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

Yoko K Takada,Michiko Shimoda,Emanual Maverakis,Brunie H Felding,R Holland Cheng,Yoshikazu Takada

doi:10.1016/j.jbc.2021.100399

Yoko K Takada, Michiko Shimoda + Show 4 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100399

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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.

Highlights

CD40L is a type II protein ligand member of the tumor necrosis factor (TNF) superfamily expressed by activated T cells
We showed that sCD40L activated soluble and cell-surface integrins αvβ3, α5β1, and α4β1 in a dose-dependent manner
Since integrins on normal blood cells (e.g., B cells) are not activated, it is possible that CD40L-mediated integrin activation by binding to site 2 is required for CD40L/CD40 signaling, in addition to site 1 binding

Summary

Introduction

CD40L is a type II protein ligand member of the tumor necrosis factor (TNF) superfamily expressed by activated T cells. CD40L can initiate inflammatory and procoagulatory responses in vascular endothelial cells [5,6,7] Findings such as these have led to the belief that CD40–CD40L interactions play a more general role in immune regulation. CD40L functions through its interactions with cell surface proteins CD40, and α5β1 and αIIbβ integrins. The recent discovery on the binding of 25-hydroxycholesterol to integrin site 2 upregulates inflammatory cytokines, TNF, and IL-6, production [17] indicates that site-2-mediated integrin activation is involved in proinflammatory signaling. We propose that CD40L acts as a ligand and an allosteric activator of several integrins and mediates proinflammatory signaling independent of CD40. We further propose that this may be a common mechanism for a group of proinflammatory proteins and that CD40L-induced integrin activation is required for CD40L signaling

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