The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

Estrogens and selective estrogen receptor (ER) modulators such as tamoxifen are known to increase uterine cell proliferation. Mounting evidence suggests that estrogen signaling is mediated not only by ERα and ERβ nuclear receptors, but also by GPR30 (GPER), a seven transmembrane (7TM) receptor. Here, we report that primary human endometriotic H-38 cells express high levels of GPR30 with no detectable ERα or ERβ. Using a novel tamoxifen analogue, STX, which activates GPR30 but not ERs, significant stimulation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways was observed in H-38 cells and in Ishikawa endometrial cancer cells expressing GPR30; a similar effect was observed in JEG3 choriocarcinoma cells. STX treatment also increased cellular pools of phosphatidylinositol (3,4,5) triphosphate, a proposed ligand for the nuclear hormone receptor SF-1 (NR5A1). Consistent with these findings, STX, tamoxifen, and the phytoestrogen genistein were able to increase SF-1 transcription, promote Ishikawa cell proliferation, and induce the SF-1 target gene aromatase in a GPR30-dependent manner. Our findings suggest a novel signaling paradigm that is initiated by estrogen activation of the 7TM receptor GPR30, with signal transduction cascades (PI3K and MAPK) converging on nuclear hormone receptors (SF-1/LRH-1) to modulate their transcriptional output. We propose that this novel GPR30/SF-1 pathway increases local concentrations of estrogen, and together with classic ER signaling, mediate the proliferative effects of synthetic estrogens such as tamoxifen, in promoting endometriosis and endometrial cancers. [Cancer Res 2009;69(13):5415–23]

Phosphatidylinositol 4,5-bisphosphate (PIP₂) is best known as a plasma membrane–bound regulatory lipid. Although PIP₂ and phosphoinositide-modifying enzymes coexist in the nucleus, their nuclear roles remain unclear. We showed that inositol polyphosphate multikinase (IPMK), which functions both as an inositol kinase and as a phosphoinositide 3-kinase (PI3K), interacts with the nuclear receptor steroidogenic factor 1 (SF-1) and phosphorylates its bound ligand, PIP₂. In vitro studies showed that PIP₂ was not phosphorylated by IPMK if PIP₂ was displaced or blocked from binding to the large hydrophobic pocket of SF-1 and that the ability to phosphorylate PIP₂ bound to SF-1 was specific to IPMK and did not occur with type 1 p110 PI3Ks. IPMK-generated SF-1–PIP₃ (phosphatidylinositol 3,4,5-trisphosphate) was dephosphorylated by the lipid phosphatase PTEN. Consistent with the in vitro activities of IPMK and PTEN on SF-1–PIP_n, SF-1 transcriptional activity was reduced by silencing IPMK or overexpressing PTEN. This ability of lipid kinases and phosphatases to directly remodel and alter the activity of a non–membrane protein–lipid complex establishes a previously unappreciated pathway for promoting lipid-mediated signaling in the nucleus.

Significance: We previously reported that lipids PI(4,5)P ₂ (PIP ₂ ) and PI(3,4,5)P ₃ (PIP ₃ ) bind NR5A nuclear receptors to regulate their activity. Here, the crystal structures of PIP ₂ and PIP ₃ bound to NR5A1 (SF-1) define a new interaction surface that is organized by the solvent-exposed PIP _n headgroups. We find that stabilization by the PIP ₃ ligand propagates a signal that increases coactivator recruitment to SF-1, consistent with our earlier work showing that PIP ₃ increases SF-1 activity. This newly created surface harbors a cluster of human mutations that lead to endocrine disorders, thus explaining how these puzzling mutations cripple SF-1 activity. We propose that this new surface acts as a PIP ₃ -regulated interface between SF-1 and coregulatory proteins, analogous to the function of membrane-bound phosphoinositides.

The crystal structure of LRH-1 ligand binding domain bound to our previously reported agonist 3-(E-oct-4-en-4-yl)-1-phenylamino-2-phenyl-cis-bicyclo[3.3.0]oct-2-ene 5 is described. Two new classes of agonists in which the bridgehead anilino group from our first series was replaced with an alkoxy or 1-ethenyl group were designed, synthesized, and tested for activity in a peptide recruitment assay. Both new classes gave very active compounds, particularly against SF-1. Structure−activity studies led to excellent dual-LRH-1/SF-1 agonists (e.g., RJW100) as well as compounds selective for LRH-1 (RJW101) and SF-1 (RJW102 and RJW103). The series based on 1-ethenyl substitution was acid stable, overcoming a significant drawback of our original bridgehead anilino-substituted series. Initial studies on the regulation of gene expression in human cell lines showed excellent, reproducible activity at endogenous target genes.