It has been reported that Src family kinases are important signaling molecules from integrin to Ras and that c-Src and Ras play a. Activation of integrin.

Agonist stimulation of integrin receptors, composed of transmembrane α and β subunits, leads cells to regulate integrin affinity (‘activation’), a process that controls cell adhesion and migration, and extracellular matrix assembly. A final step in integrin activation is the binding of talin to integrin β cytoplasmic domains. We used forward, reverse and synthetic genetics to engineer and order integrin activation pathways of a prototypic integrin, platelet αIIb β3. PMA activated αIIb β3 only after expression of both PKC α (protein kinase C α) and talin at levels approximating those in platelets. Inhibition of Rap1 GTPase reduced αIIb β3 activation, whereas expression of constitutively active Rap1A(G12V) bypassed the requirement for PKC α.

Overexpression of a Rap effector, RIAM (Rap1-GTP-interacting adaptor molecule), activated αIIb β3 and bypassed the requirement for PKC α and Rap1. In addition, shRNA (short hairpin RNA)-mediated knockdown of RIAM blocked talin interaction with and activation of integrin αIIb β3. Rap1 activation caused the formation of an ‘activation complex’ containing talin and RIAM that redistributed to the plasma membrane and activated αIIb β3. The central finding was that this Rap1-induced formation of an ’integrin activation complex’ leads to the unmasking of the integrin-binding site on talin, resulting in integrin activation.

Integrin receptors Integrin adhesion receptors are glycosylated heterodimers composed of non-covalently associated type I transmembrane α and β subunits [-]. Each subunit contains a large extracellular domain (N-terminus of >700 residues), a single transmembrane domain (>20 residues) and a usually short cytoplasmic domain (C-terminus of 13–70 residues). Membrane-proximal regions of the cytoplasmic tails are well conserved in most integrin subunits, and the boundary between the transmembrane and cytoplasmic domains is assumed to lie between the conserved tryptophan/tyrosine and lysine/arginine residues [,]. This conserved lysine/arginine residue is followed by a stretch of four to six apolar residues, resulting in ambiguity about the beginning of the integrin cytoplasmic domain [,].

The membrane-proximal sequences GFFKR and LLXXXHDRRE are conserved in the α and β subunits respectively [,]. Many integrins are expressed and remain in a low-affinity binding state until cellular stimulation transforms them into a high-affinity form, an event that can modify cell adhesion [,]. Because information travels from the inside to the outside of the cell, this is often referred to as ‘inside-out’ signalling [,,]. This form of signalling is often precisely regulated in time and space in settings such as platelet aggregation and leucocyte transmigration [,].

A major focus of recent research has been affinity-dependent regulation of integrin-mediated adhesion, a process that is operational as integrin activation [-]. Transmembrane propagation Integrin activation requires transmission of conformational rearrangements from the cytoplasmic face to the extracellular domain, thus implicating the involvement of the integrin transmembrane domain in the signal transduction process. These regions are highly conserved between integrins and are also conserved between species []. Mutational analysis and computational modelling together suggest that a helical interface between the integrin α and β subunit transmembrane domains stabilizes the inactive state, and suggest that disruption of this helical transmembrane interface leads to activation [-]. Four motions could contribute the proposed rearrangements in the transmembrane domain and the alterations of α– β transmembrane interface: separation, pistoning, twisting and hinging [,,].

Each involves some changes in orientation of the subunits relative to one another and to the membrane. Although recent work supports the separation and the pistoning models, high-resolution structures of the transmembrane domains will be required to distinguish clearly between the different models [,,]. Helical packing of integrin transmembrane regions is likely to require specific crossing angles and in-register alignment of side-chain arrays []. Furthermore, the insertion of the integrin membrane-proximal domain could vary, thus shortening or lengthening the transmembrane domain and changing the number of residues buried within the lipid bilayer [,,,]. The pistoning model suggests that intracellular activating signals could shorten the transmembrane helix, thereby changing membrane tilt angle and register with the neighbouring helix to avoid hydrophobic mismatch with the fixed width of the membrane bilayer.

This change in tilt could be the critical event in disruption of transmembrane interactions that stabilizes the low-affinity conformation, leading to integrin activation [,,]. In support of this model, the majority of activating transmembrane mutations identified by random mutagenesis are predicted to shorten the transmembrane helix [,].

Changes in the length or orientation of the integrin transmembrane domain may also lead to physiological integrin activation []. Intracellular rearrangements Changes in interactions and/or in the structures of the cytoplasmic domains of integrins within the membrane-proximal regions play crucial roles in integrin activation via inside-out signalling [,,,]. The interaction between the membrane-proximal regions of the α and β subunits is believed to be stabilized by a salt bridge between a conserved arginine residue in the α tail and an aspartate residue in the β tail and by the hydrophobic residues immediately N-terminal to the arginine and aspartate residues [,,].

This association between the α and β subunits is thought to prevent integrin activation by stabilizing the low-affinity state [,,]. Mutations that disrupt this ‘clasp’ lead to integrin activation, and integrins can be constitutively activated by deletion or mutations in these membrane-proximal sequences [,,-]. Furthermore, replacement of the cytoplasmic transmembrane regions by heterodimeric coiled-coil peptides inactivates the receptor, and cleavage of the coiled-coil activates integrins [-]. These data together strongly indicate that this membrane-proximal interaction plays an essential role as a stabilizer in the association of the α and β membrane-proximal regions, maintaining the integrins in a low-affinity state [,,].

The important role of α and β tail interaction is further supported by FRET studies that show that activation is associated with a change in separation or orientation of α and β cytoplasmic tails relative to each other [,]. The integrin cytoplasmic domains play a central role in integrin activation [,], and overexpression of particular proteins that bind to the cytoplasmic tails can result in integrin activation [,-]. Binding of the adaptor, talin, to the β cytoplasmic domain is a crucial, final step in activation of several classes of integrins [-]. This finding has been confirmed for β1 [], β2 [,,] and β3 [] integrins; hence, talin appears to play a general role in activating multiple classes of integrins.

Moreover, a study using mice harbouring point mutations in the β3 cytoplasmic tail provided the first in vivo evidence supporting the importance of talin binding for integrin activation in mammals []. Talin is a major cytoskeletal protein that co-localizes with and binds to integrins and to actin and actin-binding proteins such as vinculin [,-]. It forms an antiparallel homodimer made of ~270 kDa subunits, each of which consists of an N-terminal globular head domain of ~50 kDa and a C-terminal rod domain of ~220 kDa [,]. The head domain contains a FERM (4.1/ezrin/radixin/moesin) domain comprising three subdomains: Fl, F2 and F3.

F2 and F3 subdomains of talin bind specifically to integrin β3 tails, although F3 shows a higher affinity than F2 []. In addition, expression of F3, but not F2 or other high-affinity β tail-binding proteins, activates αIIb β3 integrins, implying that the major integrin-binding and activating fragment of talin lies within the 96-residue F3 subdomain [,]. Knockdown of talin expression in CHO (Chinese-hamster ovary) cells inhibits the activation of both β1 and β3 integrins without altering integrin expression, and this cannot be compensated for by the expression of activating molecules such as activated R-Ras or the CD98 heavy chain []. Furthermore, talin knockdown prevents agonist-stimulated fibrinogen binding to megakaryocyte integrin αIIb β3, suggesting that normal cellular activation of integrins also requires talin [].

The talin F3 structure resembles a PTB (phosphotyrosine-binding) domain, which recognizes ligands containing β turns formed by NPXY motifs []. NPXY motifs are well conserved in most integrin β tails, and mutations that disrupt this motif perturb β-turn formation, inhibiting talin binding and interfering with integrin activation [,].

Similarly, mutations within the talin PTB-like domain prevent integrin β tail binding and thereby block integrin activation [,]. Thus the integrin β tail–talin interaction represents a general mechanism for integrin activation. A major question is how talin binding activates integrins.

When the ability of activating talin fragments to bind to different regions of the β3 cytoplasmic domain was compared with that of non-activating talin fragments (i.e. The F2 domain) by monitoring the perturbation of specific NMR resonances of the β tail, F2–F3 and F3 fragments, but not F2, showed distinct perturbation of the membrane-proximal region of the β3 tail, suggesting the involvement of the β3 tail membrane-proximal region in talin-mediated integrin activation []. As noted above, mutations in this region result in integrin activation. NMR studies suggest that direct disruption of α– β tail interaction by the talin head domain result in integrin activation [].

In addition, F2–F3 and F3 also perturb the more distal region of the β3 tail []. The membrane-distal region of the integrin β tail provides a substantial fraction of the binding energy and has been suggested to contribute to integrin activation []. These data together suggest a two-step activation model: the talin head domain first recognizes the high-affinity binding site in the membrane-distal region, which provides a strong linkage between the talin and the integrin β tail, and subsequently binds to a second lower-affinity membrane proximal site that is involved in α– β association, triggering separation of the tails and integrin activation () [,]. Model of talin-induced integrin activation Many other PTB-domain-containing proteins bind to integrin β tails in a similar fashion to talin [,].

Such proteins include Numb, Dok-1, ICAP-l α (integrin cytoplasmic domain-associated protein-l α) and Kindlins [,], yet these proteins either fail to activate or are much less active than talin. Thus there must be additional unique features in the integrin–talin interaction that enable talin to cause activation []. A flexible loop between β-strands 1 and 2 of the F3 domain of talin accepts the side chains of the membrane-proximal region of the integrin β tail. This mobile loop is absent from other PTB-domain-containing proteins, which implies that this may be the unique feature that distinguishes talin from other PTB-domain-containing proteins in its ability to activate integrins. Furthermore, a mutation within this loop blocks talin binding to the membrane-proximal region and hinders integrin activation. In addition, when the talin F3 domain engages the β membrane-proximal region, additional favourable electrostatic contacts between the F3 and the lipid headgroups of the membrane bilayer could stabilize further the talin– β tail association. Mutation of one of the predicted contact residues in talin F3 also blocked activation.

These data suggest that the formation of a complex between integrin β tail membrane-proximal helix and the talin F3 domain, together with the favourable electrostatic contacts between the talin F3 and the lipid membrane, contribute to the energy required to stabilize the integrin-activated state []. Recent studies show that two other FERM-domain-containing proteins, MIG-2 (mitogen-inducible gene 2) and radixin, interact with the cytoplasmic tails of β1 and β3 and of β2 respectively [,]. Furthermore, those studies indicate that these interactions may have context-dependent roles in integrin activation [,]. Thus, although talin is an important player in integrin activation mechanisms, the possibility remains that other integrin tail-binding proteins can substitute for talin. Regulation of integrin activation As noted above, numerous different signalling pathways can regulate integrin activation, and talin binding to the β tail is often a final step. The issue now is to understand how these different signalling pathways intersect with talin binding. Previous studies have identified Rap1 GTPase as a potent activator of integrins [-].

Many cytokines and growth factors promote integrin-dependent cell adhesion through the activation of Rap1 []. Furthermore, Rap1 regulates integrins that are associated with the actin cytoskeleton, such as integrins of the β1, β2 and β3 family []. In patients with LAD-III (leucocyte adhesion deficiency III) who suffer from defects in leucocyte and platelet integrin activation, β1, β2and β3 integrins are expressed at normal levels.

Cell from these patients are impaired in their abilities to bind to integrin ligands with high affinity in response to agonists [,]. Although expression of Rap1 and talin are normal in these patients, a mutation in CalDAG-GEFI (Ca 2+ diacylglycerol guanine-nucleotide-exchange factor I), which is a key Rap1/2 GEF (guanine-nucleotide-exchange factor) [], is associated with LAD-III.

Similarly, CalDAG-GEFI −/− mice exhibit defects in activation of platelet and leucocyte β1, β2 and β3 integrins and therefore impaired inflammatory response and lack of thrombus formation []. These findings together reveal that CalDAG-GEFI is a critical regulator of inside-out integrin activation in human T-lymphocytes, neutrophils and platelets, and emphasized the importance of Rap1 signalling pathways [,]. In the last few years, many Rap1 effectors have been identified. Among these, RIAM (Rap1-GTP-interacting adaptor molecule) [,] is clearly implicated. Overexpression of RIAM induced the active conformation of integrins and enhanced cell adhesion, whereas depleting RIAM eliminated adhesion mediated by Rap1 []. Cell-type-specificity of integrin-affinity regulation by these signalling pathways is probably due to variations in expression of these GTPases or of the upstream and/or downstream elements that link them to integrin activation.

