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Twofold sila-substitution (C/Si exchange) in the saturated ring of the tetrahydronaphthalene skeleton of the RXR-selective retinoid agonist bexarotene (1a) leads to disila-bexarotene (1b). Compound 1b was synthesized in a multistep synthesis, starting from 1,2-bis(chlorodimethylsilyl)ethane. The identity of 1b was established by elemental analyses and multinuclear NMR studies, and the C/Si analogues 1a and 1b (and an intermediate in the synthesis of 1b) were structurally characterized by single-crystal X-ray diffraction. Furthermore, 1a and 1b were studied for their interaction with retinoid X receptors. Although the twofold sila-substitution of 1a resulted in significant differences in the molecular structures of 1a and 1b, disila-bexarotene (1b) was shown to be a highly potent RXR agonist.

Activation function 2 in the ligand binding domain of nuclear receptors forms a hydrophobic cleft that binds the LXXLL motif of p160 transcriptional coactivators. Here we provide evidence that activation function 2 in the androgen receptor serves as the contact site for the androgen dependent NH(2)- and carboxyl-terminal interaction of the androgen receptor and only weakly interacts with p160 coactivators in an LXXLL-dependent manner. Mutagenesis studies indicate that it is the NH(2)-/carboxyl-terminal interaction that is required by activation function 2 to stabilize helix 12 and slow androgen dissociation critical for androgen receptor activity in vivo. The androgen receptor recruits p160 coactivators through its NH(2)-terminal and DNA binding domains in an LXXLL motif-independent manner. The results suggest a novel function for activation function 2 and a unique mechanism of nuclear receptor transactivation.

Bacterially‐expressed fusion proteins containing the DNA‐(region C) or hormone‐binding (region E) domains of the chicken progesterone receptor (cPR) fused to the C terminus of Escherichia coli beta‐galactosidase were analysed for the specificity of interaction with natural and synthetic hormone‐responsive elements (HREs) and progestins, respectively. The purified fusion protein containing the progestin‐binding domain bound progesterone with an apparent Kd of 1.0‐1.5 nM and was specifically photocross‐linked with the synthetic progestin R5020 in crude bacterial lysates. Labelling of intact bacterial cells with [3H]R5020 revealed that the majority, if not all, of the bacterially produced hormone‐binding domain was active. No differences in the binding to a synthetic palindromic glucocorticoid/progestin‐responsive element (GRE/PRE) were found when the bacterially produced cPR DNA‐binding domain was compared in methylation interference assays with the full‐length chicken progesterone receptor form A expressed in eukaryotic cells. The study of dissociation kinetics, however, revealed differences in the half‐life of the complexes formed between the palindromic GRE/PRE and either the receptor form A or the fusion protein containing the cPR DNA‐binding domain. DNase I protection experiments demonstrated that the bacterially produced region C of the cPR generated specific ‘footprints’ on the mouse mammary tumour virus long terminal repeat (MMTV‐LTR) which were nearly identical to those previously reported for the rat glucocorticoid receptor.

The complete mRNA sequence of the chicken progesterone receptor (cPR) has been determined. Expression of the cloned cDNA both in vivo and in vitro produces a protein that has the same apparent mol. wt on SDS–polyacrylamide gels as the ‘natural’ cPR form B (109 kd) as determined by immunoblotting and photoaffinity labelling. When expressed in HeLa or in Cos‐1 cells the ‘cloned’ cPR displays hormone binding characteristics indistinguishable from the ‘natural’ receptor and, in the presence of progestins, exhibits ‘tight nuclear binding’. A protein corresponding in size to the cPR form A (79 kd) could be detected by expressing in vivo and in vitro an N‐terminally truncated cPR starting at methionine 128. A protein of the same apparent mol. wt results from internal initiation during in vitro translation. In contrast, such a protein was barely detectable after in vivo expression of the cPR cDNA in Cos‐1 cells. These results suggest that form A is generated by an oviduct cell specific process involving either internal initiation of translation and/or proteolysis in the vicinity of methionine‐128. The cPR contains two highly conserved regions C and E, a characteristic of the steroid/thyroid hormone receptor supergene family. By expression of a series of cPR deletion mutants, region E could be defined as the hormone binding domain whereas region C is indispensable for the tight nuclear association of the progestin‐receptor complex. In the presence of progestins, the cloned cPR efficiently trans‐activates transcription from the long terminal repeat region (LTR) of the mouse mammary tumor virus (MMTV). Deletion of the entire N‐terminal region A/B or of the hormone binding domain E results in a 100‐fold reduction of transcriptional activation. No stimulation of transcription can be detected when the C‐terminal deletion extends into region C, indicating that this region is involved in the recognition of the hormone responsive element (HRE) of the MMTV LTR.

In human HL-60 promyelocytic leukemia cells, diazepinylbenzoic acid derivatives can exhibit either antagonistic or synergistic effects on the differentiation-inducing activities of natural or synthetic retinoids, the activity depending largely on the nature of the substituents on the diazepine ring. Thus, a benzolog of the retinoid antagonist LE135 (6), 4-(13H-10,11,12,13-tetrahydro-10,10,13,13,15-pentamethyldinaphtho[2,3-b][1,2-e]diazepin-7-yl)benzoic acid (LE540, 17), exhibits a 1 order of magnitude higher antagonistic potential than the parental LE135 (6). In contrast, 4-[5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyldibenzo[b,e][1,4]diazepin-11-yl]benzoic acid (HX600, 7), a structural isomer of the antagonistic LE135 (6), enhanced HL-60 cell differentiation induced by RAR agonists, such as Am80 (2). This synergistic effect was further increased for a thiazepine, HX630 (29), and an azepine derivative, HX640 (30); both synergized with Am80 (2) more potently than HX600 (7). Notably, the negative and positive effects of the azepine derivatives on retinoidal actions can be related to their RAR-antagonistic and RXR-agonistic properties, respectively, in the context of the RAR−RXR heterodimer.

