Biomedical subjects

H Gronemeyer

Publications and source records attributed to H Gronemeyer.

At least 73 records · Page 4Linked to original sources

PubMed1994

Dimerization interfaces formed between the DNA binding domains determine the cooperative binding of RXR/RAR and RXR/TR heterodimers to DR5 and DR4 elements.

We have previously reported that the binding site repertoires of heterodimers formed between retinoid X receptor (RXR) and either retinoic acid receptor (RAR) or thyroid hormone receptor (TR) bound to response elements consisting of directly repeated PuG(G/T)TCA motifs spaced by 1-5 bp [direct repeat (DR) elements 1-5] are highly similar to those of their corresponding DNA binding domains (DBDs). We have now mapped the dimerization surfaces located in the DBDs of RXR, RAR and TR, which are responsible for cooperative interaction on DR4 (RXR and TR) and DR5 (RXR and RAR). The D-box of the C-terminal CII finger of RXR provides one of the surfaces which is specifically required for the formation of the heterodimerization interfaces on both DR4 and DR5. Heterodimerization with the RXR DBD on DR5 specifically requires the tip of the RAR CI finger as the complementary surface, while a 7 amino acid sequence encompassing the 'prefinger region', but not the TR CI finger, is specifically required for efficient dimerization of TR and RXR DBDs on DR4. Importantly, DBD swapping experiments demonstrate not only that the binding site repertoires of the full-length receptors are dictated by those of their DBDs, but also that the formation of distinct dimerization interfaces between the DBDs are the critical determinants for cooperative DNA binding of these receptors to specific DRs.

Amino Acid Sequence↗

PubMed1994

The dimerization interfaces formed between the DNA binding domains of RXR, RAR and TR determine the binding specificity and polarity of the full-length receptors to direct repeats.

Heterodimers of retinoid X receptor (RXR) and retinoic acid receptor (RAR) bind preferentially to directly repeated elements with spacing of two (DR2) or five (DR5) base pairs, due to the specific heterocooperative interaction of their DNA binding domains (DBDs) on these elements. We have demonstrated in the accompanying paper that the heterodimeric DBD interface that is responsible for the cooperative binding to DR5 elements, specifically involves the D-box of the RXR CII finger and the tip of the RAR CI finger. We show here that a second type of dimerization interface, which specifically implicates the RAR T-box and the RXR CII finger to the exclusion of the D-box, determines the selective binding to DR2 elements. Interestingly, the same type of dimerization interface (RXR T-box and CII finger) is responsible for the cooperative binding of homodimers of the RXR DBD to DR1 elements. Based on the three-dimensional structure of the glucocorticoid receptor DBD, modeling of RXR/RAR, RXR/TR and RXR/RXR DBD cooperative interactions predicts that in all cases the DBD contributing the CII finger, i.e. that of RXR, has to be positioned 5' to its cooperatively bound partner. This binding polarity of the DBDs is conferred upon the full-length receptors, since crosslinking experiments indicate that RXR is always 5' to RAR in complexes between either DR5 or DR2 and RXR/RAR heterodimers. The possible significance of these observations for transactivation by retinoic acid receptors is discussed.

Base Sequence↗

PubMed1994

Homo- and heterodimers of the retinoid X receptor (RXR) activated transcription in yeast.

