β3 or β1 decreased integrin activation (O’Toole et al., 1995). Consequently, an S752P substitution in αIIbβ3 resulted in impaired adhesion to Fn, and T788A or T789A mutations in β1 decreased cell adhesion and invasion in T-lymphoma cells (Ylänne et al., 1995; Stroeken et al., 2000). In spite of these observations however, the β1 splice variant β1D lacks the serine/threonine stretch, and binds much stronger to talin than β1A, suggesting that the serine/threonine-region negatively impacts talinintegrin binding (Belkin et al., 1997). In summary, most studies indicate that the MP-NPxY motif is crucial, whereas the MDNPxY motif only subtly affects integrin activation. Morover, it seems that phosphorylation of the NPxY motifs is not essential for activation, but instead may regulate inactivation. 2.2 Outside-in signaling Upon ligand binding, integrins cluster together and actin filaments are recruited, which mediates cell spreading over the substrate, assembly of cell-matrix adhesions, and cell migration. Collectively, these events are referred to as outside-in signaling. Three different types of cell-matrix adhesions are distinguished, being focal complexes (FCs), FAs, and fibrillar adhesions (Table IV). These adhesions differ in appearance and molecular composition, and the differences are best-exemplified in the context of the Fn-binding integrins αvβ3 and α5β1. FCs are small adhesions found at the cell periphery, generally containing αvβ3, talin, vinculin and paxillin. FAs are also located at the cell periphery and also contain αvβ3, vinculin, paxillin and talin, as well as many proteins that are highly phosphorylated on tyrosines. By contrast, fibrillar adhesions are found at more central positions and hardly contain tyrosine-phosphorylated proteins. Fibrillar adhesions are associated with Fn fibrils, and contain α5β1 and tensin. The three adhesion complexes probably represent different stages of development; FCs being early adhesions, which over time develop into FAs, whereas fibrillar adhesions develop as a result of Fn-associated α5β1 displacement from FAs (reviewed by Geiger et al; 2001). 12 Table IV. Characteristic features of different types of cell-matrix adhesions (from Geiger et al., 2001). 2.2.1 Focal adhesions Integrin connection to the cytoskeleton can occur in multiple ways and involves a number of proteins, including talin, α-actinin, vinculin, FAK, paxillin, the IPP complex, kindlin-2, and probably also kindlin-1 (Fig 7). Talin links integrins to actin filaments either directly or via additional proteins like vinculin and α-actinin. The latter crosslinks actin filaments to provide stiffness to actin filament networks. Vinculin interacts with talin, α-actinin and the actin cytoskeleton, and recruits additional proteins like paxillin and vinexin (Ziegler et al., 2006, 2008). Vinculin is believed to regulate FA dynamics, as increased levels of phosphoinositides inhibit the interaction between vinculin and actin, which promotes FA turnover and cell motility (Ziegler et al., 2006). In addition, vinculin controls the interaction between paxillin and FAK, and FAK is also believed to regulate FA turnover (Subauste et al., 2004). The IPP complex is formed in the cytosol by binding of integrin-linked kinase (ILK) to the adaptor proteins PINCH and parvin. Recruitment of the IPP complex to FAs requires all three components (Legate et al., 2006; Zhang et al., 2002; Sepulveda et al., 2005). Paxillin can bind both ILK and parvin and kindlin-2 can bind ILK (Nikolopoulos and Turner, 2000, 2001; Montanez et al., 2008). Both paxillin and kindlin-2 are required for localization of the IPP complex to FAs, since an ILK mutant that does not interact with paxillin does not localize in FAs, and FA-like structures in the absence of kindlin-2 do not contain ILK (Nikolopoulos and Turner, 2001; Montanez et al., 2008). The IPP complex binds to β1 and β3 tails, and the actin cytoskeleton and regulates several signaling pathways (Legate et al., 2006). Kindlin-2 links integrins to the actin cytoskeleton via migfilin and filamin (Tu et al., 2003). It is unclear whether this interaction is important, as the targeted deletion of migfilin in mice does not cause any abnormalities (Moik et al., 2011). Whether kindlin-1 also connects integrins to the cytoskeleton remains to be determined. 13 Figure 7. Integrin activation and ligand binding leads to assembly of FAs. An inactive integrin (A) is activated by talin (B) and then binds to ECM (C) Subsequently, integrin clustering into FAs and connection to the actin cytoskeleton occurs via talin, paxillin, vinculin, FAK, and the IPP complex (from Legate et al., 2006). 