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N L Weigel

Publications and source records attributed to N L Weigel.

72 records · Page 4Linked to original sources
PubMed1990

Regulation of in vitro transcription by progesterone receptor. Characterization and kinetic studies.

We have devised an in vitro assay system to study the transcriptional activity of native chicken progesterone receptor (cPR). Purified cPR added to cell-free extracts from HeLa cell nuclei stimulates accurate transcription from a promoter driven by two progesterone response elements. The transcriptional enhancement is entirely progesterone response element dependent and promoter specific. We have defined the appropriate conditions of template, nuclear extract, salt, and magnesium ion requirements for efficient transcriptional enhancement by cPR. Under optimized conditions, a synthesis rate of one transcript/20 promoters is achieved in the presence of saturating amounts of cPR. Kinetic studies suggest that the progesterone receptor can form a functional preinitiation complex with RNA polymerase II and other transcription factors present in unfractionated HeLa nuclear extract. Following formation of this complex, transcription can commence rapidly upon addition of nucleotides.

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

Dimerization of the chicken progesterone receptor in vitro can occur in the absence of hormone and DNA.

We have analyzed the dimerization of two forms of the chicken progesterone receptor (cPRA and cPRB) by nondenaturing gradient gel electrophoresis and chemical cross-linking with dimethylpimelimidate (DMP). We demonstrate by these two methods that the PRs assemble in vitro into dimers in the absence of DNA, and that dimerization does not require hormone. The cPRA homodimer binds quantitatively to its cognate DNA response element in our nondenaturing gradient gel assay. DMP cross-linking confirms that both forms of the receptor (cPRA and cPRB) assemble into dimers in solution. Finally, in a standard mobility shift assay, chemically cross-linked receptors bind to the progesterone DNA response element with high affinity. We conclude that the PR contains a dimerization motif, which can promote stable subunit-subunit contacts without the presence of hormone in vitro. The complex thus formed expresses sequence-specific DNA-binding activity indistinguishable from that observed in the presence of hormone.

Animals↗
PubMed1989

Expression of functional chicken oviduct progesterone receptors in yeast (Saccharomyces cerevisiae).

The cDNAs encoding full-length chicken oviduct progesterone receptor B (PRB) and a truncated receptor (C1C2) lacking the amino-terminal domain were expressed in yeast (Saccharomyces cerevisiae) using a ubiquitin fusion system. The expression of the fusion protein is under the control of a copper-responsive yeast metallothionein promoter, and the fusion protein is subsequently cleaved by the yeast host enzyme to produce receptor protein. Western immunoblot analyses of yeast extracts containing full-length PRB revealed a polypeptide co-migrating with authentic chicken oviduct PRB. Using a polyclonal antibody (907) directed against the "hinge" region of the authentic chicken progesterone receptor, a 42-kDa polypeptide was detected by Western analysis in yeast extracts containing C1C2 receptors. Standard hormone binding assays indicated that these receptors produced in yeast cells exhibited steroid binding affinity and specificity characteristic of the authentic chicken progesterone receptor. To test for progesterone receptor-mediated activation of transcription in yeast, reporter plasmids were constructed to transform yeast cells expressing PRB or C1C2 receptors. The reporter gene contained two copies of a progesterone response element upstream of the yeast proximal CYC1 promoter fused to the beta-galactosidase gene of Escherichia coli. The induction of beta-galactosidase activity by PRB and C1C2 was strictly dependent on specific ligand and the presence of a progesterone response element. However, overproduced C1C2 receptors had an adverse effect on the transcription of the lacZ gene. It was found that when overproduced C1C2 was activated by progesterone, an inhibitory effect on normal yeast cell growth was evident. These observations suggest that C1C2 is a potent trans-acting factor in yeast and that the amino-terminal domain of the chicken progesterone receptor may play a role in selective modulation of target gene activation.

Animals↗
PubMed1989

Antibodies to chicken progesterone receptor peptide 523-536 recognize a site exposed in receptor-deoxyribonucleic acid complexes but not in receptor-heat shock protein-90 complexes.

We have prepared monospecific polyclonal rabbit antibodies to the peptide sequence 523-536 of the chicken progesterone receptor. This region, located between the DNA-binding and hormone-binding domains, is predicted by hydropathic analyses to be on the surface of the protein. The synthetic peptide was coupled to keyhole limpet hemocyanin and injected into rabbits. Three rabbits produced antibodies; all three are specific for progesterone receptors, recognize both native and denatured receptor, and do not interfere with either hormone binding or receptor recognition of its DNA response element in gel retardation assays. However, the antibodies do not interact with cytosolic 8S receptor complexes which contain the heat shock protein hsp90, suggesting that this site is occluded in the 8S complex. In contrast, the antibodies recognize a type of receptor dimer which forms on the DNA response element. Thus, these antibodies are a unique tool for studying receptor protein-protein interactions.

