Plexin-semaphorin-integrin Domain Research Articles

Engineering, Characterization, and Biological Evaluation of an Antibody Targeting the HGF Receptor.

The Hepatocyte growth factor (HGF) and its receptor (MET) promote several physiological activities such as tissue regeneration and protection from cell injury of epithelial, endothelial, neuronal and muscle cells. The therapeutic potential of MET activation has been scrutinized in the treatment of acute tissue injury, chronic inflammation, such as renal fibrosis and multiple sclerosis (MS), cardiovascular and neurodegenerative diseases. On the other hand, the HGF-MET signaling pathway may be caught by cancer cells and turned to work for invasion, metastasis, and drug resistance in the tumor microenvironment. Here, we engineered a recombinant antibody (RDO24) and two derived fragments, binding the extracellular domain (ECD) of the MET protein. The antibody binds with high affinity (8 nM) to MET ECD and does not cross-react with the closely related receptors RON nor with Semaphorin 4D. Deletion mapping studies and computational modeling show that RDO24 binds to the structure bent on the Plexin-Semaphorin-Integrin (PSI) domain, implicating the PSI domain in its binding to MET. The intact RDO24 antibody and the bivalent Fab2, but not the monovalent Fab induce MET auto-phosphorylation, mimicking the mechanism of action of HGF that activates the receptor by dimerization. Accordingly, the bivalent recombinant molecules induce HGF biological responses, such as cell migration and wound healing, behaving as MET agonists of therapeutic interest in regenerative medicine. In vivo administration of RDO24 in the murine model of MS, represented by experimental autoimmune encephalomyelitis (EAE), delays the EAE onset, mitigates the early clinical symptoms, and reduces inflammatory infiltrates. Altogether, these results suggest that engineered RDO24 antibody may be beneficial in multiple sclerosis and possibly other types of inflammatory disorders.

Thiol Isomerase Activity of β3 Integrin Psi Domain and L33P Polymorphism: Implications in Blood Coagulation and Anti-Thrombotic Therapy

Background: Integrins are α and β subunit heterodimeric receptors expressed ubiquitously on metazoan cells that play key roles in cell adhesion, movement, and signaling. The αIIbβ3 integrin on platelets is essential for thrombosis and hemostasis through its binding of fibrinogen and other ligands to mediate platelet adhesion and aggregation. The plexin-semaphorin-integrin (PSI) domain is an approximately 54 amino acid sequence at the N-terminus of the β3 subunit located in close proximity to the “knee” region. Our lab recently discovered that two “CXXC” motifs within PSI domains in the integrin family exert endogenous thiol isomerase activity, and blockade of this activity in β3 integrin with our novel anti-PSI domain monoclonal antibodies (mAbs) significantly attenuated platelet adhesion and aggregation without impairing hemostasis (Blood, 2017). Interestingly, intravital microscopy studies demonstrate that our anti-PSI antibodies inhibited thrombosis in vivo 10-30 fold more potent than their effects in vitro under anti-coagulant conditions, suggesting a possible inhibitory effect on blood coagulation. Notably, A L33P polymorphism (HPA-1b) within the β3 integrin PSI domain is linked to an approximately two-fold increased risk for cardiovascular disease (CVD), however, the underlying mechanism for this remains unclear.Methods/Results: To investigate the role of PSI domain in blood coagulation, we first employed thromboelastography (TEG) to compare our anti-PSI domain mAbs with other anti-β3 mAbs that do not directly bind to β3 PSI domain. Results show that anti-PSI domain mAbs inhibited blood coagulation in human and murine whole blood or platelet-rich plasma (PRP) significantly more than anti-αIIbβ3 antibodies JAN-D1, M1, and Abciximab precursor 7E3. To address whether the L33P polymorphism affects PSI domains thiol isomerase activity, we generated L33P PSI domain via site-directed mutagenesis in E. coli. Using a scrambled RNase assay, we found that L33P polymorphism enhanced thiol-isomerase activity relative to WT PSI domain. We further corroborated these findings through an insulin β chain reduction assay, and a MPB (N-Maleimidopropionyl-biocytin) western blot assay, which quantifies thiol isomerase activity through MPB binding to free thiols that have not been oxidized into RNase. Interestingly, TEG results show that recombinant human PSI domain enhanced blood coagulation in platelet-microparticle (PMP) free plasma, which was generated through high-speed centrifugation (17,000 x g) of platelet poor plasma (PPP)for 15 minutes that removed residual platelets and microparticles .Conclusion: We have discovered a novel role of integrin β3 PSI domain in blood coagulation, which is enhanced by the L33P polymorphism (HPA-1b). These data highlight the β3 PSI domain as a suitable therapeutic target for its roles in both platelet adhesion/aggregation, and blood coagulation. Furthermore, these data may explain the increased risk of CVD such as myocardial infarction and deep vein thrombosis for individuals with the L33P polymorphism. DisclosuresNo relevant conflicts of interest to declare.

