A conserved carboxy-terminal subdomain is important for ligand interpretation and transactivation by nuclear receptors.

Nuclear receptors share a highly conserved region located at the very carboxy-terminal part of the ligand-binding domain. Site-directed mutagenesis of conserved hydrophobic residues in this region was reported to create mouse and human glucocorticoid receptors (GRs) and estrogen receptors that cannot transactivate but apparently maintain all the other functions. We constructed analogous mutations in the rat GR to compare the mechanism of deficiency to our recently generated trans-dominant-negative mutant. We found that in the rat GR these carboxy-terminal mutations do not generate trans-dominant-negative receptors. We show that these GR mutants fail to bind dexamethasone properly, and hence receptor transformation and subsequent functions are abolished. Furthermore, we report the identification of a GR mutant that is strongly responsive to the antagonist RU486 but is silent with the agonist dexamethasone. We demonstrate that the reversal of the responsiveness is restricted to GR, since analogous mutations in related receptors do not generate similar phenotypes. Contrary to the case of the progesterone receptor, we show that carboxyl-end truncated GR mutants are not activated by RU486. We conclude that sequence conservation of this subdomain does not necessarily imply functional conservation. Chimeric constructs with GAL4 revealed the importance of protein-protein interactions to exert ligand discrimination, which is mediated by the carboxy-terminal subdomain.