Interaction of glucocorticoid analogues with the human glucocorticoid receptor

Transient co-transfection of receptor cDNA and suitable reporter genes was used to study human glucocorticoid receptor (hGR) function in a neutral mammalian cell background. A variety of natural and synthetic steroids were analyzed for their ability to activate gene expression through the hGR and to bind to extracts of cells expressing the hGR cDNA. There was very good correlation between these two in vitro parameters for these compounds. Furthermore, correlation of these data with reported in vivo anti-inflammatory potencies was surprisingly close, with two exceptions. The in vitro data suggest an explanation for the discrepant compounds, consistent with published data on their metabolic fate in vivo. The co-transfection assay has utility as a quantitative predictor of in vivo glucocorticoid pharmacology.     

Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor

The H(4)R (histamine H(4) receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H(4)R is primarily expressed in eosinophils and mast cells and has the highest homology with the H(3)R. The occurrence of at least twenty different hH(3)R (human H(3)R) isoforms led us to investigate the possible existence of H(4)R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H(4)R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H(4)R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H(4)R-ligand induced signalling or constitutive activity for these H(4)R splice variants. However, when co-expressed with full-length H(4)R [H(4)R((390)) (H(4)R isoform of 390 amino acids)], the H(4)R splice variants have a dominant negative effect on the surface expression of H(4)R((390)). We detected H(4)R((390))-H(4)R splice variant hetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H(4)R splice variants were detected in various cell types and expressed at similar levels to the full-length H(4)R((390)) mRNA in, for example, pre-monocytes. We conclude that the H(4)R splice variants described here have a dominant negative effect on H(4)R((390)) functionality, as they are able to retain H(4)R((390)) intracellularly and inactivate a population of H(4)R((390)), presumably via hetero-oligomerization.     

Cell type-dependent modulation of the dominant negative action of human mutant thyroid hormone beta 1 receptors.

BACKGROUND: Mutations in the ligand-binding domain of the thyroid hormone receptor beta (TR beta) gene cause the syndrome of resistance to thyroid hormone (RTH). The clinical phenotype results from the antagonism of the normal TR alpha and the non-mutated TR beta alleles by the TR beta 1 mutants, via a dominant negative effect. There is, however, marked heterogeneity of organ resistance within and among kindreds with RTH. This study examines the potential role of cell type in modulating the dominant negative potency of human TR beta 1 (h-TR beta 1) mutants. MATERIALS AND METHODS: Transient transfections were performed in HeLa and NIH3T3 cells, using a wild type (WT) and three naturally occurring mutant h-TR beta 1 constructs, and three natural thyroid hormone response elements (TREs). Immunocytochemistry was performed to detect levels of TR beta 1 expression in these two cell types. In order to determine how TR beta 1 interacts with other cellular partners, gel-shift analyses using HeLa and NIH3T3 nuclear extracts were performed. RESULTS: Transfection studies using WT h-TR beta 1 in HeLa and NIH3T3 cells, showed that the 3,3',5-triiodothyronine (T3)-induced transactivation of the different TREs varied between cell types. Unlike the non-T3-binding h-TR beta 1 mutant, PV, mutants ED and OK displayed the expected T3-induced dose responsiveness in these two cell types. For each TRE examined, the magnitude of the dominant negative effect varied between the cell types. The levels of receptor expression in HeLa and NIH3T3 cells were identical, as determined by immunocytochemistry. Gel-shift analyses showed differences in the formation of hetero- and homodimers depending on both the cell type and TRE motif. CONCLUSIONS: The cell type in which a mutant receptor operates affects the relative amounts of hetero- and homodimers. Together with the nature of the mutation and the TRE-motif, this could modulate the dominant negative action of mutant receptors in different tissues, which, in turn, could contribute to the variable phenotypic characteristics of RTH.     

