Anti-Monomeric C-Reactive Protein Antibody Ameliorates Arthritis and Nephritis in Mice.

Conformation-specific Ags are ideal targets for mAb-based immunotherapy. Here, we demonstrate that the monomeric form of C-reactive protein (mCRP) is a specific therapeutic target for arthritis and nephritis in a murine model. Screening of >1800 anti-mCRP mAb clones identified 3C as a clone recognizing the monomeric, but not polymeric, form of CRP. The anti-mCRP mAb suppressed leukocyte infiltration in thioglycollate-induced peritonitis, attenuated rheumatoid arthritis symptoms in collagen Ab-induced arthritis model mice, and attenuated lupus nephritis symptoms in MRL/Mp-lpr/lpr lupus-prone model mice. These data suggest that the anti-mCRP mAb 3C has therapeutic potential against rheumatoid arthritis and lupus nephritis.

Soluble CD40L activates soluble and cell-surface integrin αvß3, α5ß1, and α4ß1 by binding to the allosteric ligand-binding site (site 2).

CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvß3 and α5ß1, which bind to the trimeric interface of CD40L. We previously showed that several integrin ligands can bind to the allosteric site (site 2), which is distinct from the classical ligand-binding site (site 1), raising the question of if CD40L activates integrins. In our explorations of this question, we determined that integrin α4ß1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvß3, α5ß1, and α4ß1 in cell-free conditions, indicating that this activation does not require inside-out signaling. Moreover, sCD40L activated cell-surface integrins in CHO cells that do not express CD40. To learn more about the mechanism of binding, we determined that sCD40L bound to a cyclic peptide from site 2. Docking simulations predicted that the residues of CD40L that bind to site 2 are located outside of the CD40L trimer interface, at a site where four HIGM1 (hyper-IgM syndrome type 1) mutations are clustered. We tested the effect of these mutations, finding that the K143T and G144E mutants were the most defective in integrin activation, providing support that this region interacts with site 2. We propose that allosteric integrin activation by CD40L also plays a role in CD40L signaling, and defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants.

Tunable hydrogels for mesenchymal stem cell delivery: Integrin-induced transcriptome alterations and hydrogel optimization for human wound healing.

Therapeutic applications for mesenchymal stem/stromal cells (MSCs) are growing; however, the successful implementation of these therapies requires the development of appropriate MSC delivery systems. Hydrogels are ideally suited to cultivate MSCs but tuning hydrogel properties to match their specific in vivo applications remains a challenge. Thus, further characterization of how hydrogel-based delivery vehicles broadly influence MSC function and fate will help lead to the next generation of more intelligently designed delivery vehicles. To date, few attempts have been made to comprehensively characterize hydrogel impact on the MSC transcriptome. Herein, we have synthesized cell-degradable hydrogels based on bio-inert poly(ethylene glycol) tethered with specific integrin-binding small molecules and have characterized their resulting effect on the MSC transcriptome when compared with 2D cultured and untethered 3D hydrogel cultured MSCs. The 3D culture systems resulted in alterations in the MSC transcriptome, as is evident by the differential expression of genes related to extracellular matrix production, glycosylation, metabolism, signal transduction, gene epigenetic regulation, and development. For example, genes important for osteogenic differentiation were upregulated in 3D hydrogel cultures, and the expression of these genes could be partially suppressed by tethering an integrin-binding RGD peptide within the hydrogel. Highlighting the utility of tunable hydrogels, when applied to ex vivo human wounds the RGD-tethered hydrogel was able to support wound re-epithelialization, possibly due to its ability to increase PDGF expression and decrease IL-6 expression. These results will aid in future hydrogel design for a broad range of applications.

Integrin Binding to the Trimeric Interface of CD40L Plays a Critical Role in CD40/CD40L Signaling.

