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.     

The relative influence of metal ion binding sites in the I-like domain and the interface with the hybrid domain on rolling and firm adhesion by integrin alpha4beta7

We examined the effect of conformational change at the beta(7) I-like/hybrid domain interface on regulating the transition between rolling and firm adhesion by integrin alpha(4)beta(7). An N-glycosylation site was introduced into the I-like/hybrid domain interface to act as a wedge and to stabilize the open conformation of this interface and hence the open conformation of the alpha(4) beta(7) headpiece. Wild-type alpha(4)beta(7) mediates rolling adhesion in Ca(2+) and Ca(2+)/Mg(2+) but firm adhesion in Mg(2+) and Mn(2+). Stabilizing the open headpiece resulted in firm adhesion in all divalent cations. The interaction between metal binding sites in the I-like domain and the interface with the hybrid domain was examined in double mutants. Changes at these two sites can either counterbalance one another or be additive, emphasizing mutuality and the importance of multiple interfaces in integrin regulation. A double mutant with counterbalancing deactivating ligand-induced metal ion binding site (LIMBS) and activating wedge mutations could still be activated by Mn(2+), confirming the importance of the adjacent to metal ion-dependent adhesion site (ADMIDAS) in integrin activation by Mn(2+). Overall, the results demonstrate the importance of headpiece allostery in the conversion of rolling to firm adhesion.     

Effects of altered restraints in beta1 integrin on the force-regulated interaction between the glycosylated I-like domain of beta1 integrin and fibronectin III9-10: a steered molecular dynamic study

Cytoskeletal restraints affect force-regulated integrin function in cell adhesion. However, the structural and molecular basis underlying the effect of cytoskeletal restraints on beta1 integrin binding to fibronectin is still largely unknown. In this study, we used steered molecular dynamics simulations to investigate the changes in glycosylated beta1 integrin-fibronectin binding and in conformation and structure of the glycosylated beta1 I-like domain-FN-III9-10 complex caused by altered restraints applied to beta1 I-like domain. The results revealed that imposition of the increased constraints on beta1 integrin increased resistance to force-induced dissociation of the beta1 I-like domain-fibronectin complex. Specifically, the increased constraints enhanced resistance to relative conformational changes in the RGD-synergy site in fibronectin, increased the conformational stability of fibronectin, and prevented losses in hydrogen bond occupancy of each beta-strand pair in FN-III10 resulting from external force. The increased constraints also resulted in an increase in correlated motion between residues in the beta1 I-like domain, which may directly affect the interaction of beta1 integrin with fibronectin. Results from this study provide molecular and structural insights into the effects of altered restraints in beta1 integrin on the interaction between glycosylated beta1 Integrin and fibronectin and its induced cell adhesion.     

High affinity ligand binding by integrins does not involve head separation

Conformational change in the integrin extracellular domain is required for high affinity ligand binding and is also involved in post-ligand binding cellular signaling. Although there is evidence to the contrary, electron microscopic studies showing that ligand binding triggers alpha- and beta-subunit dissociation in the integrin headpiece have gained popularity and support the hypothesis that head separation activates integrins. To test directly the head separation hypothesis, we enforced head association by introducing disulfide bonds across the interface between the alpha-subunit beta-propeller domain and the beta-subunit I-like domain. Basal and activation-dependent ligand binding by alpha(IIb)beta(3) and alpha(V)beta(3) was unaffected. The covalent linkage prevented dissociation of alpha(IIb)beta(3) into its subunits on EDTA-treated cells. Whereas EDTA dissociated wild type alpha(IIb)beta(3) on the cell surface, a ligand-mimetic Arg-Gly-Asp peptide did not, as judged by binding of complex-specific antibodies. Finally, a high affinity ligand-mimetic compound stabilized noncovalent association between alpha(IIb) and beta(3) headpiece fragments in the presence of SDS, indicating that ligand binding actually stabilized subunit association at the head, as opposed to the suggested subunit separation. The mechanisms of conformational regulation of integrin function should therefore be considered in the context of the associated alphabeta headpiece.     

