PHENYLALANINE-780 NEAR THE C-TERMINUS OF THE MOUSE GLUCOCORTICOID RECEPTOR IS IMPORTANT FOR LIGAND-BINDING AFFINITY AND SPECIFICITY

Site-directed mutagenesis was employed to make two single amino acid s ubstitutions for highly conserved amino acid residues near the C-termi nus of the 783-amino acid mouse glucocorticoid receptor. Substitution of leucine for histidine-781 caused little or no change in the concent ration of dexamethasone required for half-maximal activation of a chlo ramphenicol acetyltransferase reporter gene expressed from a mouse mam mary tumor virus promoter. However, when phenylalanine-780 was changed to alanine, the half-maximal concentrations of various agonists were increased as follows, compared with the wild-type glucocorticoid recep tor: triamcinolone acetonide by 7-fold, dexamethasone by 25-fold, and hydrocortisone and deoxycorticosterone by more than 150-fold. Binding of labeled steroids by the mutant receptor in vitro and in vivo was al so decreased. In contrast, this mutation caused a small decrease in th e concentration of RU486 required for antagonist or partial agonist ac tivity. Thus, the phenyl group of phenylalanine-780 of the mouse gluco corticoid receptor is an important determinant of ligand binding affin ity and specificity.