Recent work has ordered a pathway from agonist stimulation to integrin activation for the first time, using a synthetic approach to reconstruct an integrin activation pathway in CHO cells () []. The central finding was that Rap1 induces formation of an integrin-activation complex containing RIAM and talin, which in turn leads to the unmasking of the integrin-binding site on talin; a critical, final, step in integrin activation. Moreover, these key components of the pathway, Rap1, RIAM and talin, are widely used in many cellular contexts and with various integrins []. ΑIIb β3 is a prototypic integrin, and many of the principles of integrin function established by early studies with this integrin have proved to be widely applicable across the entire integrin family [].

Platelet agonists, including PMA, thrombin, ADP and collagen, stimulate increased affinity of αIIb β3 integrin in platelets; these agonists have failed to activate recombinant αIIb β3 expressed in CHO cells or several other nucleated cells [,]. This puzzling observation has led to the reconstruction of an integrin-activation pathway in CHO cells []. This study illustrates the principle that variations in cellular abundance of the components of the integrin-activation complex such as talin or in their regulators such as PKC α (protein kinase C α) can account for the cell-type-specificity of integrin activation [].

Moreover, this reconstructed model also provides a system that can be used to manipulate the signalling events involved in integrin activation and to integrate and rationalize the currently available literature, and to connect different agonists and signalling pathways that are known to control integrin activation []. Similarly, it will allow for quantitative and mutational analyses of signalling pathways that regulate integrin activation. The analysis may be extended further to agonist receptors [e.g. PAR1 (protease-activated receptor 1)], G-proteins (e.g.

Rap1, G αq) and tyrosine kinases (e.g. Tec kinases) that have been implicated in integrin activation.

Abstract Higher levels of focal adhesion kinase (FAK) are expressed in colon metastatic carcinomas. However, the signaling pathways and their mechanisms that control cell adhesion and motility, important components of cancer metastasis, are not well understood. How To Crack Zip File Password Protected Files On Usenet Binaries. We sought to identify the integrin-mediated mechanism of FAK cleavage and downstream signaling as well as its role in motility in human colon cancer GEO cells. Our results demonstrate that phosphorylated FAK (tyrosine 397) is cleaved at distinct sites by integrin signaling when cells attach to collagen IV. Specific blocking antibodies (clone P1E6) to integrin α2 inhibited FAK activation and cell motility (micromotion).

Ectopic expression of the FAK C-terminal domain FRNK attenuated FAK and ERK phosphorylation and micromotion. Calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal blocked FAK cleavage, cell adhesion, and micromotion.

Antisense approaches established an important role for μ-calpain in cell motility. Expression of wild type μ-calpain increased cell micromotion, whereas its point mutant reversed the effect.

Further, cytochalasin D inhibited FAK phosphorylation and cleavage, cell adhesion, locomotion, and ERK phosphorylation, thus showing FAK activation downstream of actin assembly. We also found a pivotal role for FAK Tyr 861 phosphorylation in cell motility and ERK activation. Our results reveal a novel functional connection between integrin α2 engagement, FAK, ERK, and μ-calpain activation in cell motility and a direct link between FAK cleavage and enhanced cell motility. The data suggest that blocking the integrin α2/FAK/ERK/μ-calpain pathway may be an important strategy to reduce cancer progression.

Cell adhesion is of fundamental importance in that it affects cell motility, cell differentiation, signal transduction, and cell invasion (). Changes in cell adhesion are facilitated by the integrin family of cell surface receptors of extracellular matrix (ECM) proteins. The integrins link cell surface cytoskeletal proteins like focal adhesion kinase (FAK) to actin as well as other cytoskeletal proteins. Significantly, it was shown in the past decade that integrins can directly activate intracellular signaling processes and are thus important signal transduction receptors for biological function.

The mammalian integrin receptor family is composed of at least 18 distinct α-subunits and 9 β-subunits, thereby generating 28 distinct integrins through various modes of association (, ). GEO colon cancer cells express integrin α2β1, which is formed by a noncovalent association of the α2 subunit as a monogamous partner to the promiscuous β1 subunit. Expression of integrin α2 is regulated during normal cell differentiation and is altered during tumorigenesis (). The expression of integrin α2 has been correlated with metastatic behavior in breast cancer, hepatocarcinoma, and rhabdomyosarcoma (, ).

Our results further support this linkage in colon carcinoma as well (–). Integrin α2 is an important collagen receptor on platelets and epithelial cells (). Collagen type IV (CN IV) is the major component of the basement membranes, and a metastatic cell invades the basement membranes to make its way to the target tissue or organ. There have been few mechanistic studies concerning integrin α2-mediated cellular adhesion and motility in human colon carcinomas (–, ). At present, the mechanisms underlying bidirectional signaling triggered by integrins are poorly understood. Ligand binding to integrins is generally regulated to reflect the activation state of the cell. Inside-out regulation of integrin affinity protects the host from pathological integrin-mediated adhesion such as in thrombosis, inflammation, and infectious diseases (,, ).

The phosphorylation of cytosolic proteins plays an important role in inside-out signaling. Inside-out and outside-in signaling are associated with distinct conformational changes in the integrin extracellular domain (, ). Our experiments were aimed at determining the mechanism by which human colon cancer GEO cells activate FAK by “outside-in signaling” upon attachment to CN IV, which in turn controls cell adhesion and motility (inside-out signaling). One of the earliest insights about integrin signaling was the observation that integrin-mediated adhesion and/or clustering led to enhanced tyrosine phosphorylation of a nonreceptor tyrosine kinase now called FAK (–). The N-terminal domain of FAK, including its erythrocyte band 4.1-ezrin-radixin-moesin (FERM) region, directs interactions with other molecules such as integrins, c-Src, and phospholipase γ.

This domain contains a major tyrosine autophosphorylation site at residue 397. Its C-terminal noncatalytic domain includes FAK-related nonkinase (FRNK), a domain for multiple protein-protein interactions, as well as a focal adhesion target region. Both FRNK and focal adhesion target domains act as dominant negatives for cell adhesion functions (). Recently, compelling evidence has indicated a role by FAK in the pathology of human cancer ().

High levels of FAK expression have been associated with increased invasiveness in malignant tumors. Increased levels of FAK in 17 of 20 invasive colon tumors and in all 15 metastatic tumors were reported by Weiner (), whereas FAK gene expression was not detected in normal colon tissue. Recently, it has been suggested that up-regulation of FAK occurs at an early stage of tumorigenesis (). A progressive increase in FAK mRNA levels was observed as tumor invaded and metastasized (). The phenotype of FAK-deficient mice is embryonic lethal due to delayed embryonic migration.

This phenotype is also reminiscent of fibronectin or integrin α5-deficient mice, further supporting the concept that extracellular matrix (ECM), integrins and FAK are closely linked (). FAK –/– cells exhibit reduced migration, whereas cells overexpressing FAK display increased migration on fibronectin (, ). However, the mechanisms of FAK activation, cleavage, and deregulation of motility, a critical aspect of tumor progression, are not clear, and no clear model has emerged as to how FAK signaling functions in combination with integrin α2, ERK, and calpain to promote cell motility. FAK also regulates integrin signaling by recruiting cytoskeletal and signaling proteins via multiple adaptor domains. The absence of cytoskeletal proteins like FAK or paxillin or mutations in the tyrosine residues of these components result in dramatic effects on cell adhesion and motility (). The translocation of these proteins via the actin cytoskeleton is induced independently of growth factor receptor kinase activity ().

Gaps remain in understanding the sequential events that occur to assemble a focal contact upon cell adhesion. Recently, it was reported that FAK Tyr 861 was crucial for H-Ras-induced transformation in fibroblasts through association of FAK with p130 CAS (). The mechanism(s) by which FAK tyrosine sites promote cell motility is less clear. Cell motility requires a critical balance between cell attachment and detachment, and it requires the complex integration of motility-promoting and motility-inhibiting signals.

Calpain has been shown to be a critical regulator of cell motility (). Calpain is a cysteine protease activated by increased intracellular Ca 2+ that localizes to focal adhesions, potentially causing cleavage of focal adhesion proteins. The calpain family includes at least 13 known members, of which two calpain isozymes, μ-calpain and m-calpain, are implicated in cell adhesion, spreading, and migration (–). Tumors that have metastasized have been found to have higher levels of calpain than those that are not metastatic (). The μ- and m-calpains are phosphorylated, thus enabling their role in signal transduction.

However, examination of the precise role of calpains in signal transduction has just begun. It is still obscure as to how the calpains are activated by clustering of integrins.

Whereas linkage of calpains with several pathological conditions has been reported, their role in cancer is poorly understood (). It appears that the function of calpains in cell adhesion and motility may be limited their ability to cleave components of focal complexes in a limited fashion (remodeling), which in turn may increase adhesion turnover. The molecular mechanism of such events is not known and may be deregulated in cancer.

Our data demonstrate a direct link between FAK cleavage and enhanced cell motility in colon cancer cells. Recently, we showed that in human colon carcinoma HCT116 cells, ERK plays an important role mediating endogenous cellular control of integrin α2 expression, cell adhesion, and motility (). Previously, Miyamoto et al. () have shown that integrin aggregation can initiate activation of the ERK signal transduction pathway. () have shown that integrin-mediated ERK activation was via FAK activation, whereas growth factors responded via Shc signaling in NIH3T3 fibroblast cells, suggesting important differences in the activation of ERK/mitogen-activated protein kinase signaling by different ligands. These studies did not address the role of ERK/mitogen-activated protein kinase signaling in cell adhesion and motility. Recent experiments suggest that integrin activation of mitogen-activated protein kinase may be independent of FAK or Ras, through the use of alternative or parallel routes ().

Here, we identify a role for ERK/mitogen-activated protein kinase signaling in colon cancer cell motility via a novel integrin α2/FAK/MEK/calpain pathway. EXPERIMENTAL PROCEDURES Materials—Methylthiazole tetrazolium (MTT), CN IV, Me 2SO, BSA, cytochalasin D, soy bean trypsin inhibitor, and polyclonal anti-actin antibody were purchased from Sigma. Monoclonal blocking antibodies P1E6 (α2), P1B5 (α3), and P1D6 (α5) and antibodies specific for integrin α2 and α3 subunits, μ-calpain, and m-calpain were purchased from Chemicon International (Temecula, CA), whereas the mouse isotype control IgG 1 was from R & D Systems (Minneapolis, MN). Antibodies specific for integrin α5 and β1 (clone 18) were obtained from BD Biosciences. Calbiochem provided the calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) and the proteasome inhibitor lactacystin. The MEK inhibitor U0126 was obtained through Promega (Madison, WI). Antisense phosphothiorate-linked nucleotides for μ-calpain and m-calpain were synthesized by Integrated DNA Technologies, Inc.

(Coralville, IA). The polyclonal anti-FAK phosphospecific antibodies were provided by BIOSOURCE (Camarillo, CA), whereas the rabbit anti-human C-terminal FAK (SC-558), anti-human N-terminal FAK (SC-557), ERK, phosphorylated ERK1/2, and cyclin E antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Microarrays of gold film-coated electrodes as well as the electric cell-substrate impedance-sensing (ECIS) software (model 1600R) used for cell motility (micromotion) experiments were purchased from Applied Biophysics (Troy, NY).

Mouse and rabbit peroxide-conjugated AffiniPure goat IgG (H + L) secondary antibodies were from Jackson Laboratories (West Grove, PA). The reagents for cell transfection, Oligofectamine and Lipofectamine, were purchased from Invitrogen, whereas FuGENE 6 was purchased from Roche Applied Science.

Cell Culture—Trypsinization, Replating Assays, and Immunoblotting. GEO cells were cultured in a humidified incubator at 37 °C for 3–4 days with 5% CO 2 in a chemically defined serum-free medium. At about 80% confluence, the medium was changed to supplemented McCoy's (SM) medium (). Cells were harvested with trypsin (0.125%) at 37 °C and pelleted by gentle centrifugation at 800 × g in a clinical centrifuge. The pellet was resuspended in SM medium, soy bean trypsin inhibitor (0.5 mg/ml) was added, and further recovery of the cells was allowed for 1 h at 37 °C in a shaker incubator. Cells were then kept either in suspension or replated to attach for the indicated time periods to dishes precoated with CN IV (5 μg/ml).

The precoating of culture dishes with CN IV was performed by incubating dishes for 2 h at 37°C. Subsequently, nonspecific sites were blocked with 3% BSA for 3 h at room temperature and finally washed with 10 ml of cold PBS.

After cell attachment at 37 °C, dishes were placed on ice and washed gently with cold PBS. The cells were lysed either in suspension or after attachment to precoated CN IV dishes. The lysis buffer A contained 50 m m Tris, pH 7.5, 150 m m NaCl, and 1% Nonidet P-40 along with protease inhibitors (1 m m phenylmethylsulfonyl fluoride, 2 m m EDTA, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 50 m m β-glycerophosphate, 1 m m benzamidine).