Allosteric beta1 integrin antibodies that stabilize the low affinity state by preventing the swing-out of the hybrid domain

The ligand binding function of integrins can be modulated by various monoclonal antibodies by both direct and indirect mechanisms. We have characterized an anti-beta(1) antibody, SG/19, that had been reported to inhibit the function of the beta(1) integrin on the cell surface. SG/19 recognized the wild type beta(1) subunit that exists in a conformational equilibrium between the high and low affinity states but bound poorly to a mutant beta(1) integrin that had been locked in a high affinity state. Epitope mapping of SG/19 revealed that Thr(82) in the beta(1) subunit, located at the outer face of the boundary between the I-like and hybrid domains, was the key binding determinant for this antibody. Direct visualization of the alpha (5)beta(1) headpiece fragment in complex with SG/19 Fab with electron microscopy confirmed the location of the binding surface and showed that the ligand binding site is not occluded by the bound Fab. Surface plasmon resonance showed that alpha (5)beta(1) integrin bound by SG/19 maintained a low affinity toward its physiological ligand fibronectin (Fn) whereas binding by function-blocking anti-alpha(5) antibodies resulted in a complete loss of fibronectin binding. Thus a class of the anti-beta antibodies represented by SG/19 attenuate the ligand binding function by restricting the conformational shift to the high affinity state involving the swing-out of the hybrid domain without directly interfering with ligand docking.     

The role of the specificity-determining loop of the integrin beta subunit I-like domain in autonomous expression, association with the alpha subunit, and ligand binding

Integrin beta subunits contain a highly conserved I-like domain that is known to be important for ligand binding. Unlike integrin I domains, the I-like domain requires integrin alpha and beta subunit association for optimal folding. Pactolus is a novel gene product that is highly homologous to integrin beta subunits but lacks associating alpha subunits [Chen, Y., Garrison, S., Weis, J. J., and Weis, J. H. (1998) J. Biol. Chem. 273, 8711-8718] and a approximately 30 amino acid segment corresponding to the specificity-determining loop (SDL) in the I-like domain. We find that the SDL is responsible for the defects in integrin beta subunit expression and folding in the absence of alpha subunits. When transfected in the absence of alpha subunits into cells, extracellular domains of mutant beta subunits lacking SDL, but not wild-type beta subunits, were well secreted and contained immunoreactive I-like domains. The purified recombinant soluble beta1 subunit with the SDL deletion showed an elongated shape in electron microscopy, consistent with its structure in alphabeta complexes. The SDL segment is not required for formation of alpha5beta1, alpha4beta1, alphaVbeta3, and alpha6beta4 heterodimers, but is essential for fomation of alpha6beta1, alphaVbeta1, and alphaLbeta2 heterodimers, suggesting that usage of subunit interface residues is variable among integrins. The beta1 SDL is required for ligand binding and for the formation of the epitope for the alpha5 monoclonal antibody 16 that maps to loop segments connecting blades 2 and 3 of beta-propeller domain of alpha5, but is not essential for nearby beta-propeller epitopes.     

Locking the beta3 integrin I-like domain into high and low affinity conformations with disulfides

Although integrin alpha subunit I domains exist in multiple conformations, it is controversial whether integrin beta subunit I-like domains undergo structurally analogous movements of the alpha7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the beta(3) integrin I-like domain to lock its beta6-alpha7 loop and alpha7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor alpha(IIb)beta(3)(T329C/A347C) was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded alpha(IIb)beta(3)(V332C/M335C) was locked in a high affinity state. The results suggest that activation of the beta subunit I-like domain is analogous to that of the alpha subunit I domain, i.e. that axial movement in the C-terminal direction of the alpha7-helix is linked to rearrangement of the I-like domain metal ion-dependent adhesion site into a high affinity conformation.     

