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H Gronemeyer

Publications and source records attributed to H Gronemeyer.

At least 19 recordsLinked to original sources
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↗
PubMed1991

Progestin receptors: isoforms and antihormone action.

We present evidence that the two isoforms of A and B of the chicken (cPR) and human progesterone receptor (hPR) originate from two different mRNA populations. One of these encodes the isoforms A which originate by initiation of translation at an in-frame AUG found 127 (cPR) and 165 (hPR) codons downstream of the AUG which gives rise to the isoforms B. Two estrogen-inducible hPR promoters were identified which are responsible for the generation of these two classes of transcripts. Characterization of the cPR promoter suggested the possible existence of cell-type and isoform-specific auto-regulation of cPR transcription and provided evidence that estrogen-induction of cPR expression occurs at a post-transcriptional level. Finally, we demonstrate promoter-specific transcriptional activation by the hPR isoforms A and B, and we discuss the mechanism of action of the anti-progestin RU486.

Animals↗
PubMed1991

[Nuclear receptors. Hormones, anti-hormones and regulation of the transcription].

Nuclear receptors are ligand-inducible transcription factors which respond to endocrine, paracrine and, possibly, autocrine signals, thus triggering key events in early embryonal development, differentiation and homeostasis. Here we discuss recent results which may lead to an understanding of the molecular mechanisms of transcriptional activation by a given transcription activation function (TAF) of these activators and of the receptor isoform-specific activation of transcription. In addition, we present a concept of the mechanism of action of two different types of antihormones.

Animals↗
PubMed1991

[Retinoic acid has an antiestrogenic effect on different regulated estrogen genes in different cellular types].

In this paper, we confirmed that retinoic acid is an antiestrogenic compound with respect to different chimaeric estrogenic responses and with respect to different cellular types. This was shown by transient transfection of MCF-7 cells with plasmids driving the chloramphenicol acetyl-transferase gene via different estrogenic regulatory part (pS2) and the first promotor of the progesterone receptor gene (PR1); an identical conclusion was obtained in HeLa cells by cotransfecting a plasmid expressing the estrogen receptor. In addition, the inhibitory effect of retinoic acid was not observed for genes regulated by the progesterone receptor and the glucocorticoid receptor. As the antiestrogenic effect of retinoic acid was increased by cotransfecting acid receptor(s) RAR alpha, beta, gamma, we concluded that RAR(s) is(are) involved in the antiestrogenic effect of retinoic acid.

Estrogen Antagonists↗
PubMed1990

Transient expression of human and chicken progesterone receptors does not support alternative translational initiation from a single mRNA as the mechanism generating two receptor isoforms.

Two isoforms (A and B) of the human (hPR) and chicken (cPR) progesterone receptors originate from a single PR gene. cPR form A results from initiation of translation at a downstream ATG codon (ATG2) which in the cPR cDNA-deduced open reading frame is found 128 amino acids C-terminal to and in-frame with the first ATG codon (ATG1) that gives rise to form B. Our recent observation of an abundant cPR mRNA which encodes only form A suggested to us that the two isoforms are translated from different transcripts (Jeltsch, J. M., Turcotte, B., Garnier, J. M., Lerouge, T., Krozowski, Z., Gronemeyer, H., and Chambon, P. (1990) J. Biol. Chem. 265, 3961-3974). This view is, however, at variance with data obtained by transient transfection with expression vectors containing most of the cPR cDNA (downstream of nucleotide +53) since both isoforms were generated in transiently transfected COS cells (Conneely, O. M., Kettelberger, D. M., Tsai, J. J., Schrader, W. T., and O'Malley, B. W. (1989) J. Biol. Chem. 264, 14062-14064). To further support our above conclusion, vectors containing either hPR or cPR cDNAs were introduced into HeLa and COS-1 cells. Only hPR form B originated from a vector containing the entire cDNA (containing nucleotides 1 to approximately 4400), and form A was produced only from a vector expressing hPR transcripts (nucleotides 814 to approximately 4400) lacking ATG1. Vectors expressing the 5'-untranslated and coding region of the cPR mRNA (nucleotides 29-2921) generated only traces of form A in the two cell lines. Similar traces of form A were observed in COS-1 cells transfected with a vector lacking the 5'-untranslated region. Collectively, these results do not support the hypothesis that similar amounts of the two PR isoforms are generated by alternative initiation of translation on a single PR transcript. We discuss data indicating that for hPR and cPR, isoforms A and B are in fact translated from different mRNAs.

Animals↗
PubMed1990

Characterization of multiple mRNAs originating from the chicken progesterone receptor gene. Evidence for a specific transcript encoding form A.

