A glucocorticoid/retinoic acid receptor chimera that displays cytoplasmic/nuclear translocation in response to retinoic acid. A real time sensing assay for nuclear receptor ligands

Members of the nuclear receptor superfamily play key roles in a host of physiologic and pathologic processes from embryogenesis to cancer. Some members, including the retinoic acid receptor (RAR), are activated by ligand binding but are unaffected in their subcellular distribution, which is predominantly nuclear. In contrast, several members of the steroid receptor family, including the glucocorticoid receptor, are cytoplasmic and only translocate to the nucleus after ligand binding. We have constructed chimeras between RAR and glucocorticoid receptor that selectively respond to RAR agonists but display cytoplasmic localization in the absence of ligand. These chimeric receptors manifest both nuclear translocation and gene activation functions in response to physiological concentrations of RAR ligands. The ability to achieve regulated subcellular trafficking with a heterologous ligand binding domain has implications both for current models of receptor translocation and for structural-functional conservation of ligand binding domains broadly across the receptor superfamily. When coupled to the green fluorescent protein, chimeric receptors offer a powerful new tool to 1) study mechanisms of steroid receptor translocation, 2) detect dynamic and graded distributions of ligands in complex microenvironments such as embryos, and 3) screen for novel ligands of "orphan" receptors in vivo.     

Structural features of the ligand binding site on human complement protein C8gamma: a member of the lipocalin family

Human C8 is one of five components of the cytolytic membrane attack complex of complement. It contains three subunits (C8alpha, C8beta, C8gamma) arranged as a disulfide-linked C8alpha-gamma heterodimer that is noncovalently associated with C8beta. C8gamma has the distinction of being the only lipocalin in the complement system. Lipocalins have a core beta-barrel structure forming a calyx with a binding site for a small hydrophobic ligand. A natural ligand for C8gamma has not been identified; however previous structural studies indicate C8gamma has a typical lipocalin fold that is suggestive of a ligand-binding capability. A distinctive feature of C8gamma is the division of its putative ligand binding pocket into a hydrophilic upper portion and a large hydrophobic lower cavity. Access to the latter is restricted by the close proximity of two tyrosine side chains (Y83 and Y131). In the present study, binding experiments were performed using lauric acid as a pseudoligand to investigate the potential accessibility of the lower cavity. The crystal structure of a C8gamma.laurate complex revealed that Y83 and Y131 can move to allow penetration of the hydrocarbon chain of laurate into the lower cavity. Introducing a Y83W mutation blocked access but had no effect on the ability of C8gamma to enhance C8 cytolytic activity. Together, these results indicate that the lower cavity in C8gamma could accommodate a ligand if such a ligand has a narrow hydrophobic moiety at one end. Entry of that moiety into the lower cavity would require movement of Y83 and Y131, which act as a gate at the cavity entrance.     

Receptor binding characteristics of the endocrine disruptor bisphenol A for the human nuclear estrogen-related receptor gamma. Chief and corroborative hydrogen bonds of the bisphenol A phenol-hydroxyl group with Arg316 and Glu275 residues

Bisphenol A, 2,2-bis(4-hydroxyphenyl)propane, is an estrogenic endocrine disruptor that influences various physiological functions at very low doses, even though bisphenol A itself is ineffectual as a ligand for the estrogen receptor. We recently demonstrated that bisphenol A binds strongly to human estrogen-related receptor gamma, one of 48 human nuclear receptors. Bisphenol A functions as an inverse antagonist of estrogen-related receptor gamma to sustain the high basal constitutive activity of the latter and to reverse the deactivating inverse agonist activity of 4-hydroxytamoxifen. However, the intrinsic binding mode of bisphenol A remains to be clarified. In the present study, we report the binding potentials between the phenol-hydroxyl group of bisphenol A and estrogen-related receptor gamma residues Glu275 and Arg316 in the ligand-binding domain. By inducing mutations in other amino acids, we evaluated the change in receptor binding capability of bisphenol A. Wild-type estrogen-related receptor gamma-ligand-binding domain showed a strong binding ability (K(D) = 5.70 nm) for tritium-labeled [(3)H]bisphenol A. Simultaneous mutation to Ala at positions 275 and 316 resulted in an absolute inability to capture bisphenol A. However, individual substitutions revealed different degrees in activity reduction, indicating the chief importance of phenol-hydroxyl<-->Arg316 hydrogen bonding and the corroborative role of phenol-hydroxyl<-->Glu275 hydrogen bonding. The data obtained with other characteristic mutations suggested that these hydrogen bonds are conducive to the recruitment of phenol compounds by estrogen-related receptor gamma. These results clearly indicate that estrogen-related receptor gamma forms an appropriate structure presumably to adopt an unidentified endogenous ligand.     

