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1,2,3,4-tetrahydro-2,2-dimethyl-6-(trifluoromethyl)-8-pyridono[5,6-g]quinoline (TDPQ), a selective non-steroidal ligand of the androgen receptor (AR), is a fluorescent compound we found to be photocytotoxic. We have characterized its spectral properties in comparison to the structural precursor carbostyril 151 (C151) and to its racemic structural isomer ETPQ. The absorption maximum in CH3CN of either TDPQ or ETPQ is 400 nm whereas that of C151 is 350 nm. The fluorescence lifetimes (τ) and quantum yields (ϕf) in CH3CN are typical for fluorescent dyes: TDPQ (4.2ns, 0.8) and ETPQ (4.6ns, 0.76). C151 showed lower τ and ϕf of 0.2ns, and 0.02. Thus, from the spectral point of view, TDPQ can function as a fluorescent label in the (sub)micromolar concentration range. While the fluorescence maxima of the compounds were solvent insensitive, the ϕf for ETPQ decreased in aqueous solutions regardless of the presence of albumin or DNA. The ϕf of TDPQ was less affected. The quantum yield of singlet oxygen (1O2) photosensitization (ϕso) by TDPQ and ETPQ was about 7% in CH3CN, which is sufficient to induce photocytotoxicity. We detected TDPQ fluorescence in human breast tumor cells using confocal microscopy. Using AR-negative MDA-MB-231 and AR-positive MDA-MB-453 cells, we confirmed that TDPQ was photocytotoxic in the AR-positive breast cancer cells. The combination of the well-defined AR activity with the photocytotoxic potential makes TDPQ a promising candidate for the selective targeting of AR-positive malignant cells during photodynamic therapy.

A series of androgen receptor modulators based on 8H-[1,4]oxazino[2,3-f]quinolin-8-ones was synthesized and evaluated in an androgen receptor transcriptional activation assay. The most potent analogues from the series exhibited single-digit nanomolar potency in vitro. Compound 18h demonstrated full efficacy in the maintenance of muscle weight, at 10 mg/kg, with reduced activity in prostate weight in an in vivo model of androgen action.

The androgen receptor is a member of the extended family of nuclear receptors and is widely distributed throughout the body. Androgen therapy is used to compensate for low levels of the natural hormones testosterone (T) and dihydrotestosterone and consists of administration of T, prodrugs thereof, or synthetic androgens. However, currently available androgens have many drawbacks. We identified 6-dialkylamino-4-trifluoromethylquinolin-2(1H)-ones as orally available tissue-selective androgen receptor modulators.

Holding a Ph.D. or other doctoral degree in the biological sciences used to allow for only one main career path. Faculty positions at either research-intensive universities or at predominantly teaching institutions defined the career path very well. The situation is considerably more complex now. Many individuals decide to pursue careers in for-profit organizations, either by personal preference, geographic needs, or simply as an expedient way to continue in biology in a meaningful way. This article arises from the need to define a better understanding of the nature of corporate job seeking for potential job applicants. The author draws upon his experience over a thirty-year scientific career in industry and academics. The distinctions he makes are helpful for job-seekers in this new environment.

A series of 1,2-dihydrochromeno[3,4-f]quinoline derivatives was synthesized and tested in biological assays to evaluate the nonsteroidal progesterone receptor modulator pharmacophore (4) as antiprogestins. A number of potent analogues were identified by modification of the substituents at the D-ring.

Two potent nonsteroidal progestins from the 5-aryl-1,2-dihydro-5H-chromeno[3,4-f]quinoline class (LG120746 and LG120747) were selected for scale-up, resolution, and biological evaluation of the purified enantiomers. For each quinoline, the levorotatory enantiomer was determined to be the more potent agonist of the human progesterone receptor isoform B (hPR-B) (EC50 < 3 nM), but the dextrorotatory enantiomers retained significant PR modulatory activity (EC50 < 200 nM). In two in vivo rodent models of progestational activity, a pregnancy maintenance assay and a uterine wet weight assay, the two eutomers displayed potent progesterone-like effects. In a third model for progestational activity, the mammary end bud assay, these compounds were significantly less active. These studies demonstrate that certain members of this class of selective progesterone receptor modulators display encouraging and potentially useful tissue-selective progestational effects.

