Yoshikazu Takada

IGF1 plays a critical role in cell proliferation and survival. Previous studies show that IGF1 binds to integrin αvβ3 and induces αvβ3-IGF1-IGF1R ternary complex formation. However, how IGF1 binding to αvβ3 leads to IGF1R activation is unclear. Previous studies showed that Gα13, a guanine nucleotide-binding protein of the G12 class of Gα proteins, binds to the integrin β3 tail through the EEE motif upon fibrinogen binding to integrin αIIbβ3 and induces RhoA activation. We discovered that the EEE/AAA mutation of the β3 tail inhibited IGF1-induced cell survival, suggesting that Gα13 binding to the β3 tail is required for IGF1 signaling. Since RhoA activation may not be directly involved in IGF1R activation, we studied if Gα13 binds to molecules other than RhoA. Since Gα13 binds to several cytoplasmic tyrosine kinases, we studied if Gα13 binds to the IGF1R kinase by a docking simulation. The simulation predicted that Gα13 binds to the IGF1R kinase through a new binding site. Mutating the predicted Gα13 binding site in the IGF1R kinase (residues 1020-1022) or the predicted IGF1R kinase binding site in Gα13 (residues 260-279) inhibited Gα13 binding to the IGF1R kinase, which is consistent with the docking model. Notably, the Gα13(260-279A) mutant inhibited IGF1-induced cell survival. We propose that IGF1 binding to αvβ3 induces Gα13 binding to the β3 tail and subsequent Gα13 binding to the IGF1R kinase, leading to IGF1R activation. Interestingly, Gα13(260-279A) mutation inhibited cell survival due to a constitutively active Gα13(Q226L) mutant. We propose that Gα13(Q226L) induces its effect by binding to the IGF1R kinase. We propose that the Gα13 binding site of the IGF1R kinase or the IGF1R binding site in Gα13 may be a novel therapeutic target.

Previous studies showed that IGF1 binds to α6β4 and induces α6β4-IGF1-IGF1R complex, which leads to the IGF1R kinase activation. An IGF1 mutant defective in integrin binding was defective in signaling and ternary complex formation, and acted as an antagonist, although the mutant still bound to IGF1R, suggesting that IGF1 binding to α6β4 plays a critical role in IGF1R activation. β4 has a unique long tail (>1000 residues) and deletion of part of the β4 tail containing the calx-β domain is known to reduce cell proliferation. We thus hypothesized that calx-β is involved in IGF1 signaling. Docking simulation predicted that calx-β binds to IGF1R kinase. We discovered that isolated calx-β bound to the IGF1R kinase domain. Point mutations in the predicted calx-β binding site in IGF1R kinase inhibited calx-β binding to IGF1R kinase. Notably, the isolated calx-β domain with cell-penetrating peptide (Tat-calx-β) enhanced survival of keratinocytes and non-transformed cells without IGF1. Tat-calx-β did not enhance survival of cancer cells. Several missense mutations are clustered in the predicted IGF1R kinase binding site of the calx-β domain of β4 in genetic skin blistering disease (JEB-PA). We discovered that several JEB-PA mutants were defective in calx-β binding to the IGF1R kinase and inhibited cell survival of keratinocytes, suggesting that these mutations may suppress calx-b binding to IGF1R kinase. These findings suggest that IGF1 binding to α6β4 triggers calx-β binding to the IGF1R kinase and activates IGF1R kinase.