Role of Altered Sialylation of the I-Like Domain of β1 Integrin in the Binding of Fibronectin to β1 Integrin: Thermodynamics and Conformational Analyses

N-glycosylation of the I-like domain of β1 integrin plays an essential role in integrin structure and function, and the altered sialylation of β1 integrin regulates β1 integrin binding to fibronectin. However, the structural basis underlying the effect of altered sialylation of the β1 I-like domain on β1 integrin binding to fibronectin remains largely unknown. In this study, we used a combination of molecular dynamics simulations and binding free energy analyses to investigate changes in binding thermodynamics and in conformation of the glycosylated β1 I-like domain-FN-III_9-10 complex caused by altered sialylation of the β1 I-like domain. Binding free energy analyses showed that desialylation of β1 I-like domain increased β1 integrin binding to fibronectin, consistent with experimental results. Interaction analyses showed that altered sialylation of the β1 I-like domain resulted in significant changes in the interaction of the N-glycans of the I-like domain with both the I-like domain and fibronectin, and these changes could directly affect the allosteric regulation of the interaction between the I-like domain and fibronectin. Altered sialylation of the β1 I-like domain caused significant conformational changes in key functional sites of both the β1 I-like domain and fibronectin. In addition, altered sialylation of the β1 I-like domain resulted in changes in the degree of correlated motions between residues in the I-like domain and residues in fibronectin, and in the degree of motion changes in fibronectin, which could affect β1 integrin binding to fibronectin. We believe results from this study provide thermodynamic and structural evidence for a role of altered sialylation of β1 integrin in regulating β1 integrin binding to fibronectin and it's induced cellular activities.     

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.     

A protein kinase C/Ras/ERK signaling pathway activates myeloid fibronectin receptors by altering beta1 integrin sialylation

Here we report that myeloid cells differentiating along the monocyte/macrophage lineage down-regulate the ST6Gal-I sialyltransferase via a protein kinase C/Ras/ERK signaling cascade. In consequence, the beta1 integrin subunit becomes hyposialylated, which stimulates the ligand binding activity of alpha5beta1 fibronectin receptors. Pharmacologic inhibitors of protein kinase C, Ras, and MEK, but not phosphoinositide 3-kinase, block ST6Gal-I down-regulation, integrin hyposialylation, and fibronectin binding. In contrast, constitutively active MEK stimulates these same events, indicating that ERK is both a necessary and sufficient activator of hyposialylation-dependent integrin activation. Consistent with the enhanced activity of hyposialylated cell surface integrins, purified alpha5beta1 receptors bind fibronectin more strongly upon enzymatic desialylation, an effect completely reversed by resialylation of these integrins with recombinant ST6Gal-I. Finally, we have mapped the N-glycosylation sites on the beta1 integrin to better understand the potential effects of differential sialylation on integrin structure/function. Notably, there are three N-glycosylated sites within the beta1 I-like domain, a region that plays a crucial role in ligand binding. Our collective results suggest that variant sialylation, induced by a specific signaling cascade, mediates the sustained increase in cell adhesiveness associated with monocytic differentiation.     

Effect of altered glycosylation on the structure of the I-like domain of beta1 integrin: a molecular dynamics study

Glycosylation plays an important role in the regulation of integrin function. Molecular mechanisms underlying the effects of altered glycosylation on beta1 integrin structure and function are still largely unknown. In this study, we used a molecular modeling approach to study the effects of altered glycosylation, with alpha2-6 sialic acid and without alpha2-6 sialic acid, on the structure of the I-like domain of the beta1 integrin. Our results demonstrated that altered glycosylation affected the interactions between oligosaccharides and the I-like domain, which in turn changed the accessibility of the specificity-determining loop for ligand binding. Altered glycosylation caused significant conformational changes for most of the key functional regions of the I-like domain of beta1 integrin, including the metal ion-dependent adhesion site that contains a DLSYS motif, and other critical residues for ligand binding (Asn-224, Glu-229, Asp-233, Asp-267, and Asp-295). In addition, altered glycosylation caused significant movement of the alpha1 and alpha7 helices, which are important for the activation of beta1 integrin. The results from this study offered molecular mechanisms for the experimental observations that variant glycosylation regulates integrin function.     