Updated Understanding of Platelets in Thrombosis and Hemostasis: The Roles of Integrin PSI Domains and their Potential as Therapeutic Targets.

Platelets are small blood cells known primarily for their ability to adhere and aggregate at injured vessels to arrest bleeding. However, when triggered under pathological conditions, the same adaptive mechanism of platelet adhesion and aggregation may cause thrombosis, a primary cause of heart attack and stroke. Over recent decades, research has made considerable progress in uncovering the intricate and dynamic interactions that regulate these processes. Integrins are heterodimeric cell surface receptors expressed on all metazoan cells that facilitate cell adhesion, movement, and signaling, to drive biological and pathological processes such as thrombosis and hemostasis. Recently, our group discovered that the plexin-semaphorin-integrin (PSI) domains of the integrin β subunits exert endogenous thiol isomerase activity derived from their two highly conserved CXXC active site motifs. Given the importance of redox reactions in integrin activation and its location in the knee region, this PSI domain activity may be critically involved in facilitating the interconversions between integrin conformations. Our monoclonal antibodies against the β3 PSI domain inhibited its thiol isomerase activity and proportionally attenuated fibrinogen binding and platelet aggregation. Notably, these antibodies inhibited thrombosis without significantly impairing hemostasis or causing platelet clearance. In this review, we will update mechanisms of thrombosis and hemostasis, including platelet versatilities and immune-mediated thrombocytopenia, discuss critical contributions of the newly discovered PSI domain thiol isomerase activity, and its potential as a novel target for anti-thrombotic therapies and beyond.

Dissection of \u03b14\u03b27 integrin regulation by Rap1 using novel conformation-specific monoclonal anti-\u03b27 antibodies

Integrin activation is associated with conformational regulation. In this study, we developed a system to evaluate conformational changes in α4β7 integrin. We first inserted the PA tag into the plexin-semaphorin-integrin (PSI) domain of β7 chain, which reacted with an anti-PA tag antibody (NZ-1) in an Mn2+-dependent manner. The small GTPase Rap1 deficiency, as well as chemokine stimulation and the introduction of the active form of Rap1, Rap1V12, enhanced the binding of NZ-1 to the PA-tagged mutant integrin, and increased the binding affinity to mucosal addressing cell adhesion molecule-1 (MAdCAM-1). Furthermore, we generated two kinds of hybridomas producing monoclonal antibodies (mAbs) that recognized Mn2+-dependent epitopes of β7. Both epitopes were exposed to bind to mAbs on the cells by the introduction of Rap1V12. Although one epitope in the PSI domain of β7 was exposed on Rap1-deficienct cells, the other epitope in the hybrid domain of β7 was not. These data indicate that the conversion of Rap1-GDP to GTP exerts two distinct effects stepwise on the conformation of α4β7. The induction of colitis by Rap1-deficient CD4+ effector/memory T cells suggests that the removal of constraining effect by Rap1-GDP on α4β7 is sufficient for homing of these pathogenic T cells into colon lamina propria (LP).

