An assessment of the role of domain F and pest sequences in estrogen receptor half-life and bioactivity

The estrogen receptor (ER) is a rapidly turning over protein, with a half-life of ca. 3–4 h in estrogen target cells. Sequence analysis of the human ER reveals a putative PEST sequence, sequences rich in proline (P), glutamic acid (E), serine (S) and threonine (T), in the carboxy-terminal F domain of the protein. Since PEST sequences have been implicated in the rapid turnover of some proteins, we have used site-directed mutagenesis to investigate the role of the F region containing PEST residues in the stability and bioactivity of the receptor. A truncated form of ER lacking the last 41 amino acids of the protein and encompassing the PEST sequences (amino acids 555 to 567) was made by mutagenesis of the ER cDNA. Pulse-chase experiments, involving immunoprecipitation of [³⁵S]methionine/[³⁵]Scysteine labeled receptors or of receptors covalently labeled with tamoxifen aziridine followed by gel electrophoresis, were used to determine the half-life of the wild-type and truncated ERs. These experiments showed that the turnover rate of the receptors expressed in Chinese hamster ovary and monkey kidney (COS-1) cells was 3 to 5 h and that elimination of the PEST residues did not have a significant effect on the degradation rate of the protein. Moreover, deletion of the last 41 amino acids (F domain) of the ER did not affect transactivation ability, ligand binding affinity, or the phosphorylation pattern of the receptor. Therefore, the role of domain F in ER function remains unclear, but it is not a determinant of the relatively rapid rate of ER turnover in cells.     

Probing the structure and function of the estrogen receptor ligand binding domain by analysis of mutants with altered transactivation characteristics.

We have developed a genetic screen for the yeast Saccharomyces cerevisiae to isolate estrogen receptor (ER) mutants with altered transactivation characteristics. Use of a "reverse" ER, in which the mutagenized ligand binding domain was placed at the N terminus of the receptor, eliminated the isolation of truncated constitutively active mutants. A library was screened with a low-affinity estrogen, 2-methoxyestrone (2ME), at concentrations 50-fold lower than those required for activation of the unmutagenized ER. Several mutants displaying enhanced sensitivity to 2ME were isolated. We further characterized a mutant carrying the substitution L536P, which was located immediately N terminal to the AF-2-activating domain of the receptor. Amino acid 536 corresponds to a ligand contact residue in retinoic acid receptor gamma, suggesting that key contact points are conserved among receptors. Introduction of L536P into the original ER cDNA isolate HE0, which contains the substitution G400V, rendered the receptor more sensitive to a variety of agonists. When introduced into the wild-type ER HEG0, L536P also rendered the receptor more sensitive to agonists, and, in addition, induced high levels of constitutive activity that could be inhibited by antiestrogens. Estrogens containing a keto substitution in the steroid D ring, but not those containing a hydroxyl group, were full agonists of L536P-HEG0. Limited proteolytic analysis suggested that the L536P substitution, which is located immediately N terminal to the AF-2 domain, induces a conformational change in the ER that partially mimics binding by hormone. Both HEG0 and L536P-HEG0 formed complexes with hsp90 in vitro, indicating a lack of correlation between interaction with hsp90 in vitro and hormonal regulation of ER transactivation in vivo. This supports the idea that a factor(s) acting downstream of hsp90 is important for controlling activity of the hormone-free receptor.     

WW domain binding protein-2, an E6-associated protein interacting protein, acts as a coactivator of estrogen and progesterone receptors