Amino Acid Sequence↗
PubMed1989

Hormone-dependent regulation of chicken progesterone receptor deoxyribonucleic acid binding and phosphorylation.

To further understand the structure-function relationships of the chicken oviduct progesterone receptor, the effects of in vivo and in situ progesterone treatment were studied. Immunoprecipitated receptors isolated from oviduct slices incubated in the presence of H(3)32PO4 exhibited hormone-dependent phosphorylation. This was correlated with an increase in the apparent mol wt of receptors when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increased DNA binding of total cytosolic receptors. Further, in vivo progesterone treatment resulted in dissociation of both the A and B receptor forms from nonhormone-binding proteins (such as heat shock protein-90) in vitro when analyzed by sucrose gradient ultracentrifugation. The 4S and 8S receptors were separated by phosphocellulose column chromatography, treated with ammonium sulfate to convert all receptors to DNA-binding forms, and analyzed for binding to DNA cellulose. The 4S receptor produced as a consequence of in vivo hormone treatment had a 3.35-fold higher affinity for DNA and bound to about a 3-fold greater extent than receptor that did not show altered interaction with other proteins. Thus, in vivo progesterone treatment results in increased receptor phosphorylation, altered interaction with heat shock protein-90, and increased DNA binding.

Ammonium Sulfate↗
PubMed1989

Hormone-induced changes in the in vitro DNA-binding activity of the chicken progesterone receptor.

Previous analyses have indicated that steroid hormone receptors undergo an allosteric change in structure upon binding by the steroid ligand. This structural change was envisioned as an intramolecular unmasking of the protein's DNA-binding domain, thus allowing the receptor to function in gene regulation. We report an analysis of the effect of hormone on the DNA-binding activity of the chicken progesterone receptor. Using an isocratic elution of DNA affinity columns we show that unliganded receptor (aporeceptor) can bind a 23-basepair progesterone response element with high affinity and a high degree of sequence preference. Hormone causes a 1.5-fold increase in affinity for the PRE sequence and a 2-fold decrease in affinity for non-specific DNA. Kinetic analysis of the off-rate of receptor-DNA complexes is consistent with this minor effect of hormone. In addition, gel retardation analysis of receptor-progesterone response element complexes further substantiates that hormone is not required for sequence-specific DNA binding. These results indicate that hormone is not necessary for the progesterone receptor to fold into a conformation that recognizes specific gene regulatory sequences.

Animals↗
PubMed1988

Molecular interactions of steroid hormone receptor with its enhancer element: evidence for receptor dimer formation.

A steroid hormone responsive element (GRE/PRE), sufficient to confer glucocorticoid and progesterone inducibility when linked to a reporter gene, was used in band-shift assays to examine its molecular interactions with steroid hormone receptors. Both progesterone and glucocorticoid receptors bound directly and specifically to the GRE/PRE. The purine contact sites for both form A and form B chicken progesterone receptor, as well as those for rat glucocorticoid receptor, are identical. A peptide fragment produced in bacteria that primarily contain the DNA binding domain of the glucocorticoid receptor binds first to the TGTTCT half-site of the GRE/PRE, and a second molecule binds subsequently to the TGTACA (half-site) of the GRE/PRE in a cooperative manner. Utilizing the peptide fragment and the protein A-linked fragment, we demonstrated that the receptor interacts with its cognate enhancer as a dimer.

Animals↗
PubMed1987

High-yield high-performance liquid chromatographic analysis of steroid hormone receptors on glass columns.

The HPLC characteristics of extensively purified chicken oviduct progesterone receptors were compared on TSK 3000 SW size-exclusion and DEAE-5-PW media packed in either glass or stainless-steel columns. Recoveries of [3H]progesterone-labeled receptor from size exclusion were 75-95% in glass columns and less than 10% in stainless-steel columns. Similarly, recoveries from DEAE were greater than 90% in glass columns but only approximately 45% in stainless-steel columns. Recoveries in glass columns were similar on several HPLC systems. Thus, the requisite component for high yields from extensively purified receptor preparations was the glass column itself. While receptor B exhibited ionic strength-dependent mobility similar to several standard proteins on size-exclusion glass column HPLC, receptor A was very peculiar. Resolution of receptors A and B was superior to previous reports using size exclusion open-end chromatography. We also resolved functionally active proteolytic fragments. Finally, the generality of glass column size-exclusion HPLC was demonstrated by high-yield analysis of different steroid hormone receptors from different tissues and species.