Galectin-3 (Gal3) is one of the most pro-inflammatory proteins and a biomarker of inflammatory diseases and cancer. Previous studies showed that Gal3 binds to αv and β1 integrins, but it is unclear how Gal3 binds to integrins. Here, we show that Gal3 bound to soluble αvβ3 and αIIbβ3 integrins in 1 mM Mn²⁺ in cell-free conditions in a glycan-independent manner. Docking simulation predicts that Gal3 binds to the classical RGD-binding site (site 1) of αvβ3, but the predicted Gal3-binding site does not include galactose-binding site. RGDfV or eptifibatide inhibited Gal3 binding to αvβ3 and αIIbβ3, respectively, but lactose, a pan-galectin inhibitor, did not inhibit Gal3 binding to integrins. Point mutations of the predicted site 1 binding interface of Gal3 effectively inhibited Gal3 binding to site 1. Site 2 is involved in pro-inflammatory signaling (e.g., TNF and IL-6 secretion), and we previously showed that pro-inflammatory cytokines (e.g., CCL5 and TNF) bind to site 2 and allosteric integrin activation. Docking simulation predicted that Gal3 binds to site 2 of αvβ3 and α5β1. We found that Gal3 induced allosteric activation of soluble integrins αvβ3, αIIbβ3, and α5β1 in 1 mM Ca²⁺ in cell-free conditions. Point mutations in the predicted site 2 binding interface inhibited Gal3-induced integrin activation, suggesting that Gal3 binding to site 2 is required for Gal3-induced integrin activation. Known anti-inflammatory agents, Ivermectin, NRG1, and FGF1, inhibited integrin activation induced by Gal3 in αvβ3 and αIIbβ3. These findings suggest that Gal3 binding to site 2 may be a potential mechanism of pro-inflammatory and pro-thrombotic action of Gal3.

Previous studies suggest that multiple inflammatory chemokines (e.g., CCL5, CXCL12) bind to the allosteric site of integrins (site 2) and induce allosteric integrin activation and inflammatory signals. PF4 is abundantly present in platelet granules, but PF4 levels are very low in plasma. PF4 is released from damaged platelets and is markedly increased in plasma (>1000×) in pathological conditions. PF4 (tetramer) is an inhibitory chemokine, and the specifics of PF4 signaling are unclear. Docking simulation predicted that PF4 monomer binds to site 2, but PF4 by itself did not induce allosteric integrin activation. Anti-PF4 mAbs KKO and RTO generate complexes with PF4 tetramer and monomer, respectively. We discovered that the PF4/RTO complex induced potent integrin activation, but the PF4/KKO complex did not. We hypothesize that inactive PF4 tetramer is converted by RTO to active monomer. A PF4 mutant (4E), in which four basic amino acid residues in the predicted site 2 binding site were mutated to Glu, did not induce integrin activation and acted as a dominant-negative antagonist, suggesting that the RTO/PF4 complex is required to bind to site 2 for integrin activation. Notably, RTO-like autoantibody was detected in plasma of healthy people. We propose that autoanti-PF4 in healthy controls may not be a problem since plasma PF4 levels are very low. When plasma PF4 tetramer is increased, active PF4 monomer is generated by autoanti-PF4 and plays a role in disease pathogenesis. Notably, anti-inflammatory cytokine neuregulin-1 and anti-inflammatory ivermectin bind to site 2 and suppress integrin activation induced by RTO/PF4 complex, suggesting that neuregulin-1 and ivermectin are potentially useful to suppress PF4/anti-PF4-mediated inflammatory signals.

Ivermectin (IVM), a broad-spectrum anthelmintic agent, has anti-inflammatory properties, and affects cellular and humoral immune responses. We recently showed that multiple pro-inflammatory cytokines (e.g., FGF2, CCL5, CD40L) bind to the allosteric site (site 2) of integrins and activate them. 25-Hydroxycholesterol, a pro-inflammatory lipid mediator, is known to bind to site 2 and induce integrin activation and inflammatory signals (e.g., IL-6 and TNF secretion), suggesting that site 2 is critically involved in inflammation. We showed that two anti-inflammatory cytokines (FGF1 and NRG1) bind to site 2 and inhibit integrin activation by inflammatory cytokines. We hypothesized that ivermectin binds to site 2 and inhibits inflammatory signaling by pro-inflammatory cytokines. A docking simulation predicts that ivermectin binds to site 2. Ivermectin inhibits the integrin activation induced by inflammatory cytokines, suggesting that ivermectin is a site 2 antagonist. We showed that TNF, a major pro-inflammatory cytokine, binds to integrin site 2 and induces allosteric integrin activation like other pro-inflammatory cytokines, suggesting that site 2 binding and integrin activation is a potential mechanism of the pro-inflammatory action of these cytokines. Ivermectin suppressed the activation of soluble β3 integrins by TNF and other pro-inflammatory cytokines in a dose-dependent manner in cell-free conditions. Binding to site 2 and the inhibition of binding of inflammatory cytokines may be a potential mechanism of anti-inflammatory action of ivermectin.