The polymorphic nature of sequences which act as retinoic acid response elements (RAREs and RXREs) in transactivation assays in mammalian cells, suggests that elements consisting of a direct repetition of a half site motif, separated by 1 to 5 base pairs (DR1 to DR5), are targets for retinoic acid (RA) signalling. In a previous report we showed that in yeast cells, heterodimers of the retinoic acid receptors RAR alpha and RXR alpha were required for efficient transcription of a reporter gene containing a DR5 element [Heery et al., (1993); Proc. Natl. Acad. Sci. USA, 90: 4281-4285]. Here we report that DR1 to DR5 elements containing a direct repeat of the 5'-AGGTCA-3' motif, and an inverted repeat of the same sequence with no spacer (IR0), behave as RAREs in yeast cells coexpressing RAR alpha and RXR alpha, albeit with different efficacies. Heterodimer activity was strongest on a DR5 reporter gene, and the strength of activation of the reporter series (DR5 > DR1 > DR3 > DR2 = IR0 = DR4) correlated with the ability of the heterodimer to bind to the corresponding sequences in vitro. Significantly, a reporter containing a DR1 element was selectively and efficiently activated in yeast cells expressing only RXR alpha. This activity was dependent on the induction by 9-cis retinoic acid of an activation function (AF-2) located in the RXR alpha ligand binding domain. In addition, a strong synergistic activity of RXR alpha was observed on a reporter containing the putative RXR element (RXRE) from the rat CRBPII gene promoter. Thus, RXR alpha can function independently as a transcription factor, in the absence of RARs or other heteromeric partners. Similarly, homodimers of RAR alpha selectively stimulated the transcription of a DR5 reporter in a ligand-dependent manner, but less efficiently than RAR alpha/RXR alpha heterodimers.

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PubMed1993

The patterns of binding of RAR, RXR and TR homo- and heterodimers to direct repeats are dictated by the binding specificites of the DNA binding domains.

We show here that, in addition to generating an increase in DNA binding efficiency, heterodimerization of retinoid X receptor (RXR) with either retinoic acid receptor (RAR) or thyroid hormone receptor (TR) alters the binding site repertoires of RAR, RXR and TR homodimers. The binding site specificities of both homo- and heterodimers appear to be largely determined by their DNA binding domains (DBDs), and are dictated by (i) homocooperative DNA binding of the RXR DBD, (ii) heterocooperative DNA binding of RXR/RAR and RXR/TR DBDs, and (iii) steric hindrance. No homodimerization domain exists in the DBDs of TR and RAR. The dimerization function which is located in the ligand binding domain further stabilizes, but in general does not change, the repertoire dictated by the corresponding DBD(s). The binding repertoire can be further modified by the actual sequence of the binding site. We also provide evidence supporting the view that the cooperative binding of the RXR/RAR and RXR/TR DBDs to directly repeated elements is anisotropic, with interactions between the dimerization interfaces occurring only with RXR bound to the 5' located motif. This polarity, which appears to be maintained in the full-length receptor heterodimers, may constitute a novel parameter in promoter-specific transactivation.

Amino Acid Sequence↗

PubMed1993

Efficient transactivation by retinoic acid receptors in yeast requires retinoid X receptors.

All-trans and 9-cis retinoic acids are natural derivatives of vitamin A that modulate gene expression as a consequence of binding to nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RXRs form heterodimers with RARs in vitro, and such complexes display enhanced binding affinities for cognate DNA response elements. As yeast is devoid of endogenous RARs and RXRs, we used this organism to investigate whether transactivation in vivo requires RAR/RXR heterodimers. Using a domain-swapping approach, we demonstrate that chimeric RAR alpha 1 and RXR alpha containing the DNA-binding domain of the estrogen receptor activate transcription of a cognate reporter gene in yeast, independently of each other. These activities result from an inducible transcription activation function located in the ligand-binding domains of RAR alpha 1 and RXR alpha and a constitutive activation function located in the A/B region of RAR alpha 1. The inducible activation function of RXR alpha is induced exclusively by 9-cis-retinoic acid in this system. Transactivation of a reporter gene containing a retinoic acid response element by RAR alpha was considerably increased by RXR alpha, even in the absence of ligand. Optimal induction was achieved with 9-cis-retinoic acid, which stimulates the activity of both receptors. This study illustrates the utility of yeast to investigate signal transduction by retinoids in the absence of endogenous RARs, RXRs, and detectable retinoic acid isomerization.

Animals↗

PubMed1993

Retinoic acid-response elements with a highly repetitive structure isolated by immuno-selection from genomic DNA.

