Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster

The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acid-binding residue within the ligand-binding pocket, 1323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.     

An RNA-binding domain in the thyroid hormone receptor enhances transcriptional activation

Thyroid hormone plays important roles in development, differentiation, and metabolic homeostasis by binding to nuclear thyroid hormone receptors, which regulate target gene expression by interacting with DNA response elements and coregulatory proteins. We show that thyroid hormone receptors also are single-stranded RNA binding proteins and that this binding is functionally significant. By using a series of deletion mutants, a novel RNA-binding domain was localized to a 41-amino acid segment of thyroid hormone receptor alpha1 between the second zinc finger and the ligand-binding domain. This RNA-binding domain was necessary and sufficient for thyroid hormone receptor binding to the steroid receptor RNA activator (SRA). Although SRA does not bind directly to steroid receptors, it has been identified as a steroid receptor coactivator, and was thought not to be a coactivator for thyroid hormone receptors. However, transfection studies revealed that SRA enhances thyroid hormone induction of appropriate reporter genes and that the thyroid hormone receptor RNA-binding domain is important for this enhancement. We conclude that thyroid hormone receptors bind RNA through a novel domain and that the interaction of this domain with SRA, and perhaps other RNAs, enhances thyroid hormone receptor function.     

The mouse glucocorticoid receptor DNA-binding domain is not phosphorylated in vivo

The glucocorticoid receptor is phosphorylated, but the precise location of the phosphorylated groups is unknown. We cultured AtT-20 cells in medium containing [32P]-orthophosphate and used immunoaffinity methods to isolate the intact receptor and a tryptic fragment containing the DNA binding domain. Analysis of the intact receptor, co-labeled with the affinity ligand dexamethasone-mesylate, confirmed that the receptor was phosphorylated. Isolation of the DNA binding domain by trypsinization and immunopurification showed that it was not phosphorylated. Interestingly, a non-immunoreactive phosphorylated fragment similar in size to the DNA-binding fragment was observed. Our results suggest that phosphorylation of the DNA binding domain of the glucocorticoid receptor is not essential for hormone action.     

Evidence for DNA-binding domain--ligand-binding domain communications in the androgen receptor

DNA binding as well as ligand binding by nuclear receptors has been studied extensively. Both binding functions are attributed to isolated domains of which the structure is known. The crystal structure of a complete receptor in complex with its ligand and DNA-response element, however, has been solved only for the peroxisome proliferator-activated receptor γ (PPARγ)-retinoid X receptor α (RXRα) heterodimer. This structure provided the first indication of direct interactions between the DNA-binding domain (DBD) and ligand-binding domain (LBD). In this study, we investigated whether there is a similar interface between the DNA- and ligand-binding domains for the androgen receptor (AR). Despite the structural differences between the AR- and PPARγ-LBD, a combination of in silico modeling and docking pointed out a putative interface between AR-DBD and AR-LBD. The surfaces were subjected to a point mutation analysis, which was inspired by known AR mutations described in androgen insensitivity syndromes and prostate cancer. Surprisingly, AR-LBD mutations D695N, R710A, F754S, and P766A induced a decrease in DNA binding but left ligand binding unaffected, while the DBD-residing mutations K590A, K592A, and E621A lowered the ligand-binding but not the DNA-binding affinity. We therefore propose that these residues are involved in allosteric communications between the AR-DBD and AR-LBD.     

A Charge-inverting Mutation in the “Linker” Region of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Alters Agonist Binding and Gating Kinetics Independently of Allosteric Modulators

AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728–4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.     

Oligomerization and ligand-binding properties of the ectodomain of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluRD.

The extracellular part of ionotropic glutamate receptor (iGluR) subunits can be divided into a conserved two-lobed ligand-binding domain ("S1S2") and an N-terminal approximately 400-residue segment of unknown function ("X domain") which shows high sequence variation among subunits. To investigate the structure and properties of the N-terminal domain, we have now produced affinity-tagged recombinant fragments which represent the X domain of the GluRD subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptors either alone or covalently linked to the ligand-binding domain ("XS1S2"). These fragments were expressed in insect cells as secreted soluble proteins and were recognized by a conformation-specific anti-GluRD monoclonal antibody. A hydrodynamic analysis of the purified fragments revealed them to be dimers, in contrast to the S1S2 ligand-binding domain which is monomeric. The X domain did not bind radiolabeled AMPA or glutamate nor did its presence affect the ligand binding properties of the S1S2 domain. Our findings demonstrate that the N-terminal domain of AMPA receptor can be expressed as a soluble polypeptide and suggest that subunit interactions in iGluR may involve the extracellular domains.     

