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

J Biol Chem. Jan-Jun 2021;296:100399. doi: 10.1016/j.jbc.2021.100399. Epub 2021 Feb 9.

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.

Keywords: CD40L; docking simulation; fibrinogen; fibronectin; high IgM syndrome type 1 (HIGM1); integrin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Site
  • Animals
  • CD40 Ligand / immunology
  • CD40 Ligand / metabolism*
  • Cell Line
  • Cricetinae
  • Humans
  • Integrin alpha4beta1 / immunology
  • Integrin alpha4beta1 / metabolism*
  • Integrin alpha5beta1 / immunology
  • Integrin alpha5beta1 / metabolism*
  • Integrin alphaVbeta3 / immunology
  • Integrin alphaVbeta3 / metabolism*
  • Molecular Docking Simulation
  • Protein Binding
  • Receptors, Cell Surface / chemistry*
  • Receptors, Cell Surface / metabolism
  • Signal Transduction
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism*

Substances

  • Integrin alpha4beta1
  • Integrin alpha5beta1
  • Integrin alphaVbeta3
  • Receptors, Cell Surface
  • CD40 Ligand