An Authentic Murine Model of Fetal/Neonatal Alloimmune Thrombocytopenia

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies directed against paternally-inherited antigens present on the surface of fetal platelets. The human platelet alloantigen, HPA-1a (also known as the PlA1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Caucasians. A single Leu33Pro amino acid polymorphism residing within the ~50 amino acid plexin/semaphorin/integrin (PSI) domain near the N-terminus of the integrin β3 subunit (platelet membrane glycoprotein (GP)IIIa), is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa. Neither human polyclonal nor mouse monoclonal anti-HPA-1a antibodies, however, recognize murine GPIIIa due to amino acid differences both within and surrounding the HPA-1a alloepitope. As a result, there are currently no authentic mouse models of FNAIT capable of recapitulating the human alloimmune response to this clinically important platelet alloantigen. We have recently shown that humanizing residues 30, 32, 33, and 39 within the PSI domain, as well as amino acid 470 within the linearly distant but conformationally close Epidermal Growth Factor (EGF) 1 domain, of murine GPIIIa, is sufficient to re-create the epitope recognized by most maternal anti-HPA-1a alloantibodies. We therefore used CRISPR/Cas9 gene editing technology to generate transgenic mice whose platelets express human residues A30P32L33D39Q470 on a murine GPIIIa backbone - hereafter referred to as APLDQ mice. Intraperitoneal injection of an anti-HPA-1a mAb induced severe thrombocytopenia in these APLDQ, but not wild-type, mice. Furthermore, platelets from APLDQ mice, when introduced into wild-type mice, elicited a strong polyclonal immune response that was specific for, and importantly restricted to, the epitopes created by these humanized residues, demonstrating that the APLDQ humanized form of murine GPIIIa is immunogenic in mice. Wild-type female mice pre-immunized with APLDQ platelets, when bred with APLDQ male mice, gave birth to severely thrombocytopenic pups, many of whom exhibited an accompanying bleeding phenotype. Notably, administration of intravenous immunoglobulin G (IVIG) into pregnant female mice at days 10 and 17 lowered the concentration of anti-APLDQ alloantibodies in both the maternal and fetal circulation, and importantly normalized the platelet count in the pups. Taken together, these data establish a novel murine model of FNAIT that recapitulates many of the clinically important features of FNAIT. Availability of APLDQ humanized transgenic mice should pave the way for the pre-clinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans. Disclosures No relevant conflicts of interest to declare.

Therapeutic efficacy of a novel humanized antibody-drug conjugate recognizing plexin-semaphorin-integrin domain in the RON receptor for targeted cancer therapy

BackgroundAntibody-drug conjugates (ADCs) targeting the RON receptor, a tumorigenic factor contributing to cancer malignancy, has been considered as a novel strategy for cancer therapy. Here we describe a humanized antibody recognizing the RON plexin-semaphorin-integrin (PSI) domain with increased drug delivery capability for potential clinical application.MethodMonoclonal antibody PCM5B14 specific to the human and monkey RON PSI domain was generated and characterized by various immunological methods. Humanized antibody H5B14 was created by grafting PCM5B14 complementarity-determining regions into human IgG1/κ acceptor frameworks and conjugated with monomethyl auristatin E and duocarmycin to form two H5B14-based ADCs. Stability of H5B14-based ADCs in human plasma was measured using hydrophobic interaction chromatography. Various biochemical and biological assays were used to determine ADC- regulated RON internalization, cell viability, spheroid formation, and death of cancer stem-like cells. Efficacies of H5B14-based ADCs in vivo were validated using tumor xenograft models. Maximal tolerated doses of H5B14-based ADCs were established in mice.ResultsH5B14 was highly specific to the human RON PSI domain and superior over other anti-RON ADCs in induction of RON internalization in various cancer cell lines tested. H5B14-based ADCS had a drug to antibody ratio of ~ 3.70:1 and were stable in human plasma with a minimal dissociation within a 10-day period. Functionally, H5B14-mediated drug delivery decreased cell viability at early stages with an average IC50 at ~ 20 nM in multiple cancer cell lines examined. H5B14-based ADCs also inhibited spheroid formation and caused death of cancer stem-like cells with RON+/CD44+/ESA+ phenotypes. In vivo, H5B14-based ADCs in a single injection inhibited tumor xenograft growth mediated by multiple cancer cell lines. Tumoristatic concentrations calculated from xenograft tumor models were in the range of 0.63 to 2.0 mg/kg bodyweight. Significantly, H5B14-based ADCs were capable of eradicating tumors at variable levels across multiple xenograft models regardless their malignant statuses. Toxicologically, H5B14-based ADCs were well tolerated in mice up to 60 mg/kg.ConclusionH5B14-based ADCs targeting the RON PSI domain are superior in inducing RON internalization, leading to robust drug delivery and overall inhibition and eradication of tumors in multiple xenograft models. These findings warrant H5B14-based ADCs for clinical trials in the future.