How the headpiece hinge angle is opened: New insights into the dynamics of integrin activation

How the integrin head transitions to the high-affinity conformation is debated. Although experiments link activation with the opening of the hinge angle between the betaA and hybrid domains in the ligand-binding headpiece, this hinge is closed in the liganded alpha(v)beta3 integrin crystal structure. We replaced the RGD peptide ligand of this structure with the 10th type III fibronectin module (FnIII10) and discovered through molecular dynamics (MD) equilibrations that when the conformational constraints of the leg domains are lifted, the betaA/hybrid hinge opens spontaneously. Together with additional equilibrations on the same nanosecond timescale in which small structural variations impeded hinge-angle opening, these simulations allowed us to identify the allosteric pathway along which ligand-induced strain propagates via elastic distortions of the alpha1 helix to the betaA/hybrid domain hinge. Finally, we show with steered MD how force accelerates hinge-angle opening along the same allosteric pathway. Together with available experimental data, these predictions provide a novel framework for understanding integrin activation.     

Intercellular adhesion molecule-3 binding of integrin alphaL beta2 requires both extension and opening of the integrin headpiece

The leukocyte-restricted integrin alpha(L)beta(2) is required in immune processes such as leukocyte adhesion, migration, and immune synapse formation. Activation of alpha(L)beta(2) by conformational changes promotes alpha(L)beta(2) binding to its ligands, ICAMs. It was reported that different affinity states of alpha(L)beta(2) are required for binding ICAM-1 and ICAM-3. Recently, the bent, extended with a closed headpiece, and extended with open headpiece conformations of alpha(L)beta(2), was reported. To address the overall conformational requirements of alpha(L)beta(2) that allow selective binding of these ICAMs, we examined the adhesion properties of these alpha(L)beta(2) conformers. alpha(L)beta(2) with different conformations were generated by mutations, and verified by using a panel of reporter mAbs that detect alpha(L)beta(2) extension, hybrid domain movement, or I-like domain activation. We report a marked difference between extended alpha(L)beta(2) with closed and open headpieces in their adhesive properties to ICAM-1 and ICAM-3. Our data show that the extension of alpha(L)beta(2) alone is sufficient to mediate ICAM-1 adhesion. By contrast, an extended alpha(L)beta(2) with an open headpiece is required for ICAM-3 adhesion.     

TSG-6 binds via its CUB_C domain to the cell-binding domain of fibronectin and increases fibronectin matrix assembly.

Human plasma fibronectin binds with high affinity to the inflammation-induced secreted protein TSG-6. Fibronectin binds to the CUB_C domain of TSG-6 but not to its Link module. TSG-6 can thus act as a bridging molecule to facilitate fibronectin association with the TSG-6 Link module ligand thrombospondin-1. Fibronectin binding to TSG-6 is divalent cation-independent and is conserved in cellular fibronectins. Based on competition binding studies using recombinant and proteolytic fragments of fibronectin, TSG-6 binding localizes to type III repeats 9-14 of fibronectin. This region of fibronectin contains the Arg-Gly-Asp sequence recognized by alpha5beta1 integrin, but deletion of that sequence does not prevent TSG-6 binding, and TSG-6 does not inhibit cell adhesion on fibronectin substrates mediated by this integrin. This region of fibronectin is also involved in fibronectin matrix assembly, and addition of TSG-6 enhances exogenous and endogenous fibronectin matrix assembly by human fibroblasts. Therefore, TSG-6 is a high affinity ligand that can mediate fibronectin interactions with other matrix components and modulate some interactions of fibronectin with cells.     

