To elucidate the ligand binding properties of the estrogen receptor (ER) and how ligand access to and release from the ligand binding pocket is affected by the conformational state of the receptor, we have measured the rates of estradiol association and dissociation, the equilibrium binding, and the stability of estradiol binding to denaturants, comparing wild-type human ER and a point mutant (Y537S ER) that shows full constitutive activity, i.e., the same full transcriptional activity in the absence or presence of estrogen. Ligand binding kinetics and affinity were measured with the full-length (1-595) ERs and with truncated forms of both receptors containing domains C through F (including the DNA binding, hinge, and ligand binding domains, amino acids 175-595) or domains E and F (the ligand binding domain; amino acids 304-595). With all ERs, the rates of ligand association and dissociation were considerably slower with the Y537S mutant ER than with wild-type ER (6-fold and 3-4-fold, respectively). These marked differences in ligand on and off rates for the wild-type and Y537S receptors result in a predicted (k-1/k+1) and measured Kd that is 2-fold lower for Y537S ER compared to wild-type ER. The binding of estradiol by wild-type ER was disrupted by high concentrations of urea (above 2 M), whereas the Y537S ER was distinctly more resistant to this disruption. These results are consistent with a model in which wild-type ER in the absence of ligand adopts a transcriptionally inactive collapsed pocket conformation, stabilized by specific interactions of Y537 with nearby regions of ER. When estradiol is bound, the wild-type ER adopts a transcriptionally active, closed pocket (ligand occupied) conformation. By contrast, the Y537S mutant ER favors the transcriptionally active closed pocket conformation, whether occupied by ligand or not, the latter state (closed pocket but unoccupied) accounting for its constitutive activity. Our findings suggest that the entry or exit of ligand from the binding pocket requires that ER adopt an open pocket conformation. The reduced rates of ligand association and dissociation in the constitutively active form of the ER, as well as its greater resistance to disruption of ligand binding by urea, support the supposition that the rate at which this open pocket conformation can be accessed from the unoccupied or ligand-occupied Y537S ER is slower than from the unoccupied or occupied forms of wild-type ER. Thus, the binding and release of ligand by ER require that the receptor access an open pocket state, and the ease with which this state can be accessed is affected by mutations that alter receptor conformation.

No abstract

The effectiveness of estrogens in stimulating gene transcription mediated by the estrogen receptor (ER) appears to depend on ER interactions with coactivator 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 stabilizes coactivator binding, we asked whether coactivator binding to ER causes a reciprocal stabilization of agonist ligand binding. We used a fluorescent ligand for ER, tetrahydrochrysene-ketone, to monitor the rates of ligand dissociation from ERalpha and ERbeta, 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 receptor 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 of this protein. Both the NR-2 peptide and the SRC1-NRD fragment markedly slow the rate of dissociation of the agonist ligands tetrahydrochrysene-ketone, estradiol, and diethylstilbestrol, increasing the half-life of the ER-agonist 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 maximally 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 increasing the concentration of SRC-1 in cells by transfection of an expression plasmid encoding SRC-1 causes a 17-fold increase in the potency of estradiol in an estrogen-responsive reporter gene transcription assay. Thus, there is multifactorial control over receptor-coactivator interaction, its strength being determined by the agonist vs. antagonist nature of the ligand and the particular structure of the agonist ligand, and by the receptor subtype and the NR box sequence. The stabilizing effect of coactivator on ER-agonist ligand complexes may be important in determining the potency of estrogen agonists in a cell and may also underlie the tissue-selective pharmacology of certain synthetic estrogens.

Recent clinical studies have shown a sex dimorphism of morbidity and mortality due to shock, trauma, and sepsis, with females tolerating these insults better than males. Experimental animal studies have suggested that sex hormones have a pivotal role in this dimorphism. In the present investigation, a prospective cohort study at a university level-1 trauma center was conducted to evaluate the association between sex hormones and alterations in coagulation and inflammation. Patients with an admission to the intensive care unit, injury severity score (ISS) greater than 4, and obtainable consent were included in the study. In addition to routine clinical laboratories and patient outcomes, plasma TNF-[alpha], IL-6, IL-8, estradiol, progesterone, and testosterone were measured. Sixty-two patients (71% men, 29% women) met criteria for entry. Mean age was 42 +/- 17 years, and mean ISS was 23 +/- 13, with no statistical difference in age or ISS between sexes. Estradiol levels were positively correlated with ISS (P < 0.05) and negatively correlated with TNF-[alpha] (P < 0.01). Initial estradiol levels were higher in patients who developed an infection (P < 0.05). Testosterone was negatively correlated with age (P < 0.01) and was higher in patients who developed acute respiratory distress syndrome (P < 0.05) and in patients who did not survive (P < 0.05). The estradiol-to-progesterone ratio (E2-Pr) was higher in the survivors (P < 0.05). The E2-Pr had positive correlations with fibrinogen levels, rate of fibrin deposition and cross-linking, and overall clot strength (P < 0.05). Estradiol-to-progesterone ratio was negatively correlated with partial thromboplastin times (P < 0.01). In men, the E2-Pr was also negatively correlated with the time to onset of clot formation (P = 0.03). Sex hormone levels (or their ratios) were not correlated to platelet count or international normalized ratios. These findings provide evidence that sex hormone levels in the early posttraumatic period are significantly associated with alterations in the hemostatic and inflammatory response to trauma.