title: Ligand-selective interactions of ER detected in living cells by fluorescence resonance energy transfer.
abstract: Some aspects of ligand-regulated transcription activation by the estrogen receptor (ER) are associated with the estrogen-dependent formation of a hydrophobic cleft on the receptor surface. At least in vitro, this cleft is required for direct interaction of ER with an alpha helix, containing variants of the sequence LXXLL, found in many coactivators. In cells, it is unknown whether ER interactions with the different LXXLL-containing helices are uniformly similar or whether they vary with LXXLL sequence or activating ligand. Using fluorescence resonance energy transfer (FRET), we confirm in the physiological environment a direct interaction between the estradiol (E2)-bound ER and LXXLL peptides expressed in living cells as fusions with spectral variants of the green fluorescent protein. This interaction was blocked by a single amino acid mutation in the hydrophobic cleft. No FRET was detected when cells were incubated with the antiestrogenic ligands tamoxifen and ICI 182,780. E2, diethylstilbestrol, ethyl indenestrol A, and 6,4'-dihydroxyflavone all promoted FRET and activated ER-dependent transcription. Measurement of the level of FRET of ER with different LXXLL-containing peptides suggested that the orientations or affinities of the LXXLL interactions with the hydrophobic cleft were globally similar but slightly different for some activating ligands.
authors.affiliation: Departments of Pharmaceutical Chemistry and Cellular & Molecular Pharmacology, University of California, San Francisco, California 94143-0446, USA.
authors.forename: R V
authors.initials: RV
authors.lastname: Weatherman
authors.forename: C-Y
authors.initials: CY
authors.lastname: Chang
authors.forename: N J
authors.initials: NJ
authors.lastname: Clegg
authors.forename: D C
authors.initials: DC
authors.lastname: Carroll
authors.forename: R N
authors.initials: RN
authors.lastname: Day
authors.forename: J D
authors.initials: JD
authors.lastname: Baxter
authors.forename: D P
authors.initials: DP
authors.lastname: McDonnell
authors.forename: T S
authors.initials: TS
authors.lastname: Scanlan
authors.forename: F
authors.initials: F
authors.lastname: Schaufele
mesh_terms: D002460:Cell Line; D004054:Diethylstilbestrol / Q000378:metabolism; D004735:Energy Transfer; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D000077267:Fulvestrant; D049452:Green Fluorescent Proteins; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020218:Response Elements; D020845:Selective Estrogen Receptor Modulators / Q000378:metabolism / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013629:Tamoxifen / Q000378:metabolism / Q000494:pharmacology; D014158:Transcription, Genetic; D000097573:Red Fluorescent Protein
chemical_list: D004054:Diethylstilbestrol; D004958:Estradiol; D047628:Estrogen Receptor alpha; D000077267:Fulvestrant; D049452:Green Fluorescent Proteins; D008024:Ligands; D008164:Luminescent Proteins; D010455:Peptides; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D020845:Selective Estrogen Receptor Modulators; D013629:Tamoxifen; D000097573:Red Fluorescent Protein