2.2.2 Fibrillar adhesions The high phosphorylation degree of FAs -in contrast to fibrillar adhesions- suggests that FAs are the major sites of signaling, whereas fibrillar adhesions probably provide the cells with a firm anchorage (Zamir, et al., 2000). αvβ3 forms a tight bond with vitronectin, whereas α5β1 forms a tight bond with Fn. It is thought that fibrillar adhesions arise from the specific translocation of α5β1 and tensin from FAs, which starts at the margins of the FA towards the cell centre. Of all integrins, α5β1 is most efficient in binding to soluble Fn dimers and supporting a fibroblast-like, contractile cell shape by driving cytoskeletal organization (Danen et al., 2002). The binding of α5β1 to soluble Fn dimers results in a reorientation of FAs, increased Rho activity, and subsequent assembly of Fn dimers into fibrils, a process designated Fn fibrillogenesis (Huveneers et al., 2008; Pankov et al., 2000). It is suggested that actomyosin-driven contraction drives the formation of fibrillar adhesions. αvβ3 integrins bind to immobilized vitronectin or Fn and are not much affected by contraction forces from the cytoskeleton. By contrast, α5β1 binds soluble Fn, and is translocated out of FAs because of low tensional forces in a tensin-dependent manner (Fig 8; Zamir et al., 2000; Pankov et al., 2000). Fn fibrillogenesis is important for structural support from the ECM, and probably for ‘sensing’ the environment, or (Clark et al., 2005). 14 Figure 8. Differential distribution of FN-binding integrins αvβ3 and α5β1. (A) FAs contain mainly αvβ3 and additional proteins as vinculin and paxillin. Only small amounts of α5β1 and tensin are present. B) Actomyosin-driven contraction forces drive the formation of fibrillar adhesions. Whereas αvβ3 stays in place due to its association with stretched vitronectin or Fn (providing high tension), α5β1 binding to soluble Fn (low tension) allows its translocation. While tensin co-translocates with α5β1, vinculin and paxillin stay in FAs (from Zamir et al., 2000) 2.2.3 Regulation of outside-in signaling by the NPxY motifs As discussed in the previous chapter, inside-out activation of integrins is regulated by the cytoplasmic sequences of the β-subunit. Downstream of integrin activation and ligand binding, the same sequences are likely to also mediate outside-in signaling events. Talin-binding to the MP-NpxY motif promotes cell spreading either directly, because of its association with actin filaments, or indirectly by preventing the binding of filamin. Filamin, in addition to inhibiting integrin activation, also inhibits cell spreading by recruiting FilGAP, which prevents the activation of Rac required for cell spreading (Kiema et al., 2006; Nieves et al., 2010). Because talin-binding is essential for both integrin activation and cell spreading, a recent study made use of constitutively active chimeric integrins (containing a deletion of the GFFKR sequence in the α-subunit) to address the relative contribution of talin to outside-in signaling, independent of its effects on integrin activation (Nieves et al., 2010). Interestingly, prevention of talinbinding by a Y-A substitution in the MP-NPxY motif did not impair cell spreading, FA assembly or stress fiber formation in constitutively active integrins, implying that talinbinding is not absolutely required for outside-in signaling and that other proteins that do not bind to the NPxY motif can also regulate these processes. However, cell spreading was impaired when talin was depleted in cells expressing constitutively active integrins without Y-A mutations in the β-tail, but not in the presence of these mutations (Nieves et al., 2010). This inhibition could be relieved by depletion of filamin, suggesting that talin binding to the NPxY motif is important downstream of integrin activation to prevent the binding of filamin (Nieves et al., 2010). Some difference is observed between different integrins in the ability to bind talin or filamin, providing differential regulation of integrin activation, cell spreading and FA formation by talin 15 and filamin. How the differential binding of talin and filamin to integrins is regulated is not known yet (Nieves et al., 2010). The kindlins are also thought to mediate outside-in signaling events, as kindlin-3-deficient platelets are unable to spread even in the presence of Mn2+ to induce integrin activation (Moser et al., 2008). Similarly, Mn2+ stimulation of kindlin-2-deficient cells does not completely rescue cell spreading or FA assembly, whereas the few FAs that are formed contain paxillin but not ILK, suggesting that kindlin-1 is required for recruitment of the IPP complex (Montanez et al., 2008). Thus, it seems that in addition to integrin activation, the NPxY motifs are also important for outside-in signaling. The function of tyrosine-phosphorylation is again uncertain. In platelets, tyrosine-phosphorylation of both NPxY motifs is observed during platelet aggregation, secondary to agonist-induced αIIbβ3 activation and fibrinogen binding, and is thus an outside-in effect (Law et al., 1999). Platelets with Y-F substitutions in both NxxY motifs of β3 were defective in aggregation and clot-retraction in vitro, and in mice, the same mutations cause a mild bleeding defect (Law et al., 1999). These observations suggest that tyrosine-phosphorylation of both NPxY motifs is required for αIIbβ3 functions. In contrast, phosphorylation of the MD-tyrosine disrupts kindlin-2 binding to the β3-tail, suggesting that phosphorylation rather negatively impacts β3 functions (Bledzka et al., 2010). Furthermore, phosphorylation of the MP-NPxY in β1 impaired adhesion, assembly of FCs, and migration, whereas phosphorylation of the MD-NxxY motif was important for optimal migration (Sakai et al., 1998; Sakai et al., 2001). To reconcile conflicting results, it has been suggested that the dynamic regulation of phosphorylation and dephosphorylation is important, for example for integrin cycling in and out FAs (Sakai et al., 1998, 2001). In support of this