The cells attached to CN IV were harvested with 75 μl of lysis buffer/100-mm dish, whereas those that were in suspension were lysed with 50 μl of lysis buffer. The lysates were centrifuged in a microcentrifuge at 4 °C for 20 min at 16,000 × g. The supernatants were used for protein determination by the Bio-Rad protocol, and equal amounts of protein aliquots were separated by SDS-PAGE and analyzed by Western blotting. Immunoprecipitation—GEO cells attached to CN IV were lysed as described above.

The lysates (100 μg of protein) were precleared with normal rabbit serum and protein A/G-agarose beads (Calbiochem/Oncogene) at 4 °C for 1 h. The supernatants were incubated with rabbit anti-FAK antibody (N-terminal) overnight at 4 °C with rotation. Protein-antibody complexes were incubated with 35 μl of protein A/G-agarose beads for 2 h at 4°C. After extensive washing with lysis buffer, the beads were boiled with 2× Laemmli buffer and analyzed for FAK by immunoblotting with anti-FAK antibody (N-terminal). Biotinylation—To determine cell surface expression of integrins, GEO cells were biotinylated as described previously (–). Briefly, subconfluent GEO monolayers were treated with Joklik's EDTA for 8 min at room temperature, and cells were then scraped and pelleted by centrifugation. The pellet was washed twice with cold PBS, and cells were biotinylated with NHS-LC-Biotin (Pierce), 0.1 mg/ml in Me 2SO at room temperature for 1 h.

The biotinylated cells were lysed in buffer A, sheared through a 26-gauge needle, and centrifuged at 16,000 × g for 20 min at 4 °C. Cell lysates were incubated with streptavidin agarose for 90 min at 4 °C, and beads were washed five times with lysis buffer.

The beads were boiled in 2× Laemmli buffer for 7 min, supernatant was filtered through Bio-Rad columns, and proteins were separated by SDS-PAGE. The integrins were analyzed by immunoblotting using specific antibodies against α and β1 subunits. Short-term Cell Adhesion Assays—For adhesion assays, 96-well tissue culture plates were coated for 2 h at 37°C with CN IV at the indicated concentrations, blocked with 3% bovine serum albumin for 3 h, and then rinsed once with PBS. Subsequently, the MTT procedure was followed as described previously (, ).

After trypsinization (0.125% trypsin in EDTA), GEO cells were preincubated at 37 °C with or without inhibitors for 2 h to determine cell adhesion. Cells were plated at 6 × 10 4 cells/well on precoated CN IV plates and incubated for 90 min in the absence or presence of inhibitors. Nonadherent cells were removed by washing with SM medium. The relative number of attached cells was determined by the MTT method (). Synthesis of Calpain Antisense Oligonucleotides—Based on the published effective sequences (), antisense phosphothiorate-linked nucleotides for μ-calpain (calpain I) and for m-calpain (calpain II) were synthesized by Integrated DNA Technologies (Coralville, IA). The nucleotides were dissolved in sterile water and stored at –20 °C before use.

The effects of the oligonucleotides were validated by analyzing the lysates from antisense oligonucleotide-treated cells for endogenous calpain levels by Western blotting. Plasmid Constructs and GEO Cell Transfection—The FAK and kinase-dead FAK (kinase-defective FAK with a Lys 454 → Arg mutation in the ATP binding site) constructs were provided by Drs. Jun-Lin Guan (Cornell University, Ithaca, NY), and the FRNK construct was a gift of Dr.

Schlaepfer (The Scripps Research Institute, La Jolla, CA). The HA-tagged dominant negative mutant Y861F and WT FAK were gifts of Dr. Eok-Soo Oh (Seoul, Korea) (). The hemagglutinin-tagged construct of μ-calpain and its mutant (His 272 → Ala) subcloned into the mammalian expression vector pcDNA3 were described previously () (provided by Drs. Kulkarni; Cleveland Foundation). GEO cells were grown in serum-free medium containing 4 μg/ml transferrin and 20 μg/ml insulin at 37 °C with 5% CO 2. For transient transfection experiments, cells were grown to 50–60% confluence, medium was changed to SM, and transfections were carried out using FUGENE 6 according to our previous report ().

The ratio of FUGENE 6 to DNA was maintained at 10 μl of FUGENE 6 to 2.5 μg of DNA. The DNAs were mixed with the FUGENE 6 and set at room temperature for 45 min.

The mixture was then added dropwise to the cells. The cells were harvested at 48 h post-transfection.

Cell Motility (Micromotion) Measurements by the ECIS Technique—Cell micromotion was measured using the ECIS technique previously reported (–,, ). In the current system (model 1600R), cells were seeded at 1 × 10 5 cells/well on small gold electrodes (diameter 250 μm) at the bottom of tissue culture wells (area 0.5 cm 2). The gold electrodes were preincubated with 200 μl of supplemental McCoy's medium for at least 1 h. A constant current source applied a noninvasive 1 μA, 4000 Hz AC signal between the small electrode and a much larger counter electrode (0.15 cm 2). Any variation of current due to cell movement was recorded. The ECIS software (Applied BioPhysics, Troy, NY) calculated the resistance and capacitance values of the electrode over a period of time. Attachment and movement of the cells on the electrode change the flow of the current, resulting in fluctuations in the electrode resistance and capacitance.

These cellular movements are called micromotion () and are a measure of the motile ability of the cell being measured. As the cells move on the electrode, the sensitive nature of the lock-in amplifier detects the fluctuations in the resistance and capacitance values (). These fluctuations were analyzed statistically using ECIS software to reveal the percentage variation in resistance, which in turn is a reflection of cellular micromotion on the electrode.

Since the measurements are electrical, they are quantitative and generate data that can be analyzed readily to provide sensitive measurements of changes in cell behavior. This technique detects both translational ( xy plane) and vertical ( z direction) movement of cells (). RESULTS GEO Cells Predominantly Express Integrin α 2β 1—To determine the expression of various integrin receptors, GEO cells were cultured, harvested by Joklik's EDTA, biotinylated, and lysed as described previously (–). The lysates were analyzed by immunoblotting for protein expression of integrin α2β1, α3β1, and α5β1 using specific antibodies against the subunits. Shows that GEO cells predominantly express integrin α2β1, a lower extent of integrin α3β1, and very little if any integrin α5 subunit. GEO Cells Attach to CN IV (and Not to CN I) in a Concentration-dependent Fashion—There are several reports that integrin α2 is a receptor of CN I in different cell types (, ).

To determine whether that is the case with GEO cells, we tested attachment to CN I (1–5 μg/ml) as compared with BSA. To our surprise, GEO cells that predominately express integrin α2 did not significantly attach to CN I. Instead, under identical experimental conditions, GEO cells attached to CN IV in a concentration-dependent manner (). These results show that the ligand for integrin α2 receptor is cell type-specific. Adhesion of GEO Cells to CN IV Is Mediated Predominantly by Integrin α 2—The specificity of integrin α2 in mediating adhesion of GEO cells to CN IV was further determined by treatment with specific functional blocking antibodies to inhibit binding to CN IV. Monoclonal anti-integrin α2 antibody (clone PIE6) was highly effective in preventing GEO cell adhesion to CN IV, and inhibition was concentration-dependent (). Inhibitory levels ranged from 70 to 20% at antibody dilutions ranging from 1:50 to 1:500.

Blocking antibody to the integrin α5 subunit had no effect on GEO cell adhesion to CN IV. Anti-integrin α3 subunit had much less inhibitory effect (6–30% at 1:50 dilution) on GEO cell adhesion to CN IV. Integrin subunitsα3 andα5 are the predominant cell adhesion receptors for laminin and fibronectin, respectively. Plating on CN IV Enhances Tyrosine Phosphorylation of Proteins in GEO Cells—In an effort to identify proteins that are activated by cell adhesion to CN IV, GEO cells were allowed to attach to CN IV-coated dishes for 20 or 60 min in SM medium. Cells kept in suspension served as a control for nonintegrin-mediated signaling. Antiphosphotyrosine immunoblot analyses of cell lysates using 4G10 primary antibody revealed several phosphorylated proteins. In contrast to cell suspensions ( S), cell adhesion ( A) to CN IV resulted in significantly enhanced tyrosine phosphorylation of ∼190-, 125-, 90-, 70-, and 40-kDa molecular mass proteins ().

Expression of integrin α2β1, α3β1, and α5β1( A), comparison of cell adhesion on CN I and CN IV ( B), and inhibition of cell adhesion to CN IV by antibodies to integrin receptors ( C). GEO cells were harvested at near confluence, biotinylated, and lysed by integrin lysis buffer.

The lysates were analyzed by 7.5% polyacrylamide gel electrophoresis and immunoblotting with antibodies against human integrin α subunits as detailed under “Experimental Procedures.” The membranes were stripped at 50 °C for 30 min in stripping buffer (100 m m β-mercaptoethanol, 2% SDS, 62.5 m m Tris-HCl, pH 6.8), blocked with 5% nonfat dry milk for 1 h, and immunoblotted either for integrin β1 or for actin using specific antibodies ( A). 96-well tissue culture plates were coated with different concentrations of CN I or CN IV as indicated.

GEO cells were seeded at 6 × 10 4 cells/well onto coated plates and incubated for 90 min at 37 °C. The relative number of attached cells was determined by MTT assay as described under “Experimental Procedures” ( B). 96-well tissue culture plates were coated with CN IV (5 μg/ml), and blocking monoclonal antibodies to integrin α2, α3, and α5 subunits were added at different dilutions as indicated. The control IgG 1 was used at 1:50 dilution. Lane 1, without antibody; lanes 2–4, integrin α2 antibody; lanes 5–7, integrin α3 antibody; lane 8, integrin α5 antibody ( C). Adhesion assays were performed as detailed under “Experimental Procedures.”. Cell Attachment to CN IV Leads to FAK Activation and Cleavage—To identify proteins in, equal amounts of total protein from cell suspension and cell attachment lysates were fractionated by SDS-PAGE.

Western blot analysis using specific phosphoantibodies showed that in contrast to suspended cells, attachment to CN IV enhances tyrosine phosphorylation and cleaves phosphorylated FAK (Tyr 397) to a 90-kDa fragment (). Cells in suspension showed either little or no FAK cleavage. GEO cells attached to CN IV exhibited FAK cleavage at both 20 and 60 min. At 60 min, the cleavage of FAK was greater than at the 20-min period. The 90-kDa fragment in total FAK is detected by the anti-human N-terminal FAK (, bottom). Integrin α 2 Mediates FAK Phosphorylation—We hypothesized that integrin α2 plays a pivotal role in FAK activation. To test this hypothesis, GEO cells were incubated with specific integrin α2-blocking antibodies (P1E6) for 30 min at 37 °C prior to replating on precoated CN IV dishes.

The phosphorylation of FAK was significantly blocked by P1E6 in GEO cells (). Cell Attachment to CN IV Activates ERK1 and ERK2—In addition to FAK activation and cleavage, the same membrane was stripped and analyzed by specific phosphorylation antibodies against extracellular signal-regulated kinase (ERK) activation. Western blot analysis using a specific phospho-Tyr 204 antibody showed that the kinetics of ERK activation were similar to FAK activation and cleavage (). These results suggested that integrin α2-mediated FAK activation was regulating downstream ERK signaling. Cytochalasin D Inhibits FAK Activation, Cell Adhesion, and Haptotactic Micromotion in a Parallel Fashion—Actin polymerization is critical in formation of focal adhesions when cells attach to extracellular matrix protein.

To determine the relationship between actin stress fiber organization and FAK activation and cleavage, GEO cells were treated with cytochalasin D (0.50 μ m), an agent that binds to the barbed ends of F-actin filaments and prevents actin polymerization. Shows that cyto D inhibits integrin α2-mediated activation and cleavage of FAK. These results indicate that the integrity of the endogenous actin cytoskeleton is essential for FAK activation and cleavage. These results support the hypothesis that integrin α2-stimulated FAK activation is mediated by actin cytoskeletal association.

Actin polymerization is critical in cell spreading and motility; therefore, to evaluate the significance of intact actin, we performed adhesion assays and cell motility experiments using cyto D as the inhibitor. As shown in, we observed 45% inhibition of GEO cell adhesion to CN IV with cyto D (0.25 μ m) as compared with Me 2SO-treated control cells. These results confirm that actin organization is important in GEO cell adhesion to CN IV. Next, to determine the effect of CN IV on cell motility, we evaluated haptotactic cell micromotion using CN IV as a substrate. The ECIS technique was used to quantitate cell micromotion in GEO cells. Shows that in the uncoated electrode well (control), the percentage variation in resistance measured over a period of 70 min was 0.8467% ( top) as compared with the percentage variation 2.147% of precoated CN IV electrode well ( bottom).

