Biomedical subjects

M R Yudt

Publications and source records attributed to M R Yudt.

6 recordsLinked to original sources

PubMed2006

Selective and opposing actions of progesterone receptor isoforms in human endometrial stromal cells.

Transcriptional regulation by progesterone is mediated primarily through the two progesterone receptor (PR) isoforms, PR-A and PR-B. Primary human endometrial stromal cell cultures, in which endogenous PR expression was lost, were infected with adenovirus expressing PR-A, PR-B, or both. Global gene expression analysis was conducted on vehicle and 30 nM progesterone (P4) treated cells following 12 h treatment. Interestingly, many genes regulated by PR-B alone or upon PR-A and PR-B co-expression, did not overlap with each other or with the PR-A expression group. Although many genes known to be progestin regulated in the uterus in vivo were also regulated in this study, markedly little overlap with published P4 regulated genes from human breast cancer cells was observed. Progesterone dose response curves were generated for several genes demonstrating gene selective potency and efficacy for each PR isoform. Furthermore, the PR isoforms opposed each other in regulation of tissue factor, with PR-B increasing and PR-A decreasing both mRNA and protein levels. Our data provide a view of global gene expression by PR isoforms in human endometrial cells and a comparison with other cell types. The specific genes and regulation patterns found provide groundwork to revealing the mechanism of PR isoform selectivity, and perhaps ultimately to the tissue selective properties these receptors appear to exhibit.

Cells, Cultured↗

PubMed2001

Preventing estrogen receptor action with dimer-interface peptides.

The human estrogen receptor-alpha (hER) is a ligand-activated transcription factor that functions as a homodimer. We sought to further understand the molecular processes involved in dimerization, and to develop a reagent that may function as an antiestrogen independent of the ligand binding site. To this end, we designed a 16-residue 'dimer-interface' oligopeptide derived from the helical region of the hER which is directly involved in dimerization. This peptide, termed the I-box peptide, has a high helical propensity in aqueous solution. The I-box peptide blocks hER action by causing aggregation and precipitation of both the ligand-bound and apo-hER. This effect is dependent on the helical nature of the peptide. A single Ile to Pro mutation in the helical region of the I-box peptide significantly reduces the helical content and abolishes the precipitation activity. Furthermore, the peptide activity appears to be specific for the hER. The I-box peptide does not significantly affect other proteins or steroid receptors tested. A homologous peptide derived from the nuclear receptor RXRalpha dimer interface, and a LXXLL-containing peptide from the coactivator TIF2 have no detectable in vitro effect on hER function or solubility. Our data suggest that rationally designed molecules capable of affecting steroid receptor quaternary structures may be potential avenues for the development of specific inhibitors of this class of proteins.

Amino Acid Sequence↗

PubMed2001

Molecular identification and characterization of a and b forms of the glucocorticoid receptor.

The human glucocorticoid receptor (hGRalpha) is a ligand-activated transcription factor that mediates the physiological effects of corticosteroid hormones and is essential for life. Originally cloned in 1986, the transcriptionally active hGRalpha was reported to be a single protein species of 777 amino acids (molecular mass = 94 kDa). Biochemical data, obtained using various mammalian tissues and cell lines, however, have consistently revealed an additional, slightly smaller, second hGR protein (molecular mass = 91 kDa) that is not recognized by antibodies specific for the transcriptionally inactive and dominant negative, non-hormone-binding hGRbeta isoform. We report here that when a single GR cDNA is transfected in COS-1 cells, or transcribed and translated in vitro, two forms of the receptor are observed, similar to those seen in cells that contain endogenous GR. These data suggest that two forms of the hGRalpha are produced by alternative translation of the same gene and are henceforth termed GR-A and GR-B. To test this hypothesis, we have investigated the role of an internal ATG codon corresponding to methionine 27 (M27) as a potential alternative translation initiation site for the GR. Mutagenesis of this ATG codon to ACG in human, rat, and mouse GR cDNA results in generation of a single 94-kDa protein species, GR-A. Moreover, mutagenesis of the initial ATG codon to ACG (Met 1 to Thr) also resulted in production of single, shorter protein species (91 kDa), GR-B. Mutagenesis of the Kozak translation initiation sequence strongly indicates that a leaky ribosomal scanning mechanism is responsible for generating the GR-A and -B isoforms. Western blot analysis using peptide-specific antibodies show both the A and B receptor forms are present in human cell lines. Both receptors exhibit similar subcellular localization and nuclear translocation after ligand activation. Functional analyses of hGR-A and hGR-B under various glucocorticoid-responsive promoters reveal the shorter hGR-B to be nearly twice as effective as the longer hGR-A species in gene transactivation, but not in transrepression.