is the observation that a Y-S mutation in either NPxY motif impairs integrin recruitment to FAs (Vignoud et al., 1997). Phosphorylation of the MP-NPxY motif occurs during the maturation of FAs, and promotes the binding of tensin (McCleverty et al., 2007). Phosphorylation does not increase the affinity of tensin, but promotes its binding probably indirectly, due to a decrease of talin affinity. Tensin recruits various kinases and RhoGAPs to FAs and thereby contributes to integrin signaling (Legate and Fässler, 2009). Because tensin is important for the translocation of α5β1 into fibrillar adhesions and for Fn fibrillogenesis, it has been suggested that phosphorylation of the MP-tyrosine in β1 is involved in Fn fibrillogenesis. However, a Y-F substitution in the MP-NxxY motif of β1 caused increased Fn matrix assembly, and it therefore seems that the phosphorylation of this motif negatively regulates Fn fibrillogenesis (Sakai et al., 2001). Thus, in vitro reports on the importance of tyrosine-phosphorylations of the NPxY motifs of β1 are controversial. Perhaps the best evidence that tyrosinephosphorylations are not important for β1 functions stems from mice carrying YY/FF mutations in both NPxY motifs of β1. As discussed in the previous chapter, these mice are completely normal, which does not support a role for tyrosine-phosphorylation either in integrin activation, or in outside-in signaling (Czuchra et al., 2006; Chen et al., 2006). In summary, both NPxY motifs are required for integrin-mediated events downstream of activation, and the MP-NPxY motif seems most important. The importance of tyrosine-phosphorylations of the NPxY motifs remains controversial, and probably differs between integrins; whereas for αIIbβ3 tyrosine-phosphorylation seems important, this is not the case for β1-integrins. 16 3 Integrin trafficking Internalization of integrins occurs by endocytosis (Fig 9). Endocytosed integrins travel from early endosomes (EEs) via late endosomes to multivesicular endosomes (MVEs), and ultimately end up in lysosomes for degradation. Alternatively, they are recycled to the plasma membrane. Newly synthesized integrins are delivered to the plasma membrane via the biosynthetic-secretory pathway. This pathway involves trafficking from the endoplasmic reticulum (ER) via the Golgi network to the plasma membrane. In the Golgi, integrins can be redirected to the ER, or targeted for lysosomal degradation, for example when they are not properly folded. Figure 9. A model for integrin traffic. Integrin-ECM interaction leads to clustering of integrins and formation of FCs. β1 integrins and ECM proteins are internalized with the help of dynamin and activated PKCα. Internalized β1 integrins travel to the early endosomes, from where they can be either degraded in lysosomes recycled to the PM via a Rab4-dependent mechanism (short loop) or continued to the perinuclear recycling compartment (PNRC; long loop). (from Pellinen and Ivaska, 2006) 3.1 Integrin endocytosis Endocytosis of integrins occurs mainly in a clathrin-dependent manner, although clathrin-independent mechanisms such as caveolin have been implicated as well (Table V; Shi & Sottile, 2008; Caswell et al., 2009). During clathrin-mediated endocytosis, three clathrin subunits form a clathrin triskelion. The triskelions assemble into pentagons or hexagons, thereby forming a coat around the vesicles (clathrin-coated vesicles; CCVs). Recruitment of integrins into CCVs requires the action of bridging proteins or adaptors 17 that bind both clathrin and the integrin (reviewed by Caswell et al., 2009). In addition, clathrin-mediated endocytosis requires proteins that mediate vesicle formation and fusion with target membranes. Regulators of clathrin-mediated endocytosis include dynamin, the AP1-AP4 adaptor complexes, Numb, Dab2, SNAREs (soluble NSF (Nethylmaleimide-sensitive fusion protein) attachment protein receptor), and Rabs. The GTPase dynamin assembles around the neck of the bud and influences the rate by which vesicles pinch off. AP2 is a heterotetrameric complex that is crucial for linking proteins to clathrin and for recruiting additional proteins required during endocytosis (Pearse et al., 2000). AP2 and other components of the endocytic machinery like dynamin are recruited by PIPK (Chao et al., 2009). AP2 binds directly to PIPK, and disruption of this interaction causes AP2 to recognize endocytosis signals on receptors and subsequent assembly of a clathrin coat (Bairstow et al., 2006). SNAREs regulate the docking and fusing of vesicles with target membranes and provide specificity by selecting for the appropriate target membrane. There are two types of SNAREs; vesicle-SNAREs (vSNAREs) and target-SNAREs (t-SNAREs). Together they bring the vesicle and the target membrane in close proximity to facilitate membrane fusion. Disrupting SNARE function impairs trafficking of α5β1, which interferes indirectly with integrin signaling, lamellipodium extension, cell migration, and FA turnover. Integrin signaling is probably impaired due to a lack of activation of a FAK/Src/PI3kinase-dependent pathway, which requires SNARE-dependent trafficking of Src to the membrane and subsequent FAK-Src interaction. Overexpression of membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and FA turnover in the absence of SNARE function (Skalski et