William T. Schrader

Molecular cloning of the chicken progesterone receptor

To define the functional domains of the progesterone receptor required for gene regulation, complementary DNA (cDNA) clones encoding the chicken progesterone receptor have been isolated from a chicken oviduct lambda gt11 cDNA expression library. Positive clones expressed antigenic determinants that cross-reacted with six monospecific antibodies derived from two independent sources. A 36-amino acid peptide sequence obtained by microsequencing of purified progesterone receptor was encoded by nucleotide sequences in the longest cDNA clone. Analysis of the amino acid sequence of the progesterone receptor deduced from the cDNA clones revealed a cysteine-rich region that was homologous to a region found in the estrogen and glucocorticoid receptors and to the avian erythroblastosis virus gag-erb-A fusion protein. Northern blot analysis with chicken progesterone receptor cDNAs indicated the existence of at least three messenger RNA species. These messages were found only in oviduct and could be induced by estrogens.

The A and B forms of the chicken progesterone receptor arise by alternate initiation of translation of a unique mRNA

In order to establish the origin of the A and B proteins of the chicken progesterone receptor we have expressed its cDNA in vivo in heterologous cells and in vitro in reticulocyte cell lysates. The A and B proteins were expressed from a single cDNA both in heterologous receptor negative cells and in a cell-free system. Both proteins bind progesterone and are indistinguishable from chick oviduct authentic A and B proteins in terms of size, immunoreactivity and hormone binding properties. Truncated mRNAs which lack the receptor B protein translation signal are capable of generating the receptor A protein by initiation of translation at a second internal start site. We conclude from these data that the chicken progesterone receptor A and B proteins arise most likely by alternate initiation of translation from a single mRNA transcript.

The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex

Highly purified chicken progesterone receptor (cPR) is shown to stimulate RNA synthesis directly in an in vitro transcription assay. Stimulation of transcription by cPR requires the presence of progesterone response elements (PREs) in the template and can be specifically inhibited by addition of competitor oligonucleotides containing PREs. Binding of receptor to two PREs is cooperative and leads to synergistic (27-fold) stimulation of transcription. A purified fusion protein containing the DNA binding domain of cPR linked to yeast ubiquitin was produced in E. coli and also functions in the transcription assay. Using this in vitro transcription system, we demonstrate that hormone-free cPR activated by salt treatment induces transcription of a test gene in a hormone-independent manner. Finally, we present evidence that the progesterone receptor acts by facilitating the formation of a stable preinitiation complex at the target gene promoter and thus augments the initiation of transcription by RNA polymerase II.

An Alternative Ligand-Independent Pathway for Activation of Steroid Receptors

Publisher Summary Classical steroid hormones such as estrogens, progestins, androgens, glucocorticoids, and mineralocorticoids act via binding to specific intracellular receptors. These receptors are DNA-binding transcription factors, which, in turn, regulate the amount of mRNA transcripts emanating from target genes. The cognate aporeceptors for steroid hormones are usually found in a complex with heat shock proteins before ligand-dependent activation. Ligand binds and induces an allosteric conformational change in the receptor, which causes heat shock proteins to be shed, and facilitates dimerization of the receptors. Pharmacologic inhibition of cellular phosphatases could enhance ligand-dependent activation of the chicken progesterone receptor (cPR). Also, inhibition of cellular kinases can dampen ligand-dependent activation of cPR. The steroid receptor pathway can be activated alternatively from the membrane via pathway convergence, but the average cell responses are likely to be more complicated.

Phosphorylation of hen progesterone receptor by cAMP dependent protein kinase.

Abstract Progesterone receptor A and B subunits from laying hen oviducts were highly purified and their phosphorylation by cAMP-dependent protein kinase from bovine heart was studied. Both proteins are phosphorylated by the kinase using physiological or subphysiological concentrations of the enzyme. This result indicates that the receptors are good substrates. The reaction is dependent upon exogenous enzyme; no phosphorylation is seen in the absence of protein kinase.

Purification and characterization of the chick oviduct progesterone receptor a subunit

Abstract The DNA-binding A subunit of the chick oviduct progesterone receptor has been purified approximately 20,000-fold to apparent homogeneity. The procedure employs phosphocellulose, DNA-cellulose and DEAE-cellulose column chromatography. Purity has been assessed by polyacrylamide gel electrophoresis in two denaturing systems. The protein is obtained with about 20% of the available ligand sites complexed with labeled progesterone. Dissociation kinetics and displacement studies show the protein is identical to the crude starting material. Molecular weight (Mr) determined by gel electrophoresis in l% Na dodecyl SO 4 is 79,000. Values for Stokes radius (46 A) and sedimentation coefficient (3.6 S) are in agreement with the properties of the crude A protein and give an indicated molecular weight of 72,000 for the native protein. The purified protein retains its DNA-binding ability to both DNA cellulose columns and to free DNA using a nitrocellulose filter adsorption assay. The latter assay shows no saturability nor DNA sequence specificity under the conditions employed. The binding is enhanced 8-fold by heat denaturing the DNA.

