Ligand-selective interactions of ER detected in living cells by fluorescence resonance energy transfer

R. V. Weatherman, C. Y. Chang, N. J. Clegg, D. C. Carroll, R. N. Day, J. D. Baxter, D. P. McDonnell, Thomas (Tom) Scanlan, F. Schaufele

Research output: Contribution to journalArticle

44 Citations (Scopus)

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 α 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.

Original languageEnglish (US)
Pages (from-to)487-496
Number of pages10
JournalMolecular Endocrinology
Volume16
Issue number3
DOIs
StatePublished - 2002
Externally publishedYes

Fingerprint

Fluorescence Resonance Energy Transfer
Estrogen Receptors
Ligands
Peptides
Diethylstilbestrol
Cell Fusion
Tamoxifen
Green Fluorescent Proteins
Hydrophobic and Hydrophilic Interactions
Transcriptional Activation
Estradiol
Estrogens
Amino Acids
Mutation

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

Weatherman, R. V., Chang, C. Y., Clegg, N. J., Carroll, D. C., Day, R. N., Baxter, J. D., ... Schaufele, F. (2002). Ligand-selective interactions of ER detected in living cells by fluorescence resonance energy transfer. Molecular Endocrinology, 16(3), 487-496. https://doi.org/10.1210/me.16.3.487

Ligand-selective interactions of ER detected in living cells by fluorescence resonance energy transfer. / Weatherman, R. V.; Chang, C. Y.; Clegg, N. J.; Carroll, D. C.; Day, R. N.; Baxter, J. D.; McDonnell, D. P.; Scanlan, Thomas (Tom); Schaufele, F.

In: Molecular Endocrinology, Vol. 16, No. 3, 2002, p. 487-496.

Research output: Contribution to journalArticle

Weatherman, RV, Chang, CY, Clegg, NJ, Carroll, DC, Day, RN, Baxter, JD, McDonnell, DP, Scanlan, TT & Schaufele, F 2002, 'Ligand-selective interactions of ER detected in living cells by fluorescence resonance energy transfer', Molecular Endocrinology, vol. 16, no. 3, pp. 487-496. https://doi.org/10.1210/me.16.3.487
Weatherman, R. V. ; Chang, C. Y. ; Clegg, N. J. ; Carroll, D. C. ; Day, R. N. ; Baxter, J. D. ; McDonnell, D. P. ; Scanlan, Thomas (Tom) ; Schaufele, F. / Ligand-selective interactions of ER detected in living cells by fluorescence resonance energy transfer. In: Molecular Endocrinology. 2002 ; Vol. 16, No. 3. pp. 487-496.
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