Coactivator peptides have a differential stabilizing effect on the bindingof estrogens and antiestrogens with the estrogen receptor

The effectiveness of estrogens in stimulating gene transcription mediated b y the estrogen receptor (ER) appears to depend on ER interactions with coac tivator proteins. These coactivators bind to ER when it is liganded with an estrogen agonist, but not when it is liganded with an estrogen antagonist. Because estrogen agonists are known to induce a conformation in ER that st abilizes coactivator binding, we asked whether coactivator binding to ER ca uses a reciprocal stabilization of agonist ligand binding. We used a fluore scent ligand for ER, tetrahydrochrysene-ketone, to monitor the rates of lig and dissociation from ER alpha and ER beta, and to see how this process is affected by the p160-class coactivator, steroid receptor coactivator-1 (SRC -1). We used a 15-amino acid peptide corresponding to the second nuclear re ceptor box LXXLL motif in SRC-1 (NR-2 peptide), which is known to interact with the ER ligand-binding domain, a mutant peptide with an LXXAL sequence (NR-2A peptide), and a 203-amino acid fragment of SRC-1, termed the nuclear receptor domain (SRC1-NRD), embodying all three of the internal NR boxes o f this protein. Both the NR-2 peptide and the SRC1-NRD fragment markedly sl ow the rate of dissociation of the agonist ligands tetrahydrochrysene-keton e, estradiol, and diethylstilbestrol, increasing the half-life of the ER-ag onist complex by up to 50- to 60-fold. The SRC1-NRD has much higher potency in retarding ligand dissociation than does the NR-2 peptide; it is maximal ly effective at 30 nM, and it appears to bind with the stoichiometry of one SRC1-NRD per ER dimer. The peptides had little effect on the dissociation rate of antagonist ligands. Consistent with these results, we find that inc reasing the concentration of SRC-1 in cells by transfection of an expressio n plasmid encoding SRC-1 causes a 17-fold increase in the potency of estrad iol in an estrogen-responsive reporter gene transcription assay. Thus, ther e is multifactorial control over receptor-coactivator interaction, its stre ngth being determined by the agonist vs, antagonist nature of the ligand an d the particular structure of the agonist ligand, and by the receptor subty pe and the NR box sequence. The stabilizing effect of coactivator on ER-ago nist ligand complexes may be important in determining the potency of estrog en agonists in a cell and may also underlie the tissue-selective pharmacolo gy of certain synthetic estrogens.