NEUTRAL MUTATIONS TO 3 ACIDIC AF2 RESIDUES IN THE MOUSE ESTROGEN-RECEPTOR CONFER AGONIST ACTIVITY TO A-RING ISOMERS OF ESTRADIOL

The ability of the estrogen receptor (ER) to function as a ligand-medi ated transcription factor involves the activation function-2 (AF2) in the hormone binding domain (HBD). Although several types of ligand bin d to the ER, AF2 functions selectively, as it is activated by estradio l (E-2) but not by antiestrogens. The mechanism used by AF2 to interpr et the chemical and structural information encoded in the bound ligand , and to transfer this information to other transcriptional regulatory proteins on the promoters of estrogen-regulated genes, is unknown at present. To address this issue, we have examined the activities of two mouse ERs with mutations in the AF2 region. One incorporated changes in three acidic residues (pJ3MOR, D542N/E546Q/D549N) on the polar face of the putative AF2 alpha-helix, whereas the other contained alterati ons in two hydrophobic amina, acids (pJ3MOR, L543D/L544A) on the non-p olar face. Transcriptional activity was measured with chloramphenicol acetyltransferase (CAT) reporter genes including at minimal (JA12) or a complex (pS2tkCAT) promoter. In transient cotransfection assays usin g COS-1 cells, it was found that A-ring isomers of E-2, which are inac tive or behave as antagonists with the wild-type ER, acted as agonists when the neutral AF2 mutant ER and the pS2tkCAT promoter were tested. On the other hand, when the mutant ER with changes in key hydrophobic residues was used with this same promoter, the estrogen antagonist, I CI 164,384, acted as an agonist. The findings in this report establish a role for acidic AF2 amino acids, and confirm the contributions made by hydrophobic residues, in the interpretation of ligand identity and in the transmission of this information to the transcriptional regula tory apparatus. Critical residues on both sides of the AFT alpha-helix are, therefore, likely to be involved in distinguishing between ligan ds with either extensive or subtle alterations in structure, and in me diating this information to the regulatory proteins on estrogen-respon sive genes. (C) 1997 Elsevier Science Ltd.