In vitro binding sites of retinoic acid receptors (RARs) were isolated from mouse genomic DNA by immunoprecipitation of receptor/DNA complexes. PuG(G/T)TCA half-site motifs, which constitute RA-responsive elements (RAREs), were identified in the immuno-selected fragments (ISFs), some of which contained highly repetitive arrangements of this motif. Genomic Southern analysis of a number of ISFs showed them to be of a single or low copy number. Several, but not all, ISFs acted as ligand-dependent RAREs in transient transfection assays. Two ISFs with repetitive RARE motifs responded preferentially to 9-cis retinoic acid-liganded retinoid X receptor in the presence or absence of co-transfected RAR, while little activation was seen with RAR alone in the presence of either all-trans or 9-cis retinoic acid. Another ISF, containing consensus TATA and CAAT box motifs, was shown to have RA-inducible promoter activity. The results suggest a high degree of promiscuity in response element recognition by retinoid receptors.

Animals↗

PubMed1993

Type II antagonists impair the DNA binding of steroid hormone receptors without affecting dimerization.

Two types of steroid antagonists exert their activity by different mechanisms when bound to the cognate receptor. Type I anti-progestins, such as RU486, induce DNA binding of the human progesterone receptor (hPR), while no hPR/DNA complexes were seen in gel shift assays in the presence of the type II anti-progestin ZK98,299 or RU50,331. ZK98,299-liganded hPR exerted significantly less tight nuclear binding than receptor complexes formed with RU486. Also a type II anti-glucocorticoid (RU43,044) was detected which completely abrogated DNA binding of its cognate receptor in vivo. In keeping with the existence of two different classes of anti-progestins, agonist- or RU486-induced hyperphosphorylation of the two hPR isoforms present in the T47D breast cancer cells was not induced by ZK98,299. This lack of hyperphosphorylation was, however, not the cause but most likely the consequence, of the reduced ability of the hPR/ZK98,299 complex to interact with DNA. No "mixed-ligand" heterodimers were formed in vitro between hPR isoform A bound to ZK98,299 and R5020-liganded isoform B, but nuclear co-translocation studies indicated that ZK98,299 efficiently induced hPR dimerization in vivo.

Animals↗

PubMed1993

Transcription activation by nuclear receptors.

Nuclear receptors constitute a superfamily of ligand-inducible transcription factors which respond to endocrine, paracrine and, possibly, autocrine signals. Multiple regulatory mechanisms assure that signal transduction results in an accurate regulation of the respective gene networks. Apart from selective expression of the cognate receptor and its binding to specific hormone response elements of target genes, additional mechanisms are responsible for the cell- and promoter-specific transcription activation. They are based on the "interpretation" of the signal by the multiple functional modules of a given receptor and involve a specific interplay with various factors binding to complex target gene promoters and cell-specific intermediary transcription factors that mediate the activity of the two receptor transcription activation functions, as well as homo- and heterodimerization, and interference with other signalling pathways. Moreover, a single ligand may initiate different gene programs due to the differential target gene specificities of nuclear receptor isoforms. Thus, signal transduction by nuclear receptors involves a multitude of interactive elements, as could have been expected from the central role of these signals in homeostasis, embryonic development and differentiation. Two distinct mechanisms are involved in anti-hormone action. Type I anti-hormones impair the activity of the transcription activation function, while type II antagonists impair DNA binding. Experiments aimed at an understanding of these mechanisms are discussed.

Animals↗

PubMed1992

Characterization and purification of human retinoic acid receptor-gamma 1 overexpressed in the baculovirus-insect cell system.