The structure of the 42-kilobase (kb) long chicken progesterone receptor (cPR) gene and of all six transcripts that are detectable on Northern blots was determined. The first of 8 exons encodes the N-terminal region A/B which is highly divergent among different species and contains a constitutive transcription activation function. The DNA (DBD)- and hormone-binding domains (HBD) are assembled from 2 and 5 exons, respectively, with the individual "zinc fingers" of the DBD encoded by separate exons. In addition to the previously described 4.5-kb cPR mRNA species, alternative polyadenylation, splicing variation, and "5'-truncation" lead to the generation of 5 further mRNAs. Most importantly, this 5'-truncation produces, by an as yet unidentified mechanism, an abundant transcript which encodes form A but not form B of cPR. Lack of splicing at the exon 2 splice-donor and polyadenylation due to a signal site in the second intron generates a previously undetected 3.4-kb mRNA species. The corresponding cDNA was sequenced in its entirety and shown to encode only region A/B and the N-terminal "finger" of the DBD. Alternative polyadenylation upstream of the signal site for the 4.5-kb mRNA is responsible for the appearance of a 3.3-kb mRNA. The longest cPR mRNA (8.2 kb) originates from a transcription termination point more than 3 kb downstream of the 4.5-kb mRNA 3'-end. Finally, the primary sequence of more than 2 kb upstream sequences of the cPR gene, containing several consensus hexamer progestin/glucocorticoid receptor-binding sites (PRE/GRE and putative Sp1 binding motifs, is discussed.

Amino Acid Sequence↗
PubMed1990

Agonistic and antagonistic activities of RU486 on the functions of the human progesterone receptor.

RU486 induced the binding to a palindromic progestin responsive element (PRE) in vitro of homo- and heterodimers of the human progesterone receptor (hPR) isoforms A and B, present in T47D breast cancer cells or in HeLa cells transiently expressing the recombinant proteins. The resulting complexes were indistinguishable from those induced with the agonist R5020 with respect to specificity, affinity and stability. Ligand exposure was a necessary prerequisite to observe PR/PRE complexes. Antagonist-induced complexes migrated more rapidly during electrophoresis than agonist-induced ones, and no 'mixed' PR/RU486-PR/R5020 complexes were observed, suggesting that the dimerization interfaces of agonist- and antagonist-bound molecules are non-compatible. The analysis of a series of deletion mutants and chimeric receptors revealed the presence of two transcription activation functions (TAFs), located in the N-terminal region A/B (TAF-1) and the hormone binding domain (TAF-2). In the presence of agonists, both TAFs were active in HeLa cells. In the presence of RU486 TAF-2 was inactive, while TAF-1 within the hPR form B/RU486 complex activated transcription from a reporter gene containing a single palindromic PRE. We consider this to be the most convincing evidence that the receptor/RU486-complex does in fact bind to PREs in vivo. No transcriptional activation was observed in the presence of RU486 from a reporter gene containing the complex MMTV-LTR PRE. In contrast to hPR form B, form A was not able to activate transcription from PRE/GRE-tk-CAT in the presence of RU486. In vivo competition between hPR/RU486 and either cPR/R5020 or the human glucocorticoid receptor/dexamethasone (hGR/Dex) complex further supported that hPR/RU486 bound in vivo to its cognate responsive element. Indeed, the observed inhibition of transcription was shown to be due to competition for the MMTV PRE, since no transcriptional interference by the hPR/RU486 was observed, and since no heterodimers were formed between hPR/RU486 and cPR/R5020 or hGR/Dex. That the ligand-free hPR, however, was unable to compete, demonstrated that ligand binding is the prerequisite for DNA binding of hPR in vivo.

Animals↗
PubMed1990

Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B.

The human progesterone receptor (hPR) cDNA, synthesized from T47D breast cancer cells, and the hPR gene 5'-flanking region were cloned and sequenced. Comparison of the cDNA-deduced amino acid sequence with other PR homologues demonstrated the modular structure characteristic of nuclear receptors. As in the case of the chicken homologue, there are two hPR forms, A and B, which originate from translational initiation at AUG2 (codon 165) and AUG1, respectively. Northern blot analysis of T47D mRNA using various cDNA derived probes identified two classes of hPR mRNAs, one of which could code for hPR form B, while the other one lacked the 5' region upstream of AUG1. S1 nuclease mapping and primer extension analyses confirmed that the second class of hPR transcripts are initiated between +737 and +842 and thus encode hPR form A, but not form B. By using the hPR gene 5'-flanking sequences as promoter region in chimeric genes, we show that a functional promoter (located between -711 and +31) directs initiation of hPR mRNAs from the authentic start sites located at +1 and +15. Most importantly, initiation of transcription from chimeric genes demonstrated the existence of a second promoter located between +464 and +1105. Transient co-transfection experiments with vectors expressing the human estrogen receptor showed that both promoters were estrogen inducible, although no classical estrogen responsive element was detected in the corresponding sequences. When transiently expressed, the two hPR forms similarly activated transcription from reporter genes containing a single palindromic progestin responsive element (PRE), while form B was more efficient at activating the PRE of the mouse mammary tumor virus long terminal repeat. Transcription from the ovalbumin promoter, however, was induced by hPR form A, but not by form B.

Amino Acid Sequence↗