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Soluble CD40L activates soluble and cell-surface integrin αvβ3, α5β1, and α4β1 by binding to the allosteric ligand-binding site (site 2).

ABSTRACT: CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvβ3 and α5β1, 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 α4β1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvβ3, α5β1, and α4β1 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.

SUBMITTER: Takada YK

PROVIDER: S-EPMC7960543 | biostudies-literature |

REPOSITORIES: biostudies-literature

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CD40L Activates Platelet Integrin αIIbβ3 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.

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sVEGFR-1 signaling through α5β1 integrin

Project description:Soluble VEGFR-1 (sVEGFR-1) acts both as a decoy receptor for VEGFs and as an extracellular matrix protein for α5β1 integrin. A sVEGFR-1-derived peptide that interacts with α5β1 integrin promotes angiogenesis. However, canonical signal downstream integrin activation is not induced, resulting into lack of focal adhesion maturation. We performed a gene expression profile of endothelial cells adhering on sVEGFR-1 compared to that of cells adhering on fibronectin, the principal α5β1 integrin ligand. Three protein kinase-C substrates, adducin, MARCKS, and radixin were differently modulated. Adducin and MARCKS were less phosphorylated whereas radixin was higher phosphorylated in sVEGFR-1 adhering cells, the latter leading to prolonged small GTPase Rac1 activation and induction of a pathway involving the heterotrimeric G protein α13. Altogether, our data indicated endothelial cell acquisition of an highly motile phenotype when adherent on sVEGFR-1. Finally, we indicated radixin as accountable for the angiogenic effect of α5β1 integrin interaction with sVEGFR-1 that in turn depends on an active VEGF-A/VEGFR-2 signaling.

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Project description:The prototypic acute phase reactant C-reactive protein (CRP) is not only a marker but also a potential contributor to inflammatory diseases. CRP exists as the circulating native, pentameric CRP (pCRP) and the monomeric isoform (mCRP), formed as a result of a dissociation process of pCRP. mCRP is highly pro-inflammatory, but pCRP is not. The mechanism of pro-inflammatory action of mCRP is unclear. We studied the role of integrins in pro-inflammatory action of mCRP. Docking simulation of interaction between mCRP and integrin αvβ3 predicted that mCRP binds to αvβ3 well. We found that mCRP actually bound to integrins αvβ3 and α4β1 well. Antagonists to αvβ3 or α4β1 effectively suppressed the interaction, suggesting that the interaction is specific. Using an integrin β1 mutant (β1-3-1) that has a small fragment from the ligand binding site of β3, we showed that mCRP bound to the classical RGD-binding site in αvβ3. We studied the role of integrins in CRP signaling in monocytic U937 cells. Integrins αvβ3 and α4β1 specifically mediated binding of mCRP to U937 cells. mCRP induced AKT phosphorylation, but not ERK1/2 phosphorylation, in U937 cells. Notably, mCRP induced robust chemotaxis in U937 cells, and antagonists to integrins αvβ3 and α4β1 and an inhibitor to phosphatidylinositide 3-kinase, but not an MEK inhibitor, effectively suppressed mCRP-induced chemotaxis in U937 cells. These results suggest that the integrin and AKT/phosphatidylinositide 3-kinase pathways play a role in pro-inflammatory action of mCRP in U937 cells. In contrast, pCRP is predicted to have a limited access to αvβ3 due to steric hindrance in the simulation. Consistent with the prediction, pCRP was much less effective in integrin binding, chemotaxis, or AKT phosphorylation. These findings suggest that the ability of CRP isoforms to bind to the integrins is related to their pro-inflammatory action.

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