CD40L plays a major role in immune response and is a major therapeutic target for inflammation. Integrin α5ß1 and CD40 simultaneously bind to CD40L. It is unclear if α5ß1 and CD40 work together in CD40/CD40L signaling or how α5ß1 binds to CD40L. In this article, we describe that the integrin-binding site of human CD40L is predicted to be located in the trimeric interface by docking simulation. Mutations in the predicted integrin-binding site markedly reduced the binding of α5ß1 to CD40L. Several CD40L mutants defective in integrin binding were defective in NF-κB activation and B cell activation and suppressed CD40L signaling induced by wild-type CD40L; however, they still bound to CD40. These findings suggest that integrin α5ß1 binds to monomeric CD40L through the binding site in the trimeric interface of CD40L, and this plays a critical role in CD40/CD40L signaling. Integrin αvß3, a widely distributed vascular integrin, bound to CD40L in a KGD-independent manner, suggesting that αvß3 is a new CD40L receptor. Several missense mutations in CD40L that induce immunodeficiency with hyper-IgM syndrome type 1 (HIGM1) are clustered in the integrin-binding site of the trimeric interface. These HIGM1 CD40L mutants were defective in binding to α5ß1 and αvß3 (but not to CD40), suggesting that the defect in integrin binding may be a causal factor of HIGM1. These findings suggest that α5ß1 and αvß3 bind to the overlapping binding site in the trimeric interface of monomeric CD40L and generate integrin-CD40L-CD40 ternary complex. CD40L mutants defective in integrins have potential as antagonists of CD40/CD40L signaling.

Expression of integrins to control migration direction of electrotaxis.

Proper control of cell migration is critically important in many biologic processes, such as wound healing, immune surveillance, and development. Much progress has been made in the initiation of cell migration; however, little is known about termination and sometimes directional reversal. During active cell migration, as in wound healing, development, and immune surveillance, the integrin expression profile undergoes drastic changes. Here, we uncovered the extensive regulatory and even opposing roles of integrins in directional cell migration in electric fields (EFs), a potentially important endogenous guidance mechanism. We established cell lines that stably express specific integrins and determined their responses to applied EFs with a high throughput screen. Expression of specific integrins drove cells to migrate to the cathode or to the anode or to lose migration direction. Cells expressing αMß2, ß1, α2, αIIbß3, and α5 migrated to the cathode, whereas cells expressing ß3, α6, and α9 migrated to the anode. Cells expressing α4, αV, and α6ß4 lost directional electrotaxis. Manipulation of α9 molecules, one of the molecular directional switches, suggested that the intracellular domain is critical for the directional reversal. These data revealed an unreported role for integrins in controlling stop, go, and reversal activity of directional migration of mammalian cells in EFs, which might ensure that cells reach their final destination with well-controlled speed and direction.-Zhu, K., Takada, Y., Nakajima, K., Sun, Y., Jiang, J., Zhang, Y., Zeng, Q., Takada, Y., Zhao, M. Expression of integrins to control migration direction of electrotaxis.

Stromal cell-derived factor-1 (CXCL12) activates integrins by direct binding to an allosteric ligand-binding site (site 2) of integrins without CXCR4.

Leukocyte arrest on the endothelial cell surface during leukocyte extravasation is induced by rapid integrin activation by chemokines. We recently reported that fractalkine induces integrin activation without its receptor CX3CR1 through binding to the allosteric site (site 2) of integrins. Peptides from site 2 bound to fractalkine and suppressed integrin activation by fractalkine. We hypothesized that this is not limited to membrane-bound fractalkine. We studied whether stromal cell-derived factor-1 (SDF1), another chemokine that plays a critical role in leukocyte arrest, activates integrins through binding to site 2. We describe here that (1) SDF1 activated soluble integrin αvß3 in cell-free conditions, suggesting that SDF1 can activate αvß3 without CXCR4; (2) site 2 peptide bound to SDF1, suggesting that SDF1 binds to site 2; (3) SDF1 activated integrins αvß3, α4ß1, and α5ß1 on CHO cells (CXCR4-negative) and site 2 peptide suppressed the activation; (4) A CXCR4 antagonist AMD3100 did not affect the site 2-mediated integrin activation by SDF1; (5) Cell-surface integrins were fully activated in 1 min (much faster than activation of soluble αvß3) and the activation lasted at least for 1 h. We propose that the binding of SDF1 to cell-surface proteoglycan facilitates the allosteric activation process; (6) Mutations in the predicted site 2-binding site in SDF1 suppressed integrin activation. These results suggest that SDF1 (e.g. presented on proteoglycans) can rapidly activate integrins in an allosteric manner by binding to site 2 in the absence of CXCR4. The allosteric integrin activation by SDF1 is a novel target for drug discovery.

