Tests of the extension and deadbolt models of integrin activation

Despite extensive evidence that integrin conformational changes between bent and extended conformations regulate affinity for ligands, an alternative hypothesis has been proposed in which a "deadbolt" can regulate affinity for ligand in the absence of extension. Here, we tested both the deadbolt and the extension models. According to the deadbolt model, a hairpin loop in the beta3 tail domain could act as a deadbolt to restrain the displacement of the beta3 I domain beta6-alpha7 loop and maintain integrin in the low affinity state. We found that mutating or deleting the beta3 tail domain loop has no effect on ligand binding by either alphaIIbbeta 3 or alphaVbeta3 integrins. In contrast, we found that mutations that lock integrins in the bent conformation with disulfide bonds resist inside-out activation induced by cytoplasmic domain mutation. Furthermore, we demonstrated that extension is required for accessibility to fibronectin but not smaller fragments. The data demonstrate that integrin extension is required for ligand binding during integrin inside-out signaling and that the deadbolt does not regulate integrin activation.     

Integrin inside-out signaling and the immunological synapse

Integrins dynamically equilibrate between three conformational states on cell surfaces. A bent conformation has a closed headpiece. Two extended conformations contain either a closed or an open headpiece. Headpiece opening involves hybrid domain swing-out and a 70 ? separation at the integrin knees, which is conveyed by allostery from the hybrid-proximal end of the βI domain to a 3 ? rearrangement of the ligand-binding site at the opposite end of the βI domain. Both bent-closed and extended-closed integrins have low affinity, whereas extended-open integrin affinity is 10(3) to 10(4) higher. Integrin-mediated adhesion requires the extended-open conformation, which in physiological contexts is stabilized by post-ligand binding events. Integrins thus discriminate between substrate-bound and soluble ligands. Analysis of LFA-1-ICAM-1 interactions in the immunological synapse suggests that bond lifetimes are on the order of seconds, which is consistent with high affinity interactions subjected to cytoskeletal forces that increase the dissociation rate. LFA-1 βI domain antagonists abrogate function in the immunological synapse, further supporting a critical role for high affinity LFA-1.     

Two synergistic activation mechanisms of alpha2beta1 integrin-mediated collagen binding

Activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces ligand-independent aggregation of a cell surface collagen receptor, alpha2beta1 integrin. Concomitantly, TPA increases the avidity of alpha2beta1 for collagen and the number of conformationally activated alpha2beta1 integrins. The structural change was shown using a monoclonal antibody 12F1 that recognizes the "open" (active) conformation of the inserted domain in the alpha2 subunit (alpha2I). Amino acid residue Glu-336 in alpha2 subunit is proposed to mediate the interaction between alpha2I domain and beta1 subunit. Glu-336 seems to regulate a switch between open and "closed" conformations, since the mutation alpha2E336A inhibited the TPA-related increase in the number of 12F1 positive integrins. E336A also reduced cell adhesion to collagen. However, E336A did not prevent the TPA-related increase in adhesion to collagen or alpha2beta1 aggregation. Thus, alpha2beta1 integrin avidity is regulated by two synergistic mechanisms, first an alpha2E336-dependent switch to the open alpha2I conformation, and second an alpha2E336-independent mechanism temporally associated with receptor aggregation.     

Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling

How ligand binding alters integrin conformation in outside-in signaling, and how inside-out signals alter integrin affinity for ligand, have been mysterious. We address this with electron microscopy, physicochemical measurements, mutational introduction of disulfides, and ligand binding to alphaVbeta3 and alphaIIbbeta3 integrins. We show that a highly bent integrin conformation is physiological and has low affinity for biological ligands. Addition of a high affinity ligand mimetic peptide or Mn(2+) results in a switchblade-like opening to an extended structure. An outward swing of the hybrid domain at its junction with the I-like domain shows conformational change within the headpiece that is linked to ligand binding. Breakage of a C-terminal clasp between the alpha and beta subunits enhances Mn(2+)-induced unbending and ligand binding.     

Transition from rolling to firm adhesion can be mimicked by extension of integrin alphaLbeta2 in an intermediate affinity state

AlphaLbeta2 affinity for intercellular adhesion molecule-1 (ICAM-1) is regulated by the conformation of the alphaL I domain, which is in turn controlled by the conformation and orientation of other adjacent domains. Additionally, overall integrin conformation (bent versus straightened) influences the orientation of the I domain and access to its ligands, influencing adhesive efficiency. The open or high affinity I domain conformation supports strong adhesion, whereas the closed, low affinity conformation mediates weak interactions or rolling. We have previously suggested that alphaLbeta2 can also exist on the cell surface in an intermediate affinity state. Here we have studied the adhesive properties of integrin alphaLbeta2 containing mutant I domains with intermediate affinities for ICAM-1. In an overall bent conformation, the intermediate affinity state of alphaLbeta2 is hardly detected by conventional adhesion assays, but robust adhesion is seen when an extended conformation is induced by a small molecule alpha/beta I allosteric antagonist. Intermediate affinity alphaLbeta2 supports more stable rolling than wild-type alphaLbeta2 under shear conditions. Moreover, antagonist-induced extension transforms rolling adhesion into firm adhesion in a manner reminiscent of chemokine activation of integrin alphaLbeta2. These findings suggest the relevance of intermediate affinity states of alphaLbeta2 to the transition between inactive and active states and demonstrate the importance of both I domain affinity and overall integrin conformation for cell adhesion.     