WW domain binding protein-2 (WBP-2) was cloned as an E6-associated protein interacting protein, and its role in steroid hormone receptors functions was investigated. We show that WBP-2 specifically enhanced the transactivation functions of progesterone receptor (PR) and estrogen receptor (ER), whereas it did not have any significant effect on the androgen receptor, glucocorticoid receptor, or the activation functions of p53 and VP-16. Depletion of endogenous WBP-2 with small interfering RNAs indicated that WBP-2 was required for the proper functioning of PR and ER. We also demonstrated that WBP-2 contains an intrinsic activation domain. Moreover, chromatin immunoprecipitation assays demonstrate the hormone-dependent recruitment of WBP-2 onto an estrogen-responsive promoter. Mutational analysis suggests that one of three polyproline (PY) motifs of WBP-2 is essential for its coactivation and intrinsic activation functions. We show that WBP-2 and E6-associated protein each enhance PR function, and their effect on PR action are additive when coexpressed, suggesting a common signaling pathway. In this study, we also demonstrate that the WBP-2 binding protein, Yes kinase-associated protein (YAP) enhances PR transactivation, but YAP's coactivation function is absolutely dependent on WBP-2. Taken together, our data establish the role of WBP-2 and YAP as coactivators for ER and PR transactivation pathways.     

Agonist-dependent repression mediated by mutant estrogen receptor alpha that lacks the activation function 2 core domain

Nuclear receptor corepressor (N-CoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT) form heterogeneous complexes with various histone deacetylases (HDACs). In this report, we found that ER alpha-Delta AF2, a mutant estrogen receptor alpha (ER alpha) deleted for the C-terminal activation function 2 (AF2) core domain, directs estradiol (E(2))-dependent repression and impairs E(2)-induced transactivation by wild type ER alpha. This repression required coexpressed BRG1 in SW-13 cells that lack BRG1, the ATPase constituent of the chromatin-remodeling SWI.SNF complex, and was abolished by HDAC inhibitor trichostatin A. We further demonstrated that ER alpha-Delta AF2 constitutively associates with SMRT but binds DNA in an E(2)-dependent manner in vivo. These results suggest that ER alpha-Delta AF2 and similar mutant receptors recently found associated with certain tumors may actively perturb the normal E(2) signaling via SWI/SNF, N-CoR/SMRT, and HDAC.     

P21-activated kinase-1 phosphorylates and transactivates estrogen receptor-alpha and promotes hyperplasia in mammary epithelium

Stimulation of p21-activated kinase-1 (Pak1) induces cytoskeleton reorganization and signaling pathways in mammary cancer cells. Here, we show that inhibition of Pak1 kinase activity by a dominant-negative fragment or by short interference RNA markedly reduced the estrogen receptor-alpha (ER) transactivation functions. To understand the role of Pak1 in mammary glands, we developed a murine model expressing constitutively active Thr423 glutamic acid Pak1 driven by the beta-lactoglobulin promoter. We show that mammary glands from these mice developed widespread hyperplasia associated with apocrine metaplasia and lobuloalveolar hyperdevelopment during lactation. Mammary tissues with active Pak1 also exhibited an increased activation of mitogen-activated protein kinase and stimulated transactivation functions of the ER and expression of endogenous ER target genes. Furthermore, Pak1 directly phosphorylated the activation function-2 domain of the ER at the N-terminal residue Ser305, and its mutation to Ala (S305A) abolished the Pak1-mediated phosphorylation and transactivation functions of the ER, while its mutation to glutamic acid (S305E) promoted transactivation activity of ER. These findings reveal a novel role for the Pak1-ER pathway in promoting hyperplasia in mammary epithelium.     

The steroid binding domain of porcine estrogen receptor

For the purpose of characterizing the estrogen binding domain of porcine estrogen receptor (ER), we have made use of affinity labeling of partially purified ER with [3H]tamoxifen aziridine. The labeling is very efficient and selective particularly after partial purification of ER. A 65,000-dalton (65-kDa) band was detected on the fluorogram of a sodium dodecyl sulfate-polyacrylamide gel, together with a 50-kDa band and a few more smaller bands. The 50-kDa protein appears to be a degradation product of the 65-kDa protein in view of the similar peptide map. ER was affinity labeled before or after controlled limited proteolysis with either trypsin, papain, or alpha-chymotrypsin. The labeling patterns of limited digests indicate that a fragment of about 30 kDa is relatively resistant to proteases and has a full and specific binding activity to estrogen, whereas smaller fragments have lost much of the binding activity. This fragment is very hydrophobic and probably corresponds to the carboxy half of ER.