3.9 Article

Estrogen receptor dimerization: Ligand binding regulates dimer affinity and dimer dissociation rate

Journal

MOLECULAR ENDOCRINOLOGY
Volume 16, Issue 12, Pages 2706-2719

Publisher

ENDOCRINE SOC
DOI: 10.1210/me.2002-0250

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Funding

  1. NIDDK NIH HHS [5R37 DK 15556] Funding Source: Medline

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Nuclear receptors form strong dimers that are essential for their function as transcription factors, and it is thought that ligand binding can affect dimer stability. In this report, we describe convenient fluorescence resonance energy transfer (FRET)-based methods for measuring the thermodynamic and kinetic stability of dimers of the estrogen receptor-a ligand-binding domain (ERalpha-LBD). We have developed receptors that are chemically labeled with a single fluorophore in a site-specific manner. These fluorophore-labeled ERs are functional and can be used to measure directly the affinity and stability of ERalpha-LBD dimers. Our results indicate that unliganded ERalpha-LBDs exist as very stable dimers and that the dissociation rate of these dimers is slow (t(1/2) = 39 +/- 3 min at 28 C) and is further slowed (less than or equal to7-fold) by the addition of various ligands. Estrogen antagonists provide greater kinetic stabilization of the ER dimers than agonists. In addition, coactivator peptides containing the LXXLL motif selectively stabilize agonist-bound ERalpha-LBD dimers. These fluorescence-based assays for measuring the kinetic and thermodynamic stability of ER dimers. provide a functional in vitro method for assessing the agonist or antagonist character of novel ligands.

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