Mutations in retinoid X receptor that impair heterodimerization with specific nuclear hormone receptor

Retinoid X receptor (RXR) serves as a promiscuous heterodimerization partner for many nuclear receptors through the identity box, a 40-amino acid subregion within the ligand binding domain. In this study, we randomly mutated two specific residues within the human RXRalpha identity box region previously identified as important determinants in heterodimerization (i.e. Ala(416) and Arg(421)). Interestingly, most of these mutants still retained wild type interactions with thyroid hormone receptor (TR), retinoic acid receptor, peroxisome proliferator-activated receptor alpha, small heterodimer partner, and constitutive androstane receptor. However, RXR-A416D and R421L were specifically impaired for interactions with TR, whereas RXR-A416K lost both TR and retinoic acid receptor interactions. Accordingly, RXR-A416D did not support T3 transactivation in mammalian cells, whereas RXR-A416K was not supportive of transactivation by retinoids or T3. These results provide a basis upon which to further design mutant RXRs highly selective in heterodimerization, potentially useful tools to probe nuclear receptor function in vivo.     

Impaired transactivation of the glucocorticoid receptor cloned from the Guyanese squirrel monkey

Squirrel monkeys are among a diverse group of New World primates that demonstrate unusually high levels of circulating corticosteroids and glucocorticoid receptor (GR) insensitivity. Recent evidence suggests that overexpression of an immunophilin impairs dexamethasone binding to GR in the Bolivian squirrel monkey (Saimiri boliviensis). Here we describe the cloning, expression, and functional characterization of GR from the closely related Guyanese squirrel monkey (S. sciureus). The cloned Guyanese squirrel monkey GR (gsmGR) cDNA closely resembles human GR (hGR) cDNA, and yields a high affinity dexamethasone binding receptor when expressed in COS-1 cells. Transactivation analysis of hGR and gsmGR expressed in CV-1 cells and cultured squirrel monkey kidney (SMK) cells indicates that: (1) SMK cells elaborate a functional high activity GR from human GR cDNA; (2) gsmGR is an order of magnitude less efficient than hGR at transactivation in CV-1 and SMK cells; and (3) maximal transactivation by gsmGR is attenuated in both cell lines. Glucocorticoid resistance in S. sciureus is at least partly attributable to a naturally occurring mutation in the GR gene that results in impaired GR transactivation.     

Melatonin prevents glucocorticoid inhibition of cell proliferation and toxicity in hippocampal cells by reducing glucocorticoid receptor nuclear translocation

Glucocorticoids are the main product of the adrenal cortex and participate in multiple cell functions as immunosupressors and modulators of neural function. Within the brain, glucocorticoid activity is mediated by high-affinity mineralocorticoid and low-affinity glucocorticoid receptors. Among brain cells, hippocampal cells are rich in glucocorticoid receptors where they regulate excitability and morphology. Also, elevated glucocorticoid levels suppress hippocampal neurogenesis in adults. The pineal neuroindole, melatonin, reduces the affinity of glucocorticoid receptors in rat brain and prevents glucocorticoid-induced apoptosis. Here, the ability of melatonin to prevent glucocorticoid-induced cell death in hippocampal HT22 cells was investigated in the presence of neurotoxins. Results showed that glucocorticoids reduce cellular growth and also enhance sensitivity to neurotoxins. We found a G(1) cell cycle arrest mediated by an increase of cyclin/cyclin-dependent kinase inhibitor p21(WAF1/CIP1) protein after dexamethasone treatment and incremental change in amyloid beta protein and glutamate toxicity. Melatonin prevents glucocorticoids inhibition of cell proliferation and reduces the toxicity caused by glucocorticoids when cells were treated with dexamethasone in combination with neurotoxins. Although, melatonin does not reduce glucocorticoid receptor mRNA or protein levels, it decreases receptor translocation to nuclei in these cells.     

Craniorachischisis in a squirrel monkey

A stillborn squirrel monkey had craniorachischisis. The calvarium was absent and the skin and vertebral neural arches were cleft from the foramen magnum to the midlumbar region. Cranial nerves and degenerating neural tissue were attached to the cranial base and there were remnants of spinal cord in skin-covered areas of the spinal canal. The adrenal glands were normal. This is the only reported case of craniorachischisis in a monkey fetus.     

Effects of theophylline on the activation and nuclear translocation of the hepatic glucocorticoid receptor at low temperature.

$\text{In vitro}$ nuclear binding of the (³H) dexamethasone-receptor complex from rat liver cytosol takes place at a slow rate when nuclei are incubated at low temperature. However, if theophylline is included during the incubation there is a threefold increase in the rate of nuclear binding. The activation by theophylline is independent of its known effect on cyclic AMP levels since the cyclic nucleotide has no effect on nuclear binding either in the absence or presence of theophylline. Activation ability is specific to methylxanthines and not to nucleoside derivatives. Theophylline may be acting directly on the (³H) dexamethasone-receptor complex converting it to an active form.     

A high-content glucocorticoid receptor translocation assay for compound mechanism-of-action evaluation

Ligand-induced cytoplasm to nucleus translocation is a critical event in the nuclear receptor (NR) signal transduction cascade. The development of green fluorescent proteins and their color variants fused with NRs, along with the recent developments in automated cellular imaging technologies, has provided unique tools to monitor and quantify the NR translocation events. These technology developments have important implications in the mechanistic evaluation of NR signaling and provide a powerful tool for drug discovery. The unique challenges for developing a robust NR translocation assay include cytotoxicity accompanied with chronic overexpression of NRs, basal translocation induced by serum present in culture medium, and interference from endogenous NRs, as well as subcellular dynamics. The authors have developed a robust assay system for the glucocorticoid receptor (GR) that was applied to a panel of nuclear receptor ligands. Using a high-content imaging system, ligand-induced, dose-dependent GR nuclear translocation was quantified and a correlation with other conventional assays established.     

