The phantom ligand effect: allosteric control of transcription by the retinoid X receptor

OBJECTIVE: This two-part study investigated the cognitive processing of food- and threat-related information in a non-eating-disordered population, particularly exploring the mechanisms that might explain the process of cognitive avoidance among women with bulimic attitudes. METHOD: In the first study, 30 female students solved anagrams of neutral, food, and threat words. In the second study, 50 male and female students solved anagrams of words reflecting physical threat, self-directed ego threat, and ego threat directed from others. RESULTS: In this task, there was no association between slower processing of threats and eating characteristics, but there were associations with Eating Disorders Inventory (EDI) scales that reflect "ego development." DISCUSSION: The most reliable evidence of cognitive avoidance was among those participants who show the ego development characteristics that have been identified as important in the development and etiology of eating psychopathology. The implications for models of eating psychopathology and their treatment are discussed.     

High level transactivation by a modified Bombyx ecdysone receptor in mammalian cells without exogenous retinoid X receptor

Our studies of the Bombyx mori ecdysone receptor (BE) revealed that, unlike the Drosophila melanogaster ecdysone receptor (DE), treatment of BE with the ecdysone agonist tebufenozide stimulated high level transactivation in mammalian cells without adding an exogenous heterodimer partner. Gel mobility shift and transfection assays with both the ultraspiracle gene product (Usp) and retinoid X receptor heterodimer partners indicated that this property of BE stems from significantly augmented heterodimer complex formation and concomitant DNA binding. We have mapped this "gain of function" to determinants within the D and E domains of BE and demonstrated that, although the D domain determinant is sufficient for high affinity heterodimerization with Usp, both determinants are necessary for high affinity interaction with retinoid X receptor. Modified BE receptors alone used as replication-defective retroviruses potently stimulated separate "reporter" viruses in all cell types examined, suggesting that BE has potentially broad utility in the modulation of transgene expression in mammalian cells.     

Requirements for repression of retinoid X receptor by the oncoprotein P75gag-v-erbA and the thyroid hormone receptors

Three major ionic currents, Ca2+-dependent K+ current (IK-Ca), delayed rectifier type K+ current (Ikd) and Ca2+ current (ICa), were activated by depolarization under whole-cell clamp in single smooth muscle cells isolated from guinea-pig urinary bladder. Externally applied ruthenium red (RuR) reduced the amplitude of IK-Ca and ICa at 0 mV (IC50 values were 4.2 and 5.6 muM, respectively), but did not affect IKd. Spontaneous transient outward currents (STOCs) and caffeine-induced outward currents (Icaf) at -30 mV were reduced by external 10 muM RuR. When 10 muM RuR was added to the pipette solution, IK-Ca during depolarization, STOCs and Icaf significantly decreased with time. RuR did not change the unitary current amplitude of the large-conductance Ca2+-dependent K+ (BK) channels, but reduced the open probability of the channel under excised patch-clamp recording mode. RuR reduced the channel activity more effectively from the cytosolic face than from the other. This inhibition decreased when the cytosolic Ca2+ concentration was increased. These results indicate that RuR blocks BK and Ca2+ channels in urinary bladder smooth muscle cells. The decrease in IK-Ca, STOCs and Icaf by RuR is attributable to the direct inhibition of BK channel activity, probably in addition to the inhibition of Ca2+ release from storage sites. The direct inhibition of BK channel activity by RuR may be related to the interaction of RuR with the Ca2+-binding sites of the channel protein.     

Negative feedback control of the retinoid-retinoic acid/retinoid X receptor pathway by the human TR4 orphan receptor, a member of the steroid receptor superfamily

Amino acid sequence analysis indicates that the human TR4 orphan receptor (TR4) is a member of the estrogen/thyroid receptor subfamily of the steroid/thyroid receptor superfamily and recognizes the AGGTCA direct repeat (DR) of the hormone response element. Here we demonstrate using the electrophoretic mobility shift assay that TR4 binds specifically to DR with a spacing of 1 and 5 base pairs (DR1 and DR5), which are the response elements for retinoic acid receptor (RAR) and retinoid X receptor (RXR), respectively. A reporter gene assay using chloramphenicol acetyltransferase demonstrated that TR4 repressed RA-induced transactivation in a TR4 dose-dependent manner. Inhibition of the retinoid signal pathway also occurs through natural response elements found in CRBPII and RARbeta genes. Our data suggest that the mechanism of repression may not involve the formation of functionally inactive heterodimers between TR4 and RAR or RXR. Instead, we show that TR4 may compete for hormone response elements with RAR and RXR due to its higher binding affinity. Furthermore, treatment of F9 murine teratocarcinoma (F9) cells with 10(-6) M all-trans-retinoic acid increased TR4 mRNA levels, and this change was accompanied by an increased amount of endogenous TR4 protein that can bind to RXRE in electrophoretic mobility shift assay. Our data therefore strongly suggest that the retinoid signal pathway can be regulated by TR4 in a negative feedback control mechanism, which may restrict retinoic acid signaling to certain elements in a cell-specific fashion.     

Retinoic acid receptor/retinoid X receptor heterodimers can be activated through both subunits providing a basis for synergistic transactivation and cellular differentiation

The receptor for 9-cis-retinoic acid, retinoid X receptor (RXR), forms heterodimers with several nuclear receptors, including the receptor for all-trans-retinoic acid, RAR. Previous studies have shown that retinoic acid receptor can be activated in RAR/RXR heterodimers, whereas RXR is believed to be a silent co-factor. In this report we show that efficient growth arrest and differentiation of the human monocytic cell line U-937 require activation of both RAR and RXR. Also, we demonstrate that the allosteric inhibition of RXR is not obligatory and that RXR can be activated in the RAR/RXR heterodimer in the presence of RAR ligands. Remarkably, RXR inhibition by RAR can also be relieved by an RAR antagonist. Moreover, the dose response of RXR agonists differ between RXR homodimers and RAR/RXR heterodimers, indicating that these complexes are pharmacologically distinct. Finally, the AF2 activation domain of both subunits contribute to activation even if only one of the receptors is associated with ligand. Our data emphasize the importance of signaling through both subunits of a heterodimer in the physiological response to retinoids and show that the activity of RXR is dependent on both the identity and the ligand binding state of its partner.     

