Hormone binding domain of human glucocorticoid receptor. Enhancement of transactivation function by substitution mutants M565R and A573Q.

To determine the importance of specific amino acids in the hormone-binding domain of the human glucocorticoid receptor (hGR), we have generated mutants M565R, G567A, and A573Q. In hormone binding assays using [3H]cortisol, half-maximal saturation of dexamethasone competition was achieved at 10 pM with hGR M565R and hGRA573Q compared to 10 nM with wild type hGR. Similar results were obtained in competition assays with [3H]dexamethasone and the glucocorticoid antagonist RU 486. The substitution mutants M565R and A573Q demonstrated a higher relative affinity for aldosterone compared to the wild type hGR. In CV-1 cells cotransfected with the mutant receptors, hGR M565R and A573Q showed a remarkable 6-fold elevated transcription activation of the chimeric reporter gene mouse mammary tumor virus-chloramphenicol acetyl-transferase (MMTV-CAT). The mutant hGR G567A failed to bind agonists and antagonists efficiently. Immunoblotting with hGR specific antibodies of the whole cell extract from transfected CV-1 cells revealed that these differences in hormone binding and transcription activation were not due to the decreased levels of expression. These data support that idea that Gly567 in the hGR hormone-binding domain lies in a region crucial to ligand binding and transactivation function.