Characterization and Functional Properties of the A and B Forms of Human Progesterone Receptors Synthesized in a Baculovirus System

Christensen, Kurt D.; Estes, Patricia A.; Oñate, Sergio A.; Beck, Candace A.; DeMarzo, Angelo M.; Altmann, Magda; Lieberman, Ben A.; John, Judy St.; Nordeen, Steven K.; Edwards, Dean P.

doi:10.1210/mend-5-11-1755

Cited by 101 publications

(75 citation statements)

References 51 publications

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“…As found previously when we purified endogenous T47D PR (20), alkaline elution of PR from antibody affinity resins is a relatively mild condition that retains the majority (60%) of the steroid originally bound to the native receptor (not shown). It should be noted that expression levels of PR in the present study are much higher (>10-fold) than the originally reported levels, which ranged between 0.1 and 0.2% of protein for expression of PR from these same baculovirus vectors (12). The higher levels of expression are due in part to inclusion of hormone which upregulates PR in Sf9 cells and to performing viral infection of insect cells as suspension rather than as attachment cultures (12).…”

Section: Methodscontrasting

confidence: 62%

See 1 more Smart Citation

The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences.

Oñate¹,

Prendergast²,

Wagner³

et al. 1994

Mol. Cell. Biol.

189

136

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Steroid hormone receptors are ligand-dependent transcriptional activators that exert their effects by binding as dimers to cis-acting DNA sequences termed hormone response elements. When human progesterone receptor (PR), expressed as a full-length protein in a baculovirus system, was purified to homogeneity, it retained its ability to bind hormonal ligand and to dimerize but exhibited a dramatic loss in DNA binding activity for specific progesterone response elements (PREs). Addition of nuclear extracts from several cellular sources restored DNA binding activity, suggesting that PR requires a ubiquitous accessory protein for efficient interaction with specific DNA sequences. Here we have demonstrated that the high-mobility-group chromatin protein HMG-1, as a highly purified protein, dramatically enhanced binding of purified PR to PREs in gel mobility shift assays. This effect appeared to be highly selective for HMG-1, since a number of other nonspecific proteins failed to enhance PRE binding. Moreover, HMG-1 was effective when added in stoichiometric amounts with receptor, and it was capable of enhancing the DNA binding of both the A and B amino-terminal variants of PR. The presence of HMG-1 measurably increased the binding affinity of purified PR by 10-fold when a synthetic palindromic PRE was the target DNA. The increase in binding affinity for a partial palindromic PRE present in natural target genes was greater than 10-fold. Coimmunoprecipitation assays using anti-PR or anti-HMG-1 antibodies demonstrated that both PR and HMG-1 are present in the enhanced complex with PRE. HMG-1 protein has two conserved DNA binding domains (A and B), which recognize DNA structure rather than specific sequences. The A-or B-box domain expressed and purified from Escherichia coli independently stimulated the binding of PR to PRE, and the B box was able to functionally substitute for HMG-1 in enhancing PR binding. DNA ligase-mediated ring closure assays demonstrated that both the A and B binding domains mediate DNA flexure. It was also demonstrated in competition binding studies that the intact HMG-1 protein binds to tightly curved covalently closed or relaxed DNA sequences in preference to the same sequence in linear form. The finding that enhanced PRE binding was intrinsic to the HMG-1 box, combined with the demonstration that HMG-1 or its DNA binding boxes can flex DNA, suggests that HMG-1 facilitates the binding of PR by inducing a structural change in the target DNA.

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Section: Methodscontrasting

confidence: 62%

“…PR-rich T47D human breast cancer cells and PR-negative MDA231 cells were cultured as previously described (19,20). Spodoptera frugiperda (Sf9) insect cells were grown at 27°C in Grace's insect medium (GIBCO) supplemented with 3.3 g of yeastolate (GIBCO) per liter, 3 (12,19,20).…”

Section: Methodsmentioning

confidence: 99%

The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences.

Oñate¹,

Prendergast²,

Wagner³

et al. 1994

Mol. Cell. Biol.

189

136

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“…PR-B was expressed in baculovirus-infected Sf9 insect cells as previously described (18). A detailed description of the PR-B purification and a quantitative analysis of its solution properties were also previously described (19).…”

Section: Expression and Purification Of The Full-length B-isoform Of mentioning

confidence: 99%

Cooperative DNA Binding by the B-Isoform of Human Progesterone Receptor: Thermodynamic Analysis Reveals Strongly Favorable and Unfavorable Contributions to Assembly