We showed that multiple inflammatory cytokines (e.g., CCL5, CXCL12, CX3CL1, CD40L, and FGF2) bind to the allosteric site (site 2) of integrins, distinct from the classical RGD-binding site (site 1), and allosterically activate integrins. A major inflammatory lipid mediator 25-hydroxycholesterol is known to bind to site 2 and allosterically activates integrins and induces inflammatory signals (e.g., IL-6 and TNF secretion). Thus, site 2 is involved in inflammatory signaling. Neuregulin-1 (NRG1) is known to suppresses the progression of inflammatory diseases, fibrosis, and insulin resistance. But, the mechanism of anti-inflammatory action of NRG1 is unclear. We previously showed that NRG1 binds to the classical RGD-binding site (site 1). Mutating the 3 Lys residues that are involved in site 1 binding (NRG1 3KE mutant) is defective in binding to site 1 and in ErbB3-mediated mitogenic signals. Docking simulation predicted that NRG1 binds to site 2. We hypothesized that NRG1 acts as an antagonist of site 2 and blocks allosteric activation by multiple cytokines. Here, we describe that NRG1 binds to site 2 but does not activate soluble αvβ3 or αIIbβ3 in 1 mM Ca²⁺, unlike inflammatory cytokines. Instead, NRG1 suppressed integrin activation by several inflammatory cytokines, suggesting that NRG1 acts as a competitive inhibitor of site 2. Wild-type NRG1 is not suitable for long-term treatment due to its mitogenicity. We showed that the non-mitogenic NRG1 3KE mutant still bound to site 2 and inhibited allosteric activation of soluble and cell-surface integrins, suggesting that NRG1 3KE may have potential as a therapeutic.

Previous studies showed that tetraspanins activate integrins, but the mechanism of this action is unclear. We previously showed that the extracellular-2 (EC2) domains of CD9, CD81, and CD151 bind to the classical RGD-binding site (site 1) of integrin αvβ3, suggesting that they are integrin ligands. We showed that several inflammatory cytokines (e.g., CX3CL1, CXCL12, CCL5, and CD40L) bind to the allosteric site (site 2) of integrins, which is distinct from site 1, and activate integrins (allosteric activation). 25-hydroxycholesterol, a major inflammatory lipid mediator, is known to bind to site 2 and induce inflammatory signals, suggesting that site 2 plays a role in inflammatory signaling. We hypothesized that the EC2 domains activate integrins by binding to site 2. Here we describe that docking simulation predicted that CD81 EC2 binds to site 2 of αvβ3 and more strongly to site 2 of α5β1. Peptide from site 2 bound to isolated EC2 domains, suggesting that the EC2 domains bind to site 2. The EC2 domains only weakly activated αvβ3 but more efficiently activated cell surface integrins α5β1 and α4β1 on the cell surface. These results are consistent with the previous findings that these tetraspanins preferentially interact with β1 integrins. The integrin activation by the EC2 domains was increased at low EC2 concentrations and reduced as EC2 concentrations increased (biphasic), which is consistent with the findings that the EC2 domains bind to two sites (site 1 and 2). We propose that the EC2 binding to site 2 is a novel target for drug discovery.

Several inflammatory cytokines bind to the allosteric site (site 2) and allosterically activate integrins. Site 2 is also a binding site for 25-hydroxycholesterol, an inflammatory lipid mediator, and is involved in inflammatory signaling (e.g., TNF and IL-6 secretion) in addition to integrin activation. FGF2 is pro-inflammatory and pro-thrombotic, and FGF1, homologous to FGF2, has anti-inflammatory and anti-thrombotic actions, but the mechanism of these actions is unknown. We hypothesized that FGF2 and FGF1 bind to site 2 of integrins and regulate inflammatory signaling. Here, we describe that FGF2 is bound to site 2 and allosterically activated β3 integrins, suggesting that the pro-inflammatory action of FGF2 is mediated by binding to site 2. In contrast, FGF1 bound to site 2 but did not activate these integrins and instead suppressed integrin activation induced by FGF2, indicating that FGF1 acts as an antagonist of site 2 and that the anti-inflammatory action of FGF1 is mediated by blocking site 2. A non-mitogenic FGF1 mutant (R50E), which is defective in binding to site 1 of αvβ3, suppressed β3 integrin activation by FGF2 as effectively as WT FGF1.