Direct binding to integrins and loss of disulfide linkage in interleukin-1ß (IL-1ß) are involved in the agonistic action of IL-1ß.

There is a strong link between integrins and interleukin-1ß (IL-1ß), but the specifics of the role of integrins in IL-1ß signaling are unclear. We describe that IL-1ß specifically bound to integrins αvß3 and α5ß1. The E128K mutation in the IL1R-binding site enhanced integrin binding. We studied whether direct integrin binding is involved in IL-1ß signaling. We compared sequences of IL-1ß and IL-1 receptor antagonist (IL1RN), which is an IL-1ß homologue but has no agonistic activity. Several surface-exposed Lys residues are present in IL-1ß, but not in IL1RN. A disulfide linkage is present in IL1RN, but is not in IL-1ß because of natural C117F mutation. Substitution of the Lys residues to Glu markedly reduced integrin binding of E128K IL-1ß, suggesting that the Lys residues mediate integrin binding. The Lys mutations reduced, but did not completely abrogate, agonistic action of IL-1ß. We studied whether the disulfide linkage plays a role in agonistic action of IL-1ß. Reintroduction of the disulfide linkage by the F117C mutation did not affect agonistic activity of WT IL-1ß, but effectively reduced the remaining agonistic activity of the Lys mutants. Also, deletion of the disulfide linkage in IL1RN by the C116F mutation did not make it agonistic. We propose that the direct binding to IL-1ß to integrins is primarily important for agonistic IL-1ß signaling, and that the disulfide linkage indirectly affects signaling by blocking conformational changes induced by weak integrin binding to the Lys mutants. The integrin-IL-1ß interaction is a potential target for drug discovery.

The CD9, CD81, and CD151 EC2 domains bind to the classical RGD-binding site of integrin αvß3.

Tetraspanins play important roles in normal (e.g. cell adhesion, motility, activation, and proliferation) and pathological conditions (e.g. metastasis and viral infection). Tetraspanins interact with integrins and regulate integrin functions, but the specifics of tetraspanin-integrin interactions are unclear. Using co-immunoprecipitation with integrins as a sole method to detect interaction between integrins and full-length tetraspanins, it has been proposed that the variable region (helices D and E) of the extracellular-2 (EC2) domain of tetraspanins laterally associates with a non-ligand-binding site of integrins. We describe that, using adhesion assays, the EC2 domain of CD81, CD9, and CD151 bound to integrin αvß3, and this binding was suppressed by cRGDfV, a specific inhibitor of αvß3, and antibody 7E3, which is mapped to the ligand-binding site of ß3. We also present evidence that the specificity loop of ß3 directly bound to the EC2 domains. This suggests that the EC2 domains specifically bind to the classical ligand-binding site of αvß3. αvß3 was a more effective receptor for the EC2 domains than the previously known tetraspanin receptors α3ß1, α4ß1, and α6ß1. Docking simulation predicted that the helices A and B of CD81 EC2 bind to the RGD-binding site of αvß3. Substituting Lys residues at positions 116 and 144/148 of CD81 EC2 in the predicted integrin-binding interface reduced the binding of CD81 EC2 to αvß3, consistent with the docking model. These findings suggest that, in contrast with previous models, the ligand-binding site of integrin αvß3, a new tetraspanin receptor, binds to the constant region (helices A and B) of the EC2 domain.

Ligand-induced Epitope Masking: DISSOCIATION OF INTEGRIN α5ß1-FIBRONECTIN COMPLEXES ONLY BY MONOCLONAL ANTIBODIES WITH AN ALLOSTERIC MODE OF ACTION.

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5ß1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-ß1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-ß1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5ß1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking.

Secreted Phospholipase A2 Type IIA (sPLA2-IIA) Activates Integrins in an Allosteric Manner.