Structural requirements for activation in alphaIIb beta3 integrin

Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant αIIbβ3 integrins that are constrained specifically in either a bent or an extended conformation. These mutant αIIbβ3 integrins were expressed in mammalian cells, and fibrinogen binding to these cells was examined. The bent integrins were created through the introduction of artificial disulfide bridges in the β-head/β-tail interface. Cells expressing bent integrins all failed to bind fibrinogen unless pretreated with DTT to disrupt the disulfide bridges. The extended integrins were created by introducing N-glycosylation sites in amino acid residues located close to the α-genu, where the integrin legs fold backward. Among these mutants, activation was maximized in one integrin with an N-glycosylation site located behind the α-genu. This extension-induced activation was completely blocked when the swing-out of the hybrid domain was prevented. These results suggest that the bent and extended conformers represent low affinity and high affinity conformers, respectively, and that extension-induced activation depends on the swing-out of the hybrid domain. Taken together, these results are consistent with the current hypothesis that integrin affinity is regulated by the switchblade-like movement of the integrin legs.     

Regulation of integrin αIIbβ3 ligand binding and signaling by the metal ion binding sites in the β I domain

The ability of αIIbβ3 to bind ligands and undergo outside-in signaling is regulated by three divalent cation binding sites in the β I domain. Specifically, the metal ion-dependent adhesion site (MIDAS) and the synergistic metal binding site (SyMBS) are thought to be required for ligand binding due to their synergy between Ca(2+) and Mg(2+). The adjacent to MIDAS (ADMIDAS) is an important ligand binding regulatory site that also acts as a critical link between the β I and hybrid domains for signaling. Mutations in this site have provided conflicting results for ligand binding and adhesion in different integrins. We have mutated the β3 SyMBS and ADMIDAS. The SyMBS mutant abolished ligand binding and outside-in signaling, but when an activating glycosylation mutation in the αIIb Calf 2 domain was introduced, the ligand binding affinity and signaling were restored. Thus, the SyMBS is important but not absolutely required for integrin bidirectional signaling. The ADMIDAS mutants showed reduced ligand binding affinity and abolished outside-in signaling, and the activating glycosylation mutation could fully restore integrin signaling of the ADMIDAS mutant. We propose that the ADMIDAS ion stabilizes the low-affinity state when the integrin headpiece is in the closed conformation, whereas it stabilizes the high-affinity state when the headpiece is in the open conformation with the swung-out hybrid domain.     

The A-domain of beta 2 integrin CR3 (CD11b/CD18) is a receptor for the hookworm-derived neutrophil adhesion inhibitor NIF

The A-domain is a approximately 200-amino acid peptide present within structurally diverse proadhesive proteins including seven integrins. A recombinant form of the A-domain of beta 2 integrins CR3 and LFA-1 has been recently shown to bind divalent cations and to contain binding sites for protein ligands that play essential roles in leukocyte trafficking to inflammatory sites, phagocytosis and target cell killing. In this report we demonstrate that the neutrophil adhesion inhibitor, NIF produced by the hookworm Ancyclostoma caninium is a selective CD11b A-domain binding protein. NIF bound directly, specifically and with high affinity (Kd of approximately 1 nM) to recombinant CD11b A-domain (r11bA). The binding reaction was characterized by rapid association and very slow dissociation, and was blocked by an anti-r11bA monoclonal antibody. No binding was observed to rCD11aA. The NIF-r11bA interaction required divalent cations, and was absent when the mutant r11bA D140GS/AGA (that lacks divalent cation binding capacity) was used. The NIF binding site in r11bA was mapped to four short peptides, one of which being an iC3b binding site. The interaction of NIF with CR3 in intact cells followed similar binding kinetics to those with r11bA, and occurred with similar affinity in resting and activated human neutrophils, suggesting that the NIF epitope is activation independent. Binding of NIF to CR3 blocked its ability to bind to its ligands iC3b, fibrinogen, and CD54, and inhibited the ability of human neutrophils to ingest serum opsonized particles. NIF thus represents the first example of a disintegrin that targets the integrin A-domain, and is likely to be used by the hookworm to evade the host's inflammatory response. The unique structure of NIF, which lacks a disintegrin motif, emphasizes basic structural differences in antagonists targeting A+ and A- integrins, that should be valuable in drug design efforts aimed at generating novel therapeutics. Identification of the region in NIF mediating A-domain binding should also be useful in this regard, and may, as in the case of disintegrins, unravel a new structural motif with cellular counterparts mediating important physiologic functions.     