Marked potentiation of the dominant negative action of a mutant thyroid hormone receptor beta in mice by the ablation of one wild-type beta allele

Mutations in the thyroid hormone receptor (TR) beta gene result in resistance to thyroid hormone (RTH), characterized by reduced sensitivity of tissues to thyroid hormone. To understand which physiological TR pathways are affected by mutant receptors, we crossed mice with a dominantly negative TRbeta mutation (TRbetaPV) with mice carrying a TRbeta null mutation (TRbeta(-/-)) to determine the consequences of the TRbetaPV mutation in the absence of wild-type TRbeta. TRbeta(PV/-) mice are distinct from TRbeta(+/-) mice that did not show abnormalities in thyroid function tests. TRbeta(PV/-) mice are also distinct from TRbeta(PV/+) and TRbeta(-/-) mice in that the latter shows mild dysfunction in the pituitary-thyroid axis, whereas the former exhibit very severe abnormalities, including extensive papillary hyperplasia of the thyroid epithelium, indistinguishable from that observed in TRbeta(PV/PV) mice. Similar to TRbeta(PV/PV) mice, TRbeta(PV/-) mice exhibited impairment in weight gain. Moreover, the abnormal regulation patterns of T3-target genes in the tissues of TRbeta(PV/-) and TRbeta(PV/PV) mice were strikingly similar. Using TR isoforms and PV-specific antibodies in gel shift assays, we found that in vivo, PV competed with TRalpha1 for binding to thyroid hormone response elements in TRbeta(PV/-) mice as effectively as in TRbeta(PV/PV) mice. Thus, the actions of mutant TRbeta are markedly potentiated by the ablation of the second TRbeta allele, suggesting that interference with wild-type TRalpha1-mediated gene regulation by mutant TRbeta leads to severe RTH.     

糖皮质激素受体的结构和功能

近年来已克隆出人、大鼠和小鼠糖皮质激素受体的cDNA,并利用定点突变等技术分析了受体结构和功能的关系。还利用小鼠乳腺瘤病毒、人金属硫蛋白II_A基因等详细研究了受体和DNA的相互作用,对糖皮质激素的诱导蛋白也进行了深入研究。     

Structure/function of the human glucocorticoid receptor: tyrosine 735 is important for transactivation.

Ligand-induced activation of the glucocorticoid receptor (GR) is not well understood. The GR ligand-binding domain was modeled, based on homology with the progesterone receptor. Tyrosine 735 interacts with the D ring of dexamethasone, and substitution of D ring functional groups results in partial agonist steroids with reduced ability to direct transactivation. Loss of the Tyr735 hydroxyl group by substitution to phenylalanine (Tyr735Phe) did not reduce ligand binding affinity [dissociation constant (Kd) 4.3 nM compared with Kd 4.6 nM for wild-type] and did not alter transrepression of an nuclear factor-kappaB (NF-kappaB reporter. But, there was a significant 30% reduction in maximal transactivation of a mouse mammary tumor virus (MMTV) reporter, although with an unchanged EC50 (8.6 nM compared with 6 nM). Substitution to a nonaromatic hydrophobic amino acid, valine (Tyr735Val), retained high-affinity ligand binding for dexamethasone (Kd 6 nM compared with 4.6 nM) and did not alter transrepression of NF-kappaB. However, there was a 36% reduction in MMTV activity with a right shift in EC50 (14.8 nM). The change to serine, a small polar amino acid (Tyr735Ser), caused significantly lower affinity for dexamethasone (10.4 nM). Maximal transrepression of NF-kappaB was unaltered, but the IC50 for this effect was increased. Tyr735Ser had a major shift in EC50 (118 nM) for transactivation of an MMTV reporter. Maximal transactivation of MMTV induced by the natural ligand cortisol was reduced to 60% by Tyr735Phe and Tyr735Val and was completely absent by Tyr735Ser. These data suggest that tyrosine 735 is important for ligand interpretation and transactivation.     

Truncated TrkB: beyond a dominant negative receptor

BDNF activates trkB receptors to regulate neuronal survival, differentiation, and proliferation. Mutations in the BDNF gene, altered BDNF expression, and altered trkB expression are associated with degenerative and psychiatric disorders. The full-length trkB receptor (trkB.tk(+)) undergoes autophosphorylation to activate intracellular signaling pathways. The truncated trkB receptor (trkB.t1) is abundantly expressed in the brain but lacks the catalytic tyrosine kinase domain. TrkB.t1 is a dominant-negative receptor that inhibits trkB.tk(+) signaling. While this is an important function of trkB.t1, it is only one of its many functions. TrkB.t1 sequesters and translocate BDNF, induces filopodia and neurite outgrowth, stimulates intracellular signaling cascades, regulates Rho GTPase signaling, and modifies cytoskeletal structures. TrkB.t1 is an active signaling molecule with regulatory effects on neurons and astrocytes.     