Properties of progestin-binding protein in benign hypertrophic human prostate

RU 27987 is a new ligand for progesterone receptor and binds in high affinity to nuclei of target tissues of progesterone. Using this compound, progestin-binding components in the benign hypertrophic human prostate were studied, and compared with those examined with R 5020, a conventional ligand, in the study of progesterone receptor. In cytosols, the binding affinity of RU 27987 was higher than that of R 5020, and the number of maximum binding sites for RU 27987 seemed to be large but correlated well with those of R 5020. The binder for RU 27987 sedimented at 8.6 S, and the binding was specific to progestational steroids, indicating that binding properties of this binder in the cytosols are identical to those for R 5020. Although there was no binding with R 5020 in the nuclear extract, a small amount of specific binding with RU 27987 was detected. However, the cytosol bound with RU 27987 was not retained in DNA Sepharose and no specific binder for RU 27987 in the nuclear extract was observed in a sucrose density gradient centrifugation. From these observations, it was assumed that the nuclear binding observed was attributable to contamination of the cytosolic binder. The results obtained in the present study suggest that the progestin-binding component in the benign prostatic hypertrophy is not the progesterone receptor but a high affinity binder for progestins whose physiological role is not clear at present.     

Proteasome activator subunit PA28 alpha and related Ki antigen (PA28 gamma) are absent from the nuclear fraction purified by sucrose gradient centrifugation

The aim of the present work was to attempt to partially purify PA28 (REG) alpha and gamma (Ki antigen) in the nuclear fraction from NT2/D1 cells. Nuclei were isolated by the hypertonic sucrose gradient centrifugation method and fractionated into membrane/nucleoplasmic and chromatin/nucleolar fractions. Western blotting with anti-histone and anti-beta-tubulin monoclonal antibodies confirmed the accuracy of the procedure. Proteasomes were present mainly in the cytoplasm but also in the nuclei. Disruption of the nuclear envelope released the proteasomes implying a loose or no binding with the chromatin. PA28 alpha and gamma were detected mainly in the cytosol and to a lesser extent in the crude nuclear pellet, however the purified nuclei were devoid of PA28 alpha and gamma. This indicates, that only a small fraction of the PA28 activator is present in the nuclei as detected by immunofluorescence or/and it is easily removed during nuclear purification.     

Structural basis for binding multiple ligands by the common cytokine receptor gamma-chain

The common gamma-chain (gamma(c)) that functions both in ligand binding and signal transduction is a shared subunit of the multichain receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The structural basis by which the ectodomain of gamma(c) contributes to binding six distinct cytokines is only partially defined. In the present study, epitope mapping of antagonistic anti-gamma(c) monoclonal antibodies led to the identification of Asn-128 of mouse gamma(c) that represents another potential contact residue that is required for binding IL-2, IL-7, and IL-15 but not IL-4. In addition, Tyr-103, Cys-161, Cys-210, and Cys-211, previously identified to contribute to binding IL-2 and IL-7, were also found to be involved in binding IL-4 and IL-15. Collectively, these data favor a model in which gamma(c) utilizes a common mechanism for its interactions with multiple cytokines, and the binding sites are largely overlapping but not identical. Asn-128 and Tyr-103 likely act as contact residues whereas Cys-161, Cys-210, and Gly-211 may stabilize the structure of the proposed ligand-interacting surface formed by the two extracytoplasmic domains.     

Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 triggers transient activation of NADPH oxidase activity. Continuous oxidase activation requires continuous de novo receptor cross-linking

Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 (Fc gamma RI) on U937 cells triggered superoxide anion (O-2) release. This was accomplished by the binding of an Fc gamma RI-specific monoclonal antibody, mAb 32, followed by cross-linking of the mAb on the cell with anti-mouse IgG F(ab')2 by Fc gamma RI-specific mAbs 32 and 22 used as an equimolar mixture or by Fc gamma RI-specific mAb 197 (a murine IgG2a and thus a multivalent ligand for Fc gamma RI) alone. At subsaturating concentrations of the Fc gamma RI-cross-linking ligands, O2- generation was continuous over relatively long intervals. However, saturating concentrations triggered an often substantial but always transient O2- burst. This transient burst of oxidase activity ceased with maximal ligand accumulation on the cell. Cells in which oxidase activity had ceased could be restimulated using phorbol 12-myristate 13-acetate or aggregated human IgG1, indicating that cessation of O2- generation was not due to a generalized exhaustion or inhibition of the NADPH oxidase pathway. Cells incubated in subsaturating concentrations of cross-linking antibodies continued to release O2- until binding of the ligand ceased. In addition, the rates of O2- production and ligand accumulation were the same. Thus, continuous O2- production appeared to be dependent upon continuous de novo formation of cross-linked and activated Fc gamma RI. Furthermore, the mol of O2- released in response to Fc gamma RI cross-linking by the multivalent ligand mAb 197 were directly proportional to the mol of mAb bound over a range of saturating and subsaturating concentrations. This evidence suggests a quantal relationship between each Fc gamma RI activated (cross-linked) and the resultant oxidase activity and supports a "rate" model for the activation of this response. Thus, each Fc gamma RI entering the pool of activated receptors probably makes a unitary contribution to the signal. An additional finding showed that cross-linked Fc gamma RI became associated with the cell cytoskeleton and that this association was also transient. Dissociation of Fc gamma RI from its cytoskeletal attachment occurred well after cessation of O2- production.     