VEGF-A is a key cytokine in tumor angiogenesis and a major therapeutic target for cancer. VEGF165 is the predominant isoform of VEGF-A, and it is the most potent angiogenesis stimulant. VEGFR2/KDR domains 2 and 3 (D2D3) bind to the N-terminal domain (NTD, residues 1–110) of VEGF165. Since removal of the heparin-binding domain (HBD, residues 111–165) markedly reduced the mitogenic activity of the growth factor, it has been proposed that the HBD plays a critical role in the mitogenicity of VEGF165. Here, we report that αvβ3 specifically bound to the isolated VEGF165 HBD but not to VEGF165 NTD. Based on docking simulation and mutagenesis, we identified several critical amino acid residues within the VEGF165 HBD required for αvβ3 binding, i.e., Arg123, Arg124, Lys125, Lys140, Arg145, and Arg149. We discovered that VEGF165 HBD binds to the KDR domain 1 (D1) and identified that Arg123 and Arg124 are critical for KDR D1 binding by mutagenesis, indicating that the KDR D1-binding and αvβ3-binding sites overlap in the HBD. Full-length VEGF165 mutant (R123A/R124A/K125A/K140A/R145A/R149A) defective in αvβ3 and KDR D1 binding failed to induce ERK1/2 phosphorylation, integrin β3 phosphorylation, and KDR phosphorylation and did not support proliferation of endothelial cells, although the mutation did not affect the KDR D2D3 interaction with VEGF165. Since β3-knockout mice are known to show enhanced VEGF165 signaling, we propose that the binding of KDR D1 to the VEGF165 HBD and KDR D2D3 binding to the VEGF165 NTD are critically involved in the potent mitogenicity of VEGF165. We propose that binding competition between KDR and αvβ3 to the VEGF165 HBD endows integrin αvβ3 with regulatory properties to act as a negative regulator of VEGF165 signaling.

FGF1 is known as an anti-inflammatory and has suppresses insulin resistance. Its homologue FGF2 is pro-inflammatory. Mechanism of FGF1’s anti-inflammatory action and FGF2’s pro-inflammatory action are unknown. Several inflammatory cytokines (e.g., CX3CL1, CCL5, and CXCL12, and CD40L) bind to the classical ligand (RGD)-binding site (site 1) of integrin αvβ3. In addition, they bind to the allosteric site (site 2) of αvβ3, which is distinct from site 1, and allosterically activate αvβ3. Site 2 is involved in inflammatory signals since inflammatory lipid mediator 5-hydroxycholesterol binds to site 2 and induces integrin activation and inflammatory signals (e.g., TNF and IL-6 secretion). We thus hypothesized that FGF1 and FGF2 bind to site 2 and affect activation status of integrins. Here we describe that FGF2 bound to site 2 and allosterically activated αvβ3 integrin. Point mutations in the site 2-binding interface of FGF2 suppressed this activation, indicating that FGF2 binding to site 2 is required for inducing integrin activation. In contrast, FGF1 bound to site 2 but did not activate αvβ3, and instead suppressed integrin activation induced by FGF2, indicating that FGF1 acts as an antagonist of site 2. These findings suggest that FGF1’s anti-inflammatory action is mediated by blocking site 2. FGF1 has potential as an anti-inflammatory agent, but is not appropriate for long-term use since it is potent mitogen. A non-mitogenic FGF1 mutant (R50E), which is defective in binding to site 1 of αvβ3, suppressed αvβ3 activation by FGF2 as effectively as WT FGF1. We propose that FGF1 R50E has therapeutic potential for inflammatory diseases.