Targeting β3 Integrin Psi Domain Inhibits Both Platelet Aggregation and Blood Coagulation: Two Birds with One Stone

Integrin αIIbβ3 plays key roles in thrombosis and hemostasis primarily through mediating platelet adhesion and aggregation. We recently reported that the active site of thiol-isomerase enzymes, CXXC motif, is expressed twice within the plexin-semaphorin-integrin (PSI) domain across all integrins and species, and the PSI domain of β3 integrin possesses endogenous thiol-isomerase activity, which may be a novel target for anti-thrombotic therapy (Blood, 2017).We developed four mouse anti-mouse β3 integrin PSI domain monoclonal antibodies (mAbs). These mAbs cross-react with β3 PSI domains of human, mouse, pig, rat, and rabbit tested but not other regions of β3 integrin, other integrins or other thiol-isomerase enzymes. They inhibit the thiol-isomerase activity of β3 PSI domain, decrease platelet adhesion/aggregation and thrombosis without increasing bleeding. Interestingly, the inhibitory effect of these mAbs on thrombosis in vivo (no anti-coagulant) was 10-20 times greater than their inhibitory effect on platelet aggregation in anti-coagulated platelet-rich plasma in vitro. This motivated us to explore whether this PSI domain contributes to blood coagulation.To asses blood clot formation and retraction, blood was incubated in non-stick tubes for two hours at 37°C in clot retraction assays. These assays showed less clot retraction and significantly lower dry clot weight in human and mouse whole blood treated with these anti-PSI mAbs compared to controls (p<0.05). As a visual representation, laser scanning confocal microscopy of platelet-rich plasma samples with labelled fibrinogen/fibrin showed that fibrin network formation of anti-PSI mAb treated clots was decreased. To measure blood coagulation in a global assay in the presence of platelets and natural blood constituents, thromboelastography (TEG) was performed. Blinded TEG showed that anti-PSI mAb-treated whole blood (human and mouse) and platelet rich plasma (human) decreased in coagulation compared to controls (p<0.05). We further compared our anti-PSI mAbs with other anti-β3 integrin mAbs that inhibit αIIbβ3 ligand binding and platelet aggregation much stronger than the anti-PSI mAbs. In this TEG assay we found that the anti-PSI mAbs were still significantly decreasing platelet coagulation parameters, much more than other inhibitory anti-β3 mAbs including M1 and JAN D1 as well as the Abciximab precursor 7E3 (p<0.05). This suggests the decreased coagulation may result from the anti-thiol-isomerase activity of the anti-PSI mAbs.Disulfide bond exchanges are required for blood coagulation and thiol-isomerase enzymes play important roles in thrombosis. Given that blood coagulation is rapidly amplified on the platelet surface (i.e. cell-based coagulation), many coagulation factors are in close proximity to β3 integrin PSI domains (with thiol-isomerase activity). The study of interaction between β3 PSI domain and coagulation factors is currently being exploring.This is the first evidence of an allosteric therapeutic target of β3 integrin. This work suggests that a novel interaction exists between platelets and the coagulation cascade that requires the β3 integrin PSI domain and its thiol-isomerase activity. Importantly, the PSI domain can be targeted to partially block both platelet aggregation and coagulation, resulting in less bleeding while still providing a strong overall anti-thrombotic effect. DisclosuresNo relevant conflicts of interest to declare.

High-resolution mapping of the polyclonal immune response to the human platelet alloantigen HPA-1a (PlA1)

AbstractAntibodies to platelet-specific antigens are responsible for 2 clinically important bleeding disorders: posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The human platelet-specific alloantigen 1a/1b (HPA-1a/1b; also known as PlA1/A2) alloantigen system of human platelet membrane glycoprotein (GP) IIIa is controlled by a Leu33Pro polymorphism and is responsible for ∼80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa (muGPIIIa) isoforms harboring select humanized residues within the plexin-semaphorin-integrin (PSI) and epidermal growth factor 1 (EGF1) domains and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a–specific antibodies. Humanizing the PSI domain of muGPIIIa was sufficient to recreate the HPA-1a epitope recognized by some HPA-1a–specific antibodies; however, humanizing distinct amino acids within the linearly distant but conformationally close EGF1 domain was required to enable binding of others. These results reveal the previously unsuspected complex heterogeneity of the polyclonal alloimmune response to this clinically important human platelet alloantigen system. High-resolution mapping of this alloimmune response may improve diagnosis of FNAIT and should facilitate the rational design and selection of contemplated prophylactic and therapeutic anti–HPA-1a reagents.