A novel nuclear receptor superfamily member in Xenopus that associates with RXR, and shares extensive sequence similarity to the mammalian vitamin D3 receptor.

We report the isolation of xONR1, a novel member of the nuclear receptor superfamily from Xenopus laevis. xONR1 shares a high degree of amino acid sequence identity with the mammalian receptor for 1 alpha, 25-dihydroxy vitamin D3, particularly within the DNA-binding domain, although it does not bind this ligand. xONR1 DNA binding is stimulated by association with retinoid X receptor gamma (RXR gamma).     

New insights into receptor ligand binding domains from a novel assembly assay

Previous studies have demonstrated that hormone binding stabilizes the ligand binding domain (LBD) of the nuclear hormone receptors against proteolysis. We have confirmed and extended this observation using a newly developed assembly assay. In this assay, the LBD is divided into two parts, of which one includes the first helix of this domain and the other corresponds to the remainder of the LBD. Several independent criteria demonstrate that these two fragments can assemble into a functional LBD in the presence of a ligand, but not in its absence, and that this is a reflection of the stabilizing effect of ligand. We have also used this assay to demonstrate that binding of the nuclear receptor corepressor NCoR can directly stabilize the LBD. Overall, these results highlight the dynamic nature of the LBD and suggest that current models for activation based solely on allosteric effects on the C-terminal helix may be too limited.     

Ligand-independent homomeric and heteromeric complexes between interleukin-2 or -9 receptor subunits and the gamma chain

Signaling via interleukin-2 (IL-2) and interleukin-9 receptors (IL-2R and IL-9R) involves heteromeric interactions between specific interleukin receptor subunits, which bind Janus kinase 1 (JAK1) and the JAK3 binding common gamma chain (gamma c). The potential existence and roles of homomeric and heteromeric complexes before ligand binding and their modulation by ligand and JAK3 are unclear. Using computerized antibody-mediated immunofluorescence co-patching of epitope-tagged receptors at the surface of live cells, we demonstrate that IL-2Rbeta, IL-9Ralpha, and gamma c each display a significant fraction of ligand-independent homomeric complexes (24-28% co-patching), whereas control co-patching levels with unrelated receptors are very low (7%). Heteromeric complex formation of IL2-Rbeta or IL-9Ralpha with gamma c is also observed in the absence of ligand (15-30%). Ligand binding increases this hetero-oligomerization 2-fold but does not affect homo-oligomerization. Co-expression of IL-2Ralpha does not affect the hetero-oligomerization of IL-2Rbeta and gamma c. Recruitment of gamma c into heterocomplexes is partly at the expense of its homo-oligomerization, suggesting that a functional role of the latter may be to keep the receptors inactive in the absence of ligand. At the same time, the preformed complexes between gamma c and IL-2Rbeta or IL-9Ralpha promote signaling by the JAK3 A572V mutant without ligand, supporting a pathophysiological role for the constitutive oligomerization in triggering ligand-independent activation of JAK3 (and perhaps other JAK mutants) mutants identified in several human cancers.     

Activation of nuclear receptors: a perspective from structural genomics

Crystal structures of more than two dozen different nuclear receptor ligand binding domains have defined a simple paradigm of receptor activation, in which agonist binding induces the activation function-2 (AF-2) helix to form a charge clamp for coactivator recruitment. Recent structural studies present a surprising contrast. Activation of the mouse LRH-1 receptor is independent of a bound agonist despite its large ligand binding pocket, whereas the activation of the Drosophila DHR38 receptor is dependent on ecdysteroids even though the receptor lacks a ligand binding pocket. These new findings shed light on the diverse structural mechanisms that nuclear receptors have evolved for activation, and have important implications in their respective signaling pathways.