Secreted phospholipase A2 type IIA (sPLA2-IIA) is a well-established pro-inflammatory protein and has been a major target for drug discovery. However, the mechanism of its signaling action has not been fully understood. We previously found that sPLA2-IIA binds to integrins αvß3 and α4ß1 in human and that this interaction plays a role in sPLA2-IIA's signaling action. Our recent studies found that sPLA2-IIA activates integrins in an allosteric manner through direct binding to a newly identified binding site of integrins (site 2), which is distinct from the classical RGD-binding site (site 1). The sPLA2-IIA-induced integrin activation may be related to the signaling action of sPLA2-IIA. Since sPLA2-IIA is present in normal human tears in addition to rheumatoid synovial fluid at high concentrations the sPLA2-IIA-mediated integrin activation on leukocytes may be involved in immune responses in normal and pathological conditions.

Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition.

Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of 5 years in both developed and developing countries. Human lactadherin, a milk fat globule membrane glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines, we undertook a proteomic analysis of milk fat globule membrane proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine, and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2ß1 integrin-binding motif in the N-terminal domain of donkey sequence only. Because integrin α2ß1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a minilibrary of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20-amino acid peptide containing both DGE and RGD motifs (named pDGE-RGD) showed the greatest activity, and its mechanism of antiviral action was characterized; pDGE-RGD binds to integrin α2ß1 by means of the DGE motif and inhibits rotavirus attachment to the cell surface. These findings suggest the potential anti-rotavirus activity of equine lactadherin and support the feasibility of developing an anti-rotavirus peptide that acts by hindering virus-receptor binding.

Proinflammatory secreted phospholipase A2 type IIA (sPLA-IIA) induces integrin activation through direct binding to a newly identified binding site (site 2) in integrins αvß3, α4ß1, and α5ß1.

Integrins are activated by signaling from inside the cell (inside-out signaling) through global conformational changes of integrins. We recently discovered that fractalkine activates integrins in the absence of CX3CR1 through the direct binding of fractalkine to a ligand-binding site in the integrin headpiece (site 2) that is distinct from the classical RGD-binding site (site 1). We propose that fractalkine binding to the newly identified site 2 induces activation of site 1 though conformational changes (in an allosteric mechanism). We reasoned that site 2-mediated activation of integrins is not limited to fractalkine. Human secreted phospholipase A2 type IIA (sPLA2-IIA), a proinflammatory protein, binds to integrins αvß3 and α4ß1 (site 1), and this interaction initiates a signaling pathway that leads to cell proliferation and inflammation. Human sPLA2-IIA does not bind to M-type receptor very well. Here we describe that sPLA2-IIA directly activated purified soluble integrin αvß3 and transmembrane αvß3 on the cell surface. This activation did not require catalytic activity or M-type receptor. Docking simulation predicted that sPLA2-IIA binds to site 2 in the closed-headpiece of αvß3. A peptide from site 2 of integrin ß1 specifically bound to sPLA2-IIA and suppressed sPLA2-IIA-induced integrin activation. This suggests that sPLA2-IIA activates αvß3 through binding to site 2. sPLA2-IIA also activated integrins α4ß1 and α5ß1 in a site 2-mediated manner. We recently identified small compounds that bind to sPLA2-IIA and suppress integrin-sPLA2-IIA interaction (e.g. compound 21 (Cmpd21)). Cmpd21 effectively suppressed sPLA2-IIA-induced integrin activation. These results define a novel mechanism of proinflammatory action of sPLA2-IIA through integrin activation.

Insulin-like growth factor (IGF) signaling requires αvß3-IGF1-IGF type 1 receptor (IGF1R) ternary complex formation in anchorage independence, and the complex formation does not require IGF1R and Src activation.