Molecular mechanism of action of a steroid hormone receptor.

Publisher Summary This chapter presents experimental approaches to elucidate the precise molecular mechanism by which steroid receptors regulate the initiation of target gene transcription and the role of the hormonal ligand in this process. For determining the direct actions of receptor on DNA transcription, a cell-free—reconstituted—transcription system is required. In such a system, the concentration of receptor, ligand, general transcription factors, and target genes could be manipulated. In the initial experiments, the effect of purified cPR (50–80% pure) or a purified cPR derivative expressed in Escherichia coli are measured on transcription of templates that lacked or contained progesterone response elements (PREs). To examine the mechanism by which progesterone receptor interacts with general transcription factors to stimulate RNA synthesis in vitro, it was investigated whether the receptor participates in the formation of a stable preinitiation—rapid start—complex of transcription factors. In this investigation described in the chapter, in an attempt to investigate the mechanism by which regulatory proteins such as steroid hormone receptors interact with core promoters to enhance transcription, the cell-free transcription system was used.

A review of regulation of gene expression by steroid hormone receptors.

Abstract We have investigated the manner by which progesterone receptors act to induce initiation of RNA synthesis in a cell free system derived from chick oviduct. A method utilizing rifampicin enabled us to measure the formation of binary initiation complexes between RNA polymerase and chick oviduct chromatin and allowed for the quantitative assessment of RNA chain initiation sites. RNA chain propagation rates, and RNA chain size under conditions which prevent secondary chain reinitiations. Purified progesterone-receptor complex stimulated transcription of oviduct chromatin in vitro by promoting an increase of 3000 to 5000 additional sites for RNA chain initiation. These data showed that progesterone receptor can directly increase the number of RNA polymerase initiation sites in the chromatin template in the absence of a detectable change in either the rate of RNA propagation or the size of the RNA product. The stimulatory effect of the purified progesterone receptor was time dependent (T 1 2 of 15 min) and the concentration of receptor for half maximal stimulation of RNA chain initiation was ~5 × 10 −9 M, a value which correlates directly with the receptor affinity for binding to chromatin. The stimulatory effect of the purified progesterone receptor appeared to be relatively specific for oviduct chromatin in comparison to non-target tissue chromatins or DNA. Utilizing a copy DNA probe to ovalbumin mRNA, a greater than ten-fold increase in transcription of the ovalbumin gene was detected after the incubation of progesterone receptor with oviduct chromatin. The data presented here show that steroid hormone receptor complexes can directly regulate aene transcription in vitro in a manner which mimics the events observed in vivo in target cells.

Selective binding of chicken progesterone receptor A subunit to a DNA fragment containing ovalbumin gene sequences.

Summary Progesterone receptor A protein, purified to near-homogeneity from laying hen oviducts, binds preferentially to a cloned fragment of the chicken ovalbumin gene containing a portion of transcribed gene sequences, including the transcriptional start site, as well as extensive sequences preceding the 5′ end of the ovalbumin gene. As revealed using a nitrocellulose filter adsorption assay to isolate receptor-DNA complexes, the non-specific interaction of receptor A with DNA is characterized by selective binding to the largest DNA fragments present in mixtures of bacterial plasmid DNA. In contrast, the smaller ovalbumin gene fragment is preferentially bound from a mixture containing a larger plasmid DNA fragment. Furthermore, selectivity for the ovalbumin gene fragment is retained even in the presence of a six-fold molar excess of nonradioactive cloned chicken DNA containing sequences from the analogous region of the adult chicken β-globin gene. Direct competition studies using mixtures of ovalbumin and globin 5′-DNA sequences show at least a ten-fold preference by receptor for the hormone-responsive ovalbumin gene.

Inactivation of chick oviduct progesterone receptors

Abstract We have studied the effect of sodium fluoride and sodium molybdate on temperature-dependent inactivation of the chick oviduct progesterone receptor. Both fluoride and molybdate stabilize the receptors hormone-binding capacity in crude cytosol at 25° and 30°C. However, no protective effect is seen when receptors have first been dissociated by KCl treatment, ammonium sulfate fractionation, or exhaustive dialysis. Furthermore, sucrose gradient analysis reveals that molybdate and fluoride stabilize progesterone receptors in the aggregate form. We conclude that these ions may be acting not on a phosphatase in the cytosol, but directly on the receptor itself.