The results show that GEO cells attached to CN IV had an increase in motion of 154%. Adhesion of GEO cells to CN IV induces tyrosine phosphorylation of proteins ( A), FAK cleavage ( B), FAK dephosphorylation by P1E6 ( C), and ERK activation ( D). GEO cells were trypsinized, incubated with soy bean trypsin inhibitor for 1 h at 37°C, and either kept in suspension or replated to dishes coated with CN IV (5 μg/ml) in SM medium. Cells were allowed to attach to CN IV for 20 or 60 min at 37 °C. Cellular lysates from cells in suspension ( S) and cells attached ( A) were analyzed for phosphotyrosine proteins by immunoblotting with antiphosphotyrosine antibody 4G10 ( A). The immunoblots were analyzed for activation of FAK ( B; upper left) or for FAK protein by using rabbit anti-human N-terminal FAK antibody ( B; lower left).

The right panel shows that both 125-kDa FAK and its cleaved 90-kDa form are immunoprecipitated and immunoblotted by N-terminal FAK antibody. GEO cells in SM medium were incubated either with mouse IgG 1 or with integrin α2-blocking mAb (clone P1E6) at dilution 1:50 for 30 min at 37 °C. Subsequently, cells were transferred to dishes precoated with CN IV and incubated for 1 h at 37°C. The cell lysates were analyzed by immunoblotting for pFAK and FAK using specific antibodies ( C). Cellular lysates from cells in suspension ( S) and cells attached ( A) were also analyzed for ERK (Tyr 204) by immunoblotting with specific antibody ( D).

Molecular weight standards are indicated in A. The positions of pFAK (125 kDa) and its major cleaved fragment (90 kDa), ERK1 (44 kDa), and ERK2 (42 kDa) are indicated by the arrowheads. Total FAK and ERK panels are shown for equal loading. Micromotion measured by the ECIS technique is directly related to conventional cell motility ().

The ECIS technique has been used to detect cell morphology, cell motility, and cell-ECM interactions in different systems (, ). To determine the role of actin polymerization in cell locomotion, cells (6 × 10 4) were plated on electrodes precoated with CN IV. The subconfluent cultures were treated with 0.25 and 0.50 μ m concentrations of cyto D, cells were allowed to attach for 3 h, and then micromotion was recorded. Shows that in the control GEO cells, the percentage variation in resistance was 2.309% ( DMSO; top).

Treatment of the cells with cyto D (0.25 μ m; middle) decreased the percentage variation in resistance to 1.241%, indicating a decrease (46.25%) in cell micromotion. When GEO cells were treated with higher concentrations of cyto D (0.50 μ m; bottom), we observed a further decrease (69.64%) in cell micromotion (percentage variation in resistance was 0.702%), thus demonstrating that the inhibitory effect of cyto D on cell micromotion was concentration-dependent. These results show that actin polymerization plays a pivotal role in GEO cell micromotion on CN IV.

Taken together, the data demonstrate that actin polymerization plays a critical role in FAK phosphorylation and cleavage, cell adhesion, and locomotion on CN IV. Functional Blocking mAb to Integrin α 2 Attenuates Cell Micromotion—To establish that cell micromotion on CN IV was specifically integrin α2-mediated, GEO cells were preincubated either with mouse IgG or with functional blocking mAb (clone PIE6) before recording micromotion by the ECIS technique. The percentage variations in resistance observed were 2.363% (control IgG) and 0.504% (PIE6). As shown in, the suppression in cell micromotion (resistance) of GEO cells by PIE6 was 78.7%, as compared with control IgG-treated cells. ALLN Inhibits FAK Cleavage, Cell Adhesion, and Cell Micromotion in a Parallel Fashion—To explore the possibility that integrin α2-mediated signaling may activate calpains, which in turn cleave focal adhesion kinase, GEO cells were incubated in the presence of acetyl-leucyl-leucyl-norleucinal (ALLN), a cell-permeable calpain inhibitor (), for 3 h at 37 °C prior to trypsinization and replating of cells on precoated CN IV dishes. Cell lysates were analyzed by Western blot using an antibody specific to FAK Tyr 397. There was a dramatic decrease in FAK cleavage in ALLN-treated lysates as compared with control (Me 2SO-treated) lysates ().

These results show a critical role of calpain(s) in the cleavage of FAK in GEO cells. To determine the function of FAK cleavage by calpain(s) in cell adhesion, assays were performed on CN IV-coated wells. Shows that integrin α2-mediated cell adhesion was inhibited by ALLN (46–85%) in a manner that was dependent on the concentration of CN IV. To further define the role of calpain(s) in cell motility, we used the real time ECIS technique. The electrode wells were precoated with CN IV, and GEO cells (1 × 10 5) were inoculated in SM medium. Cells were allowed to attach for 3 h, and then micromotion was measured under the same experimental conditions in the presence of either Me 2SO or ALLN. Shows that in the control Me 2SO-treated cells, the percentage variation in resistance was recorded as 1.496%.

Treatment of the cells with ALLN decreased the fluctuations such that the percentage variation in resistance was now 0.433%, indicating a 71% decrease in cell micromotion by ALLN. These results demonstrate that inhibition in FAK cleavage by calpain(s) attenuates cell motility. Alternatively, the results may be interpreted as showing that cleavage of FAK enhances cell motility. Effect of cyto D on FAK phosphorylation ( A) and on cell adhesion to CN IV ( B).

The ligand CN IV enhances cell micromotion ( C), whereas cyto D attenuates micromotion in a concentration-dependent fashion ( D). GEO cells were treated either with Me 2SO (control) or with cyto D for 24 h. After trypsinization, cells were transferred to CN IV-precoated dishes and incubated at 37 °C in the presence of Me 2SO or cyto D for 1 h. Lysates were collected and analyzed by Western blot using an antibody specific to phospho-FAK (Tyr 397) ( A). GEO cells were trypsinized after treatment with Me 2SO or with cyto D for 24 h. Cells were then inoculated at 6 × 10 4 cells/well into BSA- or CN IV-coated 96 well plates and incubated at 37 °C for 90 min in the presence of Me 2SO ( DMSO) or cyto D as indicated. Nonadherent cells were washed off, and adherent cells were determined by MTT assay as described under “Experimental Procedures” ( B).

GEO cells in SM medium were seeded (6 × 10 4) in microarray wells either uncoated ( top) or coated with CN IV ( bottom). After 3 h of attachment, micromotion was recorded ( C). Precoated CN IV (5 μg/ml) electrode arrays were used in these experiments. GEO cells were monitored for cell micromotion either in the absence ( top) or presence of cyto D (0.25 μ m ( middle), 0.5 μ m ( bottom)) ( D).

Lactacystin Does Not Attenuate FAK Cleavage—Although ALLN is widely known as a selective calpain inhibitor, it may inhibit the 26 S proteasome pathway (). To confirm that the effect of ALLN was selective in inhibiting FAK cleavage and did not inhibit cleavage via the proteasome pathway, GEO cells were treated with different concentrations of lactacystin, a selective proteasome inhibitor. The cell lysates were analyzed for cyclin E, as a positive control, by immunoblotting (). The results showed that lactacystin treatment slightly enhanced cyclin E accumulation at 10 μ m concentration, whereas 20 μ m lactacystin dramatically increased the accumulation of cyclin E (, top).

Under the same conditions, cells treated either with Me 2SO or with 20 μ m lactacystin were replated on CN IV-coated dishes for 1 h in the presence of Me 2SO or lactacystin (20 μ m). The lysates were analyzed for pFAK (Tyr 397) and total FAK. GEO cells treated with lactacystin did not show inhibition in FAK cleavage as compared with control (Me 2SO) cells (, bottom). These results show that the 26 S proteasome does not play a significant role in the cleavage of FAK in GEO cells, thus supporting the notion that inhibition of FAK cleavage by ALLN is calpain-dependent. ALLN inhibits FAK cleavage ( A) and cell adhesion ( B), whereas lactacystin does not inhibit FAK cleavage ( C). GEO cells in SM medium were preincubated either in the presence of Me 2SO (control) or calpain inhibitor ALLN (100 μ m) for 3 h prior to trypsinization.

After trypsinization, cells were replated on CN IV-coated dishes and incubated for 1 h at 37 °C in the absence or presence of ALLN. Lysates were collected and analyzed by Western blot using an antibody specific to phosphorylated FAK (Tyr 397) ( A; left). For the MTT adhesion assay ( B), GEO cells were preincubated in the presence of either Me 2SO (control) or the calpain inhibitor ALLN (100 μ m) for 3 h prior to incubation on different concentrations of CN IV as indicated and described under “Experimental Procedures.” C, GEO cells were treated either with Me 2SO or with lactacystin (5, 10, and 20 μ m) for 24 h, and cell lysates were analyzed for cyclin E, as a positive control, using specific antibodies (C; top), whereas actin was used as a loading control.

Lysates treated either with Me 2SO or lactacystin (20 μ m) were analyzed for pFAK or total FAK using specific antibodies as indicated ( bottom panel). Critical Role of μ -Calpain in Cell Adhesion and Micromotion—Cell motility is dependent on cell-substrate attachment at the leading edge of the cell in coordination with cell-substrate detachment at the rear of the cell. The attachment at the leading edge of the cell is associated with the formation of the focal adhesion complexes, whereas detachment at the rear of the cell is associated with the disassembly of the focal adhesion complexes and the proteolytic cleavage of the proteins that make up the focal adhesion complexes (,, ). The major calpains expressed by GEO cells are μ- and m-calpains. To determine whether one or both forms of calpains contribute to cell micromotion, we used two approaches. First, specific antisense nucleotides for both calpains, anti-μ-calpain (5′-ACTCCTCTGTCATCCTGGGG-3′) and anti-m-calpain (5′-TGCCCGCCATGGTAGCGATC-3′) were synthesized ().

The antisense nucleotides inhibited endogenous levels of respective calpains as determined by the Western blot analyses using specific antibodies (). The micromotion was recorded in the absence or presence of these antisense nucleotides. The percentage variation in resistance of control cells was 5.537% (, top), whereas in the presence of μ-antisense nucleotide, the percentage variation in resistance decreased to 2.674% ( middle), thus showing a 52% inhibition in cell micromotion.

Simultaneous recording of micromotion of GEO cells in the presence of m-calpain antisense nucleotides showed a percentage variation of 4.857% in resistance (, bottom), indicating a small decrease in micromotion (12%). Each set of nucleotides functioned as a control for the other. These results provide evidence for the major role of μ-calpain in micromotion. Effect of calpain antisense nucleotides on endogenous levels of calpains ( A) and on micromotion ( B) and effect of μ-calpain and mutant constructs on cell adhesion ( C) and micromotion ( D).

GEO cells were treated either with anti-μ- or with anti-m-calpain oligonucleotides (20 μ m) for 24 h in a 24-well plate. The monolayers were washed with cold PBS.

Cell lysates were analyzed by Western blot for μ-calpain and m-calpain levels using specific antibodies ( A). The immunoblot images were captured by the VersaDoc Imaging System with chemiluminescence capability (model 5000; Bio-Rad). For micromotion experiments, GEO cells in SM medium were trypsinized and seeded (6 × 10 4 cells) on CN IV-precoated microarray wells. Cells were treated either with vehicle ( top) or with μ-calpain antisense nucleotide (20 μ m; middle) or with m-calpain antisense nucleotide (20 μ m; bottom). Cells were allowed to attach for 3 h, and then micromotion was recorded ( B). For C and D, GEO cells were transfected for 48 h either with empty vector pcDNA3, WT HA-μ-calpain construct, or μ-calpain mutant in which His 272 had been mutated to Ala.

Adhesion assays were performed by MTT methodology, and micromotion experiments were monitored by the ECIS technique as explained under “Experimental Procedures.”. To further confirm the role of μ-calpain in cell adhesion and motility, we used another approach, which included transfection of cells with μ-calpain plasmid. GEO cells were transiently transfected either with empty vector (pcDNA 3) or with WT HA-μ-calpain expression vector. First we examined the effect of overexpression of μ-calpain on cell adhesion to CN IV by an MTT assay (). The results showed 69% inhibition in cell adhesion by WT μ-calpain-transfected cells as compared with control empty vector transfectants. Since ectopic WT μ-calpain expression inhibits cell adhesion, therefore, a mutant of μ-calpain in which histidine 272 has been mutated to alanine would be expected to reverse the inhibitory effect of WT μ-calpain.

