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J. Biol. Chem., Vol. 282, Issue 5, 2765-2775, February 2, 2007

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

Stanley Wang¹, Chen Zhang¹, Steven K. Nordeen, and David J. Shapiro²

From the Department of Biochemistry, University of Illinois, Urbana, Illinois 61801-3602 and the Department of Pathology, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado 80045

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 (ER) or estrogen receptor (ER) bound to a fluorescein-labeled (fl) estrogen response element (ERE). SRC1a exhibited slightly higher affinity binding to flERE·ER than to flERE·ER. Binding of SRC1a to flERE·ER and to flERE·ER was 17-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-ER and flERE·E₂-ER complexes with a t 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, 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.

Received for publication, August 7, 2006 , and in revised form, November 27, 2006.

^* This work was supported by National Institutes of Health Grants DK-071909 and HD-16720 (to D. J. S.) and DK/HD62362 (to S. K. N.) and by a National Institutes of Health predoctoral traineeship (to S. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work and are considered first authors.

² To whom correspondence should be addressed: 600 S. Mathews Ave., Urbana, IL 61801-3602. Tel.: 217-333-1788; Fax: 217-244-5858; E-mail: djshapir{at}uiuc.edu.

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