Two Functional States of the CD11b A-Domain: Correlations with Key Features of Two Mn2+-complexed Crystal Structures

In the presence of bound Mn²⁺, the three- dimensional structure of the ligand-binding A-domain from the integrin CR3 (CD11b/CD18) is shown to exist in the “open” conformation previously described only for a crystalline Mg²⁺ complex. The open conformation is distinguished from the “closed” form by the solvent exposure of F302, a direct T209–Mn²⁺ bond, and the presence of a glutamate side chain in the MIDAS site. Approximately 10% of wild-type CD11b A-domain is present in an “active” state (binds to activation-dependent ligands, e.g., iC3b and the mAb 7E3). In the isolated domain and in the holoreceptor, the percentage of the active form can be quantitatively increased or abolished in F302W and T209A mutants, respectively. The iC3b-binding site is located on the MIDAS face and includes conformationally sensitive residues that undergo significant shifts in the open versus closed structures. We suggest that stabilization of the open structure is independent of the nature of the metal ligand and that the open conformation may represent the physiologically active form.     

A 50-A separation of the integrin alpha v beta 3 extracellular domain C termini reveals an intermediate activation state

The integrin alpha(v)beta(3) has been shown to exist in low and high affinity conformations. Activation to the high affinity state is thought to depend on the "switchblade-like" opening, from a low affinity bent conformation with a closed headpiece to an extended form of the integrin with an open headpiece. Activation has been shown to depend on separation of the cytoplasmic domains. How cytoplasmic domain separation is related to separation of the transmembrane domains is unknown, and the distance of separation of the transmembrane domains required for activation has not been defined. A constrained secreted form of alpha(v)beta(3) was engineered that introduced a 50-A separation of the integrin C-terminal tails of the extracellular domains of the alpha(v) and beta(3) subunits. Receptor binding and recognition by ligand-induced binding state (LIBS) monoclonal antibodies demonstrated that the mutant receptor was locked into a low affinity state that was likely in a partially extended conformation but with a closed headpiece. In the presence of RGD peptide, the constrained receptor was able to fully extend, as determined by full exposure of LIBS epitopes. In the presence of the appropriate LIBS antibody, high affinity ligand binding of the constrained receptor was achieved. The results support the existence of transient intermediate activation states of secreted alpha(v)beta(3). Furthermore, these results with the secreted alpha(v)beta(3) receptor support a model for the full-length membrane-bound form of alpha(v)beta(3), whereby a 50-A lateral separation of the integrin alpha(v) and beta(3) transmembrane domains would be sufficient to enforce the switchblade-like opening to the extended conformation but insufficient for full receptor activation.     

Integrin-based mechanosensing through conformational deformation

Conversion of integrins from low to high affinity states, termed activation, is important in biological processes, including immunity, hemostasis, angiogenesis, and embryonic development. Integrin activation is regulated by large-scale conformational transitions from closed, low affinity states to open, high affinity states. Although it has been suggested that substrate stiffness shifts the conformational equilibrium of integrin and governs its unbinding, here, we address the role of integrin conformational activation in cellular mechanosensing. Comparison of wild-type versus activating mutants of integrin αVβ3 show that activating mutants shift cell spreading, focal adhesion kinase activation, traction stress, and force on talin toward high stiffness values at lower stiffness. Although all activated integrin mutants showed equivalent binding affinity for soluble ligands, the β3 S243E mutant showed the strongest shift in mechanical responses. To understand this behavior, we used coarse-grained computational models derived from molecular level information. The models predicted that wild-type integrin αVβ3 displaces under force and that activating mutations shift the required force toward lower values, with S243E showing the strongest effect. Cellular stiffness sensing thus correlates with computed effects of force on integrin conformation. Together, these data identify a role for force-induced integrin conformational deformation in cellular mechanosensing.     

Activation-induced conformational changes in the I domain region of lymphocyte function-associated antigen 1.

Conformational changes in integrins are important for efficient ligand binding during activation. We proposed that the I domain of the integrin lymphocyte function-associated antigen 1 (LFA-1) could exist in both open and closed conformations and generated constitutively activated LFA-1 by locking the I domain in the open conformation. Here we provide structural and biochemical evidence to validate conformational change in the I domain of LFA-1 upon activation. Two monoclonal antibodies to alpha(L), HI111 and CBR LFA-1/1, bind wild-type LFA-1 well, but their binding is significantly reduced when LFA-1 is locked in the open conformation. Furthermore, this reduction in monoclonal antibody binding also occurs when LFA-1 is activated by divalent cations. HI111 maps to the top region of the I domain that is close to the putative ligand-binding site surrounding the MIDAS (metal ion-dependent adhesion site). The epitope of CBR LFA-1/1 is at the C-terminal segment of the I domain that links to the beta-propeller, and undergoes a large movement between the open and closed conformations. Our data demonstrate that these two regions undergo significant conformational changes during LFA-1 activation and that the I domain of activated LFA-1 adopts a similar tertiary structure as the predicted locked open form.     