The human progesterone receptor form B (hPR-B) was expressed in Saccharomyces cerevisiae together with a specific reporter plasmid. To understand the mechanism underlying antagonist ligand activity, libraries of hormone binding domain (HBD)-mutated hPR-B molecules were prepared. A mutant receptor was identified that had lost the ability to bind either progesterone or R5020; it could still bind RU486 and, surprisingly, fully activated transcription in the presence of this "antagonist" and other antiprogestins. When this receptor mutant was assayed in mammalian cells, RU486 again demonstrated agonistic activity. Sequence analysis indicated that the mutant phenotype was due to truncation of the carboxy (C)-terminal 42 aa. We conclude that amino acids in the extreme C-terminal region are required for the receptor to bind progesterone, while antagonists bind to a site located more N-terminal of the HBD. Our results suggest that the extreme C-terminal region of the receptor contains an inhibitory function that silences receptor transactivation in the absence of agonist and in the presence of antagonist.The function of the hormone-binding domain of the human progesterone receptor was examined in a yeast cell system and in mammalian HeLa cells using mutant receptors, progesterone, the progesterone agonist R502, and the antagonists RU-486, Org31806, and Org31376. The hormone-binding domain, located on the carboxy terminal of the peptide, is known to initiate a conformational change in the receptor upon binding an agonist or antagonist, then shedding of associated proteins including the heat shock protein, dimerization of the receptor, and finally, binding to DNA, leading to transcription. Binding of a progesterone antagonist such as RU-486 elicits all these events except transcription. First the progesterone receptor was inserted in yeast with a plasmid, and a set of mutants were generated, using beta galactosidase as an indicator. A mutant progesterone receptor, U-P1, was selected for mechanistic studies, that binds and was activated by antagonists, but was inactive with progesterone. This receptor had a deletion at base 2636, resulting in a shift of reading frame so that a stop codon 36 nucleotides downstream caused truncation of 54 amino acids at the C-terminus and addition of 12 novel amino acids. Western blot analysis confirmed the expected molecular weight. The mutant receptor was active with RU-486, suggesting that the C-terminus may be responsible for poor transcription with RU-486, suggesting in normal receptors. 2 other truncated mutants were inactive with progesterone. These data suggested that the terminal 42 amino acids of the progesterone receptor are needed to bind progesterone, and that the antagonist is contacting different amino acids than the native receptor, possibly inducing a different conformational change. The activity of the UP-1 mutant was also confirmed in HeLa cells, with the chloramphenicol acetyltransferase reporter system. The results were interpreted to mean that progesterone agonists and antagonists contact at least some different amino acids in the hormone binding domain of the receptor, and that the conformational changes resulting from binding these agents are different. It appears that the C-terminus of the receptor contains an inhibitory domain which, when removed, turns antagonists into agonists.

The progesterone receptor (PR) in the chicken oviduct is a phosphoprotein that regulates gene transcription in the presence of progesterone. Treatment with progesterone in vivo stimulates phosphorylation of the progesterone receptor. With transient transfection assays, the present work has tested whether phosphorylation participates in the regulation of PR-mediated transcription. Treatment with 8-bromo-cyclic adenosine monophosphate (8-Br cAMP), a stimulator of cAMP-dependent protein kinase [protein kinase A (PKA)], mimicked progesterone-dependent, receptor-mediated transcription in the absence of progesterone. Inhibition of PKA blocked hormone action. Treatment with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, stimulated transcription in a manner similar to that of progesterone. These observations suggest that phosphorylation of the PR or other proteins in the transcription complex can modulate PR-mediated transcription in vivo.

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.

The present studies examine the effects of in vivo and in situ progesterone treatment in the regulation of site-specific phosphorylation of the chicken oviduct progesterone receptor (PR). By gas-phase protein sequencing we have identified three hormonally regulated phosphorylation sites: Ser-211, Ser-260, and Ser-530. We determined phosphorylation stoichiometries by analyzing the amounts of phosphorylated and dephosphorylated serine at each site. Stoichiometries of sites 211 and 260 were about 20% under basal conditions and increased 1.5-2-fold by in situ progesterone treatment. Site 530 was virtually absent under basal conditions and induced to greater than 33% by in situ progesterone treatment. We tested several protein kinases for phosphorylation of the PR in vitro on these sites or peptides containing these sites. We found that the catalytic subunit of cAMP-dependent protein kinase mimicked the in vivo, hormone-induced altered mobility of PRs in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the in vivo and in vitro alterations were reversed by alkaline phosphatase. Finally, we showed that cAMP-dependent protein kinase phosphorylated Ser-528.

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 a 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.

Using a novel Escherichia coli system we have successfully overexpressed a region of the chicken progesterone receptor which encodes both the DNA- and hormone-binding domains. The expression system produces the truncated receptor fragment as an in-frame fusion with ubiquitin. This strategy greatly enhances both the solubility and stability of fusion proteins expressed in E. coli. Synthesis has been further improved by induction of the lambda PL promoter with nalidixic acid at low growth temperatures (less than or equal to 30 degrees C) rather than use of conventional heat induction protocols. We can produce 10 mg of receptor fragment/liter of cells using this system, and we estimate that at least 0.3 mg of this receptor material is biologically active, as assessed by DNA-binding and hormone-binding assays. Receptor produced in this manner is almost indistinguishable from authentic oviduct progesterone receptor using the criteria of hormone-binding specificity and affinity and binding to a progesterone response element. This expression system offers a cheap convenient method for the production of mg amounts of biologically active derivatives of progesterone receptor for biochemical studies.