Animals↗
PubMed1987

Phosphorylation of the chicken progesterone receptor.

We have examined the phosphorylation of the chicken progesterone receptor in tissue slices and in vitro. The receptor is phosphorylated in tissue slices and this phosphorylation is stimulated by progesterone. As others have reported, partially purified receptor preparations contain a kinase activity which phosphorylates histones and receptor. We have shown that this activity can be separated from the receptor. The receptor is a substrate for several kinases, including the catalytic subunit of the cAMP-dependent protein kinase and PPdPK, a polypeptide-dependent protein kinase. Phosphorylation by the cAMP-dependent protein kinase results in an apparent increase in the molecular weight of the receptor when the receptor is analyzed by SDS-PAGE. These results are consistent with apparent changes in molecular weight observed for rabbit and human progesterone receptor upon treatment of tissue or cells with hormone.

Animals↗
PubMed1986

Amino acid sequence of a chicken heat shock protein derived from the complementary DNA nucleotide sequence.

The complete nucleotide sequence for a chicken heat shock protein (hsp108) was determined from cDNA clones isolated from hen oviduct and bursal lymphoma recombinant DNA libraries. This protein has certain biochemical similarities to the progesterone receptor, but it is clearly distinct from it. The initial cDNA clone, isolated from a chicken oviduct cDNA library, was detected by antibody screening and hybrid-selected translation [Zarucki-Schulz, T., Kulomaa, M. S., Headon, D. R., Weigel, N. L., Baez, M., Edwards, D. P., McGuire, W. L., Schrader, W. T., & O'Malley, B. W. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6358-6362]. The earlier clones were used to screen for additional cDNAs, and cDNAs that define the entire mRNA sequence of hsp108 have been obtained. The nucleotide sequence codes for peptides present in hsp108 as determined by protein microsequencing. The 5' end of the mRNA was determined by primer extension studies. The mRNA contains a noncoding region of 101 nucleotides upstream from the predicted initiation codon. The 3' untranslated region contains 244 nucleotides beyond the termination codon, and it contains a predicted polyadenylation signal 26 nucleotides from the end of the complete cDNA. The coding region of 2385 nucleotides corresponds to a polypeptide chain of 795 amino acids, giving a molecular weight of 91,555 for the hsp108 protein. In another paper, evidence is presented that hsp108 shows a high degree of amino acid sequence homology with two heat shock proteins, hsp90 (yeast) and hsp83 (Drosophila), and is indeed inducible by heat shock [Sargan, D. R., Tsai, M.-J., & O'Malley, B. W. (1986) Biochemistry (following paper in this issue)].

Amino Acid Sequence↗
PubMed1984

Structural analysis of chicken oviduct progesterone receptor using monoclonal antibodies to the subunit B protein.

Two monoclonal antibodies against the B subunit (Mr 108 000) of chick oviduct progesterone receptor (PgR) were produced by immunizing rats and fusing spleen cells with NS-1 mouse myeloma cells. The hybridoma lines designated 9G10 and 3E8 produce rat IgG2a and IgG2b, respectively. Antibody-receptor interactions were demonstrated under protein denaturing conditions. Previous studies by Weigel et al. [Weigel, N. L., Tash, J. S., Means, A. R., Schrader, W. T., & O'Malley, B. W. (1981) Biochem. Biophys. Res. Commun. 102, 513-519] have shown that chick PgR can be phosphorylated in vitro. Both antibodies, 9G10 and 3E8, were shown to displace partially denatured 32P-labeled PgR from its characteristic 4S position on high salt sucrose density gradients to a form with a higher sedimentation coefficient. Further specificity and sensitivity were demonstrated by protein immunoblotting experiments. In partially purified as well as electrophoretically pure receptor B subunit preparations antibodies reacted with the Mr 108 000 receptor B band. By immunoblot assay 9G10 was 20-fold more sensitive than 3E8, the former detecting down to 5 ng of receptor and the latter 100 ng. Because of its sensitivity 9G10 was able to detect the Mr 108 000 receptor as a single band in a crude oviduct fraction and did not cross-react with any other contaminating proteins. Receptor antigenic determinants were localized by immunoblot assay of receptor proteolytic digests.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Monoclonal antibody to the hen oviduct progesterone receptor produced following in vitro immunization.