Identification of integrin beta subunit mutations that alter affinity for extracellular matrix ligand

We examined over 50 mutations in the Drosophila βPS integrin subunit that alter integrin function in situ for their ability to bind a soluble monovalent ligand, TWOW-1. Surprisingly, very few of the mutations, which were selected for conditional lethality in the fly, reduce the ligand binding ability of the integrin. The most prevalent class of mutations activates the integrin heterodimer. These findings emphasize the importance of integrin affinity regulation and point out how molecular interactions throughout the integrin molecule are important in keeping the integrin in a low affinity state. Mutations strongly support the controversial deadbolt hypothesis, where the CD loop in the β tail domain acts to restrain the I domain in the inactive, bent conformation. Site-directed mutations in the cytoplasmic domains of βPS and αPS2C reveal different effects on ligand binding from those observed for αIIbβ3 integrins and identify for the first time a cytoplasmic cysteine residue, conserved in three human integrins, as being important in affinity regulation. In the fly, we find that genetic interactions of the βPS mutations with reduction in talin function are consistent with the integrin affinity differences measured in cells. Additionally, these genetic interactions report on increased and decreased integrin functions that do not result in affinity changes in the PS2C integrin measured in cultured cells.     

Effects of I domain deletion on the function of the beta2 integrin lymphocyte function-associated antigen-1

A subset of integrin alpha subunits contain an I domain, which is important for ligand binding. We have deleted the I domain from the beta2 integrin lymphocyte function-asssociated antigen-1 (LFA-1) and expressed the resulting non-I domain-containing integrin (DeltaI-LFA-1) in an LFA-1-deficient T cell line. DeltaI-LFA-1 showed no recognition of LFA-1 ligands, confirming the essential role of the I domain in ligand binding. Except for I domain monoclonal antibodies (mAbs), DeltaI-LFA-1 was recognized by a panel of anti-LFA-1 mAbs similarly to wild-type LFA-1. However, DeltaI-LFA-1 had enhanced expression of seven mAb epitopes that are associated with beta2 integrin activation, suggesting that it exhibited an "active" conformation. In keeping with this characteristic, DeltaI-LFA-1 induced constitutive activation of alpha4beta1 and alpha5beta1, suggesting intracellular signaling to these integrins. This "cross-talk" was not due to an effect on beta1 integrin affinity. However, the enhanced activity was susceptible to inhibition by cytochalasin D, indicating a role for the cytoskeleton, and also correlated with clustering of beta1 integrins. Thus, removal of the I domain from LFA-1 created an integrin with the hallmarks of a constitutively active receptor mediating signals into the cell. These findings suggest a key role for the I domain in controlling integrin activity.     

Jararhagin-derived RKKH peptides induce structural changes in alpha1I domain of human integrin alpha1beta1

Integrin alpha(1)beta(1) is one of four collagen-binding integrins in humans. Collagens bind to the alphaI domain and in the case of alpha(2)I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In alpha(2)I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of alpha(2)I differs significantly from the "closed" conformation seen in the structure of apo-alpha(2)I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to alpha(1)I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of alpha(1)I with Mg(2+) and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-alpha(1)I ("closed" conformation) and a model structure of the alpha(1)I ("open" conformation) based on the closely related structure of alpha(2)I reveals that the binding site is partially blocked to ligands by Glu(255) and Tyr(285) in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of alpha(2)I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of alpha(1)I and shown experimentally for alpha(1)I using a fluorescent hydrophobic probe.     

Effects of conformational activation of integrin alpha 1I and alpha 2I domains on selective recognition of laminin and collagen subtypes

Collagen receptor integrins alpha 1 beta 1 and alpha 2 beta 1 can selectively recognize different collagen subtypes. Here we show that their alpha I domains can discriminate between laminin isoforms as well: alpha 1I and alpha 2I recognized laminin-111, -211 and -511, whereas their binding to laminin-411 was negligible. Residue Arg-218 in alpha1 was found to be instrumental in high-avidity binding. The gain-of-function mutation E318W makes the alpha 2I domain to adopt the "open" high-affinity conformation, while the wild-type alpha 2I domain favors the "closed" low-affinity conformation. The E318W mutation markedly increased alpha 2I domain binding to the laminins (-111, -211 and -511), leading us to propose that the activation state of the alpha 2 beta 1 integrin defines its role as a laminin receptor. However, neither wild-type nor alpha 2IE318W domain could bind to laminin-411. alpha 2IE318W also bound tighter to all collagens than alpha 2I wild-type, but it showed reduced ability to discriminate between collagens I, IV and IX. The corresponding mutation, E317A, in the alpha 1I domain transformed the domain into a high-avidity binder of collagens I and IV. Thus, our results indicate that conformational activation of integrin alpha 1I and alpha 2I domains leads to high-avidity binding to otherwise disfavored collagen subtypes.     