The full-length cDNA for the human retinoic acid receptor-gamma 1 (RAR-gamma 1) has been expressed to high levels in Spodoptera frugiferda (Sf9) cells using the baculovirus expression system. Western blot analysis revealed that RAR-gamma 1 expression increased between 32 and 60 h post-infection. The recombinant receptor was expressed primarily as a nuclear protein and displayed a molecular mass of 50 kDa as determined by SDS/PAGE and gel-filtration chromatography, consistent with its cDNA-deduced size. Based on ligand binding, 2 x 10(6) RAR-gamma 1 molecules were expressed per Sf9 cell, a level approx. 2000 times greater than in mammalian cells. The receptor was partially purified 300-fold by sequential anion-exchange, gel-filtration and DNA affinity chromatographies. The overexpressed receptor specifically bound all-trans-retinoic acid (RA) and the synthetic retinoid CD367 with high affinity (Kd 0.15 nM and 0.23 nM respectively). The RA metabolites 4-hydroxy-RA and 4-oxo-RA were poor competitors for [3H]CD367 binding to recombinant RAR-gamma 1 (K(i) > 1 microM), indicating that 4-oxidation of RA greatly reduces its affinity for RAR-gamma 1. Gel-retardation analysis demonstrated that RAR-gamma 1 specifically bound the RA response element of the mouse RAR-beta gene. RAR-gamma 1 species expressed from recombinant baculovirus (in Sf9 cells) and vaccinia virus (in HeLa cells) exhibited similar affinities for RA and CD367 and had comparable DNA-binding properties in gel-retardation experiments. Moreover, a similar requirement for additional DNA-binding stimulatory factor(s) was observed in both cases. These results provide a basis for the use of baculovirus-expressed RAR-gamma 1 in further functional and structural studies.

Amino Acid Sequence↗

PubMed1992

A limiting factor mediates the differential activation of promoters by the human progesterone receptor isoforms.

The two transcription activation functions (TAFs) of the human progesterone receptor (hPR) have been characterized. TAF-1, located in the N-terminal region A/B, has been narrowed down to a 91-amino acid sequence, which is sufficient for transcription activation in chimeras with the GAL4 DNA binding domain. Both hPR TAF-1 and TAF-2 activate a minimal promoter in yeast. No autonomous TAF could be found in the N-terminal 164 amino acids (designated AB3) which are responsible for the differential activation of promoters by the hPR isoforms A and B. Reduction of the target gene promoter complexity did not alter this differential activation, indicating that AB3 does not require additional promoter-bound factors to exert its effect. Instead, the cell specificity of AB3 and its ability to squelch hPR-induced transcription suggest that this differential isoform activity is due to the effect of a limiting factor which binds to region AB3.

Amino Acid Sequence↗

PubMed1992

In vitro activity of the transcription activation functions of the progesterone receptor. Evidence for intermediary factors.

The human progesterone receptor (hPR) is a ligand-dependent transcription factor which contains two distinct transcription activation functions (TAFs). The full-length hPR and its individual TAFs were overexpressed in the baculovirus system and tested in a HeLa cell-derived in vitro transcription system. hPR stimulated transcription in a ligand-independent manner. When the two TAFs fused to the DNA-binding domain of GAL4 were tested, only the constitutive TAF-1 was functional in vitro, strongly suggesting that the transcriptional activity of baculovirus-expressed hPR comes solely from TAF-1. The GAL-TAF-1 activator was found to self-squelch without affecting basal transcription. A partially purified fraction relieved this self-squelching and, moreover, stimulated transcriptional activation by GAL-TAF-1, while having no influence on basal transcription. These results strongly suggest that the transcriptional activity of GAL-TAF-1 requires a factor(s) distinct from the general transcription factors.

Animals↗

PubMed1992

A single amino acid that determines the sensitivity of progesterone receptors to RU486.