Recent interest in orally available androgens has fueled the search for new androgens for use in hormone replacement therapy and as anabolic agents. In pursuit of this, we have discovered a series of novel androgen receptor modulators derived from 7H-[1,4]oxazino[3,2-g]quinolin-7-ones. These compounds were synthesized and evaluated in competitive binding assays and an androgen receptor transcriptional activation assay. A number of compounds from the series demonstrated single-digit nanomolar agonist activity in vitro. In addition, lead compound (R)-16e was orally active in established rodent models that measure androgenic and anabolic properties of these agents. In this assay, (R)-16e demonstrated full efficacy in muscle and only partially stimulated the prostate at 100 mg/kg. These data suggest that these compounds may be utilized as selective androgen receptor modulators or SARMs. This series represents a novel class of compounds for use in androgen replacement therapy.

A number of conditions, including osteoporosis, frailty, and sexual dysfunction in both men and women have been improved using androgens. However, androgens are not widely used for these indications because of the side effects associated with these drugs. We describe an androgen receptor (AR) ligand that maintains expected anabolic activities with substantially diminished activity in the prostate. LGD2226 is a nonsteroidal, nonaromatizable, highly selective ligand for the AR, exhibiting virtually no affinity for the other intracellular receptors. We determined that AR bound to LGD2226 exhibits a unique pattern of protein-protein interactions compared with testosterone, fluoxymesterone (an orally available steroidal androgen), and other steroids, suggesting that LGD2226 alters the conformation of the ligand-binding domain. We demonstrated that LGD2226 is fully active in cell-based models of bone and muscle. LGD2226 exhibited anabolic activity on muscle and bone with reduced impact on prostate growth in rodent models. Biomechanical testing of bones from animals treated with LGD2226 showed strong enhancement of bone strength above sham levels. LGD2226 was also efficacious in a sex-behavior model in male rats measuring mounts, intromissions, ejaculations, and copulation efficiency. These results with an orally available, nonaromatizable androgen demonstrate the important role of the AR and androgens in mediating a number of beneficial effects in bone, muscle, and sexual function independent from the conversion of androgens into estrogenic ligands. Taken together, these results suggest that orally active, nonsteroidal selective androgen receptor modulators may be useful therapeutics for enhancing muscle, bone, and sexual function.

A series of 5-methylidene 1,2-dihydrochromeno[3,4-f]quinoline derivatives were synthesized and tested in biological assays to evaluate scope and limitations of the nonsteroidal SPRM pharmacophore (3). A number of orally available highly potent nonsteroidal modulators were identified by modification of the substituents at 5-methylidene position.

A series of 5-benylidene-1,2-dihydrochromeno[3,4-f]quinolines (4) were synthesized and tested in bioassays to evaluate their progestational activities, receptor- and tissue-selectivity profiles as selective progesterone receptor modulators (SPRMs). Most of the new analogues exhibited as highly potent progestins with more than 100-fold receptor selectivity over other steroid hormone receptors and LG120920 (7b) demonstrated tissue selectivity toward uterus and vagina versus breasts in a rodent model after oral administration.

Two articles appearing in this month’s issue highlight the next phase in the complexities of the steroid/thyroid receptor family of gene regulatory proteins. Vegeto et al. (1) and Tung et al. (2) using related approaches, have arrived at new observations regarding the potential function of receptor subunits. Both studies add fuel to the argument that says up-regulation and downregulation by these proteins are two sides of the same coin and are entirely cell and gene-context specific. Both papers use the human progesterone receptor (PR), a protein known to be expressed as two different molecular-weight forms. Both forms bind progesterone and in fact are identical except for the N-terminus, which is 164 amino acids longer in form B (PRB) than in the smaller one (PRA). Both bind their ligand with the same kinetics, and both are capable individually of progesterone-dependent regulation of reporter genes in test cells. Now these groups find that form B (the larger form) is active in transient transfection assays in several different cell types. Moreover the report by Tung et a/. (2) shows that ligands previously regarded as “antagonists” such as RU38486 are agonists when PRB is present alone. The notion that these ligands can be agonists in certain circumstances is not new, but previous reports of this activity have shown a requirement for the receptor to be truncated at its c-terminus for agonist activity to be manifest (3-5) or for elevated intracellular phosphorylation to exist (6). The implication of this finding is that the two forms of receptor must somehow interact in the normal physiological context in a way which permutes the structure of PRB to make the RU compound behave as an antagonist. This role for PRA implies that it interacts with PRB and perturbs the structure of its companion in an important way. This latter point is dealt with experimentally very nicely in both papers. The report by Vegeto et al. (1) shows that PRA is inactive by itself in two of the three cell types they tried, but it is a powerful repressor of the activity of PRB. Furthermore, this repressive activity can be achieved with any of three different “antagonists” bound to PRA. Both groups make the remarkable observation that DNA binding activity is not required for this event, at least not sequence-specific interactions of the classical kind. Tung’s group extends the obser

