GR Dimerization and the Impact of GR Dimerization on GR Protein Stability and Half-Life

Abstract

Pharmacologically, glucocorticoids, which mediate their effects via the glucocorticoid receptor (GR), are a most effective therapy for inflammatory diseases despite the fact that chronic use causes side-effects and acquired GC resistance. The design of drugs with fewer side-effects and less potential for the development of resistance is therefore considered crucial for improved therapy. Dimerization of the GR is an integral step in glucocorticoid signaling and has been identified as a possible molecular site to target for drug development of anti-inflammatory drugs with an improved therapeutic index. Most of the current understanding regarding the role of GR dimerization in GC signaling derives for dimerization deficient mutants, although the role of ligands biased toward monomerization has also been described. Even though designing for loss of dimerization has mostly been applied for reduction of side-effect profile, designing for loss of dimerization may also be a fruitful strategy for the development of GC drugs with less potential to develop GC resistance. GC-induced resistance affects up to 30% of users and is due to a reduction in the GR functional pool. Several molecular mechanisms of GC-mediated reductions in GR pool have been described, one of which is the autologous down-regulation of GR density by the ubiquitin-proteasome-system (UPS). Loss of GR dimerization prevents autologous down-regulation of the receptor through modulation of interactions with components of the UPS and post-translational modifications (PTMs), such as phosphorylation, which prime the GR for degradation. Rational design of conformationally biased ligands that select for a monomeric GR conformation, which increases GC sensitivity through improving GR protein stability and increasing half-life, may be a productive avenue to explore. However, potential drawbacks to this approach should be considered as well as the advantages and disadvantages in chronic vs. acute treatment regimes.

Keywords: Compound A; GRdim mutant; GRmon mutant; acquired glucocorticoid resistance; biased ligands; glucocorticoid receptor dimerization; half-life; ubiquitin proteasomal system.

Figure 1

(A) Domain structure of the human GR. Above the figure is indicated the position of the post-translational modifications required for proteasomal degradation. Below the figure the DBD and LBD residues involved in the dimer interface are expanded. For the DBD, the underlined residues indicate the D-box, while red residues are those identified as important for the dimerization interface by Luisi et al. (8). In addition, in green is H472 in the lever arm that adopts one of two conformations: packed or flipped depending on whether binding to GREs or nGREs occur. For the LBD black residues are those involved in hydrogen bonds, while the green residues form hydrophobic interactions to stabilize the dimer interface as identified by Bledsoe et al. (9). (B) DNA-binding motifs determine orientation and GR monomer vs. dimer binding. Faded monomer indicates binding to low affinity site.

Figure 2

Schematic representation of the monomer-dimer equilibrium for GR^wt, the DBD-dimerization deficient mutant, GR^dim, and the LBD-dimerization deficient mutant, GR^I628A, bound to either, DEX, 21OH-6,19OP, or CpdA. In the equilibrium, green arrows represents quantitative data, while orange arrows represents semiquantitative or qualitative data (see Table 2). Dotted orange arrows represents hypothesized equilibria not yet determined.

Figure 3

Simulated dimerization curves for unliganded and liganded GR^wt and liganded GR^dim and GR^I628A. Simulations were done using GraphPad Prism version 7. K_d values from Oasa et al. (36) were used, except for liganded GR^I628A, where a 10-fold increase in the K_d of the unliganded GR^wt was used as per Bledsoe et al. (9). The figure clearly shows that ligand-binding to the GR^wt results in a left shift of the dimerization curve, while mutations in either the DBD or the LBD dimerization interfaces result a right shift of the curve relative to GR^wt, with a more pronounced shift in the case of the mutation to the LBD dimerization interface.