Cysteines 638 and 665 in the hormone binding domain of human glucocorticoid receptor define the specificity to glucocorticoids.

To understand the function of cysteines, we have substituted cysteines 638, 643, and 665 by serine in the hormone-binding domain (HBD) of the human glucocorticoid receptor (hGR). In hormone-binding assays using [3H]dexamethasone, hGR C643S and hGR C665S exhibited wild type receptor Kd of 2.5 nM and hGR C665SM666L displayed a Kd of 3.7 nM, while hGR C638S exhibited a Kd of 162 pM, a 15-fold higher affinity. The affinity of hGR C638S for RU486 was 10-fold higher, and the mutants C643S and C665S bound RU486 with a 10-fold lower affinity when compared to wild type GR. While C665S bound aldosterone with very high relative affinity, the double mutant C665SM666L failed to bind aldosterone. The expression of wild type, mutant, and truncated hGRs in vitro showed an identical level of expression of the cloned receptors. Similar levels of expression of the receptors were observed in transfected cells, both by immunoprecipitation and by Western blotting. Transcription activation of the chimeric reporter gene mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) with hGR C638S was 4-fold higher than the level observed with wild type hGR in the presence of dexamethasone. In the presence of RU486, hGR C638S induced MMTV-CAT 25-fold compared to the highest levels observed with wild type hGR and RU486. Even though the hGR C665S stimulated transcription with aldosterone, hGR C665SM666L did not. DNA-receptor interaction analyses by gel mobility shift assay demonstrated that the increased transactivation potential of hGR C638S was due to its intense interaction with DNA. These findings suggest that C638 and C665 are involved in maintaining specificity to glucocorticoids.