Human progesterone receptor (PR) is phosphorylated on multiple serine residues (at least seven sites) in a manner that involves distinct groups of sites coordinately regulated by hormone and different kinases. Progress on defining a functional role for PR phosphorylation has been hampered both by the complexity of phosphorylation and the lack of simple, nonradioactive methods to detect the influence of ligands and other signaling pathways on specific PR phosphorylation sites in vivo. Toward this end, we have produced monoclonal antibodies (MAbs) that recognize specific phosphorylation sites within human PR including a basal site at Ser 190 (MAb P190) and a hormone-induced site at Ser 294 (MAb P294). Biochemical experiments showed the differential reactivity of the P190 and P294 MAbs for phosphorylated and unphosphorylated forms of PR. Both MAbs recognize specific phosphorylated forms of PR under different experimental conditions including denatured PR protein by Western blots and PR in its native conformation in solution or complexed to specific target DNA. As detected by Western blot of T47D cells treated with hormone for different times, hormone-dependent down-regulation of total PR and the Ser 190 phosphorylation site occurred in parallel, whereas the Ser 294 phosphorylation site was down-regulated more rapidly. This difference in kinetics suggests that the Ser 294 site is more labile than basal sites and is acted upon by distinct phosphatases. A strong preferential hormone-dependent phosphorylation of Ser 294 was observed on PR-B as compared with the amino-terminal truncated A form of PR. This was unexpected because Ser 294 and flanking sequences are identical on both proteins, suggesting that a distinct conformation of the N-terminal domain of PR-A inhibits phosphorylation of this site. That Ser 294 lies within an inhibitory domain that mediates the unique repressive functions of PR-A raises the possibility that differential phosphorylation of Ser 294 is involved in the distinct functional properties of PR-A and PR-B.

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.

This study describes the phosphorylation of chicken progesterone receptor (cPR) produced in yeast, Saccharomyces cerevisiae, and examines the dependence of specific phosphorylations on hormone and DNA binding. The chicken progesterone receptor is expressed in vivo as two forms, cPRB and a smaller form, cPRA. Characterization of the phosphorylation sites in the cPRB form expressed in yeast shows that progesterone receptor is phosphorylated on the three serines (Ser211, Ser260, and Ser530) reported previously in chicken oviduct. An additional site which was phosphorylated in response to hormone was also detected and was subsequently identified as Ser367. Although cPRB and cPRA are phosphorylated identically in chicken oviduct, cPRA expressed in yeast is phosphorylated on Ser211, Ser260, and Ser367, but phosphorylation of Ser530 is almost undetectable. In contrast, cPRB expressed in yeast is phosphorylated on all four sites. No phosphorylations were found in or near the region required for hormone binding, indicating that phosphorylation is not required for hormone binding. In order to determine whether any of the phosphorylations were DNA-dependent, phosphorylation was also studied using cPRA containing a partial deletion of the DNA binding domain. Two of the sites, Ser211 and Ser367, showed reduced phosphorylation in this mutant, suggesting a possible requirement for DNA binding activity for the phosphorylation of these sites. To our knowledge, this is one of the first demonstrations that a eucaryotic protein expressed in yeast is correctly phosphorylated.

A rapidly inducible and tightly regulated system for the expression of protein in yeast is basd on a chimeric promoter constructed of two copies of a vitellogenin-estrogen-response element (ERE) which are inserted upstream from the promoter of the yeast gene encoding iso-1-cytochrome c. The chimeric promoter was inserted in a yeast expression plasmid upstream from the coding sequence of ubiquitin fused in frame to a cDNA encoding the full-length chicken progesterone receptor A (cPRA). The resultant plasmid (YEpA2) was co-transformed in Saccharomyces cerevisiae with a plasmid which encodes the human estrogen receptor. Estradiol (E2)-induced transactivation of the chimeric promoter results in transcription of the cPRA gene from YEpA2, and synthesis of cPRA. The fusion protein, ubiquitin-cPRA, is rapidly cleaved in vivo to produce cPRA. Analysis of samples by Western immunoblot shows that cPRA is almost undetectable in the absence of E2, and that treatment with 50 nM E2 results in a 500–1000-fold induction of cPRA (0.06-0.3% of the total protein) after 1 h. The plasmid-expressed soluble receptor is stable and demonstrates the correct affinity for its ligand. We have prepared yeast extracts using enzymatic digestion of the cell wall with oxalyticase followed by hypotonic shock. This has resulted in a dramatic increase in the % of receptor which binds hormone compared to previous studies which used mechanical disruption techniques. The cPRA is biologically active since it activates transcription of a co-transformed reporter gene containing its response element. This system is well suited for the regulated synthesis of proteins in yeast and may be especially useful for producing labile proteins or proteins toxic to the cells.

