Raymond D. Blind

Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

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.

Stimulating the GPR30 Estrogen Receptor with a Novel Tamoxifen Analogue Activates SF-1 and Promotes Endometrial Cell Proliferation

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 ERalpha and ERbeta 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 ERalpha or ERbeta. 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.

Differential recruitment of glucocorticoid receptor phospho-isoforms to glucocorticoid-induced genes

The human glucocorticoid receptor (GR) is phosphorylated on its N-terminus at three major sites (S203, S211 and S226) within activation function 1 (AF1). Although GR has been shown to assemble at glucocorticoid responsive elements (GREs) in the presence of hormone, the timing and specificity of GR phospho-isoform recruitment to receptor target genes has not been established. Using chromatin immunoprecipitation (ChIP) and GR phosphorylation site-specific antibodies, we examined GR phospho-isoform recruitment to several glucocorticoid-induced genes including tyrosine aminotransferase (tat) and sulfonyltransferase-1A1 (sult) in rat hepatoma cells, and the glucocorticoid-induced leucine zipper (gilz) gene in human U2OS cells. GR P-S211 and GR P-S226 isoforms were efficiently recruited to the tat, sult and gilz GREs in a hormone-dependent manner. In contrast, the GR P-S203 isoform displayed no significant recruitment to any GREs of the genes analyzed, consistent with its lack of nuclear accumulation. Interestingly, the kinetics of GR P-S211 and GR P-S226 recruitment differed among genes. Our findings indicate that GR phospho-isoforms selectively occupy GR target genes, and suggests gene specific requirements for GR phosphorylation in receptor-dependent transcriptional activation.

Structure of SF-1 Bound by Different Phospholipids: Evidence for Regulatory Ligands

Despite the fact that many nuclear receptors are ligand dependent, the existence of obligate regulatory ligands is debated for some receptors, including steroidogenic factor 1 (SF-1). Although fortuitously bound bacterial phospholipids were discovered in the structures of the SF-1 ligand-binding domain (LBD), these lipids might serve merely as structural ligands. Thus, we examined whether exogenously added phospholipids would exchange for these bacterial lipids and bind to SF-1. Here, we report the first crystal structure of the SF-1 LBD bound by the exchanged phosphatidylcholine. Although the bound phosphatidylcholine phospholipid mimics the conformation of bound bacterial phosphoplipids, two surface loops, L2-3 and L11-12, surrounding the entrance to the pocket vary significantly between different SF-1 LBD structures. Based on this observation, we hypothesized that a bound ligand might control the conformations of loops L2-3 and L11-12, and that conserved residues in these dynamic loops could influence ligand binding and the receptor function. Consistent with this hypothesis, impaired phospholipid exchange and diminished transcriptional activity were observed for loop L11-12 SF-1 mutants and for the loop L2-3 human mutant R255L. The endocrine disease associated with this L2-3 mutation coupled with our cellular and biochemical data suggest that critical residues at the mouth of the ligand-binding pocket have evolved for efficient binding of phospholipid ligands and for achieving optimal SF-1 activity.

Direct modification and activation of a nuclear receptor-PIP₂ complex by the inositol lipid kinase IPMK.

The transcriptional activity of a nuclear receptor is regulated by the phosphorylation status of a bound lipid. Targeting the Lipid Ligand to Regulate Transcription Steroidogenic factor 1 (SF-1) is a nuclear receptor that transcriptionally activates genes involved in sexual development and reproduction. Blind et al. found that phosphatidylinositol 4,5-bisphosphate (PIP2) bound to SF-1 was phosphorylated by inositol polyphosphate multikinase (IPMK) to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3). Conversely, the lipid phosphatase PTEN dephosphorylated the SF-1–PIP3 complex. The transcriptional activity of SF-1 was decreased in cells after inhibiting IPMK and increased in cells overexpressing PTEN. Thus, phosphorylation of its associated lipid, rather than the nuclear receptor itself, activates SF-1 and identifies another signaling role for nuclear phosphoinositides. 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–PIPn, 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.

Small molecule agonists of the orphan nuclear receptors steroidogenic factor-1 (SF-1, NR5A1) and liver receptor homologue-1 (LRH-1, NR5A2).

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.

The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor SF-1.