Low-affinity binding in cis to P2Y2R mediates force-dependent integrin activation during hantavirus infection.

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, β3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation β5/β3 integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y2R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant αIIbβ3 integrins and (RGD)P2Y2R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y2R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y2R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing αIIbβ3 integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation-specific mAB PAC1. The data also show that blocking of the Gα13 protein from binding to the cytoplasmic domain of the β3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y2R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force yields physiological integrin activation.

The integrin PSI domain has an endogenous thiol isomerase function and is a novel target for antiplatelet therapy

AbstractIntegrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin β subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine β3, and human β1 and β2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of β3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant β3 subunit. We further developed mouse anti-mouse β3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbβ3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbβ3 endogenous PDI-like activity reduced αIIbβ3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.

Metal ion and ligand binding of integrin α5β1.

Integrin α5β1 binds to an Arg-Gly-Asp (RGD) motif in its ligand fibronectin. We report high-resolution crystal structures of a four-domain α5β1 headpiece fragment, alone or with RGD peptides soaked into crystals, and RGD peptide affinity measurements. The headpiece crystallizes in a closed conformation essentially identical to that seen previously for α5β1 complexed with a Fab that allosterically inhibits ligand binding by stabilizing the closed conformation. Soaking experiments show that binding of cyclic RGD peptide with 20-fold higher affinity than a linear RGD peptide induces conformational change in the β1-subunit βI domain to a state that is intermediate between closed (low affinity) and open (high affinity). In contrast, binding of a linear RGD peptide induces no shape shifting. However, linear peptide binding induces shape shifting when Ca(2+) is depleted during soaking. Ca(2+) bound to the adjacent to metal ion-dependent adhesion site (ADMIDAS), at the locus of shape shifting, moves and decreases in occupancy, correlating with an increase in affinity for RGD measured when Ca(2+) is depleted. The results directly demonstrate that Ca(2+) binding to the ADMIDAS stabilizes integrins in the low-affinity, closed conformation. Comparisons in affinity between four-domain and six-domain headpiece constructs suggest that flexible integrin leg domains contribute to conformational equilibria. High-resolution views of the hybrid domain interface with the plexin-semaphorin-integrin (PSI) domain in different orientations show a ball-and-socket joint with a hybrid domain Arg side chain that rocks in a PSI domain socket lined with carbonyl oxygens.

Amino Acid Changes in Drosophila αPS2βPS Integrins That Affect Ligand Affinity

We developed a ligand-mimetic antibody Fab fragment specific for Drosophila alphaPS2betaPS integrins to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the extracellular matrix protein Tiggrin into the H-CDR3 of the alphavbeta3 ligand-mimetic antibody WOW-1. The specificity of alphaPS2betaPS binding to TWOW-1 was demonstrated by numerous tests used for other integrin-ligand interactions. Binding was decreased in the presence of EDTA or RGD peptides and by mutation of the TWOW-1 RGD sequence or the betaPS metal ion-dependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the alphaPS2 membrane-proximal cytoplasmic GFFNR sequence or by exposure to Mn2+. Although Mn2+ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn2+ could be increased further by the alphaPS2 GFFNR --> GFANA mutation. A mutation in the betaPS I domain (betaPS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by alphaPS2betaPS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin beta subunit plexin-semaphorin-integrin (PSI) and stalk domains have been shown to have activating properties. We found that complete deletion of the betaPS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because betaPS-DeltaPSI displayed an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system.