We have reported previously that chicken progesterone receptor (PR) is phosphorylated in vivo in response to progesterone administration. Three phosphorylation sites have been reported, two of which show increased phosphorylation in response to hormone and one which is phosphorylated only in response to hormone administration. We found previously that PR lacking the hormone-dependent phosphorylation is active in an in vitro transcription assay. Since the source of general transcription factors is a HeLa nuclear extract which contains many kinases, we have analyzed the receptor for phosphorylation during the in vitro transcription assay. We report here that the receptor is rapidly and efficiently phosphorylated on new sites, causing a change in receptor mobility on sodium dodecyl sulfate-gels. This phosphorylation is strictly dependent upon the presence of double stranded DNA. A DNA-activated protein kinase with similar properties has been isolated previously from HeLa cell nuclei. We find that phosphorylation of PR with this purified enzyme mimics the phosphorylation observed in the transcription assay. These data suggest that a previously undetected additional series of DNA-dependent phosphorylations may be required for activation of the PR.

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.

Steroid receptors are a class of molecules that function as both signal transducers and transcription factors. From cloned sequences it is apparent that steroid receptors and other transcription factors belong to a superfamily of proteins that appear to function by similar mechanisms. Functional domains for hormone and DNA binding, and for transcriptional activation, have been defined for several receptors. In some cases, specific amino acids required for function have been identified. The multi-functional steroid receptor molecules are modular in nature in that domains function independently of structural position in receptor molecules and can even function after insertion into unrelated transactivation proteins. The mechanism of receptor action is complex and multistage and a number of unanswered questions remain to be defined. Receptors are inactive in the absence of hormone in vivo; the proposed components of this inactive complex include several proteins and RNA. Theories on the physiological role of HSP 90 in this complex range from an artifactual interaction to an absolute conformational requirement for hormone binding. Although its function has not been demonstrated clearly yet, there is a consensus that one major function is to inactivate receptor by blocking DNA binding. Most of the steroid receptors appear to be nuclear in the absence of hormone. The transformation process produces a receptor molecule that is capable of specific DNA binding and transcriptional activation. The specificity of DNA binding is conferred by as few as three amino acids in the first finger of the C1 region. Receptors appear to bind to DNA as dimers although whether dimers are preformed in cytoplasm remains unknown. Although the DNA binding domain is required for gene activation, other regions of the molecule in the carboxyl and amino terminus enhance activation function. Important interactions of steroid receptors with other receptors and unrelated transcription factors has been proposed and most certainly occurs. Finally, posttranslational modifications such as phosphorylation have been postulated to modulate several functional properties of steroid receptors.

Chick progesterone receptor subunits A and B have been photoaffinity-labeled using [3H]R5020 ([17 alpha-methyl-3H]17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione) by a modification of the procedures previously reported by our laboratory (Dure, L. S., IV, Schrader, W. T., and O'Malley, B. W. (1980) Nature (Lond.) 283, 784-786). [3H]R5020 binds to the same receptor sites as authentic progesterone, and has an apparent Kdiss of 2.0 nM. Use of a CuSO4 filter raised the coupling efficiency to 5% and labeled exclusively the receptor proteins. Smaller labeled macromolecules were found to be proteolytic fragments of receptors. The protease(s) could not be inhibited by any of the commonly used protease inhibitors. However, the proteolytic activity was completely removed by passage of crude receptor preparations through phosphocellulose columns. Receptor preparations, photoaffinity-labeled after this procedure, showed exclusively one radioactive band at Mr = 79,000 (subunit A) or Mr = 108,000 (subunit B) with no detectable side-reaction products. Labeled receptors A and B were digested with Staphylococcus aureus V8 protease to yield smaller [3H]R5020-protein fragments derived from both. Molecular weight estimates (Mr = 9,500) and apparent isoelectric points indicate similarities of these regions of both A and B. The photoaffinity protocol described here thus provides a method for study of the hormone-binding domain of progesterone receptors and of receptor proteolysis in crude extracts.