Under same experimental conditions, we observed only 32% inhibition in cell adhesion to CN IV by the mutant (as compared with 69% inhibition by WT μ-calpain). These experiments strongly support the role of μ-calpain in integrin α2-mediated GEO cell adhesion. Next, we demonstrated a cause and effect relationship between calpain-mediated adhesion and motility triggered by integrin α2 signaling. GEO cells were transfected either with empty vector or with the construct WT μ-calpain or its mutant. The transfectants were seeded at a density of (1 × 10 5) cells/well on CN IV-precoated microarray wells.

Cells were allowed to attach for 3 h, and then micromotion was monitored by ECIS (). The percentage variation in resistance of μ-calpain plasmid-transfected cells (2.224%;, middle) was 167.3% higher than empty vector-transfected cells (percentage variation 0.832%;, top). The increase in micromotion by μ-calpain transfectant was blocked by the μ-calpain mutant where histidine 272 was mutated to alanine (percentage variation 0.839%;, bottom).

FRNK Inhibits FAK Activation and Cell Micromotion, whereas Kinase-dead Mutant Increases Cell Micromotion—The mechanism(s) of FAK phosphorylation and its cleavage in cell motility is not well understood. To determine the role of FAK in cell motility, GEO cells were transiently transfected either with empty vector (pcDNA 3.1) or with HA-tagged FRNK (gift of Dr. Schlaepfer), which acts as a dominant negative mutant of FAK and interferes with FAK signaling. The validity of FRNK function is demonstrated by Western blot analysis as shown in. The effect of FRNK transfection on cell micromotion () shows that the percentage variation in resistance (2.297%) of empty vector-transfected cells was reduced by 60.62% in FRNK-transfected cells (percentage variation in resistance 0.905%).

These results show that the carboxyl domain of FAK contributes to increased cell motility. In contrast to the inhibitory effect of FRNK on cell micromotion, the FAK kinase-dead plasmid transfectants (; percentage variation in resistance 4.537%) showed enhanced micromotion (68%) on CN IV as compared with control empty vector-transfected GEO cells (percentage variation in resistance 2.701%). These results indicate that in GEO cells, the kinase domain of FAK is at least partly dispensable for micromotion on CN IV. FRNK inhibits FAK phosphorylation and cell micromotion ( ), whereas kinase-dead FAK enhances micromotion ( ). GEO cells were transfected with appropriate empty vectors, WT FRNK, or kinase-dead FAK constructs in SM medium. Cell lysates from empty vector or WT FRNK transfectants were analyzed by Western blot using specific antiphospho-FAK antibody ( top) or with FAK antibody ( bottom). Shows a comparison of micromotion between empty vector (pcDNA3.1) and WT FRNK transfectants and a comparison of empty vector (pKH3) and kinase-dead FAK construct.

Micromotion experiments were performed in precoated CN IV microarray wells. Dominant Negative Mutant Y861F Inhibits Cell Adhesion and Haptotactic Cell Micromotion—The tyrosine 861 site is located in between the proline 1- and proline 2-rich regions of the FAK carboxyl-terminal domain.

The contribution of Tyr 861 to cell motility is poorly understood. First, to determine whether the Tyr 861 site is activated by integrin α2-mediated signaling, GEO cells were either kept in suspension or replated on CN IV-precoated dishes. We observed enhanced Tyr 861 phosphorylation with attachment of cells to CN IV (). Therefore, we examined the role of Tyr 861 phosphorylation in cell adhesion and motility. GEO cells were transfected either with empty vector (pRC/CMV) or with the mutant Y861F (where Tyr 861 has been mutated to a phenylalanine residue). Adhesion assays showed that cell attachment to CN IV by the mutant was suppressed by 52–57%, depending on the concentration of CN IV as compared with empty vector transfectants ().

The effect of the mutant on cell micromotion was similar to that of cell adhesion. We observed that the percentage variation in resistance of control cells was 2.007%, whereas the mutant Y861F-transfected GEO cells recorded percentage variation in resistance 0.969% (). These values indicate attenuation of cell micromotion by 51.7% by the mutant.

These results show that phosphorylation of the Tyr 861 site promotes cell motility in GEO cells, thereby playing an important role in cell adhesion. Western blot analysis () shows that overexpression of WT FAK enhances Tyr 861 phosphorylation, as compared with control empty vector transfectants, whereas cotransfection with dominant negative mutant Y861F blocks phosphorylation. Role of FAK Tyr 861 in cell adhesion and micromotion.

GEO cells cultured in SM medium were trypsinized, incubated with soy bean trypsin inhibitor, and kept either in suspension or replated on precoated CN IV dishes. Cell lysates were analyzed by Western blot analysis using site-specific phospho-FAK (Tyr 861) antibody ( top) or FAK antibody for equal loading control ( bottom) ( A). GEO cells were transfected either with empty vector (pRC/CMV) or with the mutant Y861F. The transfectants were seeded in a 96-well plate precoated with CN IV as indicated and analyzed by the MTT methodology as described under “Experimental Procedures” ( B). GEO cells were transfected with empty vector ( C; lane 1), WT FAK ( lane 2), or cotransfected with WT FAK and mutant Y861F ( lane 3) as described under “Experimental Procedures.” Cell lysates were analyzed by Western blotting for Tyr 861 using specific phosphoantibodies. Actin was used as a loading control.

For micromotion experiments, data are presented in, and experiments were performed as described under “Experimental Procedures.”. Inhibition of ERK Activation and Cell Micromotion by U0126—Since GEO cells enhance ERK phosphorylation upon attachment to CN IV (), we examined the role of downstream ERK/mitogen-activated protein kinase signaling in motility. GEO cells were allowed to attach to collagen IV-precoated electrode wells in the absence or presence of U0126, a well known MEK inhibitor (). Those cells treated with MEK inhibitor showed about 48% attenuation in micromotion (; right; percentage variation in resistance 0.676%) as compared with those in the Me 2SO-treated control cells (; left; percentage variation in resistance 1.311%). It is noteworthy that inhibition in micromotion was directly linked to inhibition in ERK activation (Tyr 204) by U0126 as determined by Western blotting (, top). To further illustrate the mechanism of integrin α2-mediated ERK phosphorylation, we performed the following experiments. Effect of Cyto D, Dominant Negative FRNK, FAK Y861F Mutant, and μ -Calpain Mutant on ERK Phosphorylation—Our results show that cyto D dephosphorylates activated FAK ().

To determine the functional link between FAK and downstream ERK phosphorylation, GEO cells were treated with cyto D, trypsinized, and replated on CN IV-coated dishes for 1 h. The cell lysates were analyzed for ERK phosphorylation by Western blot using specific antibodies. The inhibitory effect of cyto D on ERK phosphorylation showed that in GEO cells, intact actin is important for both FAK and downstream ERK activation (Figs. To confirm the role of FAK in ERK activation, GEO cells were transiently transfected either with empty vector (pcDNA 3.1) or with dominant negative FRNK plasmid. After transfection, cells were trypsinized and replated on CN IV for 1 h.

The cell lysates were analyzed for ERK phosphorylation by Western blotting using specific antibodies. Shows that DN FRNK diminished activation of ERK. The results confirm that downstream ERK activation, in response to integrin α2 stimulation, is mediated by FAK signaling.

The experiments described above showed that FRNK attenuates ERK activation. Therefore, to determine the role of FAK Tyr 861 (located in the FRNK domain) in ERK activation, GEO cells were transfected either with empty vector pRC/CMV or with the mutant Y861F.

Cell lysates were analyzed for ERK by Western blotting with a specific phosphorylation antibody (Tyr 204;, top) or with ERK antibody (, bottom). Results showed that the mutant Y861F suppressed activation of ERK, demonstrating that the Tyr 861 phosphorylation site plays an essential role in FAK-mediated downstream ERK activation in GEO cells. To confirm the link between μ-calpain activation and ERK signaling, GEO cells were transfected either with empty vector or with the dominant negative calpain mutant in which histidine 272 has been mutated to alanine. Immunoblotting with specific antibodies (Tyr 204) showed that the mutant attenuated ERK phosphorylation (). These results show that μ-calpain regulates downstream ERK phosphorylation.

These experiments establish a direct link between FAK signaling and downstream ERK activation in GEO cells. DISCUSSION To define the mechanism of integrin-mediated signaling (outside-in signaling) in cell motility (inside-out signaling), we first determined the expression of integrins by human colon cancer GEO cells. Western blot analysis showed that the major integrin expressed by GEO cells was integrin α2β1. GEO cells also express integrin α3β1 to a smaller extent and do not express integrin α5 protein.

To determine the ligand for integrin α2, adhesion assays were performed using CN I or CN IV as substrates. The results showed that GEO cells attached to CN IV in a concentration-dependent fashion, whereas under the same conditions, they did not significantly attach to CN I. Previously, CN I was reported as a substrate for integrin α2 in various types of cells (, ). Our experiments indicate that the ligand for integrin α2 receptor is a function of cellular context. Integrins are believed to recognize their ligands on the basis of the amino acid sequence within an inserted domain (I domain) at the N terminus of the α subunit of integrin (). In GEO cells, the specificity of integrin α2 in mediating cell adhesion to CN IV was confirmed by treating cells with functional blocking antibodies to integrin α2 (clone P1E6).

The inhibition of cell adhesion to CN IV was dependent on the concentration of P1E6. Effect of U0126, cyto D, and dominant negative mutants on ERK activation. GEO cells were treated either with Me 2SO or with MEK inhibitor, U0126, for 24 h in SM medium.

After trypsinization, cells were seeded on CN IV-coated microarray wells. Cells were allowed to attach for 3 h, and then micromotion was recorded by the ECIS technique. Results show the inhibitory effect of U0126 on micromotion ( right) as compared with control ( left)( A). B shows the inhibitory effect of U0126 on ERK phosphorylation as determined by Western blot analysis using either phosphospecific antibody (Y204; top) or ERK antibody ( bottom). In C, GEO cells were treated either with Me 2SO (control) or with cyto D for 24 h.

After trypsinization, cells were transferred to CN IV-precoated dishes and incubated at 37 °C in the presence of Me 2SO or cyto D for 1 h. Lysates were collected and analyzed by Western blot using an antibody specific to phospho-ERK or total ERK, as indicated.

In D–F, GEO cells were transfected either with empty vector or with the dominant negative FRNK, Y861F, or μ-calpain mutant for 48 h, respectively. The cell lysates were analyzed by using specific antibodies for phospho-ERK ( top) or ERK ( bottom). Next we set out to determine the nature of proteins that are phosphorylated when GEO cells attach to CN IV, a major component of the basement membrane collagen. Western blot analysis using phosphotyrosine specific antibody (4G10) showed that relative to cell suspension lysates, cells attached to CN IV showed enhanced phosphorylation of FAK (Tyr 397) and its major cleaved fragment (90 kDa). The 90-kDa fragment further cleaves into 50- and 40-kDa products ().

Under similar experimental conditions, the cleavage of activated FAK was either not observed or observed to a small extent in cell lysates prepared from cell suspensions. Previous investigators using human Caco-2 intestinal epithelial cells did not observe cleavage of FAK upon cell attachment to CN IV (). Our results indicate that the limited cleavage of FAK is cell type-specific. Limited FAK cleavage may have a physiological function for cytoskeletal remodeling and may contribute to signal transduction as an alternative mechanism to phosphorylation or dephosphorylation events.

The cell motility consequences of FAK cleavage have not been directly examined previously. To determine the mechanism of phosphorylated FAK cleavage, we first investigated the role of integrin α2 signaling.

GEO cells were preincubated with functional blocking monoclonal antibody P1E6 before attachment to precoated CN IV dishes. Western blot analysis of the cell lysates showed that the activation of FAK was significantly reduced by P1E6. To determine the factor contributing to FAK cleavage, we studied the role of calpains, which play an important role in cell adhesion. In initial experiments, we used ALLN, a well characterized inhibitor of calpains (). GEO cells were pretreated with ALLN and subsequently allowed to attach on CN IV-coated dishes. ALLN blocked FAK cleavage ().

These results indicated that integrin α2-mediated signaling activates calpain(s), which in turn cleave FAK in a limited fashion. The possibility of proteasome-dependent cleavage being responsible for FAK cleavage was determined by using lactacystin as a proteasome-specific inhibitor. We followed the well characterized cyclin E protein stability as an independent positive marker of proteasome inhibition ().