et al. 2006

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Progesterone receptors (PR) play critical roles in eukaryotic gene regulation, yet the mechanisms by which they assemble at multi-site promoters are poorly understood. Here we present a thermodynamic analysis of the interactions of the PR B-isoform (PR-B) with promoters containing either one or two progesterone response elements (PREs). Utilizing quantitative footprinting, we have resolved the microscopic energetics of PR-B binding, including cooperativity terms. The results of this analysis challenge a number of assumptions found in traditional models of receptor function. First, PR-B interactions at a single PRE can be equally well described by mechanisms invoking either the receptor monomer or the dimer as the active DNA binding species. If, as is commonly accepted, PR-B interacts with response elements only as a pre-formed dimer, then its intrinsic binding affinity is not the typically observed nanomolar, but is rather picomolar. This high affinity binding is opposed, however, by a large energetic penalty. The penalty presumably pays for costly structural rearrangements of the receptor dimer and/or response element that are needed to form the protein-DNA complex. If PR-B assembles at a single response element via successive monomer binding reactions, then this penalty minimizes cooperative interactions between adjacent monomers. When binding to two response elements, the receptor exhibits strong inter-site cooperativity. Although this phenomenon has been observed before, the present work demonstrates that the energetics reach levels seen in highly cooperative systems such as λ cI repressor. This first quantitative dissection of cooperative receptor-promoter interactions suggests PR-B function is more complex than traditionally envisioned.Progesterone receptors (PR) are members of the nuclear receptor superfamily of ligandregulated transcription factors(1). An understanding of PR function is complicated by the fact that the receptor exists as two naturally occurring isoforms: an 83 kDa A-receptor (PR-A) and a 99 kDa B-receptor (PR-B). The two proteins are identical except for a 164 amino acid extension at the N-terminus of PR-B (See Figure 1). Both isoforms are characterized by a centrally located DNA binding domain (DBD) and a C-terminal hormone binding domain (HBD). The two domains are linked by a 50 amino acid "hinge" sequence of unclear function. Transcriptional activation functions are located N-terminal to the DBD (AF-1) and within the HBD (AF-2). The 164 residues unique to PR-B contain a context-dependent transcriptional activation function (AF-3) (2). ‡ This work was supported by NIH grants R01-DK061933 to DLB and F32-DK070519 to AFH, and the Tissue Culture Core Laboratory of the UCHSC Cancer Center. * To whom correspondence should be addressed: Department of Pharmaceutical Sciences, C-238, University of Colorado Health Sciences Center, 4200 E. 9 th Ave, Denver, CO 80262. Phone: 303-315-1416. Fax: 303-315-0274. E-mail: David.Bain@UCHSC.edu. NIH Public Access Author ManuscriptBiochemistry. Auth...

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“…A general description of the different groups is given in the text. In group I, GR, MR, and PR are bound to HSP in the cytoplasm while estrogen receptor (ER), PR, and androgen receptor (AR) are reportedly bound to HSP in the nucleus (14,21,77). In group II, a ligand (stoichiometry not specified) may promote the association or dissociation of homo-and heterodimers in solution and on DNA depending on the ligand, the receptors, and the response elements involved.…”

Section: Vol 15 1995 Homodimerization Of Hnf-4 5139mentioning

confidence: 99%

Exclusive Homodimerization of the Orphan Receptor Hepatocyte Nuclear Factor 4 Defines a New Subclass of Nuclear Receptors

Jiang

Nepomuceno

Hopkins

et al. 1995

Molecular and Cellular Biology

184

162

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Hepatocyte nuclear factor 4 (HNF-4), a highly conserved member of the steroid hormone receptor superfamily critical for development and liver-specific gene expression, is very similar to another superfamily member, retinoid X receptor ␣ (RXR␣), in overall amino acid sequence and DNA binding specificity. Since RXR␣ is known to heterodimerize with many other nuclear receptors, the formation of heterodimers between HNF-4 and RXR␣ was examined. With the electrophoretic mobility shift assay, coimmunoprecipitation, and transient transfection assays, it is shown that, unlike other nuclear receptors, HNF-4 does not form heterodimers with RXR␣ either in the presence or in the absence of DNA. We also show that in vitro-translated HNF-4 does not form heterodimeric complexes on DNA with a number of other receptors, including RXR␤, RXR␥, retinoic acid receptor ␣, or thyroid hormone receptor ␣. To investigate the hypothesis that the lack of heterodimerization between HNF-4 and RXR␣ is due to a strong homodimerization activity of HNF-4, glycerol gradient sedimentation and kinetic analysis were used to show that HNF-4 is in fact a stable homodimer in solution. Finally, immunohistochemistry is used to show that the HNF-4 protein is found exclusively in the nuclei in both HepG2 cells, which express endogenous HNF-4, and transfected COS cells, which overexpress HNF-4. These findings lead us to propose that HNF-4 defines a new subclass of nuclear receptors which reside primarily in the nucleus and which bind DNA and regulate transcription as homodimers.Hepatocyte nuclear factor 4 (HNF-4) is a positive-acting transcription factor which is expressed very early in embryo development and is essential to liver development and function (reviewed in references 84 and 85). Mouse HNF-4 mRNA appears in the primary endoderm of implanting blastocysts at embryonic day 4.5 and in the liver and gut primordia at day 8.5 (20), while mice deficient in HNF-4 do not survive past day 9 postcoitus (12). HNF-4 has also been proposed to be responsible for the final commitment for cells to differentiate into hepatocytes (68). In adult rodents, HNF-4 is located primarily in the liver, kidney, and intestine, and in insects HNF-4 is found in the equivalent tissues (86, 95). HNF-4 is known to activate a wide variety of essential genes, including those involved in cholesterol, fatty acid, and glucose metabolism; blood coagulation; detoxification mechanisms; hepatitis B virus infections; and liver differentiation (reviewed in references 84 and 85).HNF-4 is a member of the superfamily of ligand-dependent transcription factors, which includes the steroid hormone receptors, thyroid hormone receptor (TR), vitamin A receptor, and vitamin D receptor (VDR), as well as a large number of receptors for which ligands have not yet been identified, the so-called orphan receptors (reviewed in references 56, 72, 73, and 88). All receptors are characterized by two conserved domains: the zinc finger region, which mediates DNA binding, and a large hydrophobic domain which mediate...

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Characterization and Functional Properties of the A and B Forms of Human Progesterone Receptors Synthesized in a Baculovirus System

Cited by 101 publications

References 51 publications

The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences.

The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences.

Cooperative DNA Binding by the B-Isoform of Human Progesterone Receptor: Thermodynamic Analysis Reveals Strongly Favorable and Unfavorable Contributions to Assembly

Exclusive Homodimerization of the Orphan Receptor Hepatocyte Nuclear Factor 4 Defines a New Subclass of Nuclear Receptors

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