Alternative Splicing↗

PubMed1999

Function of estrogen receptor tyrosine 537 in hormone binding, DNA binding, and transactivation.

The human estrogen receptor (hER) is a ligand-activated transcription factor which, like many other members of the nuclear receptor protein family, exhibits a dimerization-dependent transcriptional activation. Several previous reports have provided evidence of the phosphorylation of the hER at tyrosine 537 (Y537). However, the exact function of a putative phosphorylation at this site remains controversial. Using a yeast transactivation assay, and in vitro biochemical approaches, we show that phosphorylation of tyrosine 537 is not required for the hER to bind hormone, or to activate transcription. An hER tyrosine 537 to phenylalanine (Y537F) mutant retains 70-75% of the transactivation potential of wild type hER in a yeast reporter system. Furthermore, the mutated receptor exhibits wild type hormone and DNA binding affinities. However, this mutation results in a decrease in receptor stability as measured by a decrease in the extent of hormone binding over time. The most striking difference between the wild type and Y537F hER is in the estradiol binding kinetics. Whereas the off-rate for estradiol exhibits a two-state binding mechanism, the Y537F mutant hER exhibits a monophasic estradiol off-rate. On the basis of these data and other reports describing the structure and activity of Y537 mutations, as well as knowledge of the three-dimensional structure of the hER ligand binding domain, we propose an alternate model wherein Y537F mutation favors an "open" pocket conformation, affecting the estrogen binding kinetics and stability of the hormone-bound, transcriptionally active "closed" pocket conformation. Although its phosphorylation is not essential for function of the hER, Y537 is nevertheless a critical residue intricately involved with the conformational changes of the hER and its ability to activate transcription.

Amino Acid Sequence↗

PubMed1999

The dominant negative activity of the human glucocorticoid receptor beta isoform. Specificity and mechanisms of action.

Alternative splicing of the human glucocorticoid receptor gene generates a nonhormone binding splice variant (hGRbeta) that differs from the wild-type receptor (hGRalpha) only at the carboxyl terminus. Previously we have shown that hGRbeta inhibits the transcriptional activity of hGRalpha, which is consistent with reports of elevated hGRbeta expression in patients with generalized and tissue-specific glucocorticoid resistance. The potential role of hGRbeta in the regulation of target cell sensitivity to glucocorticoids prompted us to further evaluate its dominant negative activity in other model systems and to investigate its mode of action. We demonstrate in multiple cell types that hGRbeta inhibits hGRalpha-mediated activation of the mouse mammary tumor virus promoter. In contrast, the ability of the progesterone and androgen receptors to activate this promoter is only weakly affected by hGRbeta. hGRbeta also inhibits hGRalpha-mediated repression of an NF-kappaB-responsive promoter but does not interfere with homologous down-regulation of hGRalpha. We show that hGRbeta can associate with the heat shock protein hsp90 although with lower affinity than hGRalpha. In addition, hGRbeta binds GRE-containing DNA with a greater capacity than hGRalpha in the absence of glucocorticoids. Glucocorticoid treatment enhances hGRalpha, but not hGRbeta, binding to DNA. Moreover, we demonstrate that hGRalpha and hGRbeta can physically associate with each other in a heterodimer. Finally, we show that the dominant negative activity of hGRbeta resides within its unique carboxyl-terminal 15 amino acids. Taken together, our results suggest that formation of transcriptionally impaired hGRalpha-hGRbeta heterodimers is an important component of the mechanism responsible for the dominant negative activity of hGRbeta.

Alternative Splicing↗

PubMed1995

In vivo and in vitro phosphorylation of the human estrogen receptor.

We report here that the human estrogen receptor (hER) overexpressed in Sf9 insect cells is phosphorylated similarly to hER from the human MCF-7 mammary carcinoma cell line. The recombinant and native hER labeled to steady-state with [32P]phosphate were purified to homogeneity using specific DNA-affinity chromatography followed by SDS-gel electrophoresis. Resolution of the hER tryptic digests by reverse phase-high performance liquid chromatography revealed that five [32P]phosphopeptides from the hER expressed in the Sf9 cells had retention times identical to five of the seven [32P]phosphopeptides from the hER in MCF-7 cells. Uniquely, a dephosphorylation of a single 32P-labeled peptide occurred in response to estradiol treatment of MCF-7 cells. In vitro protein kinase assays with the purified recombinant hER revealed that the DNA-dependent protein kinase (DNA-PK) phosphorylated the receptor and induced a decrease in the receptor's mobility as demonstrated by SDS-gel electrophoresis. In contrast, protein kinases A and C did not phosphorylate the purified recombinant hER. These results suggest that in the process of becoming transcriptionally active the estrogen receptor undergoes a dephosphorylation after estrogen-binding and subsequent phosphorylations, in part by the DNA-PK.

Animals↗