In GEO cells, we observed that cyclin E reaches high levels when treated with 20 μ m lactacystin, indicating that the proteasome system can be significantly blocked with this concentration of lactacystin. Under the same conditions, we did not observe attenuation of FAK cleavage by lactacystin. Together, these experiments exclude involvement of the proteasome in the calpain-mediated limited cleavage of FAK in GEO cells. These results provide further support for calpain as an important candidate for modulation of cell motility through its capacity to regulate focal adhesion dynamics. Next we investigated the significance of FAK cleavage in biological functions such as cell motility.

The mechanism and function of FAK cleavage in cell motility have not been clear. Cell motility is a multistep process that requires a critical balance between cell attachment and cell detachment on the dynamic ECM. These events are largely mediated by ECM receptors, integrins, and other proteins that form focal adhesions. Integrin clustering results in increased protein-tyrosine phosphorylation of FAK. The attachment of the leading edge of the cell is associated with the formation of focal adhesion complexes, whereas detachment at the rear of the cell is associated with the disassembly of focal adhesion complexes and the cleavage of the proteins that make up the focal adhesion complexes. The cleavage of FAK has been assumed to be associated with the disassembly of focal adhesions (); however, a direct relationship of FAK cleavage with cell motility has not been previously demonstrated.

To determine the potential role of FAK activation and its limited cleavage in cancer cell motility, we used an objective real time and quantitative ECIS technique. First, to determine whether CN IV affects cell micromotion, we compared cell micromotion on CN IV-coated and -uncoated gold electrodes, which are deposited in tissue culture wells.

Shows that the percentage variation in resistance on the CN IV-coated electrode (2.147%) was higher than on the uncoated electrode (percentage variation in resistance 0.847%). These results show that FAK activation and cleavage on CN IV enhanced cell micromotion by 154%. To establish that cell micromotion on CN IV was specifically integrin α2-mediated, GEO cells were preincubated either with mouse IgG 1 (control) or with integrin α2 blocking monoclonal antibody P1E6 before recording micromotion. Based on the percentage variation in resistance, we found that the reduction in cell locomotion of GEO cells by P1E6 was 79% as compared with control IgG-treated cells (). These results again support the concept that integrin α2-mediated FAK cleavage enhances cell motility.

To explore other factors that may contribute to FAK cleavage and motility, we examined the hypothesis that calpains are activated in adherent cells plated on ECM and that calpains may be involved in integrin α2-induced remodeling of the cytoskeleton. For these experiments, we used the small molecule calpain-specific inhibitor ALLN () which reduced GEO cell adhesion to CN IV (46–85%) (). To further define the role of calpains in cell motility, we measured micromotion under identical conditions, treating GEO cells either with Me 2SO (control) or with ALLN.

Treatment of the cells with ALLN decreased micromotion by 71% (). This is probably due to the blockade of integrin α2-CN IV detachment, which appears to be dependent on the breakdown of the focal adhesion complexes following FAK cleavage by calpains that is substantially reduced by ALLN. These results are consistent with a mechanism involving calpains in a pivotal role for control of GEO cell motility and suggest that inhibiting calpain-mediated cleavage of FAK may be a potential therapeutic approach to control pathological cell motility, such as cancer metastasis. Further in unraveling the signaling pathways critical to cell motility, we asked whether both typical isoforms of calpain, μ-calpain (calpain I) and m-calpain (calpain II), expressed by GEO cells are required for integrin α2-mediated cell micromotion. To determine whether one or both calpain isoforms contribute to cell micromotion, we used two approaches. First, specific antisense nucleotides to μ-calpain and m-calpain were synthesized according to the known sequences (). The micromotion was monitored simultaneously in the presence or absence of either μ-calpain or m-calpain antisense nucleotides.

The percentage variations in resistance observed were 5.54, 2.67, and 4.86%, respectively. Shows that, relative to control, about 52% inhibition in micromotion was observed in μ-calpain antisense-treated cells, whereas only 12% inhibition in cell micromotion was observed in cells treated with m-calpain antisense nucleotides. These results show that although GEO cells express both forms of calpain, it is μ-calpain that primarily contributes to enhanced cell micromotion. The above results demonstrate for the first time a physiological role for calpains in modulating integrin α2-mediated FAK cleavage, cell adhesion, and micromotion in colon cancer cells. As an additional approach to test the idea that the action of antisense calpain nucleotides resulted directly from inhibition of a specific type of calpain, GEO cells were transfected either with empty vector or with wild type μ-calpain expression vector. Shows an increase (167%) in cell micromotion in μ-calpain-transfected cells as compared with controls.

These results were further supported by transfecting cells with a μ-calpain mutant in which histidine 272 was mutated to alanine. ( bottom) shows that the increase in locomotion by the WT μ-calpain construct was blocked by the mutant. The mutant inhibited ERK phosphorylation as well (). New adhesions form preferentially at the front of the cell. Studies indicate that organized integrin first appears in small aggregates at the leading edge (). The rear of the cell ( heel) is functionally and structurally distinct from the front ( toe), generating an adhesive asymmetry. Extension of the leading edge is the first step of cell motility.

The motile cell phenotype requires dynamic cytoskeletal reorganization, and actin is an important component of the cytoskeleton. Cyto D, which selectively disrupts the network of actin filaments, inhibited FAK phosphorylation and cleavage, demonstrating a relationship between FAK activation, cleavage, and upstream of this, the actin cytoskeleton. Results indicating that intact actin is required for FAK activation and its limited cleavage were generated.

In addition, since actin polymerization is critical in cell spreading and motility, we performed adhesion assays and cell micromotion experiments. The results showed that cyto D inhibited cell adhesion to CN IV (45%), suggesting that actin polymerization is important in GEO cell adhesion to CN IV (). Similarly, treatment of cells with cyto D in micromotion experiments showed concentration-dependent inhibition (46–70%). The interruption of the actin network inhibits the ability of the cell to undergo movement; therefore, there is less necessity for the activation and cleavage of FAK. These results show that linkage between FAK phosphorylation, cleavage, and actin polymerization plays an important role in cell micromotion. Ectopic expression of the FAK dominant negative FRNK in GEO cells inhibited cell micromotion, suggesting that FAK is an important regulator of motility in colon carcinogenesis. Our observation that kinase-defective FAK enhances motility is consistent with previous reports ().

This phenomenon may be due to the transphosphorylation of the kinase-defective FAK by endogenous FAK on Tyr 397, allowing its binding to c-Src and leading to increased micromotion on CN IV. However, Lim et al. () have reported decreased migration in kinase-defective FAK transfected NIH3T3 fibroblasts. The difference in results from normal and cancer cells suggests that the motility function of GEO cells is dysregulated. Signaling events downstream of FAK activation and cleavage may contribute to motility of human colon carcinoma cells. There are different schools of thought concerning the mechanism by which integrin-mediated signaling activates ERK. Earlier investigators used fibroblasts, endothelial cells, and 293T cells with fibronectin activation of integrin α5β1 as a model system (, ).

It is not clear that the same mechanism(s) would be valid for other cell types or other integrins. Our initial experiments showed that when GEO cells were plated on CN IV, the kinetics of activation of ERK corresponded to upstream FAK activation (), and both kinases were dephosphorylated by cyto D. These results further support a direct link between actin, FAK, and ERK signaling. Moreover, suppression of ERK activation by the ectopic expression of dominant negative FRNK is consistent with downstream activation of ERK by FAK (). The important role of ERK in cell micromotion was confirmed by treating GEO cells with a small molecule MEK inhibitor U0126, which suppressed micromotion (48%) and ERK phosphorylation ().

The results described above suggest that integrin α2-mediated signaling controls cell motility via a FAK/ERK cascade. We sought further evidence by transient transfection of GEO cells with FRNK, an endogenous inhibitor of FAK, and subsequent determination of effects on cell micromotion.

As compared with control, attenuated micromotion was generated by FRNK transfection (), which also suppressed ERK phosphorylation. These results further confirm that the carboxyl domain of FAK regulates cell motility via ERK signaling in GEO cells. ERK activation by integrins has been proposed to be essential for focal adhesion disassembly. Activated ERK in turn phosphorylates myosin light chain kinase, which then phosphorylates the myosin light chain, resulting in cell contraction, thus facilitating cell locomotion (, ). Alternatively, activated ERK is believed to phosphorylate integrins directly and affect avidity to the ECM (). Since the FRNK domain contains the Tyr 861 site, we asked the question whether this phosphorylation site has any role to play in cell adhesion and motility.

The functional significance of this phosphorylation site is poorly understood. Recently, it was shown that phosphorylation of Tyr 861 was linked with the process of detransformation of ras-transformed fibroblasts (). The role of Tyr 861 activation by integrin-dependent signaling pathways has been controversial.

Previous studies (, –) reported a relationship between FAK Tyr 861 and tumorigenesis, although the mechanisms have not been directly addressed. () noted that the phosphorylation of Tyr 861 may not be regulated by integrin signaling in prostate cancer cell lines. Our experiments demonstrate that GEO cells show enhanced phosphorylation of FAK Tyr 861 upon attachment to CN IV. We also report that integrin α2-stimulated FAK Tyr 861 phosphorylation plays a pivotal role in cell adhesion and motility in GEO cells. Interestingly, we observed that mutation of Y861F (tyrosine mutated to phenylalanine) inhibited cell adhesion on CN IV (52–57%) as compared with empty vector-transfected cells ().

Similarly, the significance of phosphorylation site Tyr 861 was confirmed in cell micromotion, where the dominant negative mutant suppressed micromotion on CN IV by 52% (). The mutant FAK Y861F decreased phosphorylation of ERK as well (). Our experiments establish for the first time the significance of the Tyr 861 site of FAK in integrin α2-mediated colon cancer cell motility. In summary, our examination of the role of FAK in cell motility provides compelling evidence that a novel signaling pathway integrin α2/FAK/ERK/μ-calpain plays a critical role in tumor cell motility. These results present an opportunity that would allow interruption of FAK function at the early stages of colon tumorigenesis. Footnotes • 3 The abbreviations used are: ECM, extracellular matrix; ALLN, acetyl-leucyl-leucyl-norleucinal; BSA, bovine serum albumin; CN IV, collagen type IV; CN I, collagen type I; cyto D, cytochalasin D; ECIS, electric cell-substrate impedance sensing; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FRNK, FAK-related nonkinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MTT, methylthiazole tetrazolium; PBS, phosphate-buffered saline; SM, supplemental McCoy's; WT, wild type; HA, hemagglutinin. • * This work was supported by National Institutes of Health Grants CA 16056, 54807, 34432, and 50457, by the Oncologic Foundation of Buffalo/Alliance Foundation, and by the Shelby Rae Tengg Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “ advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. • Received January 25, 2006. • The American Society for Biochemistry and Molecular Biology, Inc.

Abstract Integrins are large, membrane-spanning, heterodimeric proteins that are essential for a metazoan existence. All members of the integrin family adopt a shape that resembles a large “head” on two “legs,” with the head containing the sites for ligand binding and subunit association. Most of the receptor dimer is extracellular, but both subunits traverse the plasma membrane and terminate in short cytoplasmic domains. These domains initiate the assembly of large signaling complexes and thereby bridge the extracellular matrix to the intracellular cytoskeleton. To allow cells to sample and respond to a dynamic pericellular environment, integrins have evolved a highly responsive receptor activation mechanism that is regulated primarily by changes in tertiary and quaternary structure.

This review summarizes recent progress in the structural and molecular functional studies of this important class of adhesion receptor. The name “integrin” was suggested for an integral membrane protein complex first characterized in 1986 (). The name was devised because the protein identified linked the extracellular matrix to the cytoskeleton (early developments in this field have been well described []).

In the 25 years since that first characterization, a vast amount of work has been performed, with consequent increased understanding. The essential role of integrins in tissue organization and cell development, their signal transduction mechanisms (from outside to in and inside to out!), and their potential as therapeutic targets is now established. In this article, we provide an overview of the structure of integrins, the conformational changes that determine activation state, and the mechanisms of ligand binding.

Overall Structure Integrins are heterodimers of non-covalently associated α and β subunits. In vertebrates, there are 18 α and 8 β subunits that can assemble into 24 different receptors with different binding properties and different tissue distribution (; ). The α and β subunits are constructed from several domains with flexible linkers between them. Each subunit has a single membrane-spanning helix and, usually, a short unstructured cytoplasmic tail.

The size varies but typically the α- and β-subunits contain around 1000 and 750 amino acids, respectively. Numerous reviews on integrin structure and function have been published (;;; ) so here we concentrate mainly on the implications of recent structural work that includes studies of intact ectodomains, membrane spanning regions, cytoplasmic tails and their ligands.