The progesterone analog RU486, an abortifacient, inhibits the action of progestins in humans but not in chickens or hamsters. Substitution of cysteine at position 575 by glycine in the hormone binding domain (HBD) of the chicken progesterone receptor (cPR) generated a cPR that binds RU486 and whose activity is antagonized by that compound. In fact, all receptors that bind RU486 have a glycine at the corresponding position. The hamster PR, like cPR, has a cysteine. Only glycine--not methionine or leucine--at position 575 allowed binding of RU486 to cPR. Substitution of this glycine by cysteine in the human PR (hPR) abrogated binding of RU486 but not that of an agonist. The corresponding mutation in the human glucocorticoid receptor resulted in a loss of binding of both dexamethasone and RU486. Examination of a series of 11 beta-substituted steroids showed that antagonism is not an intrinsic property of an antihormone, because one hPR antagonist acted as an agonist for a mutated hPR. The positioning of an aromatic 11 beta-substitution in the PR HBD appears to be critical for generating agonistic or antagonistic activity.

Amino Acid Sequence↗

PubMed1992

Cooperation of proto-signals for nuclear accumulation of estrogen and progesterone receptors.

Multiple proto-signals (p-NLSs) for nuclear targeting, none of which suffices on its own, cooperate in the estrogen (ER) and progesterone (PR) receptors. In the ER, an estrogen-inducible p-NLS was found in the hormone binding domain (HBD), in addition to three lysine/arginine-rich motifs resembling prototype constitutive nuclear localization signals (NLSs). The inducible and the constitutive ER p-NLSs cooperate in the presence of estrogen and hydroxy-tamoxifen, but not in the presence of ICI 164,384. In the PR, three p-NLSs, two of which are located within and directly adjacent to the second zinc finger, cooperate with each other and a weak hormone-inducible p-NLS in the PR HBD. No 'masking' of p-NLSs by the HBD was observed for ER and PR, while the ligand-free glucocorticoid receptor HBD inhibited the activity of both homologous and heterologous NLSs. Nuclear co-translocation experiments indicated that in vivo the stability of ER and PR dimers is hormonally controlled, but that, in the absence of the cognate ligand, ER dimers are more stable than PR dimers. This is likely to account for the differential hormone requirement of ER and PR DNA binding in vitro.

Amino Acid Sequence↗

PubMed1992

Mechanisms of antihormone action.

The mechanisms of action of two types of anti-hormones is discussed. Type I anti-hormones comprise the antiestrogen hydroxy-tamoxifen and the antiprogestin RU486, both of which promote DNA binding of the cognate receptors and, due to the activity of one of the two transcription activation functions of the estrogen and progesterone receptors, act as mixed agonist/antagonists. Evidence supporting that ICI 164,384 is also a member of the same group is presented. Type II antagonists impair DNA binding of the corresponding receptor in vitro and, in some cases, also in vivo. Ligand-mapping, an approach to identify the site of interaction of a steroid substitution within the hormone-binding domain of the receptor has been used to identify the 11 beta-pocket of the progesterone receptor and revealed that a single amino acid is responsible for the differential antagonistic effect of RU486 in man, chicken and hamster.

Animals↗

PubMed1992

Control of transcription activation by steroid hormone receptors.

Multiple regulatory mechanisms assure that signal transduction, involving the nuclear receptor interface, results in an accurate regulation of the respective gene networks. These mechanisms involve selective expression of the cognate receptor and its binding to specific hormone response elements of target genes. However, superimposed onto this "simple" control of activity is interpretation of the signal by the multiple functional modules of a given receptor, based on a specific interplay with 1) various factors binding to complex target gene promoters, 2) cell-specific transcription factors that mediate its enhancer function, and 3) other signaling pathways. This interpretation can be further modulated by the differential target gene specificities of receptor isoforms and, according to in vitro evidence, by factors that increase the efficiency of the receptor to interact with its response element. Thus, steroid hormone-regulated gene transcription involves a multitude of interactive elements, as is expected from the central role of nuclear receptors in homeostasis, embryonic development, and differentiation.

Animals↗

PubMed1992

Switching agonistic, antagonistic, and mixed transcriptional responses to 11 beta-substituted progestins by mutation of the progesterone receptor.

