In Vitro Fluorescence Anisotropy Analysis of the Interaction of Full-length SRC1a with Estrogen Receptors α and β Supports an Active Displacement Model for Coregulator Utilization

Stanley Wang,Chen Zhang,Steven K Nordeen,David J Shapiro

doi:10.1074/jbc.m607531200

Abstract

Binding of full-length P160 coactivators to hormone response element-steroid receptor complexes has been difficult to investigate in vitro. Here, we report a new application of our recently described fluorescence anisotropy microplate assay to investigate binding and dissociation of full-length steroid receptor coactivator-1a (SRC1a) from full-length estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta) bound to a fluorescein-labeled (fl) estrogen response element (ERE). SRC1a exhibited slightly higher affinity binding to flERE.ERbeta than to flERE.ERalpha. Binding of SRC1a to flERE.ERalpha and to flERE.ERbeta was 17beta-estradiol (E2)-dependent and was nearly absent when ICI 182,780, raloxifene, or 4-hydroxytamoxifen were bound to the ERs. SRC1a binds to flERE.E2-ERalpha and flERE.E2-ERbeta complexes with a t1/2 of 15-20 s. Short LXXLL-containing nuclear receptor (NR) box peptides from P160 coactivators competed much better for SRC1a binding to flERE.E2-ER than an NR box peptide from TRAP220. However, approximately 40-250-fold molar excess of the P160 NR box peptides was required to inhibit SRC1a binding by 50%. This suggests that whereas the NR box region is a primary site of interaction between SRC1a and ERE.E2-ER, additional contacts between the coactivator and the ligand-receptor-DNA complex make substantial contributions to overall affinity. Increasing amounts of NR box peptides greatly enhanced the rate of dissociation of SRC1a from preformed flERE.E2-ER complexes. The data support a model in which coactivator exchange is facilitated by active displacement and is not simply the result of passive dissociation and replacement. It also shows that an isolated coactivator exhibits an inherent capacity for rapid exchange.

Highlights

Diverse biological effects by binding to the intracellular proteins, estrogen receptor (ER)3 ␣ and ER␤ [6]
To extend the utility of the fluorescence anisotropy/polarization microplate assay (FAMA), we tested its ability to measure binding of full-length SRC1a to a pre-formed flERE1⁄7E2-ER complex
Based on logarithmic trend lines fitted to the data, the times (t1⁄2) required for 50% of the pre-bound SRC1a to dissociate from flERE1⁄7E2-ER␣ were ϳ1 and ϳ3 min for the SRC1a nuclear receptors (NRs) box 2 and box 4 peptides, respectively (Fig. 6A)

Summary

Introduction

Diverse biological effects by binding to the intracellular proteins, estrogen receptor (ER)3 ␣ and ER␤ [6]. The ability of NR box peptides to stimulate dissociation of full-length SRC1a from preformed flERE1⁄7E2-ER␣1⁄7SRC1a and flERE1⁄7E2-ER␤1⁄7SRC1a complexes suggests that rapid coactivator exchange results from active displacement of the coactivator from the receptor-DNA complex.

Results

Conclusion