An internal ligand-bound,metastable state of a leukocyte integrin, αXβ2

How is massive conformational change in integrins achieved on a rapid timescale? We report crystal structures of a metastable, putative transition state of integrin α_Xβ₂. The α_Xβ₂ ectodomain is bent; however, a lattice contact stabilizes its ligand-binding αI domain in a high affinity, open conformation. Much of the αI α7 helix unwinds, loses contact with the αI domain, and reshapes to form an internal ligand that binds to the interface between the β propeller and βI domains. Lift-off of the αI domain above this platform enables a range of extensional and rotational motions without precedent in allosteric machines. Movements of secondary structure elements in the β₂ βI domain occur in an order different than in β₃ integrins, showing that integrin β subunits can be specialized to assume different intermediate states between closed and open. Mutations demonstrate that the structure trapped here is metastable and can enable rapid equilibration between bent and extended-open integrin conformations and up-regulation of leukocyte adhesiveness.     

Regulation of integrin α5β1 conformational states and intrinsic affinities by metal ions and the ADMIDAS

Activation of integrins by Mn²⁺ is a benchmark in the integrin field, but how Mn²⁺ works and whether it reproduces physiological activation is unknown. We show that Mn²⁺ and high Mg²⁺ concentrations compete with Ca²⁺ at the ADMIDAS and shift the conformational equilibrium toward the open state, but the shift is far from complete. Additionally, replacement of Mg²⁺ by Mn²⁺ at the MIDAS increases the intrinsic affinities of both the high-affinity open and low-affinity closed states of integrins, in agreement with stronger binding of Mn²⁺ than Mg²⁺ to oxygen atoms. Mutation of the ADMIDAS increases the affinity of closed states and decreases the affinity of the open state and thus reduces the difference in affinity between the open and closed states. An important biological function of the ADMIDAS may be to stabilize integrins in highly discrete states, so that when integrins support cell adhesion and migration, their high and low affinity correspond to discrete on and off states, respectively.     

Real-time Analysis of Conformation-sensitive Antibody Binding Provides New Insights into Integrin Conformational Regulation

Integrins are heterodimeric adhesion receptors that regulate immune cell adhesion. Integrin-dependent adhesion is controlled by multiple conformational states that include states with different affinity to the ligand, states with various degrees of molecule unbending, and others. Affinity change and molecule unbending play major roles in the regulation of cell adhesion. The relationship between different conformational states of the integrin is unclear. Here we have used conformationally sensitive antibodies and a small LDV-containing ligand to study the role of the inside-out signaling through formyl peptide receptor and CXCR4 in the regulation of α₄β₁ integrin conformation. We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope. Occupancy of the ligand binding pocket without cell activation was sufficient to induce epitope exposure. EC₅₀ for HUTS-21 binding in the presence of LDV was identical to a previously reported ligand equilibrium dissociation constant at rest and after activation. Furthermore, the rate of HUTS-21 binding was also related to the VLA-4 activation state even at saturating ligand concentration. We propose that the unbending of the integrin molecule after guanine nucleotide-binding protein-coupled receptor-induced signaling accounts for the enhanced rate of HUTS-21 binding. Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding. Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.In the bloodstream circulating leukocytes respond to inflammatory signals by rapid changes of cell adhesive properties. These include cell tethering, rolling, arrest, and firm adhesion, all of which are well described steps of leukocyte recruitment to the sites of inflammation (1). Leukocyte arrest and firm adhesion are mediated exclusively by integrin receptors (2). At the same time integrins can also mediate tethering and rolling (3). These largely diverse cell adhesive properties are achieved by sophisticated conformational regulation; multiple states of the same molecule with different affinity for its ligand and different degrees of molecular unbending are attributed to various types of “cellular behavior.” It is proposed that the low affinity bent state translates