The study of transcription activation by a series of RU486-related 11 beta-substituted progestins revealed three types of ligands: agonists, antagonists, and a novel type of compounds that exerted a mixed activity. These ligands conferred to the human progesterone receptor (hPR) only weak activation properties despite high affinity binding and, hence, acted as agonists and, at the same time, as partial antagonists of pure agonists. When the same series of ligands was tested with mutant PRs, transcriptional activation/inactivation profiles were different from those seen with the wild-type PR, since several steroids initially classified as antagonists switched to mixed responses. One compound that acted as an antagonist for the hPR was an agonist for a mutated PR in which 15 amino acids of the hormone-binding domain were replaced by the corresponding divergent residues of the chicken homolog. In analyzing a series of steroids with wild-type and mutant PRs, we observed that a phenyl group (or a phenyl derivative) in the 11 beta position of RU486-related steroids generates antagonism with hPR, but has to be bound in a critical position in the hormone-binding domain to exert its antagonistic activity. Apparently, a deviation from this positioning by mutations in the hormone-binding domain can generate mixed or even agonistic activities.

Animals↗

PubMed1991

Control of transcription of the chicken progesterone receptor gene. In vitro and in vivo studies.

To study the promoter of the chicken progesterone receptor (cPR) gene and the relevance of several progestin-responsive elements therein, chimeric genes were constructed which contained the 5'-flanking region of the cPR gene linked to promoterless globin or chloramphenicol acetyltransferase sequences. Cell-specific initiation of transcription was observed in transiently transfected chicken embryo fibroblasts when using 876 base pairs of the cPR gene upstream region. Transcription from these reporter genes could be induced by progestins in the presence of cPR form A but not of form B. In keeping with these data, three in vitro progesterone receptor (PR)-binding sites were identified in the cPR promoter region by DNase I protection assays. However, in vivo, nuclear run-on transcription demonstrated that neither primary stimulation with progestins, nor treatment of secondarily estrogen-stimulated chicks with progestins, glucocorticoids, or androgens resulted in any significant change of cPR gene transcription in the oviduct, thus suggesting a cell- and/or development-specific role for these progestin-responsive elements. Although estrogen is known to increase PR levels in the chick oviduct, this effect does not involve stimulation of PR gene transcription, as demonstrated here by nuclear run-on experiments, the analysis of DNase I hypersensitive sites, and transient cotransfection studies. Since acute withdrawal from estrogen-stimulation markedly decreased the level of cPR mRNAs in chick oviduct when analyzed by Northern blotting, we conclude that estrogen-dependent stimulation of PR levels in the oviduct is a post-transcriptional process.

Animals↗

PubMed1991

Cell-specific inhibitory and stimulatory effects of Fos and Jun on transcription activation by nuclear receptors.

We investigated the effect of c-Fos and/or c-Jun co-expression on transcription activation by the progesterone (PR), glucocorticoid (GR) or androgen (AR) receptors using three different reporter genes and four different cell lines. We found that c-Fos could only inhibit, while c-Jun could either inhibit or further stimulate receptor-induced transcription. All these effects were receptor, promoter, and cell type specific, and, importantly, the steroid receptors had non-reciprocal effects on the transactivation ability of c-Jun in the presence or absence of c-Fos. Collectively, these results argue against heterodimer formation as a mechanism to explain the phenomena. Transactivation by the endogenous PR in T47D cells could be inhibited by increasing the intracellular c-Fos level with forskolin as well as by co-expressing c-Fos; no such effect was seen in MCF-7 cells. The inhibition by c-Fos of PR-induced transcription involves a competitive mechanism, which requires the presence of the intact c-Fos leucine zipper and is directed mainly at the transcription activation function (TAF) located in the PR and GR hormone binding domains (TAF-2). However, the co-expression of c-Fos did not alter the 'squelching/transcriptional interference' by the PR of estrogen receptor (ER)-induced transcription. Multiple mechanisms are discussed which may be involved in the crosstalk between the two signal transduction pathways.

Base Sequence↗