Cytoplasmic progesterone receptors of chick oviduct have been purified in 8% yield by steroid affinity and ion exchange chromatography. The affinity resin, deoxycorticosterone-bovine serum albumin-Sepharose, binds progesterone receptors with high affinity (KD equals 8 times 10-minus 10 M) and its use resulted in a greater than 2000-fold purification over the starting material in a single step. DEAE-Sephadex A-50 chromatography was then used to achieve final purification. NA dodecyl-SO4 gel electrophoresis and DEAE-cellulose chromatography showed that the purified receptors contained both of the previously described 4 S progesterone binding components in near equal amounts. Na dodocyl-SO4 gel electrophoresis also showed that these components consisted of single polypeptide chains with molecular weights of 110, 000 (A component) and 117, 000 (B component). There was no evidence for subunits of lower molecular weight. The purified materials have identical hormone-binding kinetics and steroid specificity to crude cytosol receptors. The isolated receptors retain the three biologically important properties exhibited by progesterone binding components present in cruder preparations: they bind specifically to (a) nuclei (KD equals 1.1 times 10-minus 9 M, 10, 000 sites per nucleus); (b) chromatin (KD equals 3 times 10-minus 9 M, 2000 sites per pg of DNA-);and (C) DNA.

Chicken oviduct progesterone receptor has been purified to homogeneity. The protein consists of two dissimilar hormone-binding subunits, A and B, present in equal amounts in the complex. They have molecular weights of 79,000 and 108,000, respectively, as shown by both SDS-gel electrophoresis of the purified proteins and photoaffinity labeling of both with a labeled synthetic progestin. The two subunits show considerable homology (or identity) of structure at the hormone-binding domain, located at the N-terminus of the proteins. Considerable divergence of sequence must exist elsewhere in A and B, as shown by tryptic peptide mapping and by the fact that subunit A has a strong DNA-binding site lacking in B. Both are phosphorylated in vitro by cAMP-dependent protein kinase; this phosphorylation appears to be responsible for creation of a second, weaker progesterone-binding site on each subunit.

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.

Many cellular regulatory processes are described by ligand interactions with macromolecules. Steroid complexing with specific cytoplasmic receptor proteins and the subsequent binding of these complexes to nuclear constituents are two such processes which are fundamental to the understanding of steroid hormone action. These reactions are characterized by the binding parameters k, the equilibrium association constant, and M0, the binding site molarity. The current methods used to determine these two parameters are discussed in relation to the general theory of binding parameter measurement. A number of practical aspects of binding parameter measurement are considered. These include problems encountered by working at dilute non-uniform protein concentrations, non-specific binding, and the limitations of all such experimental systems. An exact solution to the non-linear Scatchard plot is presented for the case when only two non-interacting classes of binding sites need be considered. Finally, a set of specific conclusions is offered which should enable investigators to obtain the most accurate information possible whenever carrying out steroid hormone or receptor binding studies.

The differentiated chick oviduct is a target tissue for progesterone. Administration of the hormone to estrogen-primed chicks causes a rapid induction of new messenger RNA, resulting in the appearance in cytoplasm of specific mRNA species. One of these species is the mRNA coding for the egg white protein avidin, which is produced only in response to progesterone. The cytoplasm contains receptors which bind progesterone specifically and transport it as a complex into oviduct nuclei. Only a small fraction of the receptors are active in vitro ; this fraction is increased to 30% by brief warming of the receptor-hormone complexes before incubation with the nuclei. The binding reaction is slow, and the receptors cannot be released from nuclei by DNase treatment. The reaction occurs preferentially with oviduct nuclei, which contain at least twice as many acceptor sites (8000/nucleus) as other chick tissues ( K d ~ 10 −8 M), and are identical to the constant for receptor binding to isolated chromatin. We have isolated the progesterone receptor component which binds to the chromatin. Oviduct cytosol from laying hens was prepared and progesterone receptors were precipitated with ammonium sulfate (30% sat.). The re-dissolved pellet was eluted from a steroid-affinity column (Sepharose 4B-BSA-deoxycorticosterone) with 3M urea. The receptors were reconstituted by dialysis and labeled with [ 3 H]-progesterone. The [ 3 H]-progesterone-receptor complexes were then purified by sequential chromatography and elution with the indicated slats at pH 74 from DEAE-cellulose (0.2 M KCl), phosphocellulose (0.26 M KCl), and hydroxylapatite (0.15 M K x PO 4 ). The peak fraction was finally chromatographed on an agarose A-1.5 M column ( K av = 0.28). Yield was 1 %, and purity approached the theoretical maximum specific activity, 10 9 d.p.m./mg protein.

