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Analysis of Ligand-Dependent Recruitment of Coactivator Peptides to Estrogen Receptor Using Fluorescence Polarization

Invitrogen Corporation, Madison, Wisconsin 53719

Address all correspondence and requests for reprints to: Mary Ozers, Ph.D., Invitrogen Corporation, 501 Charmany Drive, Madison, Wisconsin 53719. E-mail: Mary.Ozers{at}invitrogen.com.

Ligand-dependent recruitment of coactivators to estrogen receptor (ER) plays an important role in transcriptional activation of target genes. Agonist-bound ER has been shown to adopt a favorable conformation for interaction with the LXXLL motifs of the coactivator proteins. To further examine the affinity and ligand dependence of the ER-coactivator interaction, several fluorescently tagged short peptides bearing an LXXLL motif (LXXLL peptide) from either natural coactivator sequences or random phage display sequences were used with purified ER or ERß in an in vitro high-throughput fluorescence polarization assay. In the presence of saturating amounts of ligand, several LXXLL peptides bound to ER and ERß with affinity ranging from 20–500 nM. The random phage display LXXLL peptides exhibited a higher affinity for ER than the natural single-LXXLL coactivator sequences tested. These studies indicated that ER agonists, such as 17ß-estradiol or estrone, promoted the interaction of ER with the coactivator peptides, whereas antagonists such as 4-hydroxytamoxifen or ICI-182,780 did not. Different LXXLL peptides demonstrated different affinities for ER depending on which ligand was bound to the receptor, suggesting that the peptides were recognizing different receptor conformations. Using the information obtained from direct measurement of the affinity of the ER-LXXLL peptide interaction, the dose dependency (EC₅₀) of various ligands to either promote or disrupt this interaction was also determined. Interaction of ER with the LXXLL peptide was observed with ligands such as 17ß-estradiol, estriol, estrone, and genistein but not with ICI-182,780, 4-hydroxytamoxifen, clomiphene, or tamoxifen, resulting in distinct EC₅₀ values for each ligand and correlating well with the ligand biological function as an agonist or antagonist. Ligand-dependent recruitment of the LXXLL peptide to ERß was observed in the presence of the ERß-selective agonist diarylpropionitrile, but not the ER-selective ligand propyl pyrazole triol. This assay could be used to classify unknown ligands as agonists, antagonists, or partial modulators, based on either the receptor-coactivator peptide affinities or the dose dependency of this interaction in comparison with known compounds.

NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  ERβ

Coregulators:   RIP140  |  TRAP220

Ligands:   17β-Estradiol  |  4-Hydroxytamoxifen

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