The Ectodomains The breakthrough crystal structure of αVβ3 () started a deluge of structural information about integrin ectodomains. Structures of αVβ3, with and without ligand (, ), αIIbβ3 (), and αxβ2 () are all now available. These crystal structures are all in a similar overall “bent” conformation that would place the ligand binding site near the membrane surface. The overall topology and structure of integrin ectodomains is illustrated in for the case of αxβ2 (), which has an inserted α-I domain. Current knowledge of ectodomains has also been enhanced by structures of various integrin fragments including isolated α-I domains () and β2-leg fragments (;; ). Because of observed flexibility in studies by electron microscopy (EM) and the existence of conformationally sensitive antibody recognition sites (), there is a general acceptance that conformations other than the bent one are possible and are functionally relevant (see below).

The structural studies of intact ectodomains (;; ) all postulated that an upright structure of the sort illustrated in C could exist as well as the bent structure. There has, however, been controversy about whether this large change between bent and upright structures—the “switchblade” model ()—has to take place or if more conservative changes around the bent structure can explain the data—the “deadbolt” model (). The Structure of α Subunit Ectodomains The α-chain consists of four or five extracellular domains: a seven-bladed β -propeller, a thigh, and two calf domains. Nine of the 18 integrin α chains have an α -I domain of around 200 amino-acids, inserted between blades 2 and 3 of the β-propeller (). The I domain, a copy of which also appears in the β-chain, has five β-sheets surrounded by seven α helices; it is similar to von Willebrand A domains. The last three or four blades of the β-propeller contain domains that bind Ca 2+ on the lower side of the blades facing away from the ligand-binding surface. Ca 2+ binding to these sites has been shown to influence ligand binding (; ).

The thigh and calf domains have similar, immunoglobulin-like, β-sandwich folds (). They have 140–170 residues with more β-strands than typical Ig-like domains (∼100 residues).

There are two main regions of interdomain flexibility. One is the linker between the β-propeller and the thigh; the other is the “genu” or knee at the bend between the thigh and the calf-1 domain. The α-subunit genu is located close to the similar bend in the β subunit, thereby allowing extension by a hinging at the knees. The α-I domain in αxβ2 is inserted in the β-propeller domain with flexible linkers (C). Unlike the other four α-leg domains, which have relatively rigid structures, I domains show conformational changes within the domain that are important for regulating binding affinity (see below and ). The Structure of β Subunit Ectodomains The β-leg has seven domains with flexible and complex interconnections.

A β -I domain is inserted in a hybrid domain, which is, in turn, inserted in a plexin-semaphorin-integrin (PSI) domain. These domains are followed by four cysteine-rich epidermal growth factor (EGF) modules and a β -tail domain. The hybrid domain in the upper β-leg has a β-sandwich construction. The β-I domain, which is homologous to the α-I domain, is inserted into the hybrid domain. The small PSI domain, with an α/β fold, is also split into two portions (; ) connected, in β3, by a long-range Cys-13 to Cys-435 disulfide bond. Unusual EGF module boundaries were first proposed in the αVβ3 structure (), but recent crystal structures suggest that each EGF module has an even number of eight cysteines, bonded in a C1-C5, C2-C4, C3-C6, and C7-C8 pattern except for EGF1, which lacks the C2-C4 disulfide. The αIIbβ3 structure shows that all 56 cysteines in the integrin β3 subunit are disulfide bonded ().

The β-tail domain has an α + β fold (). The weak electron density of this domain observed in the αIIbβ3 crystal structure was taken to suggest a flexible connection to other regions of the β-leg by a mobile “ankle” (). A contact between the CD loop of the β-tail domain and the α7 helix of the β-I domain has been proposed to inhibit integrin activation—the “deadbolt” model ().

This contact is, however, small and no such contact is observed in the αIIbβ3 or αxβ2 structures. In general, the β-leg seems to be more flexible than the α-leg. Evidence for this comes from the ten different structures of αXβ2 observed in three different crystal lattices ().

The EGF domain region is relatively plastic, especially between EGF1 and EGF2, the β knee, and at the PSI/hybrid and hybrid/I-EGF1 junctions. There is evidence for important conformational changes occurring in the β-I/hybrid region. A transition from a “closed” to an “open” conformation of the β-I domain has been observed when the β-I α7-helix moves toward the hybrid domain (). The connecting rodlike motion of the α7-helix causes the hybrid domain to swing-out by ∼60 o (see ).

Cation Binding Sites As described below, ligand binding in α-I less integrins takes place at the largest interface between the two subunits (the β-propeller/β-I domain interface); binding is dependent on the cations Mg ++, Ca 2+, and Mn ++. From a structure of an α-I domain it was suggested that integrin ligand binding involves a Mg ++ ion, a “metal-ion-dependent adhesion site” (MIDAS) (); a crystal form with bound Mn ++ also showed considerable movement of the α7-helix on activation (). In the recent αIIbβ3 structure () strong electron densities were ascribed to cations at three sites formed by loops in the β-I domain (); Mg ++ was assigned to the central MIDAS site with Ca 2+ at the two flanking sites. One of these adjacent sites (ADMIDAS) binds an inhibitory Ca 2+ ion; binding of Mn ++ here results in a structural change that gives an active integrin ().

The second Ca 2+-binding site has been called the synergistic metal ion binding site (SyMBS) (). Mutational studies show that the SyMBS site is responsible for Ca 2+ synergy (; ). As mentioned above, the β-I domain has distinct closed and open conformational states, involving movement of the α7 helix, in α-I-less integrins (; ). Similar conformational changes are seen in α-I-domains.

When integrin ligands, such as Arg-Gly-Asp, bind the open state, the MIDAS Mg ++ ion coordinates the Asp side chain of the ligand. In α-I integrins it has been suggested that the β-I MIDAS may bind an intrinsic ligand, an invariant Glu, Glu-318 in αX (). Support for this model comes from Glu mutations that abolish integrin activation (; ); the observed flexibility of the αx α-I domain would also facilitate such interdomain interactions ().

The Membrane Spanning Helices The current view is that association of integrin α and β transmembrane (TM) segments, results in an inactive resting receptor (). Evidence for this includes experiments using EM (), disulfide cross-linking (), activating mutations (), and FRET of labeled cytoplasmic tails (). Recent studies have given new insight into the structure of the resting state.

The structures of β3 and αIIb TM segments in phospholipid bicelle model membranes have been solved by NMR separately (,) and in complex (). A similar structure was obtained for the TM region in intact αIIbβ3 using disulfide-based distance restraints combined with protein modeling (). A recent structure of a complex in organic solvents has been described () but the relevance of a system without a phospholipid/water interface is questionable. A bacterial reporter system has been used to define the sequence motif required for TM helix-helix interactions in β1 and β3 integrin subfamilies (). Several modeling studies of the TM regions have also been published (;; ). [Note that whereas most evidence supports the heterodimeric form, there is also evidence that homomeric TM oligomers can form in vitro (; ); nevertheless, the evidence for homodimeric forms mainly comes from experiments or simulations performed without the ectodomains, whose presence would be expected to favor the heterodimeric form.] The NMR structure of the αIIbβ3 TM complex is shown in.

The αIIb helix is perpendicular to the membrane whereas the β3 helix is tilted. There are glycines at the helix-helix interface in the membrane and an unusual αIIb backbone reversal that packs a consecutive pair of Phe residues against the β3 TM helix, promoting electrostatic interactions between αIIb(D723) and β3(R995). The two TM segments have essentially the same structure when studied separately suggesting that the topological features of the TM segments will remain unchanged in the separated, active state. ( A) NMR structure of the complex between the αIIb (blue) and β3 (red) TM domains (PDB: 2K9J). The approximate position of the membrane glycerol backbone is shown by gray lines.

( B) The talin F2 (blue)/F3 (yellow) domain pair in complex with a β integrin tail (red). The salt bridge that forms between K324 on F3 and D723 in the tail is shown; some key Lys and Arg residues are indicated in blue near the putative membrane interface with the F2 domain.

B was constructed from a composite of coordinates of the talin/β complex (PDB: 3G9W; []) and the membrane complex (PDB:2K9J []). Images made using PyMOL (DeLano Scientific). The ectodomain-TM linkers seem to be flexible (; ) and are thus unlikely to constrain the orientation between the ectodomain and the membrane. Although αXβ2 is bent in a similar way to integrins without an α-I domain, the terminal domains of the α-legs and β-legs, calf-2, and β-tail domains, are oriented differently (). These observations are all consistent with the flexible transmembrane domain separation model of activation rather than stiff pistons or levers. The TM complex is also likely to be stabilized by the resting ectodomain. The Cytoplasmic Tails Several NMR studies of cytoplasmic tails have been published, although there is little agreement among them.

Some studies could not detect an interaction between α and β tails, whether they were connected by a coiled coil construct () or inserted in a membrane with TM segments (). A study of isolated mixed peptides found two distinct structures of the αβ complex (), both significantly different from the most detailed published structure of the αβ tail complex ().

The latter structure does not seem to be consistent with the recently solved structures of the TM regions (; ). The most obvious explanation for these discrepancies is that the tails are rather flexible, only forming transient structures in solution in the absence of a protein interaction partner and that complexes between the tails and their binding proteins are likely to provide the most significant insights into transduction events. Cytoplasmic Tail Ligands Because they are extended and flexible, the cytoplasmic tails, especially β, can “flycast” () and reach out to form “hub” interactions with a number of proteins (; ). In particular, PTB domains bind to one of the two conserved NPXY motifs in β-tails. Especially important for activation of integrins from inside the cell are the proteins talin and kindlin.

The talin PTB domain (the head F3 subdomain) binds the first β-NPXY and the membrane proximal helix () whereas kindlin binds the second NPXY (). It has been proposed that the F2.F3 subdomains of talin make a defined contact with the membrane surface via numerous lysine and arginine residues especially in the F2 domain (B; ). Binding of F3 to the β-tail promotes tail dissociation by breaking the salt bridge between the α and β tails (αIIb[D723]-β3[R995]); a new salt bridge is formed with K324 on the F3 domain. Binding to β plus the F2.F3 contact with the membrane can also influence the orientation of the β-helix, again helping promote separation of the TM and cytoplasmic domains. Structural Studies of Intact Integrins Although crystallography of ectodomains and NMR of TM domains have provided detailed information about integrin structure, other techniques have been applied to intact integrins in attempts to distinguish among current models, such as “switchblade” and “deadbolt.” In principle this should be relatively easy because the switchblade model predicts a near doubling in molecular height on activation (C), whereas the deadbolt model predicts a modest change. In general, however, the various studies have not been in good agreement, possibly because of multiple integrin conformations in solution. Cryoelectron tomography of ice-embedded specimens was used to obtain three-dimensional images of full-length αIIbβ3 incorporated into small liposomes ().

No significant height change was observed between the inactive state, and the active state induced by Mn ++. In a FRET study, Mn ++ activation caused an increase of 5 nm in the separation between membrane and ligand binding site, consistent with a conformational change to the upright configuration (). However, FRET between fluorescently labeled Fab fragments and the β3 β-I domain indicated only small changes on platelet activation ().

A small angle neutron scattering (SANS) study of intact αIIbβ3 in Ca 2+/detergent solutions found “arched” and “handgun” forms () although a Mn ++ activated form was not studied. Analytical ultracentrifugation and EM were used to investigate αIIbβ3; measurement of frictional changes under different conditions suggested considerable plasticity in the structure (). EM studies of ectodomains are also not entirely consistent although, on balance, they favor the switchblade model. Single-particle reconstructions of the negatively stained αvβ3 ectodomain bound to a fibronectin (FN) fragment suggested that the bent conformation can bind its physiological ligand (). EM of negatively stained αvβ3 ectodomain with a cleavable clasp engineered into the carboxyl terminus showed a majority of molecules in the bent conformation when inactive, and a majority in the upright conformation when active (). Negative stain EM of a shorter ectodomain construct of α5β1 bound to FN showed similar results ().

The recent x-ray structure papers of intact ectodomains contain EM results consistent with the switchblade model (; ). A recent study of integrin activation using EM and other studies gives useful insight (). Membrane nanodiscs were synthesized with a single lipid-embedded integrin. The majority (∼90%) of the class-averaged integrin nanodisc EM images in the absence of ligand and the talin head domain had a compact structure with a height of 11±1 nm; i.e., corresponding to the bent conformation in C. In the presence of talin, ∼25% of unliganded integrins had an extended structure with a height of 19±1 nm. In contrast, at least 40% of the fibrin-bound integrins were extended. This study provides evidence that talin binding is sufficient to activate and extend membrane-embedded integrin αIIbβ3 without applied force or clustering.

