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 ER alpha. 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 tamoxifen. 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 glucocorticoid 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 ER alpha action in vivo.
Schaufele
F
F
Department of Medicine, University of California San Francisco, 94143, USA.
Chang
C Y
CY
Liu
W
W
Baxter
J D
JD
Nordeen
S K
SK
Wan
Y
Y
Day
R N
RN
McDonnell
D P
DP
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D002451:Cell Compartmentation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D000077267:Fulvestrant; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D051958:Nuclear Receptor Coactivator 2; D010455:Peptides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011960:Receptors, Estrogen / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology; D013629:Tamoxifen / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*
D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D007202:Indicators and Reagents; D008024:Ligands; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D051958:Nuclear Receptor Coactivator 2; D010455:Peptides; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D020845:Selective Estrogen Receptor Modulators; D014157:Transcription Factors; C409504:blue fluorescent protein, Aequorea victoria; D013629:Tamoxifen; D049452:Green Fluorescent Proteins; D000077267:Fulvestrant; D004958:Estradiol