al., 2011). Different SNAREs act in different parts of the endocytic or the biosynthetic-secretory pathway (Skalski et al., 2010; Xu et al., 2002; Mallard et al., 2002). The Rabs are proteins from the Ras superfamily of GTPases, which are essential for both the delivery of newly synthesized proteins to the plasma membrane, as well as for the endocytosis and recycling of plasma membrane proteins. Rab-dependent trafficking occurs along microtubules (Pellinen et al., 2006). The two main Rabs involved in Table V. Pathways for the internalization of integrins. (from Caswell et al., 2009) ‡ ‡A direct physical association with integrins has been shown, §Active conformation. AP2, adaptor protein 2; DAB2, disabled 2; HAX1, HCLS1-associated protein X1; L1CAM, L1 cell adhesion molecule; NRP1, neuropilin 1; GIPC, GAIP carboxy terminus-interacting protein; PKC, protein kinase C 18 integrin recycling are Rab4 and Rab11, which regulate a short-loop or a long-loop recycling route, respectively. The growth factor PDGF caused Rab4-dependent rapid recycling of αvβ3 but not α5β1, while in serum-starved cells internalized αvβ3 is trafficked from the Rab4-positive EEs to the Rab11-positive perinuclear recycling compartment (PNRC) and then recycled back to the plasma membrane, thus following the long-loop (Roberts et al., 2001). Protein kinase B (PKB)/Akt and PKC- are involved in the Rab11-dependent delivery of integrins to the plasma membrane, by promoting the release of integrins from the PNRC (Caswell & Norman, 2006). Only a small fraction of all integrins is present in CCVs, since around 0.1 to 1.3% (depending on the integrin) of the plasma membrane pool is internalized every minute, and CCVs have a half-life of 1-3 minutes (Bretscher 1992; Teckchandani et al., 2009; Puthenveedu & von Zastrow, 2006). A particularly actively cycling integrin is α5β1 (Bretscher, 1992). A fraction of the endocytosed α5β1 is trafficked from EEs via MVEs to lysosomes for degradation (Ng et al., 1999). This fraction is probably small and consists of ligand-bound integrins, since α5β1 and Fn colocalize in MVEs, whereas integrins that did not colocalize with Fn were mostly detected in EEs, suggesting that these are recycled to the plasma membrane. The α5-tail of α5β1 is ubiquitinated upon binding of Fn, which is required for the degradation, probably by constituting a sorting-signal in MVEs (Lobert and Stenmark, 2010). Probably, not all ligand-bound integrins are ubiquitinated for degradation, but a part may recycle via the long-loop, after being released from their ligand. Further experiments are needed to determine whether all ubiquitinated integrins are degraded or not, whether this is general for all integrins or cell types, and if and how integrin signaling proceeds after internalization. Ubiquitination-induced degradation ensures that occupied integrins are freed from their ligand, and is required for proper cell migration FA disassembly, whereas integrin recycling induces the formation of new adhesions to the ECM. Integrin activation is necessary for degradation but not for endocytosis, suggesting that both active and inactive integrins are internalized (Lobert and Stenmark, 2010; Teckchandani et al., 2009). Indeed, internalization of unligated integrins may be particularly important in migrating cells, to transfer them to the leading edge in order to form new adhesion sites (Jones et al., 2006; Caswell et al., 2009; Nishimura et al., 2007). This process does not seem to involve trafficking from the lagging to the leading edge, but rather the localized transport of integrins over short distances towards the leading edge (Rappoport and Simon, 2003; Nishimura et al., 2007). Dab2 may be required for bulk endocytosis of inactive integrins, since it colocalizes with β1 integrins spread over the entire cell surface, instead of only with active integrins residing in FAs (Teckchandani, et al. 2009). Endocytosis of unligated integrins is significantly delayed compared to the rapid endocytosis of active integrins after microtubule-induced FA disassembly (Chao et al., 2009). Thus, integrin trafficking is emerging as an important regulator of adhesion assembly and disassembly, and in this way it regulates cell adhesion and migration. Furthermore, remodeling of the matrix also requires endocytosis, and it seems that for the turnover of matrix-Fn, endocytosis of Fn-binding integrins via caveolae is important (Shi and Sottile, 2008). Caveolae are plasma membrane invaginations containing lipid rafts, the structural protein caveolin, and caveolin-associated cavins (Hansen and Nichols, 2010). At present, caveolin-mediated integrin endocytosis is poorly understood, but it seems to involve a direct association of PKCα with the β1-tail (Ng et al., 1999; Upla et al., 2004). 