into a non-adhesive resting cell, the low affinity unbent or extended state of integrin results in cell rolling, and the high affinity state promotes cell arrest (4, 5). However, the exact sequence of conformational events and the relationship between integrin conformational and functional activity remain key questions (6).Integrin conformation is regulated through G-protein-coupled receptors by a signaling pathway which is initiated by ligand binding to a GPCR,³ propagated inside the cell, and results in the binding of signaling proteins (such as talin and others) to cytoplasmic domains of integrin subunits. This binding leads to a separation of the integrin cytoplasmic domains and inside-out activation (6). Chemokines (chemotactic cytokines) as well as “classical” chemoattractants (such as formyl peptide) preferentially signal through heterotrimeric G-proteins coupled to the Gα_i subunit (1). Activation by these ligands results in up-regulation of integrin affinity and/or conformational unbending (extension) of the integrin molecule. These conformational changes lead to cell arrest and firm adhesion. G-protein receptors coupled to Gα_s-coupled subunit (adenylyl cyclase/cAMP signaling pathway) can actively down-regulate the affinity state of the ligand binding pocket without changing integrin conformational unbending. This provides an anti-adhesive signal and results in cell de-adhesion (7). Thus, interaction of multiple G-protein-coupled receptors on a single cell creates a plethora of conformational states. Understanding of the relationship between inside-out signaling through GPCRs and integrin conformational regulation will provide valuable insight into the dynamic regulation of cell adhesion.One technique to study conformational changes of integrins uses conformationally sensitive mAbs that bind to epitopes which are hidden in one conformation and exposed under certain conditions. Lately, it has been accepted that integrins exhibit two major conformations, resting and activated. A number of mAbs for “activated” integrins have been described, and the epitopes have been mapped. Together with mapping of these epitopes into three-dimensional structures of integrin (8), epitope exposure can provide helpful information about integrin conformational changes upon signaling. Moreover, because integrin inside-out activation through different signaling pathways can result in different activation states, the use of previously mapped mAbs can help dissect conformational changes upon activation.Although it is clear that inside-out activation results in a conformational rearrangement of the integrin molecule, the relationship between affinity state of the ligand binding pocket and overall molecule conformation is still debated. Currently, two contrasting models of integrin inside-out integrin activation are described. The “switchblade” model implies that an open head structure with swung-out β-hybrid domain represents the high (or at least intermediate) affinity state. A feature of this model is that integrin extension provides space for hybrid domain swing. The “deadbolt” model proposes that the movement of β-hybrid domain is not related to the inside-out signal. Ligand binding by itself can provide the energy for the hybrid domain swing out (for details, see Ref. 9 and references therein). Because these two models assign different roles to the hybrid domain motion, we evaluated the exposure of VLA-4 hybrid domain epitopes upon activation through two Gα_i-coupled GPCRs (FPR and CXCR4) and ligand binding using the conformationally sensitive HUTS-21 mAb with an epitope mapped to the hybrid domain of β1-integrin (10).We found that contrary to previous reports, where these mAbs were reported to bind or used for the detection of activated integrin (10–13), formyl peptide or SDF-1 treatment alone did not result in any significant exposure of HUTS-21 epitope despite the fact that the VLA-4 affinity up-regulation was detected in parallel on the same batch of cells. Quantitative analysis of mAb binding in real time on live cells suggests that for both the low (resting) and high affinity (induced by inside-out pathway) states, occupancy of the ligand binding pocket rather than inside-out signaling by itself causes the conformational change. Thus, these data support the idea that the hybrid domain movement, which results in the exposure of the mAb epitope, and the high affinity state of the binding pocket are regulated separately and independently of each other, a feature of the deadbolt model of inside-out activation.     

