Human glucocorticoid-induced TNF receptor ligand regulates its signaling activity through multiple oligomerization states

Abstract

Ligation between glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand (GITRL) provides an undefined signal that renders CD4(+)CD25(-) effector T cells resistant to the inhibitory effects of CD4(+)CD25(+) regulatory T cells. To understand the structural basis of GITRL function, we have expressed and purified the extracellular domain of human GITR ligand in Escherichia coli. Chromotography and cross-linking studies indicate that human GITRL (hGITRL) exists as dimers and trimers in solution and also can form a supercluster. To gain insight into the nature of GITRL oligomerization, we determined the crystallographic structures of hGITRL, which revealed a loosely associated open trimer with a deep cavity at the molecular center and a flexible C-terminal tail bent for trimerization. Moreover, a tetramer of trimers (i.e., supercluster) has also been observed in the crystal, consistent with the cross-linking analysis. Deletion of the C-terminal distal three residues disrupts the loosely assembled trimer and favors the formation of a dimer that has compromised receptor binding and signaling activity. Collectively, our studies identify multiple oligomeric species of hGITRL that possess distinct kinetics of ERK activation. The studies address the functional implications and structural models for a process by which hGITRL utilizes multiple oligomerization states to regulate GITR-mediated signaling during T cell costimulation.

Fig. 1.

Biophysical characterization of wild-type and C-terminal deletion mutant hGITRL. (a) Wild-type hGITRL was eluted from Superdex 75 column as two peaks, with calculated molecular masses of ≈27 and ≈43 kDa, corresponding to dimer and trimer, respectively, whereas mutant hGITRL appeared as a single peak of ≈27 kDa, corresponding to a dimer (b). Surface plasmon resonance analysis shows that wild-type hGITRL has modest receptor binding activity (c), whereas mutant hGITRL does not have significant receptor binding at the concentration tested (d).

Fig. 2.

Crystal structures of hGITRL. Residues of particular interest were labeled. (a) Comparison of human and mouse GITRL monomer structures shows that they share a conserved A′AHCF β-sheet but have different C-terminal orientations. The zoom image shows the molecular surface of the A′AHCF β-sheet. (b) The asymmetric assembly of hGITRL open trimer. The dotted spheres represent solvent molecules that are unequally distributed on the trimeric interface. The zoom image shows that the hook-shaped C terminus is involved in the trimeric packing. (c) The cavity in the trimeric center of hGITRL. The zoom image shows the weakened hydrophobic packing around the threefold.

Fig. 3.

Cross-linking analysis of wild-type and mutant hGITRL. After cross-linking using BS3, equal amounts of samples were loaded for SDS/PAGE analysis. Sample names were labeled on the upper side. Note that wild-type hGITRL can form high-molecular-weight superclusters binding to hGITR, whereas mutant hGITRL forms can only form dimer and shows weak receptor binding. Different oligomeric states of hGITRL were labeled. WT-hGITRL, wild-type hGITRL; Cdel-hGITRL, C-terminal deletion mutant hGITRL; WT-hGITR, wild-type hGITR.

Fig. 4.

Tetramer of trimers formed in hGITRL crystals. (a) The cluster viewed along the threefold axis. (b) Image obtained by rotating a 90° along the y axis. Notably, the organization of the cluster allows all of the subunits to be anchored on a same putative membrane.

Fig. 5.

hGITRL stimulation leads to activation of ERK. (a) Flow cytometry analysis of human THP-1 cells to confirm GITR expression. (b) Western blot analysis of ERK phosphorylation. BS-hGITRL, Cdel-hGITRL, and wt-hGITRL represent BS3-treated wild-type hGITRL, mutant hGITRL, and wild-type hGITRL, respectively. Phosphorylated forms and total ERK are indicated with arrows.

Fig. 6.

A hypothetical model for the regulation of hGITRL activity. In this model, hGITRL adopts multiple oligomerization states to progressively control GITR-mediated signaling intensity during T cell costimulation. The hGITRL dimer and the supercluster are shown representing inhibitory and hyperstimulatory species, respectively.