Temporally distinct and ligand-specific recruitment of nuclear receptor-interacting peptides and cofactors to subnuclear domains containing the estrogen receptor

Ligand binding to estrogen receptor (ER) is presumed to regulate the type and 'timing of ER interactions with different cofactors. Using fluorescence microscopy in living cells, we characterized the recruitment of five different green fluorescent protein (GFP)-labeled ER-interacting peptides to the distinct subnuclear compartment occupied by blue fluorescent protein (BFP)-labeled ERα. Different ligands promoted the recruitment of different peptides. One peptide was recruited in response to estradiol (E₂), tamoxifen, raloxifene, or ICI 182,780 incubation whereas other peptides were recruited specifically by E₂ or tamoxi'fen. Peptides containing different sequences surrounding the ER-interacting motif LXXLL were recruited with different time courses after E₂ addition. Complex temporal kinetics also were observed for recruitment of the full-length, ER cofactor glucocorficoid receptor-interacting protein 1 (GRIP1); rapid, E₂-dependent recruitment of GRIP1 was blocked by mutation of the GRIP1 LXXLL motifs to LXXAA whereas slower E₂ recruitment persisted for the GRIP1 LXXAA mutant. This suggested the presence of multiple, temporally distinct GRIP 1 recruitment mechanisms. E₂ recruitment of GRIP1 and LXXLL peptides was blocked by coincubation with excess ICI 182,780. In contrast, preformed E₂/ER/GRIP1 and E₂/ER/LXXLL complexes were resistant to subsequent ICI 182,780 addition whereas ICI 182,780 dispersed preformed complexes containing the GRIP1 LXXAA mutant. This suggested that E₂-induced LXXLL binding altered subsequent ligand/ER interactions. Thus, alternative, ligand-selective recruitment and dissociation mechanisms with distinct temporal sequences are available for ERα action in vivo.