Estrogen receptor microarrays: subtype-selective ligand binding.

We present the first example of a nuclear hormone receptor microarray, using for illustration the ligand-binding domains of the two estrogen receptors, ERalpha-LBD and ERbeta-LBD. The proteins are printed and allowed to attach to aldehyde slides; the efficiency of attachment depends on whether the LBD is liganded with agonists (low attachment) versus liganded with antagonists or unliganded (high attachment). This suggests that attachment is orientation specific and involves principally a single lysine residue. The attached ERs retain good ligand-binding activity that can be assessed using an estradiol-fluorophore conjugate, and specific and ER subtype-selective binding of ligands can be determined conveniently in competitive binding assays. This powerful new, high-throughput technique to study ligand binding to ER-LBDs can be extended to other nuclear hormone receptors and adapted to assay the recruitment of coregulator proteins.

Estrogen receptor dimerization: ligand binding regulates dimer affinity and dimer dissociation rate.

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-alpha 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 (< or =7-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.