Analysis of membrane protein self-association in lipid systems by fluorescence particle counting: application to the dihydropyridine receptor

Fluorescence particle counting (FPC) is employed to analyze the distribution of a purified membrane protein, the dihydropyridine receptor (DHP-R), in detergent micelles, in lipid vesicles, and in lipid monolayers generated from the vesicles. The method was used to identify conditions for which DHP-Rs occur singly distributed in micelles and in vesicles. In monolayers, the DHP-R showed self-association, starting from monomeric distribution at concentrations (c) of typically 10 DHP-R/microm2. The average cluster size [m(t)] of associates was followed by FPC in time and the dependence of the lateral diffusion constant [D(lat)(m,pi)] of the associates on the surface pressure (pi) was determined. By studying the dependence of m(t) on c, pi, D(lat)(pi), and salt concentration (c(s)), we derived an empirical expression for the association rate constant (k(a)) and for m(t) that fits the experimental m(t) relations. Theoretical justification for these dependencies is obtained from collision theory, leading to a mechanistic picture of the aggregation process. DHP-R association is irreversible. Its rate is not diffusion-limited. A large number of collisions is required to overcome an interaction energy barrier of about 6-11 kT, depending on m and c(s) but not on pi. The increase in association rate with increasing average cluster size m is related to increasing van der Waals attraction, while the increase in rate with increasing c(s) relates to decreasing electrostatic repulsion. Van der Waals and electrostatic forces represent, however, only part of the interaction energy. The main contribution was not dependent on the variables studied and, most likely, reflects hydration forces which need to be overcome for association.     

Discriminative stimulus properties of the atypical neuroleptic clozapine in rats: tests with subtype selective receptor ligands

The interoceptive stimulus induced by clozapine (5 mg/kg, i.p.) has been characterized in an operant drug discrimination procedure in the rat using a wide range of receptor subtype-selective agonists and antagonists. Only the muscarinic receptor antagonist scopolamine generalized fully to clozapine (>80%). Partial generalization (defined here as 40% maximal generalization) was seen with the D1 receptor antagonist SCH 23390 (43% maximal generalization), the alpha1-adrenoceptor antagonist prazosin (67%) and the alpha2-adrenoceptor antagonist methoxyidazoxan (42%). All other specific agents tested induced <25% maximal generalization, including the alpha2-adrenoceptor antagonist yohimbine (24%), the histamine H1 receptor antagonist mepyramine (21%), the D2 antagonist typical neuroleptic haloperidol (23%), the D4 receptor antagonist L-745,870 (14%), the 5-hydroxytryptamine-1A (5-HT1A) receptor agonist S-14506 (8%), the 5-HT2A receptor antagonists ketanserin (0%) and M100907 (12%), the 5-HT2B/2C receptor antagonists SB 200646A (8%) and SDZ SER 082 (6%), and the 5-HT3 receptor antagonist ondansetron (0%). The clozapine discriminative stimulus was not blocked by the dopamine D1 receptor antagonist SCH 23390, or by the 5-HT1A receptor antagonist WAY 100635, when given concomitantly with clozapine. Although the results suggest that muscarinic antagonism plays a major role in the clozapine cue, the results have to be considered in the light of the full generalization to clozapine seen with various antipsychotic agents which have very low affinity for muscarinic receptors, including zotepine, quetiapine, JL13 and PNU 96415 (a finding replicated in rats from the same breeding colony as those which generalized to scopolamine). Thus, generalization to clozapine for antipsychotics with multiple affinities but with low muscarinic affinity is probably mediated by additive or perhaps supra-additive actions at other receptors, although extensive studies with various combinations of drug mixtures are required to validate this hypothesis.     

G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins

G-protein-coupled receptors (GPCRs) represent a large family of proteins that transduce extracellular signals to the interior of cells. Signalling through these receptors rapidly desensitized primarily as the consequence of receptor phosphorylation, but receptor sequestration and downregulation can also contribute to this process. Two families of serine/threonine kinases, second messenger dependent protein kinases and receptor-specific G-protein-coupled receptor kinases (GRKs), phosphorylate GPCRs and thereby contribute to receptor desensitization. Receptor-specific phosphorylation of GPCRs promotes the binding of cytosolic proteins referred to as arrestins, which function to further uncouple GPCRs from their heterotrimeric G-proteins. To date, the GRK protein family consists of six members, which can be further classified into subgroups according to sequence homology and functional similarities. The arrestin protein family also comprises six members, which are subgrouped on the basis of sequence homology and tissue distribution. While the molecular mechanisms contributing to GPCR desensitization are fairly well characterized, little is known about the mechanism(s) by which GPCR responsiveness is reestablished, other than that receptor sequestration (internalization) might be involved. The goal of the present review is to overview current understanding of the regulation of GPCR responsiveness. In particular, we will review new evidence suggesting a pleiotropic role for GRKs and arrestins in the regulation of GPCR responsiveness. GRK-mediated phosphorylation and arrestin binding are not only involved in the functional uncoupling of GPCRs but they are also intimately involved in promoting GPCR sequestration and as such likely play an important role in mediating the subsequent resensitization of GPCRs.