Significance We previously reported that lipids PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind NR5A nuclear receptors to regulate their activity. Here, the crystal structures of PIP2 and PIP3 bound to NR5A1 (SF-1) define a new interaction surface that is organized by the solvent-exposed PIPn headgroups. We find that stabilization by the PIP3 ligand propagates a signal that increases coactivator recruitment to SF-1, consistent with our earlier work showing that PIP3 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 PIP3-regulated interface between SF-1 and coregulatory proteins, analogous to the function of membrane-bound phosphoinositides. The signaling phosphatidylinositol lipids PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind nuclear receptor 5A family (NR5As), but their regulatory mechanisms remain unknown. Here, the crystal structures of human NR5A1 (steroidogenic factor-1, SF-1) ligand binding domain (LBD) bound to PIP2 and PIP3 show the lipid hydrophobic tails sequestered in the hormone pocket, as predicted. However, unlike classic nuclear receptor hormones, the phosphoinositide head groups are fully solvent-exposed and complete the LBD fold by organizing the receptor architecture at the hormone pocket entrance. The highest affinity phosphoinositide ligand PIP3 stabilizes the coactivator binding groove and increases coactivator peptide recruitment. This receptor-ligand topology defines a previously unidentified regulatory protein-lipid surface on SF-1 with the phosphoinositide head group at its nexus and poised to interact with other proteins. This surface on SF-1 coincides with the predicted binding site of the corepressor DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region on chromosome X), and importantly harbors missense mutations associated with human endocrine disorders. Our data provide the structural basis for this poorly understood cluster of human SF-1 mutations and demonstrates how signaling phosphoinositides function as regulatory ligands for NR5As.

Disentangling biological signaling networks by dynamic coupling of signaling lipids to modifying enzymes

An unresolved problem in biological signal transduction is how particular branches of highly interconnected signaling networks can be decoupled, allowing activation of specific circuits within complex signaling architectures. Although signaling dynamics and spatiotemporal mechanisms serve critical roles, it remains unclear if these are the only ways cells achieve specificity within networks. The transcription factor Steroidogenic Factor-1 (SF-1) is an excellent model to address this question, as it forms dynamic complexes with several chemically distinct lipid species (phosphatidylinositols, phosphatidylcholines and sphingolipids). This property is important since lipids bound to SF-1 are modified by lipid signaling enzymes (IPMK & PTEN), regulating SF-1 biological activity in gene expression. Thus, a particular SF-1/lipid complex can interface with a lipid signaling enzyme only if SF-1 has been loaded with a chemically compatible lipid substrate. This mechanism permits dynamic downstream responsiveness to constant upstream input, disentangling specific pathways from the full network. The potential of this paradigm to apply generally to nuclear lipid signaling is discussed, with particular attention given to the nuclear receptor superfamily of transcription factors and their phospholipid ligands.

Structure of Liver Receptor Homolog-1 (NR5A2) with PIP3 hormone bound in the ligand binding pocket.

The nuclear receptor LRH-1 (Liver Receptor Homolog-1, NR5A2) is a transcription factor that regulates gene expression programs critical for many aspects of metabolism and reproduction. Although LRH-1 is able to bind phospholipids, it is still considered an orphan nuclear receptor (NR) with an unknown regulatory hormone. Our prior cellular and structural studies demonstrated that the signaling phosphatidylinositols PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind and regulate SF-1 (Steroidogenic Factor-1, NR5A1), a close homolog of LRH-1. Here, we describe the crystal structure of human LRH-1 ligand binding domain (LBD) bound by PIP3 - the first phospholipid with a head group endogenous to mammals. We show that the phospholipid hormone binds LRH-1 with high affinity, stabilizing the receptor LBD. While the hydrophobic PIP3 tails (C16/C16) are buried inside the LRH-1 ligand binding pocket, the negatively charged PIP3 head group is presented on the receptor surface, similar to the phosphatidylinositol binding mode observed in the PIP3-SF-1 structure. Thus, data presented in this work reinforce our earlier findings demonstrating that signaling phosphatidylinositols regulate the NR5A receptors LRH-1 and SF-1.

Inositol polyphosphate multikinase (IPMK) in transcriptional regulation and nuclear inositide metabolism.

Inositol polyphosphate multikinase (IPMK, ipk2, Arg(82), ArgRIII) is an inositide kinase with unusually flexible substrate specificity and the capacity to partake in many functional protein-protein interactions (PPIs). By merging these two activities, IPMK is able to execute gene regulatory functions that are very unique and only now beginning to be recognized. In this short review, we present a brief history of IPMK, describe the structural biology of the enzyme and highlight a few recent discoveries that have shed more light on the role IPMK plays in inositide metabolism, nuclear signalling and transcriptional regulation.