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¹ Departments of Environmental & Occupational Medicine,
² Medicine,
³ Neuroscience and Cell Biology,
⁴ Environmental and Occupational Health Sciences Institute,
⁵ The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ 08903, USA
⁶ Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ 07102, USA

(Requests for offprints should be addressed to T Thomas, Department of Environmental and Occupational Medicine, 125 Paterson Street, Clinical Academic Building, Room 7092, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ 08903, USA; Email: thomasth{at}umdnj.edu)

Estrogen receptors (ER and ERß) are ligand-activated nuclear receptors that mediate the action of estrogens. These receptors activate transcription by similar mechanism(s), although the overall amino acid sequence identity is only 47%. In order to compare the structural and conformational features of ER and ERß, we monitored their intrinsic tryptophan fluorescence during thermal unfolding. The 50% unfolding temperatures (T_M) of ER and ERß were 39±1 and 40±2°C, respectively. Estradiol had no significant effect on the T_M of ER or ERß. In contrast, binding of the estrogen-response element increased the T_M of ER and ERß by 10 °C. Thermal unfolding of estradiol-bound ER and ligand-free ERß showed two-step transitions, with the formation of intermediates that were stable between 36–48 and 34–42°C, respectively. We confirmed the presence of intermediate states during thermal unfolding by circular dichroism spectroscopy. Atomic force microscopy showed that the ERß intermediate consisted of discrete globular particles, whereas the ER intermediate showed a speckled appearance, with sparse well-defined particles. Fluorescence-quenching studies showed the presence of two classes of tryptophan in unliganded ER and ERß. Binding of estradiol to ERß exposed its tryptophans, whereas estradiol reduced the accessibility of the tryptophans of ER. Our results illustrate the differential effects of ligands on the unfolding of ER and ERß, and identify partially unfolded intermediates. Differences in the conformational flexibility and stability of ER and ERß may represent functional differences of ligand-bound ERs in recruiting coactivator proteins and initiating transcription.

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				V. Vijayanathan, S. Venkiteswaran, S. K. Nair, A. Verma, T.J. Thomas, B. T. Zhu, and T. Thomas Physiologic levels of 2-methoxyestradiol interfere with nongenomic signaling of 17beta-estradiol in human breast cancer cells. Clin. Cancer Res., April 1, 2006; 12(7 Pt 1): 2038 - 2048. [Abstract] [Full Text] [PDF]