IDENTIFICATION OF A 3RD AUTONOMOUS ACTIVATION DOMAIN WITHIN THE HUMANESTROGEN-RECEPTOR

Using a genetic selection system established in the yeast Saccharomyce s cerevisiae, we have isolated, by random mutagenesis of the human est rogen receptor (ER), six mutants that display constitutive transcripti onal activity, All of the mutants identified contained single base ins ertions or deletions leading to frameshift mutations, resulting in rec eptor truncations within the hormone-binding domain between amino acid s (aa) 324-351, Interestingly, an ER mutant (aa 1-282) was transcripti onally inactive in yeast, suggesting that a domain important for trans criptional activity lies between aa 282 and 351 within human ER, Delet ions representative of the mutants isolated in the yeast system were c reated in mammalian expression vectors and examined for transcriptiona l activity in animal cells to determine the physiological relevance of this domain, Receptors truncated at aa 282 were either weakly active or inactive; however, an ER deletion at aa 351 was approximately 50% a s active as wild type ER (induced with estrogen), Furthermore, a chime ric receptor consisting of the DNA binding domain of GAL4 fused to aa 282-351 of the human ER was transcriptionally active on a GAL4 reporte r, We conclude, therefore, that an autonomous activation domain (refer red to as AF2a), functional in both yeast and mammalian cells, lies be tween aa 282-351 of the human ER.