The Crystal Structure of the Plexin-Semaphorin-Integrin Domain/Hybrid Domain/I-EGF1 Segment from the Human Integrin β2 Subunit at 1.8-Å Resolution

Integrins are modular (alphabeta) heterodimeric proteins that mediate cell adhesion and convey signals across the plasma membrane. Interdomain motions play a key role in signal transduction by propagating structural changes through the molecule, thus controlling the activation state and adhesive properties of the integrin. We expressed a soluble fragment of the human integrin beta2 subunit comprising the plexin-semaphorin-integrin domain (PSI)/hybrid domain/I-EGF1 fragment and present its crystal structure at 1.8-A resolution. The structure reveals an elongated molecule with a rigid architecture stabilized by nine disulfide bridges. The PSI domain is located centrally and participates in the formation of extended interfaces with the hybrid domain and I-EGF1 domains, respectively. The hybrid domain/PSI interface involves the burial of an Arg residue, and contacts between PSI and I-EGF1 are mainly mediated by well conserved Arg and Trp residues. Conservation of key interacting residues across the various integrin beta subunits sequences suggests that our structure represents a good model for the entire integrin family. Superposition with the integrin beta3 receptor in its bent conformation suggests that an articulation point is present at the linkage between its I-EGF1 and I-EGF2 modules and underlines the importance of this region for the control of integrin-mediated cell adhesion.

Dual Functionality of the Anti-β1 Integrin Antibody, 12G10, Exemplifies Agonistic Signalling from the Ligand Binding Pocket of Integrin Adhesion Receptors

Although integrins are known to mediate connections between extracellular adhesion molecules and the intracellular actin cytoskeleton, the mechanisms that are responsible for coupling ligand binding to intracellular signaling, for generating diversity in signaling, and for determining the efficacy of integrin signaling in response to ligand engagement are largely unknown. By characterizing the class of anti-integrin monoclonal antibodies (mAbs) that stimulate integrin activation and ligand binding, we have identified integrin-ligand-mAb complexes that exhibit differential signaling properties. Specifically, addition of 12G10 mAb to cells adhering via integrin alpha4beta1 was found to trigger disruption of the actin cytoskeleton and prevent cell attachment and spreading, whereas mAb addition to cells adhering via alpha5beta1 stimulated all of these processes. In contrast, soluble ligand binding to either alpha4beta1 or alpha5beta1 was augmented or unaffected by 12G10. The regions of the integrin responsible for differential signaling were then mapped using chimeras. Surprisingly, a chimeric alpha5 integrin containing the beta-propeller domain from the ligand binding pocket of alpha4 exhibited the same signaling properties as the full-length alpha4 integrin, whereas exchanging or removing cytoplasmic domains had no effect. Thus the mAb 12G10 demonstrates dual functionality, inhibiting cell adhesion and spreading while augmenting soluble ligand binding, via a mechanism that is determined by the extracellular beta-propeller domain of the associating alpha-subunit. These findings therefore demonstrate a direct and variable agonistic link between the ligand binding pocket of integrins and the cell interior that is independent of the alpha cytoplasmic domains. We propose that either ligand-specific transmembrane conformational changes or ligand-specific differences in the kinetics of transmembrane domain separation underlie integrin agonism.

Evidence That Monoclonal Antibodies Directed against the Integrin β Subunit Plexin/Semaphorin/Integrin Domain Stimulate Function by Inducing Receptor Extension

The overall structure of integrins is that of a ligand-binding head connected to two long legs. The legs can exhibit a pronounced bend at the "knees," and it has been proposed that the legs undergo a dramatic straightening when integrins transit from a low affinity to a high affinity state. The knee region contains domains from both alpha and beta subunits, including the N-terminal plexin/semaphorin/integrin (PSI) domain of the beta subunit. The role played by the knee domains in the regulation of integrin-ligand binding is uncertain. Here we show that: (i) monoclonal antibodies (mAbs) N29 and 8E3 have epitopes in the beta(1) subunit PSI domain and stimulate ligand binding to alpha(5)beta(1); (ii) N29 and 8E3 cause long range conformational changes that alter the ligand binding activity of the head region; (iii) the stimulatory action of these mAbs is dependent on the calf-1 domain, which forms part of the alpha subunit knee; and (iv) the epitopes of 8E3 and N29 map close to the extreme N terminus of the PSI and are likely to lie on the side of this domain that faces the alpha subunit. Taken together, our data suggest that the binding of these mAbs results in a levering apart of the PSI and calf-1 domains, and thereby causes the alpha and beta subunit knees to separate. Several major inferences can be drawn from our findings. First, the PSI domain appears to form part of an interface with the alpha subunit that normally restrains the integrin in a bent state. Second, the PSI domain is important for the transduction of conformational changes from the knee to head. Third, unbending is likely to provide a general mechanism for control of integrin-ligand recognition.