Integrin αvß3 plays a role in insulin-like growth factor 1 (IGF1) signaling (integrin-IGF1 receptor (IGF1R) cross-talk) in non-transformed cells in anchorage-dependent conditions. We reported previously that IGF1 directly binds to αvß3 and induces αvß3-IGF1-IGF1R ternary complex formation in these conditions. The integrin-binding defective IGF1 mutant (R36E/R37E) is defective in inducing ternary complex formation and IGF signaling, whereas it still binds to IGF1R. We studied if IGF1 can induce signaling in anchorage-independent conditions in transformed Chinese hamster ovary cells that express αvß3 (ß3-CHO) cells. Here we describe that IGF1 signals were more clearly detectable in anchorage-independent conditions (polyHEMA-coated plates) than in anchorage-dependent conditions. This suggests that IGF signaling is masked by signals from cell-matrix interaction in anchorage-dependent conditions. IGF signaling required αvß3 expression, and R36E/R37E was defective in inducing signals in polyHEMA-coated plates. These results suggest that αvß3-IGF1 interaction, not αvß3-extracellular matrix interaction, is essential for IGF signaling. Inhibitors of IGF1R, Src, AKT, and ERK1/2 did not suppress αvß3-IGF-IGF1R ternary complex formation, suggesting that activation of these kinases are not required for ternary complex formation. Also, mutations of the ß3 cytoplasmic tail (Y747F and Y759F) that block ß3 tyrosine phosphorylation did not affect IGF1R phosphorylation or AKT activation. We propose a model in which IGF1 binding to IGF1R induces recruitment of integrin αvß3 to the IGF-IGF1R complex and then ß3 and IGF1R are phosphorylated. It is likely that αvß3 should be together with the IGF1-IGF1R complex for triggering IGF signaling.

An integrin binding-defective mutant of insulin-like growth factor-1 (R36E/R37E IGF1) acts as a dominant-negative antagonist of the IGF1 receptor (IGF1R) and suppresses tumorigenesis but still binds to IGF1R.

Insulin-like growth factor-1 (IGF1) is a major therapeutic target for cancer. We recently reported that IGF1 directly binds to integrins (αvß3 and α6ß4) and induces ternary complex formation (integrin-IGF1-IGF1 receptor (IGF1R)) and that the integrin binding-defective mutant of IGF1 (R36E/R37E) is defective in signaling and ternary complex formation. These findings predict that R36E/R37E competes with WT IGF1 for binding to IGF1R and inhibits IGF signaling. Here, we described that excess R36E/R37E suppressed cell viability increased by WT IGF1 in vitro in non-transformed cells. We studied the effect of R36E/R37E on viability and tumorigenesis in cancer cell lines. We did not detect an effect of WT IGF1 or R36E/R37E in cancer cells under anchorage-dependent conditions. However, under anchorage-independent conditions, WT IGF1 enhanced cell viability and induced signals, whereas R36E/R37E did not. Notably, excess R36E/R37E suppressed cell viability and signaling induced by WT IGF1 under anchorage-independent conditions. Using cancer cells stably expressing WT IGF1 or R36E/R37E, we determined that R36E/R37E suppressed tumorigenesis in vivo, whereas WT IGF1 markedly enhanced it. R36E/R37E suppressed the binding of WT IGF1 to the cell surface and the subsequent ternary complex formation induced by WT IGF1. R36E/R37E suppressed activation of IGF1R by insulin. WT IGF1, but not R36E/R37E, induced ternary complex formation with the IGF1R/insulin receptor hybrid. These findings suggest that 1) IGF1 induces signals under anchorage-independent conditions and that 2) R36E/R37E acts as a dominant-negative inhibitor of IGF1R (IGF1 decoy). Our results are consistent with a model in which ternary complex formation is critical for IGF signaling.

Integrins αvß3 and α4ß1 act as coreceptors for fractalkine, and the integrin-binding defective mutant of fractalkine is an antagonist of CX3CR1.

