Ligand-specific regulation of proteasome-mediated proteolysis of estrogen receptor-␣. Am J Physiol Endocrinol Metab 282: E891-E898, 2002. First published December 18, 2001 10.1152/ajpendo.00353.2001.-Proteasome-mediated proteolysis modulates the cellular concentration of estrogen receptor-␣ (ER␣) and is induced by treatment of cells with 17␤-estradiol. Herein, we show that multiple receptor agonists, including 17␣-estradiol and estriol as well as the antagonist ICI-182780, stimulate proteasome-dependent proteolysis of ER␣ in a process that requires ligand binding to the receptor. Proteolysis of receptor depends on ligand concentration, and there exists a direct correlation between ligand-binding affinity and the half-maximal dose of ligand required to stimulate receptor degradation. Furthermore, introduction of a point mutation into the receptor ligand-binding pocket yields a stable receptor resistant to proteolysis. Interestingly, although all ligands stimulate receptor degradation, the extent to which overall ER levels are affected varies with each ligand and is not related to ligand-binding affinity or activation of transcription. These results demonstrate ligand-specific regulation of ER␣ proteolysis, and they introduce the concept that cellular receptor concentration is governed not only at the level of induction of proteolysis but also by the efficiency with which the receptor is degraded. steroid; nuclear receptor; antagonist; estriol; pituitary CELLULAR ESTROGEN RECEPTOR LEVELS are dynamic and are particularly sensitive to changes in circulating levels of 17␤-estradiol. It has been demonstrated through a number of studies that the decline in estrogen receptor-␣ (ER␣) upon exposure to 17␤-estradiol results from a combination of mechanisms that control both receptor synthesis and degradation through transcriptional, posttranscriptional, and posttranslational mechanisms (20,27,32,33,35,36). The most rapid of these regulatory mechanisms is the direct loss of ER␣ protein brought about by the induction of proteasome-mediated proteolysis (1,21,28).Regulated proteolysis by proteasomes accounts for the turnover of most short-lived proteins, including many nuclear receptors (9,16,23,29,41,43,45). Through a series of three enzymatic reactions, ubiquitin moieties are attached to a protein substrate, which targets it to the 26S proteasome. The molecular events that direct ER␣ into this pathway have not been clearly established. However, earlier studies that examine changes in receptor-binding capacity have shown that receptor levels can be controlled by both receptor agonists and antagonists, suggesting the possibility that receptor occupation by ligand may provide specificity (3,4,15,18,22,33).In our original report of ER␣ protein regulation by proteolysis, we utilized a pituitary lactotrope model system, the PR1 cell line. The lactotrope cell population of the anterior pituitary is a major target of estrogen action. Animals that lack ER␣ show a decrease in lactotrope cell density and prolactin expression (37). In ...

A common phenotype in breast cancer is the expansion of the estrogen receptor-alpha (ER+) cell population and an inappropriate elevation of ERalpha protein, the latter predisposing patients for a poorer prognosis than those with lower levels of the receptor. A tetracycline-inducible ERalpha overexpression model was developed in the MCF-7 cell line to assess induction of endogenous gene activation and growth in response to elevations in ERalpha protein. Heightened levels of ERalpha resulted in aberrant promoter occupancy and gene activation in the absence of hormone, which was independent of ligand and AF-2 function. This increased receptor activity required the amino-terminal A/B domain and was not inhibited by tamoxifen, which supports an enhancement of AF-1 function, yet was independent of serine-104, 106, and 118 phosphorylation. Ligand-independent transcription was accompanied by an increase in growth in the absence of hormonal stimulation. The results suggest that elevated levels of ERalpha in breast cancer cells can result in activation of receptor transcriptional function in a manner distinct from classical mechanisms that involve ligand binding or growth factor-induced phosphorylation. Further, they describe a potential mechanism whereby increases in ERalpha concentration may provide a proliferative advantage by augmenting ERalpha function regardless of ligand status.

Proteolysis by the 26S proteasome is an important regulatory mechanism that governs the protein stability of several steroid/nuclear receptors and that has been implicated in the control of receptor transcriptional activation function. Herein, we report that thyroid hormone can prevent estrogen-induced proteolysis of estrogen receptor-alpha (ERalpha) protein in lactotrope cells of the pituitary. The stabilization of ERalpha protein by thyroid hormone represents a selective blockade against estradiol-stimulated degradation, because thyroid hormone (but not glucocorticoid) can protect estrogen-activated ERalpha. Moreover, thyroid hormone treatment does not interfere with signal-induced proteolysis of a separate proteasome target, IkappaBalpha or ERalpha proteolysis induced by ICI182780. Using thyroid hormone as a tool to inhibit ERalpha proteolysis, we examined the effect of loss of this regulatory function on estrogen-induced transcriptional responses. Consistent with earlier reports, estrogen activation of an idealized estrogen response element reporter gene was inhibited. However, thyroid hormone did not prevent induction of prolactin gene expression or the ability of ERalpha to stimulate proliferation. These results demonstrate that estrogen-induced proteolysis of ERalpha is not a general requirement for receptor transcriptional activation function, and they demonstrate that proteolytic regulation is a means by which other endocrine factors can indirectly modulate ERalpha activity.