Pathogenic hantaviruses bind plexin–semaphorin–integrin domains present at the apex of inactive, bent αvβ3 integrin conformers

The alphavbeta3 integrins are linked to human bleeding disorders, and pathogenic hantaviruses regulate the function of alphavbeta3 integrins and cause acute vascular diseases. alphavbeta3 integrins are present in either extended (active) or dramatically bent (inactive) structures, and interconversion of alphavbeta3 conformers dynamically regulates integrin functions. Here, we show that hantaviruses bind human alphavbeta3 integrins and that binding maps to the plexin-semaphorin-integrin (PSI) domain present at the apex of inactive, bent, alphavbeta3-integrin structures. Pathogenic hantaviruses [New York-1 virus (NY-1V) and Hantaan virus (HTNV)] bind immobilized beta3 polypeptides containing the PSI domain, and human (but not murine) beta3 polypeptides inhibit hantavirus infectivity. Substitution of human beta3 residues 1-39 for murine beta3 residues directed pathogenic hantavirus infection of nonpermissive CHO cells expressing chimeric alphavbeta3 receptors. Mutation of murine beta3 Asn-39 to Asp-39 present in human beta3 homologues (N39D) permitted hantavirus infection of cells and specified PSI domain residue interactions with pathogenic hantaviruses. In addition, cell-surface expression of alphavbeta3 locked in an inactive bent conformation conferred hantavirus infectivity of CHO cells. Our findings indicate that hantaviruses bind to a unique domain exposed on inactive integrins and, together with prior findings, suggest that this interaction restricts alphavbeta3 functions that regulate vascular permeability. Our findings suggest mechanisms for viruses to direct hemorrhagic or vascular diseases and provide a distinct target for modulating alphavbeta3-integrin functions.

A Novel Adaptation of the Integrin PSI Domain Revealed from Its Crystal Structure

Integrin beta-subunits contain an N-terminal PSI (for plexin-semaphorin-integrin) domain that contributes to integrin activation and harbors the PI(A) alloantigen associated with immune thrombocytopenias and susceptibility to sudden cardiac death. Here we report the crystal structure of PSI in the context of the crystallized alphaVbeta3 ectodomain. The integrin PSI forms a two-stranded antiparallel beta-sheet flanked by two short helices; its long interstrand loop houses Pl(A) and may face the EGF2 domain. The integrin PSI contains four cysteine pairs connected in a 1-4, 2-8, 3-6, 5-7 pattern. An unexpected feature of the structure is that the final, eighth cysteine is located C-terminal to the Ig-like hybrid domain and is thus separated by the hybrid domain from the other seven cysteines of PSI. This architecture may be relevant to the evolution of integrins and should help refine the current models of integrin activation.

Amino Acid Residues in the PSI Domain and Cysteine-rich Repeats of the Integrin β2 Subunit That Restrain Activation of the Integrin αxβ2

The leukocyte integrin alpha(X)beta(2) (p150,95) recognizes the iC3b complement fragment and functions as the complement receptor type 4. alpha(X)beta(2) is more resistant to activation than other beta(2) integrins and is inactive in transfected cells. However, when human alpha(X) is paired with chicken or mouse beta(2), alpha(X)beta(2) is activated for binding to iC3b. Activating substitutions were mapped to individual residues or groups of residues in the N-terminal plexin/semaphorin/integrin (PSI) domain and C-terminal cysteine-rich repeats 2 and 3. These regions are linked by a long range disulfide bond. Substitutions in the PSI domain synergized with substitutions in the cysteine-rich repeats. Substitutions T4P, T22A, Q525S, and V526L gave full activation. Activation of binding to iC3b correlated with exposure of the CBR LFA-1/2 epitope in cysteine-rich repeat 3. The data suggest that the activating substitutions are present in an interface that restrains the human alpha(X)/human beta(2) integrin in the inactive state. The opening of this interface is linked to structural rearrangements in other domains that activate ligand binding.