The membrane-bound chemokine fractalkine (FKN, CX3CL1) on endothelial cells plays a role in leukocyte trafficking. The chemokine domain (FKN-CD) is sufficient for inducing FKN signaling (e.g., integrin activation), and FKN-CD binds to its receptor CX3CR1 on leukocytes. Whereas previous studies suggest that FKN-CD does not directly bind to integrins, our docking simulation studies predicted that FKN-CD directly interacts with integrin α(v)ß(3). Consistent with this prediction, we demonstrated that FKN-CD directly bound to α(v)ß(3) and α(4)ß(1) at a very high affinity (K(D) of 3.0 × 10(-10) M to α(v)ß(3) in 1 mM Mn(2+)). Also, membrane-bound FKN bound to integrins α(v)ß(3) and α(4)ß(1), suggesting that the FKN-CD/integrin interaction is biologically relevant. The binding site for FKN-CD in α(v)ß(3) was similar to those for other known α(v)ß(3) ligands. Wild-type FKN-CD induced coprecipitation of integrins and CX3CR1 in U937 cells, suggesting that FKN-CD induces ternary complex formation (CX3CR1, FKN-CD, and integrin). Based on the docking model, we generated an integrin-binding defective FKN-CD mutant (the K36E/R37E mutant). K36E/R37E was defective in ternary complex formation and integrin activation, whereas K36E/R37E still bound to CX3CR1. These results suggest that FKN-CD binding to CX3CR1 is not sufficient for FKN signaling, and that FKN-CD binding to integrins as coreceptors and the resulting ternary complex formation are required for FKN signaling. Notably, excess K36E/R37E suppressed integrin activation induced by wild-type FKN-CD and effectively suppressed leukocyte infiltration in thioglycollate-induced peritonitis. These findings suggest that K36E/R37E acts as a dominant-negative CX3CR1 antagonist and that FKN-CD/integrin interaction is a novel therapeutic target in inflammatory diseases.

The heparin-binding domain of VEGF165 directly binds to integrin αvß3 and VEGFR2/KDR D1: a potential mechanism of negative regulation of VEGF165 signaling by αvß3.

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 αvß3 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 αvß3 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 αvß3-binding sites overlap in the HBD. Full-length VEGF165 mutant (R123A/R124A/K125A/K140A/R145A/R149A) defective in αvß3 and KDR D1 binding failed to induce ERK1/2 phosphorylation, integrin ß3 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 ß3-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 αvß3 to the VEGF165 HBD endows integrin αvß3 with regulatory properties to act as a negative regulator of VEGF165 signaling.

FGF9, a Potent Mitogen, Is a New Ligand for Integrin αvß3, and the FGF9 Mutant Defective in Integrin Binding Acts as an Antagonist.

FGF9 is a potent mitogen and survival factor, but FGF9 protein levels are generally low and restricted to a few adult organs. Aberrant expression of FGF9 usually results in cancer. However, the mechanism of FGF9 action has not been fully established. Previous studies showed that FGF1 and FGF2 directly bind to integrin αvß3, and this interaction is critical for signaling functions (FGF-integrin crosstalk). FGF1 and FGF2 mutants defective in integrin binding were defective in signaling, whereas the mutants still bound to FGFR suppressed angiogenesis and tumor growth, indicating that they act as antagonists. We hypothesize that FGF9 requires direct integrin binding for signaling. Here, we show that docking simulation of the interaction between FGF9 and αvß3 predicted that FGF9 binds to the classical ligand-binding site of αvß3. We show that FGF9 bound to integrin αvß3 and generated FGF9 mutants in the predicted integrin-binding interface. An FGF9 mutant (R108E) was defective in integrin binding, activating FRS2α and ERK1/2, inducing DNA synthesis, cancer cell migration, and invasion in vitro. R108E suppressed DNA synthesis and activation of FRS2α and ERK1/2 induced by WT FGF9 (dominant-negative effect). These findings indicate that FGF9 requires direct integrin binding for signaling and that R108E has potential as an antagonist to FGF9 signaling.

Cross-talk between integrin α6ß4 and insulin-like growth factor-1 receptor (IGF1R) through direct α6ß4 binding to IGF1 and subsequent α6ß4-IGF1-IGF1R ternary complex formation in anchorage-independent conditions.

