Functional analysis of ligand recognition by the glucocorticoid receptor

Abstract: The glucocorticoid receptor (GR) is a ligand-inducible transcription factor that belongs to the superfamily of nuclear hormone receptors. It contains three structural and functional domains. The C-terminal domain binds the hormone ligand, the central domain binds to DNA in glucocorticoid-regulated genes and the N-terminal domain contains the major transactivation function. The ligand-binding domain (LBD) consists of about 250 amino acids. The structure of the GR LBD is not yet solved but it most likely resembles the structure of other nuclear hormone receptor LBD:s that have been recently solved and that share a common fold. To identify amino acids that are involved in ligand recognition and to understand their function, we have performed site-directed mutagenesis of the GR LBD. In the first study we mutated C736, that had previously been shown to be affinity labelled, and used a yeast system to screen for mutants with altered function. Two such mutants were identified, C736T and C736S, and further characterised in COS-7 cells. C736S bound all ligands with reduced affinity whereas C736T bound the synthetic ligand triamcinolone acetonide with higher affinity and cortisol with lower affinity and thus had altered binding specificity. These results suggested that C736 interacted directly with the ligand. In the second study, mutagenesis of a region likely to be involved in glucocorticoid versus mineralocorticoid selectivity was performed. Amino acids in GR LBD were mutated to the corresponding amino acids found in the mineralocortiocid receptor. Mutations with increased activity with the mineralocorticoid aldosterone were selected using the yeast screening system and further characterised in COS-7 cells. Interestingly, all multiple mutations selected for their higher activity with aldosterone in the yeast screen contained the mutation V571M, which was also found as a single point mutation. We concluded that the region around V571 probably plays an important role in an interplay between different regions of the receptor that determine aldosterone binding selectivity. In the third study we used an homology model of the GR LBD based on the ER[alpha] LBD crystal structure to select four amino acids that made possible contacts with the D-ring of the steroid. The structure of the D-ring varies between different classes of steroids and D-ring interactions are therefore likely to be involved in determining binding specificity. Mutation of Q642 indicated that it made complex interactions around the 16 and 17 positions of the steroid and that T739 most likely interacts with the 21-OH group. Results from mutagenesis of M560 and M639 indicated that they made non-specified hydrophobic interactions with the steroid. In the fourth study, we identified and characterised mutations in patients with generalised glucocortiocid resistance. We found one new mutation in the LBD (G679S) that had lower affinity for ligand and a novel mutation in the DNA-binding domain (R477H) that is postulated to have a diminished interaction with DNA, based on model studies. In summary, we have identified amino acids in the ligand-binding domain of GR that are important for ligand-binding specificity and affinity and that play a functional role in the recognition of the steroid.