Modulators of the glucocorticoid receptor also regulate mineralocorticoid receptor function.

Modulators are proposed to be novel ether aminophosphoglycerides that stabilize unoccupied and occupied glucocorticoid receptor steroid binding and inhibit glucocorticoid receptor complex activation. Two isoforms, modulator 1 and modulator 2, have been purified from rat liver cytosol [Bodine, P.V., & Litwack, G. (1990) J. Biol. Chem. 265, 9544-9554]. Since the mineralocorticoid receptor is relatively resistant to activation, modulator's effect on rat distal colon mineralocorticoid receptor function was examined. Warming of unoccupied receptor decreased residual specific [3H]aldosterone binding by 86 +/- 2%. Both modulator isoforms completely prevented this destabilization with Km's of 2 +/- 1 microM modulator 1 and 24 +/- 5 microM modulator 2. Warming of occupied mineralocorticoid receptors decreased [3H]aldosterone binding by 56 +/- 3%. Modulator only partially stabilized occupied receptor binding with Km's of 10 +/- 2 microM modulator 1 and 68 +/- 8 microM modulator 2. Modulator inhibited receptor activation with Km's of 3 +/- 1 microM modulator 1 and 33 +/- 10 microM modulator 2. Double-reciprocal analysis showed linear kinetics, and mixing modulator isoforms together had additive effects on unoccupied and occupied receptor steroid binding stabilization and activation inhibition. Colon cytosol contained a low molecular weight, heat-stable factor(s) which inhibited receptor activation and stabilized occupied receptor steroid binding. Molybdate completely stabilized unoccupied mineralocorticoid receptor steroid binding and inhibited activation with half-maximal effects at 3-4 mM but only stabilized occupied receptor binding by approximately 40%. These data indicate that (i) apparent physiologic concentrations of modulator stabilize mineralocorticoid receptor steroid binding and inhibit receptor activation, (ii) an aldosterone-responsive tissue contains a modulator-like activity, and (iii) molybdate mimics the effects of modulator.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular evolution and modern human origins

While molecular evolutionists may be fascinated by the features and history of a particular gene or DNA segment, evolutionary anthropologists are often more interested in the activities and history of groups of people. We may want to know, for instance, when and where humans have migrated, how much exchange between groups has taken place, and how population sizes have changed. Population genetic theory provides the hope that through analyses of genetic data we will gain insight into the history of populations. Genetic data from extant human populations are now accruing at a remarkable rate. We might, therefore, expect to have answers in hand. There remains, however, a wide gap between the available theory and data; too often we fail to draw firm conclusions because our interpretation of analytic results requires that we make myriad assumptions about our data. In any one instance, these assumptions might include estimates of mutation rate, mutational mechanism, population sizes, the role that natural selection has played, and the rate of migration among groups. Often these assumptions are implicit, invisible to most. How, then, are we to make any progress? © 1998 Wiley-Liss, Inc.     

Cloning of the human glucocorticoid receptor cDNA.

We show that the human glucocorticoid receptor (GR), isolated from the breast cancer cell line MCF-7, has an apparent molecular weight identical to that of rat liver GR (94 kDa) and reacts with antibodies raised against the latter. These antibodies were used to clone cDNA sequences corresponding to the human GR from a lambda gt11 expression library constructed using MCF-7 poly(A)+ RNA. Three non-homologous cDNA clones with inserts of 125, 220 and 350 bp, which express epitopes recognised by the rat liver GR antibodies, were isolated. Rat liver GR antibodies, immunopurified using the immobilised purified beta-galactosidase fusion proteins, detect partially purified rat liver and human GRs on Western blots. In addition, these antibodies immuno-adsorb rat liver and human GRs affinity-labelled with [3H] triamcinolone acetonide. Northern blot analysis, using all three cDNA probes, reveals the presence of a major MCF-7 poly(A)+ RNA species of approximately 7 kb.