The Drosophila ecdysone receptor (EcR) gene is required maternally for normal oogenesis

Oogenesis in Drosophila is regulated by the steroid hormone ecdysone and the sesquiterpenoid juvenile hormone. Response to ecdysone is mediated by a heteromeric receptor composed of the EcR and USP proteins. We have identified a temperature-sensitive EcR mutation, EcR(A483T), from a previously isolated collection of EcR mutations. EcR(A483T) is predicted to affect all EcR protein products (EcR-A, EcR-B1, and EcR-B2) since it maps to a common exon encoding the ligand-binding domain. In wild-type females, we find that both EcR-A and EcR-B1 are expressed in nurse cells and follicle cells throughout oogenesis. EcR mutant females raised at permissive temperature and then shifted to restrictive temperature exhibit severe reductions in fecundity. Oogenesis in EcR mutant females is defective, and the spectrum of oogenic defects includes the presence of abnormal egg chambers and loss of vitellogenic egg stages. Our results demonstrate a requirement for EcR during female reproduction and suggest that EcR is required for normal oogenesis.     

Farnesyl pyrophosphate is a novel transcriptional activator for a subset of nuclear hormone receptors

In silico docking of a chemical library with the ligand-binding domain of thyroid hormone nuclear receptor-beta (TRbeta) suggested that farnesyl pyrophosphate (FPP), a key intermediate in cholesterol synthesis and protein farnesylation, might function as an agonist. Surprisingly, addition of FPP to cells activated TR as well as the classical steroid hormone receptors but not peroxisome proliferative-activating receptors, farnesoid X receptor, liver X receptor, or several orphan nuclear receptors the ligands of which are unknown. FPP enhanced receptor-coactivator binding in vitro and in vivo, and elevation of FPP levels in cells by squalene synthetase or farnesyl transferase inhibitors leads to activation. The FPP effect was blocked by selective receptor antagonists, and in silico docking with 143 nuclear receptor ligand-binding domain structures revealed that FPP only docked with the agonist conformation of those receptors activated by FPP. Our results suggest that certain nuclear receptors maintain a common structural feature that may reflect an action of FPP on an ancient nuclear receptor or that FPP could function as a ligand for one of the many orphan nuclear receptors the ligands of which have not yet been identified. This finding also has potential interesting implications that may, in part, explain the pleotropic effects of statins as well as certain actions of farnesylation inhibitors in cells.     

Identification and characterization of PWWP domain residues critical for LEDGF/p75 chromatin binding and human immunodeficiency virus type 1 infectivity

Lens epithelium-derived growth factor (LEDGF)/p75 functions as a bimodal tether during lentiviral DNA integration: its C-terminal integrase-binding domain interacts with the viral preintegration complex, whereas the N-terminal PWWP domain can bind to cellular chromatin. The molecular basis for the integrase-LEDGF/p75 interaction is understood, while the mechanism of chromatin binding is unknown. The PWWP domain is homologous to other protein interaction modules that together comprise the Tudor clan. Based on primary amino acid sequence and three-dimensional structural similarities, 24 residues of the LEDGF/p75 PWWP domain were mutagenized to garner essential details of its function during human immunodeficiency virus type 1 (HIV-1) infection. Mutating either Trp-21 or Ala-51, which line the inner wall of a hydrophobic cavity that is common to Tudor clan members, disrupts chromatin binding and virus infectivity. Consistent with a role for chromatin-associated LEDGF/p75 in stimulating integrase activity during infection, recombinant W21A protein is preferentially defective for enhancing integration into chromatinized target DNA in vitro. The A51P mutation corresponds to the S270P change in DNA methyltransferase 3B that causes human immunodeficiency, centromeric instability, and facial anomaly syndrome, revealing a critical role for this amino acid position in the chromatin binding functions of varied PWWP domains. Our results furthermore highlight the requirement for a conserved Glu in the hydrophobic core that mediates interactions between other Tudor clan members and their substrates. This initial systematic mutagenesis of a PWWP domain identifies amino acid residues critical for chromatin binding function and the consequences of their changes on HIV-1 integration and infection.