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

CD40L is a protein expressed by activated T cells and a member of the tumor necrosis factor superfamily. It is a costimulatory molecule that plays an important role in several T cell–antigen presenting cell interactions. CD40L has a transmembrane form and is also released as a soluble ligand (sCD40L), which allows it to interact at more distant sites.  Available evidence suggests that CD40L can modulate other cell types asides T and B cells. In vascular endothelial cells, CD40L can initiate inflammatory and procoagulatory responses. Dysregulations in the CD40L pathway are prominently featured in human diseases ranging from inflammatory conditions to systemic autoimmunity and tissue-specific autoimmune diseases. CD40L binds to and signals through several integrins, including αvβ3 and α5β1. A previous study showed that several integrin ligands can bind to the allosteric site (site 2), which is different from the known ligand-binding site (site 1). This has raised the question of if CD40L activates integrins.

To answer this question, scientists from the University of California Davis School of Medicine, Dr. Yoko Takada and, Dr. Michiko Shimoda, Professor Emanual Maverakis, Professsor Holland Cheng and Professor Yoshikazu Takada together with Dr. Brunie Felding from The Scripps Research Institute showed that CD40L is an allosteric activator of integrins αvβ3, α5β1 and α4β1 and can bind to an allosteric ligand-binding site (Site 2), which is different from αvβ3’s classical ligand-binding site (site 1). The original research is now published in the Journal of Biological Chemistry.

The research team found that sCD40L activated soluble integrin αvβ3 in cell-free conditions, and also activated αvβ3 on the cell surface. They also showed that in integrin αvβ3, CD40L bound to another ligand-binding site (site 2). The location of this site-2-binding interface of sCD40L was predicted through docking simulations to be outside of the CD40L trimer interface. The docking simulations also predicted that four hyper-IgM syndrome type 1 (HIGM1) mutations are clustered in the predicted site-2-binding interface of CD40L. Two mutants (K143T and G144E) were found to be defective in activating both soluble and cell surface αvβ3.

The sCD40L activated both soluble and cell-surface integrin α5β1. In addition, sCD40L bound to site 2 of α5β1 in a manner comparable to that of αvβ3. The four HIGM1 were found to be defective in activating cell surface α5β1, with K143T being the most defective.

In a series of molecular experiments the authors demonstrated that sCD40L activated both soluble and cell-surface integrin α4β1. CD40L also served as a ligand for α4β1 that binds to site 1. Furthermore, CD40L bound to site 2 and activated α4β1 in a manner similar to those of αvβ3 and α5β1. The four HIGM1 mutants clustered around the predicted site-2-binding interface of sCD40L were found to be defective in activating cell surface α4β1 and K143T and G144E were the most defective.

Of the four mutants, two (S128R/E129G and L155P) were found to be defective in their ability to bind to site 1. Whereas, the other two mutants (K143T and G144E) with the ability to bind to site 1 were found to be defective in integrin activation by binding to site 2.

Through elegant experiments, the authors have identified integrin α4β1 as a new receptor for CD40L. They have demonstrated that sCD40L is an allosteric activator of integrins by binding to site 2. They have also shown that four HIGM1 mutants were defective in activating integrins αvβ3, α5β1 and α4β1. Based on their findings they proposed that the integrin activation mediated by site 2 and inside-out signaling occur independently and complement to each other. They also proposed that the allosteric integrin activation by CD40L plays a role in CD40L signaling, and that the defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants. The study will provide better understanding to develop next generation of CD40L therapeutics which will advance new treatment of cancer and autoimmune diseases.