We have cloned and sequenced 4.5 kilobases (Kb) of cDNA encoding the chicken progesterone receptor. The complete cDNA contains an open reading frame of 2361 nucleotides in length and encodes a polypeptide of 787 amino acids with a mol wt of 85.9 K. At least four mRNA species have been detected in chick oviduct cells. Direct sequencing of variant cDNAs has suggested that two of the mRNAs (4.5 Kb and 3.6 Kb) differ only in the length of their 3'-untranslated regions. A third mRNA (1.8 Kb) produces a truncated polypeptide which encodes the immunoreactive NH2 terminal sequence of the receptor but lacks the hormone binding regional and half of the DNA-binding domain. The polypeptide expressed from the receptor cDNA in progesterone receptor negative Cos M-6 cells is indistinguishable from oviduct progesterone receptor in terms of hormone binding and antibody reactivity. Furthermore, the cloned receptor is capable of activating transcription of a target gene. This activation is progesterone dependent (with half-maximal stimulation at approximately 3.3 x 10(-10) M) and specific for the target gene.

A monoclonal antibody described previously by us (Edwards, D. P., Weigel, N. L., Schrader, W. T., O'Malley, B. W., and McGuire, W. L. (1984) Biochemistry 23, 4427-4435) was used to study progesterone receptor B subunits of chick and hen oviduct. We find that the antibody does not recognize the form of receptor B able to bind [3H]progesterone in vitro. Rather, it reacts exclusively with a homologous protein of the same molecular weight, termed B antigen. The antigen is present in both immature estrogen-treated chicks and in egg-laying hens. This antigen is indistinguishable from the hormone-binding receptor species (termed receptor B) as shown by peptide mapping techniques using either Staphylococcus aureus V8 protease or trypsin. The B antigen and the hormone binder can be resolved by ion-exchange chromatography. Sedimentation velocity data show that the two proteins are present in distinct, separable cytosolic entities. The functional relationship between the two proteins has not been established.

Several reports have appeared recently which contradict our earlier findings on the identity and characterization of chick progesterone receptor. Puri et al. (Puri, R. K., Grandics, P., Dougherty, J. J., and Toft, D. O. (1982) J. Biol. Chem. 257, 10831-10837) have isolated a protein with a molecular weight of Mr approximately 88,000 and have reported that this protein is the "nontransformed" molybdate-stabilized receptor. Because of this discrepancy from our own work, we have repeated their methods and obtained purified samples of this protein. In this paper we show that the Mr = 88,000 +/- 2,000 protein is not a receptor but is apparently an artifact of the use of particular steroid affinity resins. Evidence presented to show that this protein is not the authentic receptor include: 1) it is obtained from affinity resins in 10-fold molar excess over the mass of authentic receptor; 2) its tryptic map does not resemble either authentic receptor subunit; 3) it can be resolved from authentic hormone-binding receptors on DEAE-Sephadex A-25 chromatography; 4) the protein does not bind progesterone; and finally 5) its elution from affinity matrices is not biospecific for progesterone and can even be eluted in equivalent yields with buffer alone. We conclude that the Mr = 88,000 protein is not a progesterone-binding protein.

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 receptor's 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.

The purified DNA binding component (receptor A) of the chick oviduct progesterone receptor has been analyzed for its ability to bind to the cloned ovalbumin gene and to plasmid DNA of various structural compositions. The rapid equilibrium filter adsorption assay of Riggs et al. [Riggs, A. D., Suzuki, H., & Bourgeois, S. (1970) J. Mol. Biol. 48, 67] has been used to demonstrate high affinity binding of the protein to DNA (Kdiss = 10(-10) M at 50 mM KCl, pH 7.2). Studies of association rates are consistent with equilibrium measurements (t 1/2 = 40-80 min). Association of purified receptor with DNA and the kinetics of the interaction have been verified independently by velocity sedimentation techniques. Direct binding assays were performed with the ovalbumin structural gene (cDNA), the entire natural ovalbumin gene containing seven intervening sequences, and various ovalbumin gene fragments coding for the 5' end of the nuclear precursor RNA, intron-exon junctions, and the 3'-noncoding region of the gene. No DNA-sequence specificity was identified for the binding of the receptor protein to any region of ovalbumin gene DNA. In contrast, the structural integrity of the DNA template greatly affected receptor binding. The poorest affinity was to supercoiled DNA and to blunt end, linear duplex gene fragments. The receptor bound saturably to DNA containing limited nicks but became nonsaturable as nicks were increased. Binding of the protein to double-stranded DNA increased susceptibility of the DNA to digestion by the enzyme S1, a single strand specific nuclease. On the basis of preferential receptor binding to single-stranded DNA, a possible mechanism involving DNA helix destabilization is discussed.

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.

