Subnuclear trafficking of estrogen receptor-α and steroid receptor coactivator-1

We have analyzed ligand-dependent, subnuclear movements of the estrogen receptor-alpha (ER alpha) in terms of both spatial distribution and solubility partitioning. Using a transcriptionally active green fluorescent protein-ER alpha chimera (GFP-ER alpha), we find that 17 beta-estradiol (E-2) changes the normally diffuse nucleoplasmic pattern of GFP-ER alpha to a hyper-speckled distribution within 10-20 min. A similar reorganization occurs with the partial antagonist 4-hydroxytamoxifen only a subtle effect was observed with the pure antagonist ICI 182,780, To examine the influence of ligand upon ER alpha association with nuclear structure, MCF-7 cells were extracted to reveal the nuclear matrix (NM), Addition of E-2, 4-hydroxytamoxifen, or ICI 182,780 causes ER alpha to partition with the NM-bound fraction on a similar time course (10-20 min) as the spatial reorganization suggesting that the two events are related. To determine the effects of E-2 on the redistribution and solubility of GFP-ER alpha, individual cells were directly examined during both hormone addition and NM extraction and showed that GFP-ER alpha movement and NM association were coincident, Colocalization experiments were performed with antibodies to identify sites of transcription (RNA pol Ilo) and splicing domains (SRm160), Using E-2 treated MCF-7 cells, minor overlap was observed with transcription sites and a small amount of the total ER alpha pool. Experiments performed with bioluminescent derivatives of ER alpha and steroid receptor coactivator-l (SRC-1) demonstrated both proteins colocalize to the same NM-bound foci in response to E-2 but not the antagonists tested. Deletion mutagenesis and in situ analyses indicate intranuclear colocalization requires a central SRC-1 domain containing LXXLL motifs, Collectively, our data suggest that ER alpha transcription function is dependent upon dynamic early events including intranuclear rearrangement, NM association, and SRC-1 interactions.