The progesterone receptor of the hen oviduct is composed of two non-identical hormone-binding polypeptide subunits, A (Mr = 79,000) and B (Mr = 108,000). We used a highly purified preparation of B to immunize mouse spleen cells in vitro. After 5 days in culture, the cells were fused with SP2/0-Ag 14 myeloma cells. The resultant hybridomas were screened using an enzyme linked immunosorbent solid phase assay, and those hybridomas producing antibodies binding to the immunogen were cloned by limiting dilution. One such clone, 9B3-12, secreted an antibody of immunoglobulin class IgM, which binds to B. This was indicated by the ability of the antibody to increase the rate of sedimentation coefficient of the B subunit. Further, when the proteins in the B preparation were separated by electrophoresis and blotted onto nitrocellulose filters the antibody bound to a protein of 108,000 daltons. The antibody produced by 9B3-12 also reacted with subunit A and with the human progesterone receptor but failed to bind to the chick liver glucocorticoid receptor or to progesterone in the absence of its receptor.

Animals↗
PubMed1984

Molecular cloning of a cDNA for the chicken progesterone receptor B antigen.

A cDNA for the chicken progesterone receptor B subunit antigen (Mr, 108,000) has been isolated from a cDNA library prepared from size-selected chicken oviduct poly(A)+RNA. A specific monoclonal antibody raised against hen progesterone receptor B subunit (alpha PR-B) was used to screen the library. Recombinant clones reacting with the antibody by virtue of antigen expression were used in hybrid-selected translation. A single clone, pPRB-1, hybridized specifically to a mRNA that yielded a Mr 108,000 protein when translated in vitro and which was immunoprecipitable by the alpha PR-B antibody. This cDNA represents a 470-base-pair portion of the PR-B nucleotide sequence. Additional clones have been subsequently isolated from the recombinant library using the insert from pPRB-1 as a specific probe. A mRNA size of approximately 3000 nucleotides was determined for the chicken progesterone receptor B subunit by formaldehyde/agarose gel electrophoresis and blot hybridization using pPRB-1 as a probe. Preliminary studies show that withdrawal of hormone from chickens treated chronically with estrogen leads to a dramatic decrease in the cellular RNA concentration of receptor B, indicating that target tissue levels of receptor B RNA are under hormonal control.

Animals↗
PubMed1981

Analysis of chicken progesterone receptor structure using a spontaneous sheep antibody.

A spontaneous sheep antibody to chick progesterone receptor was characterized and used as a tool to study the receptor structure. The antibody, which is present to some extent in sera from about one-third of the sheep tested, binds to Staphylococcus aureus protein A--Sepharose and therefore appears to be an Igg. It is specific for the chick progesterone receptor and does not react with free progesterone or with any of the other proteins tested, including other receptors and corticosteroid binding globulin. The antibody os nonprecipitating and has a vary low titer (equivalence point = 2.5 pmol of receptor/mL of serum). The interaction of the receptor with the antibody was measured, and an apparent dissociation constant of 2 x 10(-9) M was determined from these studies. The antibody reacts equally well with the two receptor subunits A and B but does not appear to react with the native aggregate form found in the cytosol. Thus, the immunologic site is occluded in the aggregate, and therefore the antibody will be a useful probe for this important region of the proteins. The antibody recognition sites on the receptors were further characterized by analysis of a proteolytic digest of receptors by using an endogenous Ca2+-activated neutral protease. Competition studies using native receptor and receptor digests demonstrated that the antigenic site was not destroyed in the digest and was separated from the hormone binding fragment. We conclude that receptor subunits A and B have a cross-reactive immunologic site on a portion of the molecule other than the hormone binding domain.

Animals↗
PubMedDate not supplied

Differential expression of uterine progesterone receptor forms A and B during the menstrual cycle.

Recent studies suggest that the progesterone receptor isoforms (PR-A and PR-B) activate genes differentially and that PR-A may act as a repressor of PR-B function. Hence, the absolute and relative expression of the two isoforms will determine the response to progesterone. We have measured their relative expression in the uterus of cycling women who underwent endometrial biopsy. PR isoforms were identified on blots of SDS-PAGE gels by reaction with the AB-52 antibody after immunoprecipitation from endometrial extract. Both isoforms were highest in the peri-ovulatory phase, but levels of PR-A were always higher than those of PR-B. The ratio of PR-A to PR-B changed during the menstrual cycle. Between days 2 and 8, PR-B is almost undetectable and the A:B ratio is >10:1. From days 9 to 13, the ratio is about 5:1, and it is about 2:1 between days 14 and 16. Thereafter, PR-B dwindles rapidly and is virtually undetectable at the end of the cycle. In various hypoestrogenic environments, PR-B expression was reduced. However, exogenous estrogens in the follicular phase in the form of oral contraceptives, enhanced PR-B expression. These data support the possibility that progesterone acts through cycle-specific PR isoforms.

Contraceptives, Oral, Hormonal↗