19 3.2 Anterograde integrin transport Both the biosynthetic-secretory pathway and the recycling of internalised integrins deliver integrins to the plasma membrane, and the combination of the two is referred to as anterograde transport. Protein kinase D1 (PKD1) binds specifically to the cytoplasmic tail of β3 and thus regulates anterograde transport of β3 integrins. Interestingly, depletion of PKD1 impairs all anterograde αvβ3 transport, whereas prevention of phosphorylation on residue 916 specifically prevents integrin trafficking via the Golgi network, but not integrin recycling (Liljedahl et al., 2001; Woods et al., 2004; White et al., 2007). Interestingly, talin is also involved in anterograde transport of newly synthesized integrins (Albiges-Rizo et al., 1995; Martel et al., 2000). Talin controls the exit of integrins from the ER to the Golgi, for which different explanations are given. One explanation is that talin might link integrin-containing vesicles to myosin VII or myosin X and thereby regulates the transport along the actin cytoskeleton. Alternatively, talin binding exposes a GFFKR sequence in the α-subunit that may act as an integrin export signal (Martel et al., 2000; O’Toole et al., 1991, 1994). It remains unclear how talin contributes to transport of newly synthesized integrins, and also how talin-integrin interaction at the ER are regulated. 3.3 Regulation of integrin trafficking by the β-subunit cytoplasmic tails Internalization of plasma membrane receptors is regulated by internalization signals in the cytoplasmic tails. Three types of internalization signals are distinguished; tyrosinebased sorting signals like NPxY or YxxØ motifs (Ø is a residue with a bulky hydrophobic side chain), dileucine-based sorting signals like [DE]xxxL[LI] or DxxL motifs, and ubiquitination of lysine residues (Bonifacino and Traub, 2003). Whereas NPxY motifs are required for the endocytosis of several receptors, it is controversial how integrin internalization is regulated by these motifs. Initial studies using Y-S substitutions in either one of the NPxY motifs did not impair endocytosis of α5β1, which may be explained because these mutations still allow formation of β-turns and thus not prevent binding of PTB-containing proteins, as discussed above (Vignoud et al., 1994). In β3, the MD-NITY motif seems to stimulate internalization. Of the three β3 integrin variants (β3A-C), only β3A contains NxxY motifs, and β3A is expressed at lower levels on the cell surface than the other isoforms (Gawaz et al., 2001). Internalization of β3A integrins is mediated by binding of endonexin to the MD-NITY motif, and a Y-A substitution in this motif or expression of dominant-negative endonexin increase the surface levels of β3A integrins. In another study it was shown that Y-A substitutions in the NPxY motifs of β3 did not diminish constitutive endocytosis whereas the internalization of ligand-coated particles was impaired suggesting that these motifs mainly regulate endocytosis of ligand-bound integrins (Ylänne et al., 1995). In addition, substituting either one of the NPxF motifs in β2 integrins for NPxA results in a loss of internalization, and Y-F mutations in both NpxY motifs of β1 also reduces endocytosis, probably as a result of a defect in clathrin binding (Rabb et al., 1993; Pellinen et al., 2008). The latter defect could be rescued by overexpression of Rab21, suggesting a clathrin-independent pathway of endocytosis (Pellinen et al., 2008). 20 3.4 Functional consequences of integrin trafficking It is becoming increasingly clear that regulation of integrin trafficking is important for optimal integrin functions during cell adhesion, cell spreading, and cell migration (Caswell et al., 2009). For example, inhibition of integrin recycling impairs cell adhesion and migration (Proux-Gillardeaux et al., 2005). The importance of integrin transport for cell migration has been studied in most detail for the Fn-binding integrins αvβ3 and α5β1. When αvβ3 is actively recycled, recycling of α5β1 is slow and vice versa, and it is not until the recycling of αvβ3 is diminished that α5β1 recycling increases (Caswell and Norman, 2008; White et al., 2007). As described above, PKD1 regulates both the shortloop recycling of αvβ3 as well as the transport of new αvβ3 to the membrane (Woods et al., 2004). PKD1 contributes to directionally-persistent migration by controlling recycling of αvβ3 integrins, whereas the PKD1-mediated regulation of integrin transport to the membrane probably enhances the speed of migration (White et al., 2007). Random cell migration occurs when α5β1 is actively recycled. If both αvβ3 integrins and α5β1 integrins are blocked, cells migrate in a directionally-persistent manner (White et al., 2007). Figure 10. Effect of integrin endocytosis on cell migration. A) Phosphorylation of Numb results in loss of Numb binding to integrin and subsequent loss of endocytosis, leading to directional persistent migration. B/C) Active recycling of α5β1 integrins results in random cell migration, while active recycling of αvβ3 integrins leads to directional persistent migration. (from Petrie et al., 2009) Numb localizes at FAs near CCVs and associates with several intracellular proteins involved in integrin endocytosis (Nishimura et al., 2007). Numb binds to the MP-NPxY motif, although one study showed binding to β1 and β3 integrins, while another only finds Numb associated with motif of β3 and β5 but not with β1A (Nishimura et al., 2007; Calderwood et al., 2003). Numb probably forms a complex with the polarity protein Par3 and atypical-PKC (aPKC), and phosphorylation of Numb by aPKC prevents association of Numb with integrins. Endocytosis is impaired when Numb is not associated with the integrin, leading to directionally persistent cell migration (Petrie et al., 2009; Nishimura et al., 2007). It is unclear whether this is caused only by impaired β1 