Molecular dissection of the alpha-dystroglycan- and integrin-binding sites within the globular domain of human laminin-10

The adhesive interactions of cells with laminins are mediated by integrins and non-integrin-type receptors such as alpha-dystroglycan and syndecans. Laminins bind to these receptors at the C-terminal globular domain of their alpha chains, but the regions recognized by these receptors have not been mapped precisely. In this study, we sought to locate the binding sites of laminin-10 (alpha5beta1gamma1) for alpha(3)beta(1) and alpha(6)beta(1) integrins and alpha-dystroglycan through the production of a series of recombinant laminin-10 proteins with deletions of the LG (laminin G-like) modules within the globular domain. We found that deletion of the LG4-5 modules did not compromise the binding of laminin-10 to alpha(3)beta(1) and alpha(6)beta(1) integrins but completely abrogated its binding to alpha-dystroglycan. Further deletion up to the LG3 module resulted in loss of its binding to the integrins, underlining the importance of LG3 for integrin binding by laminin-10. When expressed individually as fusion proteins with glutathione S-transferase or the N-terminal 70-kDa region of fibronectin, only LG4 was capable of binding to alpha-dystroglycan, whereas neither LG3 nor any of the other LG modules retained the ability to bind to the integrins. Site-directed mutagenesis of the LG3 and LG4 modules indicated that Asp-3198 in the LG3 module is involved in the integrin binding by laminin-10, whereas multiple basic amino acid residues in the putative loop regions are involved synergistically in the alpha-dystroglycan binding by the LG4 module.     

Probing the integrin-binding site within the globular domain of laminin-511 with the function-blocking monoclonal antibody 4C7.

In an attempt to elucidate the integrin-binding site within laminin-511 (alpha5beta1gamma1), we mapped the epitope for mAb 4C7, which recognizes the globular (G) domain of the laminin alpha5 chain and inhibits binding of integrin alpha6beta1 to laminin-511, using a series of recombinant laminin-511 mutants with deletions or substitutions in the G domain. Deletion of the LG2-5 modules only partially compromised the 4C7 binding activity, while deletion of all 5 LG modules completely abrogated the activity, indicating that the epitope for 4C7 resides in the LG1 module. In support of this conclusion, 4C7 reactivity was abolished when the LG1 module of laminin-511 was swapped with the corresponding module of laminin-111, but the reactivity was retained after swapping the LG2 or LG3 module. Despite the requirement of LG1 for 4C7 binding, a recombinant LG1 module failed to bind to 4C7 when expressed alone or in tandem with LG2, but exhibited significant 4C7 binding activity when expressed as an array of LG1-3. These results indicate that 4C7 recognizes an epitope in the LG1 module, whose active conformation is stabilized in the context of the LG1-3 modules. Despite their 4C7 binding activities, neither the recombinant LG1-3 fragment nor the LG2 and LG3 swap mutants were capable of binding to integrin alpha6beta1. Thus, the integrin binding activity does not necessarily parallel the 4C7 reactivity, and possibly requires a strictly defined conformation of the LG1 module which can only be attained within an array of the intact LG1-3 modules connected to the preceding coiled-coil domain.     

Conformational regulation of the fibronectin binding and alpha 3beta 1 integrin-mediated adhesive activities of thrombospondin-1

The recognition of extracellular matrix components can be regulated by conformational changes that alter the activity of cell surface integrins. We now demonstrate that conformational regulation of the matrix glycoprotein thrombospondin-1 (TSP1) can also modulate its binding to an integrin receptor. F18 1G8 is a conformation-sensitive TSP1 antibody that binds weakly to soluble TSP1 in the presence of divalent cations. However, binding of the antibody to melanoma cells was strongly stimulated by adding exogenous TSP1 in the presence of calcium, suggesting that TSP1 undergoes a conformational change following its binding to the cell surface. This conformation was not induced by known cell surface TSP1 receptors, whereas binding of F18 was stimulated when TSP1 bound to fibronectin but not to heparin or fibrinogen. Conversely, binding of F18 to TSP1 enhanced TSP1 binding to fibronectin. Exogenous fibronectin also stimulated TSP1-dependent binding of F18 to melanoma cells. Binding of the fibronectin-TSP1 complex to melanoma cells was mediated by alpha4beta1 and alpha5beta1 integrins. Furthermore, binding to F18 or fibronectin strongly enhanced the adhesive activity of immobilized TSP1 for some cell types. This enhancement of adhesion was mediated by alpha3beta1 integrin and required that the alpha3beta1 integrin be in an active state. Fibronectin also enhanced TSP1 binding to purified alpha3beta1 integrin. Therefore, both fibronectin and the F18 antibody induce conformational changes in TSP1 that enhance the ability of TSP1 to be recognized by alpha3beta1 integrin. The conformational and functional regulation of TSP1 activity by fibronectin represents a novel mechanism for extracellular signal transduction.     