There is evidence that mechanical force is important for regulating integrin adhesiveness (; ). Talin contains both integrin and actin binding sites (), and therefore the cytoskeleton could exert a lateral force on the β subunit. Steered molecular dynamics was applied to a complete ectodomain to mimic effects of cell generated tension. Evidence was found that lateral force could be transmitted through the β leg to the hybrid domain and promote the active form (). LIGAND BINDING Historically, the pairing of integrins and their ligands has been uncovered either by ligand affinity chromatography or through the use of subunit-specific monoclonal antibodies (mAbs) to block ligand-mediated cell adhesion.

In most cases, protein-protein binding assays have confirmed the associations established by these biochemical or cell biological approaches. A characteristic feature of most integrin receptors is their ability to bind a wide variety of ligands. Conversely, many extracellular matrix (ECM) and cell surface adhesion proteins bind to multiple integrin receptors (;; ). One molecular explanation for this complexity is the evolutionary selection of common acidic peptide motifs in ECM proteins that mediate integrin binding via coordination to a divalent cation-containing binding pocket.

Integrin-ligand combinations can be clustered into four main classes, based on the nature of the molecular interaction. All five αV integrins, two β1 integrins (α5, α8), and αIIbβ3 recognize ligands containing an RGD tripeptide active site. Crystal structures of αVβ3 and αIIbβ3 complexed with RGD ligands have been reported and they reveal an identical atomic basis for this interaction (; ).

RGD binds at an interface between the α and β subunits, with the basic residue fitting into a cleft in a β-propeller module in the α subunit, and the acidic residue coordinating a cation bound in the β-I-domain. RGD-binding integrins bind to a large number of ECM and soluble vascular ligands, but with different affinities that presumably reflect the preciseness of the fit of the ligand RGD conformation with the specific α,β active site pockets. Although RGD is an essential element of the ligand binding process, macromolecular ligands can contain other binding sites, the best characterized of which is a synergy sequence in fibronectin that also binds the α5 β-propeller (, ). Α4β1, α4β7, α9β1, the four members of the β2 subfamily, and αEβ7 recognize related sequences in their ligands. Α4β1, α4β7, and α9β1 bind to an acidic motif, termed “LDV,” that is functionally related to RGD.

Fibronectin contains the prototype LDV ligand in its type III connecting segment region, but other ligands (such as VCAM-1 and MAdCAM-1) employ related sequences. Structures of this integrin subfamily are lacking, but it is highly likely that LDV peptides bind similarly to RGD at the junction between the α and β subunits. The β2 family employs a similar mode of ligand binding, but the major interaction takes place via an inserted I-domain in the α subunit (). Despite this fundamental mechanistic difference, the characterized sites within ligands that bind β2 integrins are structurally homologous to the LDV motif (). The major difference is that β1/β7 ligands employ an aspartate residue for cation coordination, whereas β2 integrins use glutamate.

Four α subunits containing an α-I-domain (α1, α2, α10, and α11) combine with β1, and form a distinct laminin/collagen-binding subfamily. A crystal structure of a complex between the α2 I-domain and a triple-helical collagenous peptide has revealed the structural basis of the interaction, with a critical glutamate within a collagenous GFOGER motif providing the key cation-coordinating residue (). The mechanism of laminin binding is less well understood, although a recent study has suggested that the extreme carboxyl terminus of the γ chain and an undefined site within α subunit laminin G domains together constitute an integrin-binding site (). Three β1 integrins (α3, α6, and α7), plus α6β4, are highly selective laminin receptors. Analysis of laminin fragments indicates that these receptors and the α-I-domain-containing β1 integrins bind to different regions of the ligands. In neither case has the active site been narrowed down to a particular sequence or residue. ACTIVATION For the interaction of integrins with their ligands to be meaningful for cellular function, the binding event must be able to regulate signal transduction.

However, adhesion is highly dynamic, with cells continuously sampling their pericellular environment, and responding by rapidly changing their position and state of differentiation, and therefore a highly responsive receptor activation mechanism is required. As integrins lack enzymatic activity, signaling is instead induced by the assembly of signaling complexes on the cytoplasmic face of the plasma membrane.

Formation of these complexes is achieved in two ways; first, by receptor clustering, which increases the avidity of molecular interactions thereby increasing the on-rate of binding of effector molecules, and second, by induction of conformational changes in receptors that creates or exposes effector binding sites. Current evidence suggests that conformational regulation is the primary mode of affinity regulation of integrins. In turn, this demands a mechanism for conveying conformational changes between the cytoplasmic tails and the ligand-binding head domain over a relatively large distance (∼20 nm).

Evidence for Conformational Regulation Gross conformational changes in integrins have been monitored by a variety of techniques, and for almost all of these studies, αIIbβ3 has served as a prototype. Treatment with RGD peptides elicited alterations in sedimentation coefficient and Stokes radius (), and receptor activation on platelets triggered changes in intramolecular FRET and cross-linking (). MAbs have proven particularly useful probes of integrin function. Early studies reported activation-dependent changes in mAb binding to αIIbβ3 that were attributed to conformational changes (; ), and these were followed by the identification of a subset of anti-αIIbβ3 mAbs, the epitopes for which were induced in response to ligand binding (, ). The acronym LIBS was coined to describe these epitopes as ligand-induced binding sites. In most cases that have been examined, activating mAbs appear to function by increasing the affinity of ligand binding. Most LIBS mAbs have epitopes that are regulated by divalent cations, and because cations also regulate ligand binding, it appears that many cation-responsive, activating mAbs recognize naturally occurring conformers of integrins.

These mAbs may therefore displace a conformational equilibrium in favor of these forms that leads to an increase in the proportion of high affinity integrin. Some other activating mAb epitopes are unaffected by either ligand or cation binding and here the most likely mechanism of action is through inducing an activated conformation in the integrin (). The location of LIBS epitopes has contributed significantly to our understanding of the process of receptor activation. The overwhelming majority of activating mAbs recognize the β subunit, and their epitopes are distributed throughout the polypeptide (; ).

This is suggestive of a large-scale alteration in the conformation of the whole integrin during activation. The regions recognized include the β-I-domain, the extreme amino terminus of the β subunit in the PSI domain, the hybrid domain, the β-subunit knee region, and distal EGF-like repeats. A few activating anti-α subunit mAbs have been reported, the epitopes for which are found in the β-propeller, the heavy-light chain border and close to the transmembrane domain, suggestive of conformational changes in these regions of the molecule (; ). How Are Conformational Changes Coupled?

As discussed above, the various structural studies in the last 10 years have greatly stimulated functional analyses. The relevance of the observed bend in the legs of the integrin dimer has been a highly contentious issue. It has been proposed that integrins are always bent, but several lines of evidence indicate that bent integrins are inactive, and extended integrins are primed. In the original crystal structure of αVβ3, the integrin was bent at an angle of 135°. Locking integrins in this state through disulphide bond engineering abolishes ligand binding by cell surface-expressed receptors (). Furthermore, when the gross structure of integrins was examined by electron microscopy under conditions in which ligand binding was low, e.g., in Ca 2+-containing buffers or following the introduction of intersubunit covalent bond constraints, predominantly bent structures were observed (; ). In bent integrins, the ligand-binding pocket may be oriented toward the plasma membrane, thereby impeding ligand engagement, but flexibility at the juxtamembrane domain could enable a “breathing” movement for the conversion of bent to extended integrin (; ).

In this context, a cryo-EM reconstruction of unstimulated αIIbβ3 indicated a partially extended conformation (). The binding of stimulatory mAbs might then displace a conformational equilibrium, leading to activation. Similarly, breaking the interactions between the α and β cytoplasmic tails appears to lead to a loss of the interactions among the leg regions, disruption of an interface between the head and legs, and a repositioning of the head to point away from the cell surface. Major support for this model comes from studies of soluble recombinant integrins by electron microscopy () and from the large number of epitopes of stimulatory mAbs that have now been shown to lie in the knee or leg regions (). Exposure of these epitopes is low in the bent state of the integrin (where they are masked) but high in the extended state (). The pathway of conformational change from the interior of the cell to the ligand-binding site of the integrin is incompletely understood, but movement of the hybrid domain appears to be a central feature of the conformational changes accompanying unbending ().

By EM, an acute angle between the hybrid domain and β-I was observed in the bent state, and an obtuse angle in the extended, ligand-occupied state (). In the bent conformation, any movement of the hybrid domain relative to the β-I domain is prevented, and therefore unbending is probably an essential prerequisite to hybrid domain motion. A central role for hybrid domain movement in affinity regulation has been established by a number of approaches.

Activating mAb epitopes in the hybrid domain map close to an interface between the hybrid domain and the α subunit β-propeller (). These epitopes would be masked in bent integrins, but would become exposed when the hybrid domain swings away from the propeller. Furthermore, engineering of glycosylation sites between the hybrid domain and the β-I-domain produces a putative wedge that leads to integrin activation (). Conformational changes in the head are the key determinant of ligand-binding activity, specifically, the conformation of the β-I domain, which, in turn, is determined by the position of the hybrid domain. Thus, a swing-out of the hybrid domain away from the α-subunit pulls downward on the α7 helix of the β-I domain and favors the upward movement of the α1 helix (). The motion of these two helices shifts the β-I domain from a low-affinity into a high-affinity conformation by backbone movements of loops that contain cation-coordinating residues.

Mutations that favor a downward shift of the α7 helix (;; ) also result in a high-affinity state. Although current models of integrin function strongly suggest that conversion to a high-affinity receptor requires extension, there is some evidence to suggest that ligand-bound integrin can adopt a bent conformation. Crystallized αVβ3 can bind a cyclic RGD peptide in the bent conformation () and electron microscopy images also show bent αVβ3 in complex with a fragment of fibronectin (). In addition, studies that have either used FRET or competition ELISA to measure the distance between a fluorescently tagged ligand peptide and labeled cell membrane or between mAbs directed against the head piece and leg regions of αIIbβ3 on platelets have revealed partial unbending (;, ). Nevertheless, when FRET-FLIM is employed to analyze the conformation of α5β1 in adherent cells, by measuring FRET between a fluorescently labeled Fab bound to α5β1 and fluorescent dye intercalated into the cell membrane, it has been shown that integrins are extended in focal adhesions and bent elsewhere (). Integrin Antagonists as Therapeutic Agents The short acidic peptides that serve as ligand active sites are essentially pro-drugs, and both RGD and LDV peptides have been converted into small molecules therapeutics. RGD-based, peptidomimetic antagonists of αIIbβ3, such as eptifibatide (from 1998) and tirofiban (from 1998), are now used widely as antithrombotic agents (), and LDV-based compounds are in development for treatment of asthma and multiple sclerosis.

In addition, mAbs that block integrin function and cell adhesion have been developed as therapeutic agents. These agents were originally assumed to compete with ligands for receptor binding, but this now appears not to be the case, with many anti-integrin mAbs having been shown to function via allosteric mechanisms. Current evidence suggests that mAbs inhibit ligand binding either by stabilizing the unoccupied state of the receptor or by preventing a conformational change necessary for ligand occupancy. In turn, the allosteric inhibition of ligand binding by anti-functional anti-integrin mAbs implies that it may be feasible to synthesize small molecule inhibitors that function in the same way. Such inhibitors could have advantages over competitive inhibitors in that a partial inhibition of function may be obtained and therefore adhesion may be more easily controlled, and they may not possess the agonistic properties of ligand mimetics, and may therefore not suffer from mechanism-related side-effects. Small molecule allosteric inhibitors that bind to α-I-domains have now been reported. These molecules appear to stabilize the low affinity conformation of the α-I-domain by blocking downward movement of the terminal α7-helix and thereby preventing rearrangements at the ligand-binding pocket necessary for high affinity ligand binding ().

CONCLUSION There has been remarkable progress in our understanding of integrin structure and function in the last 10 years. The basis of much previous work on conformation, which was performed with conformationally sensitive antibodies, and ligand binding, which was largely based on mutational analyses, can now be modeled at atomic resolution.

A unifying biophysical model of integrin function, which incorporates features such as catch bonds, extreme flexibility at the knees and the on- and off-rates of ligand and effector binding is therefore within reach. The process of integrin activation from inside the cell is also now quite well understood at a structural level. However, a number of major questions remain unresolved. These include outside-in signaling, which is much less well understood compared with inside-out signaling and it is unclear how similar or different the two processes are. We still do not know how an integrin allows a cell to interpret the binding of different ligands, and therefore how microenvironmental sensing is achieved at a molecular level.

Looking further ahead, the process of “inactivation,” where integrins return to their resting state, is not understood, and we are just starting to develop approaches to measure force transduction at adhesion sites.