endocytosis, as displayed in figure 10, or whether it is due to impaired endocytosis of 21 both β1 and β3 integrins (Nishimura et al., 2007; White et al., 2007). Suppression of Numb impairs clathrin-dependent integrin endocytosis, but to a lesser extent than suppression of AP2 and clathrin together, demonstrating that in addition to Numb, other clathrin-associated proteins such as Dab2 regulate integrin internalization (Nishimura et al., 2007). Like Numb, Dab2 also binds directly to the MD-NPxY motif of β-tails; according to one study binding does occur to β3 and β5 integrins, but not β1A integrins, while binding to β1 integrins was observed in two other studies (Calderwood et al., 2003; Teckchandani et al., 2009; Prunier & Howe, 2005). Dab-2 depletion leads to increased integrin surface expression, but reduction of the integrin intracellular pool. Reduced migration but not excess adhesion is observed in Dab2-deficient cells, and it is thought that maintenance of the intracellular integrin pool by Dab2 normally enables trafficking of integrins to the leading edge of the cell surface during cell migration to form new adhesion sites (Teckchandani et al., 2009). Numb and Dab2 differ in localization; whereas Numb localizes in FAs near CCSs at the leading edge, Dab2 is diffusely distributed over the cell surface (Nishimura et al., 2007; Teckchandani et al., 2009). In conclusion, integrins can follow different routes inside the cell, resulting in either recycling or degradation. It is likely that both active and inactive integrins can be internalized, although there may be some difference in the proteins involved in endocytosis. Since several studies give contradicting results, it remains unclear whether the NxxY motifs are important as internalization signals. 22 4 Discussion Integrins exist on the membrane in a conformation of low, intermediate, or high affinity for ligand, and the shift from low to high affinity is called integrin activation. This conformational change can occur from the inside, which is referred to as ‘inside-out’ signaling or activation. After ligand binding, integrins connect to the actin cytoskeleton, which is accompanied by the formation of adhesion plaques, cell spreading over the substrate, and the initiation of several signaling cascades (‘outside-in’ signaling). The paradigm of inside-out integrin activation has been developed mainly from studies on the platelet integrin αIIbβ3. Integrin αIIbβ3 and other integrins on non-adherent cells are ‘locked’ in the low-affinity conformation by a salt bridge between the cytoplasmic sequences of the - and the -subunits. Activation of αIIbβ3 occurs by GPCR agonists that trigger an intracellular pathway, of which the final step is talin binding to the -tail, which then induces a conformational change across the membrane, exposing the ligandbinding site. Although the mechanisms of αIIbβ3 activation are often assumed to be generic to all integrins, several findings especially from in vivo studies indicate otherwise. For example, YY/FF substitutions in the NPxY motifs of β3 in mice impair in vivo integrin function, while these substitutions do not affect the functions of β1 integrins in mice (Law et al., 1999; Czuchra et al., 2006; Chen et al., 2006). In addition, the salt bridge between the α- and β-subunit that stabilizes the inactive conformation is crucial for αIIbβ3, while mice carrying a D759A substitution in β1, which impairs the formation of a salt bridge, do not have any obvious defects (Anthis et al., 2009; Czuchra et al., 2006). Differences in the regulation of activation may reflect differences in specific integrin heterodimers, or differential modes of regulation existing between integrins on non-adherent versus integrins on adherent cells. The MP-NPxY and the MD-NxxY motifs in the cytoplasmic tail sequence of integrins are implicated both in integrin activation and outside-in signaling (table VI). Many studies have demonstrated that the MP-NPxY is important for talin binding, and that loss of talin binding impairs integrin activation (Calderwood et al., 2002; García-Alvarez et al., 2003; Bouaouina et al., 2008; Anthis et al., 2009; Tadokoro et al., 2003; Wegener et al., 2007). It seems that the talin-head is both necessary and sufficient for activation, while full-length talin (including the rod) is required for downstream events including FA assembly and cell adhesion and migration (Calderwood et al., 2003; Bouaouina et al., 2008; Zhang et al., 2008; Monkley et al., 2000; Nieswandt et al., 2007; Petrich et al., 2007). Interestingly, talin disruption or expression of only the talin-head in Drosophila does not impair integrin-ligand interaction but results nevertheless in an integrin-knockout phenotype, suggesting that inside-out events are less important than outside-in events in adherent cells in tissue. This is further underlined by studies that show that the conformational change can also be triggered by ligand, irrespective of cytoplasmic events (Du et al., 1991). It is thus unclear to what extent inside-out mechanisms contribute to integrin activation in adherent cells. The MD-NxxY motif is bound by kindlins, which are essential modulators of integrin function, as is clearly illustrated by the human disorders KS and LAD-III, as well as the phenotypes of kindlin-knockout mice (Moser et al., 2008; Ussar