Integrin αvß3 plays a role in insulin-like growth factor-1 (IGF1) signaling (integrin-IGF1 receptor (IGF1R) cross-talk). The specifics of the cross-talk are, however, unclear. In a current model, "ligand occupancy" of αvß3 (i.e. the binding of extracellular matrix proteins) enhances signaling induced by IGF1 binding to IGF1R. We recently reported that IGF1 directly binds to αvß3 and induces αvß3-IGF1-IGF1R ternary complex formation. Consistently, the integrin binding-defective IGF1 mutant (R36E/R37E) is defective in inducing ternary complex formation and IGF signaling, but it still binds to IGF1R. Like αvß3, integrin α6ß4 is overexpressed in many cancers and is implicated in cancer progression. Here, we discovered that α6ß4 directly bound to IGF1, but not to R36E/R37E. Grafting the ß4 sequence WPNSDP (residues 167-172), which corresponds to the specificity loop of ß3, to integrin ß1 markedly enhanced IGF1 binding to ß1, suggesting that the WPNSDP sequence is involved in IGF1 recognition. WT IGF1 induced α6ß4-IGF1-IGF1R ternary complex formation, whereas R36E/R37E did not. When cells were attached to matrix, exogenous IGF1 or α6ß4 expression had little or no effect on intracellular signaling. When cell-matrix adhesion was reduced (in poly(2-hydroxyethyl methacrylate-coated plates), IGF1 induced intracellular signaling and enhanced cell survival in an α6ß4-dependent manner. Also IGF1 enhanced colony formation in soft agar in an α6ß4-dependent manner. These results suggest that IGF binding to α6ß4 plays a major role in IGF signaling in anchorage-independent conditions, which mimic the in vivo environment, and is a novel therapeutic target.

Identification of inhibitors against interaction between pro-inflammatory sPLA2-IIA protein and integrin αvß3.

Increased concentrations of secreted phospholipase A2 type IIA (sPLA2-IIA), have been found in the synovial fluid of patients with rheumatoid arthritis. It has been shown that sPLA2-IIA specifically binds to integrin αvß3, and initiates a signaling pathway that leads to cell proliferation and inflammation. Therefore, the interaction between integrin and sPLA2-IIA could be a potential therapeutic target for the treatment of proliferation or inflammation-related diseases. Two one-bead-one-compound peptide libraries were constructed and screened, and seven target hits were identified. Herein we report the identification, synthesis, and biological testing of two pyrazolylthiazole-tethered peptide hits and their analogs. Biological assays showed that these compounds were able to suppress the sPLA2-IIA-integrin interaction and sPLA2-IIA-induced migration of monocytic cells and that the blockade of the sPLA2-IIA-integrin binding was specific to sPLA2-IIA and not to the integrin.

CD40L Activates Platelet Integrin αIIbß3 by Binding to the Allosteric Site (Site 2) in a KGD-Independent Manner and HIGM1 Mutations Are Clustered in the Integrin-Binding Sites of CD40L.

CD40L is expressed in activated T cells, and it plays a major role in immune response and is a major therapeutic target for inflammation. High IgM syndrome type 1 (HIGM1) is a congenital functional defect in CD40L/CD40 signaling due to defective CD40L. CD40L is also stored in platelet granules and transported to the surface upon platelet activation. Platelet integrin αIIbß3 is known to bind to fibrinogen and activation of αIIbß3 is a key event that triggers platelet aggregation. Also, the KGD motif is critical for αIIbß3 binding and the interaction stabilizes thrombus. Previous studies showed that CD40L binds to and activates integrins αvß3 and α5ß1 and that HIGM1 mutations are clustered in the integrin-binding sites. However, the specifics of CD40L binding to αIIbß3 were unclear. Here, we show that CD40L binds to αIIbß3 in a KGD-independent manner using CD40L that lacks the KGD motif. Two HIGM1 mutants, S128E/E129G and L155P, reduced the binding of CD40L to the classical ligand-binding site (site 1) of αIIbß3, indicating that αIIbß3 binds to the outer surface of CD40L trimer. Also, CD40L bound to the allosteric site (site 2) of αIIbß3 and allosterically activated αIIbß3 without inside-out signaling. Two HIMG1 mutants, K143T and G144E, on the surface of trimeric CD40L suppressed CD40L-induced αIIbß3 activation. These findings suggest that CD40L binds to αIIbß3 in a manner different from that of αvß3 and α5ß1 and induces αIIbß3 activation. HIGM1 mutations are clustered in αIIbß3 binding sites in CD40L and are predicted to suppress thrombus formation and immune responses through αIIbß3.