A large-scale purification of the progesterone receptor from laying hens is described which yields apparently homogeneous form A and form B receptor in denatured form. The purification procedure is based initially on differential DNA affinity chromatography of both form A and B receptors. Under the conditions of preparation and activation described, progesterone receptor form B binds to DNA-cellulose even in the presence of 100 mM salt. This binding cannot be observed after thermal activation. Receptors obtained at 5% purity using conventional chromatographic purification steps were covalently cross-linked with radioactive ligand by photoaffinity labeling and purified to homogeneity using preparative gel electrophoresis systems under denaturing conditions. This material has been successfully used to generate polyclonal antibodies in rabbits. Immunoblots demonstrated a high degree of cross-reaction between anti-A antibodies and progesterone receptor form B, as well as between anti-B antibodies and progesterone receptor form A, using homogeneous as well as 5% pure receptors as probes. Implications of the immunological data and the novel DNA-binding characteristics of form B are discussed with respect to topological conformation of the progesterone receptor and the structural similarity between forms A and B.

Using a yeast two‐hybrid system we report the isolation of a novel mouse protein, mSUG1, that interacts with retinoic acid receptor alpha (RAR alpha) both in yeast cells and in vitro in a ligand‐ and AF‐2 activating domain (AF‐2 AD)‐dependent manner and show that it is a structural and functional homologue of the essential yeast protein SUG1. mSUG1 also efficiently interacts with other nuclear receptors, including oestrogen (ER), thyroid hormone (TR), Vitamin D3 (VDR) and retinoid X (RXR) receptors. By comparing the interaction properties of these receptors with mSUG1 and TIF1, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with mSUG1 and TIF1; (ii) the amphipathic alpha‐helix core of the AF‐2 AD is differentially involved in interactions of RAR alpha with mSUG1 and TIF1; (iii) the AF‐2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus, the interaction interfaces between the different receptors and either mSUG1 or TIF1 may vary depending on the nature of the receptor and the putative mediator of its AF‐2 function. We discuss the possibility that mSUG1 and TIF1 may mediate the transcriptional activity of the AF‐2 of nuclear receptors through different mechanisms.

The retinoid X receptor (RXR) regulates target gene transcription via its association with cognate DNA response elements either as a homodimer or as a heterodimer with a number of other nuclear receptors. We previously demonstrated that, in solution, RXR forms tetramers with a high affinity and that ligand binding leads to dissociation of receptor tetramers to smaller species. Here it is shown that RXR tetramers form stable complexes with direct repeats (DR-1 or DR-5) or palindromic (TREpal) response elements. Binding of RXR tetramers to cognate DNA occurs with a significantly higher affinity as compared with dimers. Ligand binding by DNA-bound RXR tetramers results in their dissociation to DNA-bound dimers, a process that is completely reversed upon removal of the ligand. Formation of stable tetramer-DNA complexes requires binding of two oligonucleotides/tetramer. It is proposed that ligand-dependent modulation of the oligomeric state of RXR is a regulatory feature of this nuclear receptor.

Surface-enhanced Raman scattering and fluorescence were used to investigate the interactions of all-trans retinoic acid with the gamma-type retinoic acid receptor. Raman data revealed a significant attenuation in intensity of the bands originating from the retinoic acid polyenic chain upon receptor binding, with the spectrum being dominantly that of the β-ionone ring. Fluorescence measurements supported the hydrophobic character of the ligand binding. These novel spectroscopic results are fully consistent with the published X-ray crystallographic data and suggest that these techniques may be valuable additional tools to characterize the interactions of agonists and antagonists with residues in the ligand-binding pockets of retinoid receptor homo- and heterodimers. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: 297–302, 1998

Two rapid and high yield purification methods for the rat liver glucocorticoid receptor based on differential DNA affinity (method A) and ligand affinity (method B) chromatography are described. In method A, the amount of receptor in rat liver cytosol that can be activated and subsequently eluted from a DNA-cellulose column has been increased to 80% by introducing a second heat activation step. Using this method, 1.5 nmol of 25% pure glucocorticoid receptor can be routinely obtained per day from 15-20 rat livers. Method B yields about 2.2 nmol of 60% pure receptor with an overall yield of congruent to 60%. The quality of these purifications has been controlled by affinity labeling. In each case, more than 95% of purified binding activity represented the intact 92,000 +/- 400-Da glucocorticoid receptor polypeptide as shown by sodium dodecyl sulfate-gel electrophoresis and fluorography. No difference in the labeling pattern was observed using either [3H]triamcinolone acetonide (photoaffinity labeling) or [3H]dexamethasone 21-mesylate (electrophilic labeling). The electrophilic labeling step was performed in the cytosol prior to purification by method A to compare the labeled components thus purified with those obtained when the photoaffinity labeling was performed after the purification. Using this approach, distinct breakdown products of the glucocorticoid receptor were revealed, co-purifying during DNA affinity chromatography. Cross-linked receptor obtained by method A has been further purified to homogeneity by preparative sodium dodecyl sulfate-gel electrophoresis and successfully used as immunogen to raise glucocorticoid receptor antibodies in rabbits. These antibodies raised against glucocorticoid receptor, as well as those previously obtained using affinity chromatography-purified receptor, react with the receptor molecules irrespective of their method of purification. Glucocorticoid receptors purified by methods A and B have been analyzed for specific DNA-binding properties by the nitrocellulose filter binding assay.