Functional analysis of R651 mutations in the putative helix 6 of rat glucocorticoid receptors

Abstract

Trypsin digestion of steroid-free, but not steroid-bound, rat glucocorticoid receptor (GR) has recently been reported to occur at arginine-651 (R651). This residue is close to the affinity labeled Cys-656 and thus could be a sensitive probe of steroid binding. This hypothesis is supported by the current model of the GR ligand binding domain (LBD), which is based on the X-ray structures of several related receptor LBDs and places R651 in the middle of the putative α-helix 6 (649-EQRMS-653 of rat GR), close to the bound steroid. To test this model, R651, which could be involved in hydrophilic and/or hydrogen bonding, was mutated to alanine (A), which favors α-helices, the helix breakers proline (P) and glycine (G), or tryptophan (W). All receptors were expressed at about the same level, as determined by Western blots, but the cell-free binding activity of R651P was reduced twofold. The cell-free binding affinities were all within a factor of 10 of wild type receptors. Whole cell biological activity with transiently transfected receptors was determined with a variety of GR agonists (dexamethasone and deacylcortivazol) or antagonists (dexamethasone mesylate, RU486, and progesterone). Reporters containing both simple (GRE) and complex (MMTV) enhancers were used to test for alterations in GR interactions with enhancer/promoter complexes. Surprisingly, no correlation was observed between biological activity and ability to preserve α-helical structures for each point mutation. Finally, similar trypsin digestion patterns indicated no major differences in the tertiary structure of the mutant receptors. Collectively, these results argue that the polar/ionizable residue R651 is not required for GR activity and is not part of an α-helix in the steroid-free or bound GR. The effect of these mutations on GR structure and activity may result from a cascade of initially smaller perturbations. These LBD alterations were the most varied for interactions with deacylcortivazol and RU 486, which have recently been predicted to be sub-optimal binders due to their large size. However, further analyses of ligand size versus affinity suggest that there is no narrowly defined optimal size for ligand binding, although larger ligands may be more sensitive to modifications of LBD structure. Finally, the changes in GR activity with the various mutations seem to result from altered LBD interactions with common, as opposed to enhancer specific, transcription factors.