Chemokine arrest signals to leukocyte integrins trigger bi-directional-occupancy of individual heterodimers by extracellular and cytoplasmic ligands

Integrin heterodimers acquire high affinity to endothelial ligands by extensive conformational changes in both their α and β subunits. These heterodimers are maintained in an inactive state by inter-subunit constraints. Changes in the cytoplasmic interface of the integrin heterodimer (referred to as inside-out integrin activation) can only partially remove these constraints. Full integrin activation is achieved when both inter-subunit constraints and proper rearrangements of the integrin headpiece by its extracellular ligand (outside-in activation) are temporally coupled. A universal regulator of these integrin rearrangements is talin1, a key integrin-actin adaptor that regulates integrin conformation and anchors ligand-occupied integrins to the cortical cytoskeleton. The arrest of rolling leukocytes at target vascular sites depends on rapid activation of their α4 and β2 integrins at endothelial contacts by chemokines displayed on the endothelial surface. These chemotactic cytokines can signal within milliseconds through specialized Gi-protein coupled receptors (GPCRs) and Gi-triggered GTPases on the responding leukocytes. Some chemokine signals can alter integrin conformation by releasing constraints on integrin extension, while other chemokines activate integrins to undergo conformational activation mainly after ligand binding. Both of these modalities involve talin1 activation. In this opinion article, I propose that distinct chemokine signals induce variable strengths of associations between talin1 and different target integrins. Weak interactions of the integrin cytoplasmic tail with talin1 (the cytoplasmic integrin ligand) dissociate unless the extracellular ligand can simultaneously occupy the integrin headpiece and transmit, within milliseconds, proper allosteric changes across the integrin heterodimer back to the tail-talin1 complex. The fate of this bi-directional occupancy of integrins by both their extracellular and intracellular ligands is likely to benefit from immobilization of both ligands to cortical cytoskeletal elements. To properly anchor talin1 onto the integrin tail, a second integrin partner, Kindlin-3 may be also required, although an evidence that both partners can simultaneously bind the same integrin heterodimer is still missing. Once linked to the cortical actin cytoskeleton, the multi-occupied integrin complex can load weak forces, which deliver additional allosteric changes to the integrin headpiece resulting in further bond strengthening. Surface immobilized chemokines are superior to their soluble counterparts in driving this bi-directional occupancy process, presumably due to their ability to facilitate local co-occupancy of individual integrin heterodimers with talin1, Kindlin-3 and surface-bound extracellular ligands.Key words: adhesion, migration, endothelium, cytoskeleton, shear stress, immunityFirm adhesion of leukocytes to blood vessels is tightly regulated by integrins and their cognate ligands.^1,2 These include the α₄ integrins, VLA-4 (α₄β₁) and α₄β₇, and the β₂ integrins, LFA-1 (α_Lβ₂) and Mac-1 (α_Mβ₂). Accumulated data suggest that these counter-receptors are structurally adapted to operate under disruptive blood-derived shear forces.³ A remarkable feature of leukocyte integrins is that their affinity state and microclustering can be regulated within fractions of seconds.^4,5 The most robust signals for leukocyte integrins are transduced by chemoattractants, mostly chemokines displayed on the vessel wall.⁶ A growing body of evidence suggests that the Gi protein coupled receptors of these endothelial chemokines elicit diverse signaling pathways in distinct leukocyte subtypes,^2,22 which use two common downstream elements to coactive all leukocyte integrins: talin1 and Kindlin-3.⁷ In this review, I will describe a model explaining how chemokine signals to these elements regulate the conformation of all leukocyte integrins by facilitating a coupled bi-directional occupancy and activation via both their cytoplasmic and headpiece domains.Recent structural and biophysical studies suggest that leukocyte integrins can alternate between inactive bent conformers, which are clasped heterodimers, and variable unclasped heterodimers with extended ectodomains exhibiting intermediate and high affinity to ligand.⁵ Most leukocyte integrins are maintained in an inactive resting state,² whereas in situ chemokine-stimulated integrins unfold and extend 10–25 nm above the cell surface, allowing their headpiece to readily recognize immobilized ligand on a counter surface.⁸ These extended integrins must undergo extensive rearrangements of their headpiece I-domains induced by external endothelial-displayed ligands in order to arrest rolling leukocytes on blood vessel walls. In leukocytes, these two canonical switches are very short-lived, implying the necessity for a stabilization. It is therefore likely that any type of robust integrin activation must involve bi-directional occupancy of the integrin by both its extracellular ligand and one or more cytoplasmic partners.⁹The main cytoplasmic integrin-activating adaptor in leukocytes and platelets is talin1.^10,11 Talin knock down in multiple cell types results in nearly total loss of integrin activation.^12,13 This actin binding adaptor binds different integrin β subunit tails with low affinity,¹⁴ which can be locally increased by in situ generated PI(4,5)P2 (PIP2). This phosphoinositide presumably binds to the FERM domain within the talin head and thereby enhances talin binding to a membrane proximal NPXY motif on the β integrin tail, a key event in integrin heterodimer unclasping.^15,16 Recent studies suggest, however, that mere talin association may be insufficient to unclasp and activate the integrin heterodimer. Thus, the beta subunit tail may need to get co-occupied by the integrin co-activator, Kindlin, in order to optimize talin association with this integrin subunit.^17,18 In leukocytes, Talin1 and the Kindlin family member, Kindlin 3 co-activate both VLA-4 and LFA-1 and this co-activation is dramatically enhanced by multiple chemokine triggered effectors, the nature of which has begun to unfold¹⁹ (Fig. 1). I would like to propose that talin1-Kindlin-3 co-binding to the β tails of these and other leukocyte integrins is insufficient to switch these integrins to a conformation able to bind their soluble extracellular ligands due to fast dissocia-tion of PIP2-activated talin1 from the integrin cytoplasmic tail complex. This short lived talin-integrin complex may, on the other hand, get stabilized, if the integrin headpiece can simultaneously bind an immobilized extracellular ligand and undergo immediate outside-in activation, before the talin1 has dissociated from the integrin beta tail (Fig. 1). Such full confor-mational switch can result in additional allosteric changes in the integrin-bound talin which may expose vinculin binding sites and further increase talin-actin associations that reinforce this bi-directional allosteric integrin activation.