Small molecule inhibitors induce conformational changes in the I domain and the I-like domain of lymphocyte function-associated antigen-1. Molecular insights into integrin inhibition.

The beta(2) integrin lymphocyte function-associated antigen-1 (LFA-1) is a conformationally flexible alpha/beta heterodimeric receptor, which is expressed on the surface of all leukocytes. LFA-1 mediates cell adhesion crucial for normal immune and inflammatory responses. Intracellular signals or cations are required to convert LFA-1 from a nonligand binding to a ligand binding state. Here we investigated the effect of small molecule inhibitors on LFA-1 by monitoring the binding of monoclonal antibodies mapped to different receptor domains. The inhibitors were found to not only induce epitope changes in the I domain of the alpha(L) chain but also in the I-like domain of the beta(2) chain depending on the individual chemical structure of the inhibitor and its binding site. For the first time, we provide strong evidence that the I-like domain represents a target for allosteric LFA-1 inhibition similar to the well established regulatory L-site on the I domain of LFA-1. Moreover, the antibody binding patterns observed in the presence of the various inhibitors establish a conformational interaction between the LFA-1 I domain and the I-like domain in the native receptor that is formed upon activation. Differentially targeting the binding sites of the inhibitors, the L-site and the I-like domain, may open new avenues for highly specific therapeutic intervention in diseases where integrins play a pathophysiological role.     

Interdomain tilt angle determines integrin-dependent function of the ninth and tenth FIII domains of human fibronectin

Integrins are an important family of signaling receptors that mediate diverse cellular processes. The binding of the abundant extracellular matrix ligand fibronectin to integrins alpha(5)beta(1) and alpha(v)beta(3) is known to depend upon the Arg-Gly-Asp (RGD) motif on the tenth fibronectin FIII domain. The adjacent ninth FIII domain provides a synergistic effect on RGD-mediated integrin alpha(5)beta(1) binding and downstream function. The precise molecular basis of this synergy remains elusive. Here we have dissected further the function of FIII9 in integrin binding by analyzing the biological activity of the FIII9-10 interdomain interface variants and by determining their structural and dynamic properties in solution. We demonstrate that the contribution of FIII9 to both alpha(5)beta(1) and alpha(v)beta(3) binding and downstream function critically depends upon the interdomain tilt between the FIII9 and FIII10 domains. Our data suggest that modulation of integrin binding by FIII9 may arise in part from its steric properties that determine accessibility of the RGD motif. These findings have wider implications for mechanisms of integrin-ligand binding in the physiological context.     

Urokinase-type plasminogen activator receptor induces conformational changes in the integrin alphaMbeta2 headpiece and reorientation of its transmembrane domains

The glycosylphosphatidylinositol-linked urokinase-type plasminogen activator receptor (uPAR) interacts with the heterodimer cell adhesion molecules integrins to modulate cell adhesion and migration. Devoid of a cytoplasmic domain, uPAR triggers intracellular signaling via its associated molecules that contain cytoplasmic domains. Interestingly, uPAR changes the ectodomain conformation of one of its partner molecules, integrin alpha(5)beta(1), and elicits cytoplasmic signaling. The separation or reorientation of integrin transmembrane domains and cytoplasmic tails are required for integrin outside-in signaling. However, there is a lack of direct evidence showing these conformational changes of an integrin that interacts with uPAR. In this investigation we used reporter monoclonal antibodies and fluorescence resonance energy transfer analyses to show conformational changes in the alpha(M)beta(2) headpiece and reorientation of its transmembrane domains when alpha(M)beta(2) interacts with uPAR.