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 substitutions for highly conserved amino acid residues near the C-terminus of the 783-amino acid mouse glucocorticoid receptor. Substitution of leucine for histidine-781 caused little or no change in the concentration of dexamethasone required for half-maximal activation of a chloramphenicol acetyltransferase reporter gene expressed from a mouse mammary 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 receptor: 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 also decreased. In contrast, this mutation caused a small decrease in the concentration of RU486 required for antagonist or partial agonist activity. Thus, the phenyl group of phenylalanine-780 of the mouse glucocorticoid receptor is an important determinant of ligand binding affinity and specificity.
Chen
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D
Department of Pathology, University of Southern California, Los Angeles 90033.
Kohli
K
K
Zhang
S
S
Danielsen
M
M
Stallcup
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003900:Desoxycorticosterone / Q000378:metabolism / Q000494:pharmacology; D003907:Dexamethasone / Q000378:metabolism / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006854:Hydrocortisone / Q000378:metabolism / Q000494:pharmacology; D008024:Ligands; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D051379:Mice; D015735:Mifepristone / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D011987:Receptors, Steroid / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014222:Triamcinolone Acetonide / Q000378:metabolism / Q000494:pharmacology
D008024:Ligands; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; C028697:dexamethasone receptor; D015735:Mifepristone; D003900:Desoxycorticosterone; D010649:Phenylalanine; D003907:Dexamethasone; D015500:Chloramphenicol O-Acetyltransferase; D014222:Triamcinolone Acetonide; D006854:Hydrocortisone