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.     

Orthocephalization in the postweaning squirrel monkey

Twenty male squirrel monkeys (Saimiri sciureus boliviensis) raised in captivity were allotted to one of the following groups: weanling control (C6) sampled at 6 months of age; young control (C24) fed ad libitum on a control diet and killed at 24 months of age; and malnourished (M24) fed ad libitum on a low-protein diet and sampled at 24 months of age. Cranial points and the lateral semicircular canals were marked. On each skull, a strict lateral teleradiograph was taken, and the lengths of the midsagittal chords and their angles with respect to the vestibular line were measured. Age changed the lengths in about 70% of the chords and more than 50% of the angles. Malnutrition arrested about 50% of the lengths, but the angles were practically not affected. It is concluded that the postweaning Saimiri sciureus undergoes orthocephalization according to a general pattern already observed in rodents and suggested for pongids. Postweaning malnutrition affected growth in size but not shape changes related to the orthocephalization of the Saimiri skull. © 1996 Wiley-Liss, Inc.     

Visual sensitivity in the squirrel monkey

Several indices of visual sensitivity have been obtained from behavioral experiments conducted on the squirrel monkey (Saimiri sciureus). In this species, the photopic spectral sensitivity functions determined by increment-threshold and flicker discrimination procedures are substantially different; the involvement of two different neural processes in the two tasks is suggested. When tested similarly, the thresholds for rod and cone-based vision are not substantially different for squirrel monkeys and humans; however, above cone threshold, for a 500 nm test light, increment threshold is some 0.3 to 0.4 log₁₀ units higher for the squirrel monkey. Rod saturation has also been demonstrated to occur in the squirrel monkey.     

Isolation and characterization of rat liver nuclear glucocorticoid receptor

The rat liver nuclear glucocorticoid receptor has a molecular weight of 90 000. Using antibody bound to the stationary matrix, the cytosol and nuclear glucocorticoid receptors from rat liver were purified. The translocation of glucocorticoid receptor from rat liver cytosol into the nucleus was studied using immunoaffinity chromatography. Immediately after the intraperitoneal injection of rats with the hormone, the receptor translocation started and was complete within 10 min. The 90 000 dalton nuclear receptor component is identical to the 90 000 dalton cytosol component. They have identical molecular weights in the same gel electrophoresis system and produce identical peptide fragments after digestion with Staphyolococcal aureus V8 protease. The receptor component enriched by immunoaffinity chromatography from cytosol of adrenalectomised rats contained mainly a 45 000 dalton component.     

Mutations in Hydin impair ciliary motility in mice

Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility, and mice with Hydin defects develop lethal hydrocephalus. To determine if defects in Hydin cause hydrocephalus through a mechanism involving cilia, we compared the morphology, ultrastructure, and activity of cilia in wild-type and hydin mutant mice strains. The length and density of cilia in the brains of mutant animals is normal. The ciliary axoneme is normal with respect to the 9 + 2 microtubules, dynein arms, and radial spokes but one of the two central microtubules lacks a specific projection. The hydin mutant cilia are unable to bend normally, ciliary beat frequency is reduced, and the cilia tend to stall. As a result, these cilia are incapable of generating fluid flow. Similar defects are observed for cilia in trachea. We conclude that hydrocephalus in hydin mutants is caused by a central pair defect impairing ciliary motility and fluid transport in the brain.     

Microwaves modify thermoregulatory behavior in squirrel monkey

Squirrel monkeys (Saimiri sciureus) trained to regulate environmental temperature (T_a) behaviorally were exposed in the far field of a horn antenna to ten-minute periods of 2,450 MH_z CW microwaves. Incident power density ranged from 1 to 22 mW/cm². The corresponding specific absorption rate (SAR), derived from temperature increments in saline-filled styrofoam models, ranged from 0.15 to 3.25 W/kg. Controls included exposure to infrared radiation of equivalent incident energy and no radiation exposure. Normal thermoregulatory behavior produces tight control over environmental and body temperatures; most monkeys select a T_a of 34–36°C. Ten-minute exposures to 2,450 MH_z CW microwaves at an incident power density of 6–8 mW/cm² stimulated all animals to select a lower T_a. This threshold energy represents a whole-body SAR of 1.1 W/kg, about 20% of the resting metabolic rate of the monkey. Thermoregulatory behavior was highly efficient, and skin and rectal temperatures remained stable, even at 22 mW/cm² where the preferred T_a was lowered by as much as 4°C. No comparable reduction in selected T_a below control levels occurred during exposure to infrared radiation of equal incident power density.     

Auditory frequency discrimination in the squirrel monkey

Four squirrel monkeys (Saimiri sciureus) were tested for their frequency discrimination capacity using an eyeblink classical conditioning procedure, with air puff against the eye as unconditioned stimulus and 600-ms pure tones as conditioned stimuli. Absolute frequency difference thresholds showed a minimum (20-41 Hz, mean 30 Hz) at 4,000-8,000 Hz and increased towards higher as well as lower frequencies (70-90 Hz, mean 80 Hz at 300 Hz; 44-120 Hz, mean 82 Hz at 16,000 Hz). Relative frequency difference thresholds increased from higher to lower frequencies, with values as low as 0.3-0.8% (mean 0.5%) at 16,000 Hz and as large as 24-30% (mean 27%) at 300 Hz. The squirrel monkey's frequency discrimination function thus shows a severe deviation from Weber's law. The frequency difference thresholds are comparable to human's in the 4,000-8,000 Hz range, but are 65-80 times higher in the 500- to 300-Hz range. Individuals with high auditory thresholds do not necessarily also have high frequency difference thresholds.