et al., 2008; Montanez et al., 2008). Whereas kindlin-3 is clearly essential for talin-induced activation of integrins on non-adherent cells, it is controversial whether kindlins are essential for inside-out activation in adherent cells. For instance, kindlin-3 does not enhance β1 activation in CHO cells, and kindlin-2 in αIIbβ3-expressing CHO cells has been reported to either 23 weakly activate or inhibit αIIbβ3 activation (Shi et al., 2007; Ma et al., 2008; Montanez et al., 2008; Harburger et al., 2009). In addition, talin binding alone can activate integrins without additional binding of kindlin, whereas kindlin binding alone cannot activate integrins (Ma et al., 2008). Similarly, a Y-A substitution in the MD-NxxY motif, which inhibits kindlin binding, does not prevent activation of αIIbβ3, but a Y-A mutation in the M-P-NpxY completely abolishes activation (Ye et al., 2010). An interesting possibility is that kindlins have integrin-specific effects, as suggested by a number of studies. Both kindlin-1 and kindlin-2 can activate αIIbβ3 with talin, while they inhibit talin-mediated α5β1 activation, and knockdown of kindlin-2 but not kindlin-3 decreases adhesion to vitronectin, while adhesion to fibronectin is affected by knockdown of kindlin-3 but not kindlin-2 (Harburger et al., 2009; Bialkowska et al., 2010). Accumulating evidence suggests that the kindlins regulate cell surface expression of integrins. Kindlin-3-deficient platelets express low surface levels of αIIbβ3 and α5β1 compared to wild-type platelets, and kindlin-3-deficient macrophages also express less integrins than wild-type macrophages (Moser et al., 2008; Schmidt et al., 2011). Similarly, expression of αIIbβ3 and β1 decreases upon knockdown of kindlin-2, and expression of β1 integrins in the epidermis of KS patients is low with respect to normal individuals (Lai-Cheong et al., 2009; Qu et al., 2011). Conversely, overexpression of kindlin-1 and kindlin-2 boosts the surface expression of both α5β1 and αIIbβ3 (Harburger et al., 2009). Interestingly, surface expression of β1 integrins in mouse keratinocytes isolated from mice carrying a YY/AA substitution in the epidermis is also significantly reduced, which is most likely due to a loss of kindlin binding, in light of the observations described above (Czuchra et al., 2006). Reduction of total integrin levels, in the absence of events that disrupt activation, was studied in hypomorphic mice expressing reduced β1 integrin levels in the epidermis. These mice develop a skin phenotype similar to mice that completely lack β1 in the epidermis, albeit delayed and less severe (Piwko-Czuchra et al., 2009). In vitro, reduced β1 integrin levels were sufficient to support keratinocyte adhesion, whereas outside-in signaling events as cytoskeletal reorganization, cell spreading and proliferation were impaired. This indicates that downstream of ligand binding and initial cell adhesion, maintenance of optimal integrin levels is required for a number of cell functions. Cell surface levels are maintained by trafficking mechanisms, and it is indeed increasingly recognized that regulation of integrin trafficking is important for integrin-mediated processes including cell adhesion, cell spreading, and cell migration (Caswell et al., 2009). A role for kindlins in the control of integrin trafficking may also explain the integrin-specific effects of kindlins, as the α-subunits are very important in determining the trafficking properties of the heterodimer. For example, the α-subunit GFFKR sequence, present in all αsubunits, is suggested to serve as an export signal, and mutations in this sequence cause reduced surface expression, as observed in some GT patients (Peyruchaud et al., 1998; Martel et al., 2000). Interestingly, exposure of the GFFKR upon talin-binding has been postulated to regulate export of newly synthesized integrins from the ER to the plasma membrane (Martel et al., 2000). In addition to membrane delivery, endocytosis and recycling likely depend on the α-subunits. For instance, whereas rapid and constitutive endocytosis occurs for α5β1 and αMβ2, no or low endocytosis is observed for α3β1, α4β1 and αLβ2 (Bretscher et al., 1992). In addition, the stabilizing effect of Dab2 on surface expression is restricted to α1β1, α2β1, and α3β1, but does not apply to α5β1 or αv-containing integrins (Teckchandani et al., 2009). The α-subunit may also stimulate endocytosis and lysosomal degradation by ubiquitination, as observed for α5 (Lobert et al., 2010). 24 In conclusion, it seems that talin-binding to the MP-NPxY motif is crucial for both insideout integrin activation and connection to the cytoskeleton. Whereas the importance of inside-out activation in non-adherent cells is clear, its relevance in adherent cells remains questionable. Kindlin-3 binding to the MD-NxxY motif is also essential for inside-out activation of integrins in non-adherent cells, whereas the effects of kindlins on talin-dependent activation in adherent cells are controversial, and can be stimulatory as well as inhibitory. Alternatively, the kindlins may control integrin functions by stabilizing their cell surface expression, probably by regulating integrin trafficking. Ultimately, integrin function will depend on the sum of integrin activation mechanisms -both from the inside and the outside- as well as efficient integrin turnover. The specific requirements for optimal integrin function are likely different for each specific heterodimer. 