The 8S progesterone receptor from chick oviduct has been partially purified (500-fold) as the apoprotein lacking hormone. The procedure employs polyethylene glycol precipitation, phosphocellulose, DNA cellulose, DEAE cellulose, hydroxylapatite and ethyl agarose column chromatography. As shown for the native cytosolic receptor, the purified apoprotein can be dissociated into A and B subunits. Qualitative and quantitative analysis of subunit structure shows no difference between purified and cytosolic native progesterone receptor. The A subunit dissociated from the purified receptor maintains its DNA binding activity as shown by the “Western” protein blotting technique and sedimentation velocity analysis. Moreover, purified progesterone receptor has steroid binding specificity very close to the native material. The purified progesterone receptor is free of DNase and RNase contaminating activities and represents a useful tool for in vitro studies of hormone action.

DNA binding of chick progesterone receptor B form (PRB) has been examined and compared to that of the A form (PRA). We found that the elution profiles of the two receptors overlap on DNA-cellulose columns. Both PRA or PRB could bind to plasmid DNA equivalently as assayed by sedimentation velocity studies. However, DNA-binding activity of the two receptor forms showed differential sensitivity to reducing agents and to sulfhydryl (SH) reactive reagents. Reducing agents stabilized DNA-binding activity of PRA more efficiently than they stabilized PRB. Moreover, removal of reducing agents from receptor preparations caused preferential loss of DNA binding by PRB compared to the PRA. DNA-binding activity of PRA was readily destroyed by sulfhydryl modifying reagents such as N-ethylmaleimide and iodoacetamide while PRB was 3-4 times less sensitive to these reagents. We conclude the DNA-binding activity of PRB is less stable due to altered accessibility of SH groups despite the amino acid sequence identity of the DNA-binding domains of PRA and PRB.

Previous purifications of the progesterone receptor have yielded inadequate amounts of pure protein along with significant amounts of a nonreceptor contaminant. We have taken advantage of the high yield provided by an affinity chromatography method for partial purification and after the incorporation of additional steps, we obtained purified progesterone receptor devoid of detectable contaminants and suitable for chemical analysis. A polyclonal antibody was obtained using the pure receptor as the antigen. The antibody was specific for progesterone-binding receptor. Tissue distribution of cross-reacting material, analyzed by immunoblotting, confirmed the presence of the receptor protein only in the two tissues where progesterone binding has been described in the chick: the oviduct and the bursa of Fabricius. It was absent in receptor-negative tissues such as liver and lung. The receptor was cleaved with cyanogen bromide and trypsin to obtain fragments that were partially sequenced.

Partial proteolytic fragmentation of the two chick oviduct progesterone receptor subunits was used to identify structural features shared by the two proteins. Both subunits can be photoaffinity labeled at their hormone-binding sites (Birnbaumer, M., Schrader, W. T., and O'Malley, B. W. (1983) J. Biol. Chem. 258, 1637-1644) using the radioactive steroid [methyl-3H] 17 alpha, 21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione. Native subunits A (Mr = 79,000) and B (Mr = 108,000) were partially purified, photoaffinity-labeled, and then subjected to various mild proteolytic digestions. Labeled fragments were analyzed by fluorography after electrophoresis of the digests under denaturing conditions. Digestion patterns were characteristic for each protease tested. However, fragments from both A and B were indistinguishable for all peptides of less than Mr = 60,000. Time course studies demonstrated the sequential production of progressively smaller discrete fragments in a manner consistent with a precursor-product relationship among them and established the existence of similar structural domains resistant to proteolysis in both proteins. Autoradiographic peptide maps were obtained by 125I-labeling of pure A and B protein isolated by two-dimensional gel electrophoresis followed by exhaustive tryptic digestion and two-dimensional separation. These studies revealed that a significant proportion of the smaller A protein differs in its primary sequence from that of the B protein which excludes the possibility of their sharing a precursor-product relationship. We conclude that B and A subunits are separate proteins with common structural features in the native state, but with considerable amino acid sequence differences. The simplest hypothesis consistent with these findings is that B and A are the products of two separate genes which have diverged to give rise to two different but related proteins that fold in such a manner as to be almost indistinguishable by proteolytic attack of their native conformation.

We have prepared monospecific polyclonal rabbit antibodies to the peptide sequence 523–536 of the chicken progesterone receptor. This region, located between the DNAbinding 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 proteinprotein interactions.

To define the functional domains of the chicken progesterone receptor (CPR) and to establish the origin of the receptor A and B proteins, we have cloned and sequenced the complete cDNA encoding the CPR. The receptor A and B proteins arise by alternate initiation of translation from a single mRNA transcript. Using deletion mutants in the receptor A cDNA, we have determined that the DNA binding and gene activator regions reside in the central portion of the protein, while the hormone binding domain is located in the C-terminal region. The target gene activator site of the receptor is modulated by interaction with the hormone binding domain.

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 cDNA's indicated the existence of at least three messenger RNA species. These messages were found only in oviduct and could be induced by estrogens.

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.

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.