²⁰Open in a separate windowFigure 1Bi-directional integrin activation requires simultaneous co-occupancy of the integrin heterodimer by extracellular and cytoplasmic ligands. A proposed scheme for chemokine-triggered integrin activation on leukocytes. Integrin conformation is allosterically modulated in a bidirectional manner by at least two sets of ligands, extracellular and cytoplasmic. The degree of activation is dictated primarily by unclasping of the integrin heterodimer, a process dependent on the binding of the activated talin FERM domain to a specific site on the integrin β tail. (1) Inactive integrin. (2–5) Four postulated integrin conformations triggered by distinct chemokine signals. (2) Talin FERM domain activation close to the target integrin is a rate limiting step in integrin activation. This activation is triggered by PIP2 locally generated by talin-associated PIP5Kγ (purple rectangle) stimulated by a nearby Gi-coupled chemokine receptor. (3) Kindlin-3 binding to the integrin β tail stabilizes the otherwise weak talin1-integrin tail complex. The activated integrin can bind a soluble extracellular ligand with a low affinity due to a high k_off of the soluble ligand from the integrin headpiece. (4) In the absence of Kindlin-3, chemokine triggered, PIP2-activated talin1 binds only transiently the integrin tail (High k_off). The semiactivated integrin, even if occupied by an immobilized extracellular ligand, cannot undergo full bi-directional activation. (5) When both the extracellular ligand and talin are properly anchored, their escape from the integrin is dramatically reduced, lowering the k_off. Low k_off increases the probability of simultaneous bi-directional occupancy of both the integrin headpiece by the extracellular ligand and of the integrin tail by talin1 and Kindlin-3. This results in optimal bi-directional integrin activation and unclasping of the heterodimer. Stable linkages also allow this bi-directionally occupied integrin to undergo extensive mechanical strengthening by low forces applied on the headpiece; this further activates the headpiece I domains, further separates the β and α subunits from each other, and maximally stabilizes the unclasped integrin. Force application through the high affinity-talin complex can stretch the talin rod domain and expose vinculin binding sites (VBS). Since integrin ligands are generally multivalent, rapid integrin dimerization can take place to further stabilize the focal adhesion (not shown). Additional cytoplasmic partners of specific leukocyte integrins like a-actinin, L-plastin and RAPL may further strengthen subsets of focal adhesions. These and other cytosolic partners bind different integrin targets with different affinities. Therefore the effect of each of these partners on both the kinetics and stability of the talin1-integrin tail complex may vary with the cell type, the integrin type, the strength of the chemokine signal and the proximity between the integrin and its stimulatory GPCR.How can such postulated simultaneous bi-directional occupancy of a leukocyte integrin can be so rapidly triggered by a chemokine signal encountered during leukocyte rolling on blood vessels? An attractive mechanism for in situ facilitation of talin1 binding to the integrin β tail by chemokine signals involves chemokine triggered Gi stimulated RhoA and Rac1 GTPases and their downstream target, the PIP2 generating enzyme PIP5Kγ in the vicinity of the in situ activated integrin¹⁹ (Fig. 1). Additional talin1 molecules may also be recruited to the vicinity of this initially stimulated integrin by RIAM,²¹ an effector that associates with activated Rap-1, one of the key chemokine stimulated GTPases involved in rapid integrin mediated activation in both leukocytes and platelets.^22,23 To bidirectionally bind and activate their integrin targets, both the cytoplasmic integrin ligands, Talin1 and Kindlin-3 and the extracellular integrin ligand may need to achieve low dissociation rates from the integrin tail and headpiece, respectively. Why would an immobilized extracellular ligand be superior to soluble extracellular ligand in capacity to bi-directionally bind and activate a leukocyte integrin? The probability that a given surface-bound ligand, rather than a soluble integrin ligand would escape from its cognate integrin receptor following its dissociation is very small, since reactants in viscous medium are more likely to recombine than to diffuse apart.²⁴ Thus, surface-immobilized single integrin ligands may rebind the integrins they recenty dissociated from much more frequently than their soluble counterparts. Similarly, the cytoplasmic ligands talin1 and Kindlin may need to remain immobile once occupying their target integrin tail. Such immobilization of talin1 can be optimized by talin anchoring to the cortical cytoskeleton.²⁵ Talin may be also restricted from immediate dissociation from the integrin tail by Kindlin-3. An optimal integrin activating chemokine signal would therefore not only need to recruit and induce talin1 association with the β subunit of the target integrin and opening of the integrin clasp, but also need to keep the talin in an immobile state, and thereby maintain its low dissociation rate from its integrin tail sites.As both the integrin headpiece and the integrin subunits are predicted to undergo faster opening and separation in the presence of applied forces,^26,27 another tradeoff of this postulated immobilization of both the intracellular and extracellular integrin ligands is optimal force sensing of the integrin heterodimer. Application of force on the bidirectionally occupied integrin and its cognate ligands would be possible only if the integrin, its extracellular ligand, and talin1 are all properly anchored.^3,28,29 Force transduction through the integrin-talin1 complex can transmit additional conformational changes on the integrin-occupied talin by exposing vinculin binding sites on the talin rod.³⁰ Additional chemokine signals may induce talin rod phosphorylation and other changes in actin-talin associations (Fig. 1) that may further facilitate talin anchorage to the cortical cytoskeleton and subsequent microclustering of adjacent ligand-occupied integrins. It is well recognized that ligand occupancy anchors integrins to the cortical cytoskeleton.³¹ Thus, the anchorage states of both the extracellular and the cytoplasmic ligands of a given integrin may facilitate bidirectional integrin occupancy and optimize force driven bi-directional activation of the integrin-ligand complex and subsequent dimerization of ligand-occupied integrins. The ability of different integrin-ligand complexes to undergo diverse mechanochemical rearrangements provides a broad spectrum of integrin-ligand bond strengths, accounting for the unique capacity of chemokine stimulated leukocyte integrins to support both firm and reversible adhesions of leukocytes to their endothelial ligands.