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¹ Department of Pathology, University of Southern California, Los Angeles, California, USA
² C&C Research Laboratories, Kyunggi-do, Korea
³ Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, California, USA

(Requests for offprints should be addressed to Jeong Hoon Kim, Department of Pathology, HMR 301, University of Southern California, 2011 Zonal Avenue, Los Angeles, California 90089-9092, USA; Email: jeongkim{at}usc.edu.)

Estrogen-dependent transcriptional activation by estrogen receptor (ER) depends on the conformation of helices 3 and 12 in the ligand-binding domain. To better understand the function of helix 3 in ER, we examined the role of charged residues, which are conserved in most steroid receptors in helix 3, in estrogen-dependent transactivation. The replacement of Asp-351 with lysine (D351K) or leucine (D351 L) completely abolished estrogen-dependent transactivation without affecting estrogen-binding, DNA-binding and homodimerization activities in ER. The mutations dramatically reduced the ligand-dependent activation function 2 activity and impaired the ability of ER to bind p160 coactivators. In addition, the D351K mutant effectively inhibited the transcriptional activation activity of wild-type ER. Furthermore Asp-351 was required not only for the estrogen-dependent conformational change of wild-type ER but also for the constitutive transcriptional activity and ligand-independent active conformation of ER mutant Y537N. Similarly, in the orphan nuclear receptor called estrogen-related receptor 3 (ERR3), the replacement of Asp-273 (the corresponding amino acid to Asp-351 in ER) with lysine abolished constitutive transcriptional activity of ERR3 without affecting DNA-binding activity and impaired the ability of the receptor to interact with p160 coactivators. These data suggest a role of Asp-351 in inducing and stabilizing the active conformation of ER, and our results experimentally confirm the concept that Asp-351 in helix 3 interacts with the amide hydrogen of L540 in helix 12 to form a transcriptionally competent surface for binding p160 coactivators.