25 β1 Mutation /β3 In vivo β1 D759A β1 Y783F Phenotype Mice/cell lines Author viable, normal β1 integrin function viable, normal β1 integrin function mice mice β1 Y795F viable, normal β1 integrin function mice β1 YY783,795FF viable, normal β1 integrin function mice Czuchra et al., 2006 Czuchra et al., 2006; Chen et al., 2006 Czuchra et al., 2006; Chen et al., 2006 Czuchra et al., 2006; Chen et al., 2006 Czuchra et al., 2006; Chen et al., 2006 Czuchra et al., 2006 β1 YY783,795AA not viable β1 YY783,795AA inactive β1 integrins, reduced expression mice mice; basal keratinocytes β1 YY783,795FF impaired function, active β1 integrins, normal expression mice; in platelets only β1 YY783,795AA inactive β1 integrins, reduced expression mice; in platelets only β1 YY783,795FF reduced invasion and adhesion, normal metastatic capacity ESb lymphoma cells β1 T788,789A impaired invasion, adhesion, metastatis to liver, normal ESb lymphoma cells metastasis to spleen β3 YY747,759FF tendency to rebleed, normal β3 expression, normal mice platelet count In vitro β1 D759A normal β1 integrin function mouse keratinocytes β1 YY783,795FF normal β1 integrin function mouse keratinocytes β1 YY783,795FF normal-improved adhesion to Fn, laminin and collagen mouse ES cells β1 YY783,795AA inactive β1 integrins, impaired adhesion to Fn, laminin and mouse ES cells collagen β1 Y783F normal β1 integrin function ESb lymphoma cells β1 Y795F failed to express Y795F in Esb lymphoma cells ESb lymphoma cells β1 YY783,795FF reduced invasion, poor adhesion ESb lymphoma cells β1 TT788,789AA impaired invasion, reduced adhesion ESb lymphoma cells β1 Y795F normal β1 integrin function Jurkat T cell A1 β1 Y795A adhesion to Fn reduced, inactive β1 integrins, normal Jurkat T cell A1 expression β1 del MP-NPxY adhesion to Fn reduced, normal β1 integrin expression Jurkat T cell A1 β1 del MD-NPxY adhesion to Fn reduced, normal β1 integrin expression Jurkat T cell A1 β1 D759A active β1 integrin, normal migration GD25 cells β1 Y783F active β1 integrin, normal adhesion, Fn matrix formation, GD25 cells reduced migration β1 Y795F active β1 integrin, normal adhesion, Fn matrix formation, GD25 cells reduced migration β1 YY783,795FF active β1 integrin, normal adhesion, Fn matrix formation, GD25 cells impaired migration β1 Y783S lack of recruitment to FAs CHO cells β1 Y795S lack of recruitment to Fas CHO cells β1 YY783,795SS lack of recruitment to FAs CHO cells β1 Y783F active β1 integrin CHO cells β1 Y783A inactive β1 integrin CHO cells β1 Y795F active β1 integrin CHO cells β1 Y795A active β1 integrin CHO cells β3 Y747A inactive β3 integrin, impaired recruitment to FAs, impaired CHO cells cell spreading on Fn β3 Y759A β3 integrin activation reduced, normal recruitment to FAs, CHO cells reduced cell spreading on Fn β3 YY747,759FF no aggregation upon thrombin-stimulation mouse platelets Chen et al., 2006 Chen et al., 2006 Stroeken et al., 2000 Stroeken et al., 2000 Law et al., 1999 Czuchra et al., 2006 Czuchra et al., 2006 Chen et al., 2006 Chen et al., 2006 Stroeken et al., 2000 Stroeken et al., 2000 Stroeken et al., 2000 Stroeken et al., 2000 Romzek et al., 1998 Romzek et al., 1998 Romzek et al., 1998 Romzek et al., 1998 Sakai et al., 1998 Sakai et al., 1998, 2001 Sakai et al., 1998, 2001 Sakai et al., 1998, 2001 Vignoud et al., 1997 Vignoud et al., 1997 Vignoud et al., 1997 O'Toole et al., 1995 O'Toole et al., 1995 O'Toole et al., 1995 O'Toole et al., 1995 O'Toole et al., 1995; Ylänne et al., 1995 O'Toole et al., 1995; Ylänne et al., 1995 Law et al., 1999 Table VI. Implications of mutations in the NPxY motifs on integrin functions in vivo and in vitro. 26 5 Abbreviations βTD CHO Col CCV Dab DAG E-Cadherin ECM EE EGF ER FA FAK Fb FC FERM Fg Fn FX GT GPCR HD ICAM iC3b ILK KS LAD LAP-TGF-β Ln MAdCAM-1 MD MIDAS MP MVE Nn Op PECAM-1 PIPK PI4,5P2 PKC PKD1 PNRC PSI PTB Rap-GEF RGD TGN Tn TSP VCAM-1 Vn vWF = β-tail domain = Chinese Hamster ovary = collagen = clathrin-coated vesicles = disabled = diacylglycerol = epithelial cadherin = extracellular matrix = early endosome = epidermal growth factor = endoplasmic reticulum = focal adhesion = focal adhesion kinase = fibrillin = focal complex = four-point-one, ezrin, radixin, moesin = fibrinogen = fibronectin = factor X = Glanzmann thrombasthenia = G-protein coupled receptor = hemidesmosome = intercellular adhesion molecule = inactive complement factor 3b = integrin-linked kinase = Kindler syndrome = leukocyte adhesion deficiency = latency-associated peptide of transforming growth factor-β = laminin = mucosal addressin cell adhesion molecule-1 = membrane-distal = metal ion-dependent adhesion site = membrane-proximal = multivesicular endosome = nephronectin = osteopontin = platelet endothelial cell adhesion molecule-1 = phosphatidylinositol phosphate kinase = phosphatidylinositol-4,5-biphosphate = protein kinase C = protein kinase D1 = perinuclear recycling compartment = plexin/semaphorin/integrin = phosphotyrosine-binding domain = Rap-guanine exchange factor = Arg-Gly-Asp = trans Golgi network = tenascin = thrombospondin = vascular cell adhesion molecule-1 = vitronectin = von Willebrand Factor 27 6 Reference list 1. 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