Tamrazi Anobel, Carlson Kathryn E, Daniels Jonathan R, Hurth Kyle M, Katzenellenbogen John A
Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Mol Endocrinol. 2002 Dec;16(12):2706-19. doi: 10.1210/me.2002-0250.
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.
核受体形成强大的二聚体，这对于它们作为转录因子的功能至关重要，并且据认为配体结合可影响二聚体稳定性。在本报告中，我们描述了基于荧光共振能量转移（FRET）的便捷方法，用于测量雌激素受体α配体结合域（ERα-LBD）二聚体的热力学和动力学稳定性。我们开发了以位点特异性方式用单个荧光团化学标记的受体。这些荧光团标记的雌激素受体具有功能，可用于直接测量ERα-LBD二聚体的亲和力和稳定性。我们的结果表明，未结合配体的ERα-LBD以非常稳定的二聚体形式存在，并且这些二聚体的解离速率很慢（在28℃下t(1/2)=39±3分钟），并且通过添加各种配体进一步减慢（≤7倍）。雌激素拮抗剂比激动剂能更有效地稳定ER二聚体的动力学。此外，含有LXXLL基序的共激活剂肽选择性地稳定与激动剂结合的ERα-LBD二聚体。这些基于荧光的测定ER二聚体动力学和热力学稳定性的方法提供了一种用于评估新型配体激动剂或拮抗剂特性的功能性体外方法。
