CYSTEINE-849 AND CYSTEINE-942 OF HUMAN MINERALOCORTICOID RECEPTOR ARECRUCIAL FOR STEROID-BINDING

TO assess the role of each of the four cysteine residues in the hormon e binding domain (HBD) of the human mineralocorticoid receptor (hMR), we have separately substituted C808, C849, C910, and C942 into serine. These mutations were created in the G595-K984 hMR receptor fragment w hich encompasses the DNA binding domain, the hinge region, and the hor mone binding domain. Each mutant was further analyzed by steroid bindi ng assays and transactivation assays using wild-type and mutant protei ns expressed in vitro in the reticulocyte lysate. While the C910S muta nt exhibited similar wildtype G595-K984 receptor binding properties fo r aldosterone, the C808S mutant affinity was 3.5-fold higher. In contr ast, the C849S mutant showed a drastic drop in affinity for aldosteron e and the mutant C942S was unable to bind the steroid. Affinities for the antagonist progesterone were also determined. C808S, C849S, and C9 10S were found to bind progesterone better than aldosterone (about a 2 -fold increase in their affinities). Mutant C942S failed to bind any s teroid tested (aldosterone, progesterone, cortisol, and the synthetic antagonist RU26752). No change in the specificity toward various agoni sts and antagonists could be observed with the mutants when compared t o the wild-type G595-K984. When transactivation assays were performed, the properties df mutants C808S and C910S were similar to those of th e wild-type G595-K984, while mutant C849S showed reduced sensitivity a nd C942S was devoid of any transcriptional activity. Our data indicate that C849 and C942 are critical for the Ligand binding process of hMR . Moreover, C942 might be involved in a direct contact with the 20-ket o group of the steroid.