Differential Requirements of Hsp90 and DNA for the Formation of Estrogen Receptor Homodimers and Heterodimers

Wei Xu,David J. Shapiro,Emily Powell,Yidan Wang

doi:10.1074/jbc.m110.104356

Abstract

The two estrogen receptor (ER) subforms, ERalpha and ERbeta, are capable of forming DNA-binding homodimers and heterodimers. Although binding to DNA is thought to stabilize ER dimers, how ERalpha/alpha, ERbeta/beta, and ERalpha/beta dimerization is regulated by DNA and the chaperone protein Hsp90 is poorly understood. Using our highly optimized bioluminescence resonance energy transfer assays in conjunction with assays for transcriptional activation of ERs, we determined that DNA binding appears to play a minor role in the stabilization of ER dimers, especially in the case of ERbeta/beta homodimers. These findings suggest that ER dimers form before they associate with chromatin and that DNA binding plays a minor role in stabilizing ER dimers. Additionally, although Hsp90 is essential for the proper dimerization of ERalpha/alpha and ERalpha/beta, it is not required for the proper dimerization of ERbeta/beta. Despite this, Hsp90 is critical for the estrogen-dependent transcriptional activity of the ERbeta/beta homodimer. Thus, Hsp90 is implicated as an important regulator of distinct aspects of ERalpha and ERbeta action.

Highlights

Direct DNA binding to estrogen response elements (EREs) is thought to stabilize estrogen receptor (ER) dimers via the dimerization interface located within the DNA-binding domains (DBDs), this dimerization interface is thought to be substantially weaker than the ligand-binding domain (LBD) dimerization interface (14 –17)
Upon the addition of the Renilla luciferase (RLuc) substrate coelenterazine h, which causes RLuc to emit at a peak wavelength of 470 nm, energy was transferred to yellow fluorescent protein (YFP), exciting it at 515 nm and causing it to emit at a peak wavelength of 530 nm
The three ER dimer pairs exhibit diverse biological profiles in the presence of different ligands and DNA sequence elements; 16130 JOURNAL OF BIOLOGICAL CHEMISTRY. Both dimerization and DNA binding are critical steps determining the transcriptional outcome and cellular response to endogenous and exogenous estrogenic ligands. This highly regulated process is kept silent in the absence of ligand via the interaction of ERs with the molecular chaperone protein Hsp90, which holds ERs poised for ligand binding but prevents both their activation and degradation

Summary

Introduction

Whereas the interaction of ER␣ and other NRs with Hsp molecular chaperone complexes is well documented, substantially less data are available on the role of this molecular chaperone in transcriptional regulation by ER␤ Both ER␣ and ER␤ have been shown to interact with Hsp in the absence of endogenous and exogenous estrogens, and exposure of cells to Hsp inhibitors results in proteasome-mediated degradation of both receptor isoforms [13]. Upon ligand binding and dissociation from the Hsp molecular complex, ER␣ and ER␤ may form DNA-binding homodimers or heterodimers depending on the context of the bound ligand These estrogen-responsive DNA-binding sequences may be canonical estrogen response elements (EREs) with which ERs directly interact; ERs activate the MAY 21, 2010 VOLUME 285 NUMBER 21. Despite the well recognized mechanism of ER signaling via interaction with the Hsp molecular chaperone complex, ligand binding, interaction with coactivators, and recognition of EREs to activate the transcription of target genes, the order in which these events occur in the cellular context remains controversial

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