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Simple alkyl phenols have been tested for their ability to prevent the binding of [3H]estradiol and to displace the prebound hormone from estrogen receptors of uterine cytosols. Tetrahydronaphthol, an analog of the A and B rings of estradiol, is highly effective in preventing the forward bindng of estradiol. p-sec-Amyl phenol (pSAP) with a flexible alkyl chain corresponding to the B ring of estradiol is highly effective at 0 C in displacing estradiol which had been prebound by the receptor. Both compounds were more effective at 0 C than at 23 C. The data are discussed in terms of sequential conformational changes which might be required for the binding and release of the natural hormones and their possible relevance to receptor action.

Specific polyclonal antisera to the rat estrogen receptor (rER) were developed using a synthetic peptide as the antigen. The peptide corresponds to amino acids 270-284 deduced from the cloned rER cDNA and has no homology to other steroid hormone receptors. Antipeptide antisera raised in rabbits specifically recognize a 67,000 mol wt protein, shown to be the rER, in Western blot experiments. In immunoprecipitation experiments, one tested antiserum bound unoccupied as well as 17 beta-estradiol-occupied rERs, indicating that this region is exposed in both receptor forms. This antiserum shows no cross-reactivity with rat progesterone or glucocorticoid receptors. Cross-reactivity with rabbit, human, and, to a lesser extent, bovine ERs was observed.

Upon binding estrogen, the estrogen receptor (ER) is proposed to undergo some form of conformational transition leading to increased transcription from estrogen-responsive genes. In vitro methods used to study the transition often do not separate heat-induced effects on the ER from estrogen-induced effects. The technique of affinity partitioning with PEG-palmitate was used to study the change in the hydrophobic surface properties of the ER upon binding ligand with and without in vitro heating. Upon binding estradiol (E2), the full-length rat uterine cytosolic ER undergoes a dramatic decrease in surface hydrophobicity. The binding of the anti-estrogen 4-hydroxytamoxifen (4-OHT) results in a similar decrease in surface hydrophobicity. These effects are independent of any conformational changes induced by heating the ER to 30 degrees C for 45 min. The use of the human ER steroid binding domain overproduced in Escherichia coli (ER-C) and the trypsin-generated steroid binding domain from rat uterine cytosolic ER demonstrates that the decrease in surface hydrophobicity upon binding E2 or 4-OHT is localized to the steroid binding domain. Gel filtration analysis indicates that the change in surface hydrophobicity upon binding ligand is an inherent property of the steroid binding domain and not due to a ligand-induced change in the oligomeric state of the receptor. The decrease in surface hydrophobicity of the steroid binding domain of the ER upon binding E2 or 4-OHT represents an early and possibly a necessary event in estrogen action and may be important for "tight" binding of the ER in the nucleus.