Regulation of progesterone receptor-mediated transcription by phosphorylation.

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.

Cyclins, cyclin dependent kinases, and regulation of steroid receptor action.

Steroid receptors (SR), which are ligand activated transcription factors, and their coactivators are phosphoproteins whose activities are regulated by cell signaling pathways. Many of the identified phosphorylation sites in these proteins contain Ser/Thr-Pro motifs suggesting that they are substrates for cyclin dependent kinases and/or for mitogen activated protein kinases. An analysis of the roles of cyclins and their kinases in regulating receptor action has revealed that there are both stimulatory and inhibitory actions of cyclins, that some of the actions are independent of the partner kinases and that these activities are receptor specific. Consistent with this finding, the limited analyses of receptor activity as a function of cell cycle reveal distinct patterns of activation. SR often regulate cell proliferation. Thus, the cross-talk between cyclins and their kinases and the SR provides a means for integrating the actions of the SR with the cell cycle status of cells.

8-Bromo-cyclic AMP induces phosphorylation of two sites in SRC-1 that facilitate ligand-independent activation of the chicken progesterone receptor and are critical for functional cooperation between SRC-1 and CREB binding protein.

Elevation of intracellular 8-bromo-cyclic AMP (cAMP) can activate certain steroid receptors and enhance the ligand-dependent activation of most receptors. During ligand-independent activation of the chicken progesterone receptor (cPR(A)) with the protein kinase A (PKA) activator, 8-bromo-cAMP, we found no alteration in cPR(A) phosphorylation (W. Bai, B. G. Rowan, V. E. Allgood, B. W. O'Malley, and N. L. Weigel, J. Biol. Chem. 272:10457-10463, 1997). To determine if other receptor-associated cofactors were targets of cAMP-dependent signaling pathways, we examined the phosphorylation of steroid receptor coactivator 1 (SRC-1). We detected a 1.8-fold increase in SRC-1 phosphorylation in transfected COS-1 cells incubated with 8-bromo-cAMP. Phosphorylation was increased on two mitogen-activated protein kinase (MAPK) sites, threonine 1179 and serine 1185. PKA did not phosphorylate these sites in vitro. However, blockage of PKA activity in COS-1 cells with the PKA inhibitor (PKI) prevented the 8-bromo-cAMP-mediated phosphorylation of these sites. Incubation of COS-1 cells with 8-bromo-cAMP resulted in activation of the MAPK pathway, as determined by Western blotting with antibodies to the phosphorylated (active) form of Erk-1/2, suggesting an indirect pathway to SRC-1 phosphorylation. Mutation of threonine 1179 and serine 1185 to alanine in COS-1 cells coexpressing cPR(A) and the GRE(2)E1bCAT reporter resulted in up to a 50% decrease in coactivation during both ligand-independent activation and ligand-dependent activation. This was due, in part, to loss of functional cooperation between SRC-1 and CREB binding protein for coactivation of cPR(A). This is the first demonstration of cross talk between a signaling pathway and specific phosphorylation sites in a nuclear receptor coactivator that can regulate steroid receptor activation.

The nuclear corepressors NCoR and SMRT are key regulators of both ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of the human progesterone receptor.

Previously, we defined a novel class of ligands for the human progesterone receptor (PR) which function as mixed agonists. These compounds induce a conformational change upon binding the receptor that is different from those induced by agonists and antagonists. This establishes a correlation between the structure of a ligand-receptor complex and its transcriptional activity. In an attempt to define the cellular components which distinguish between different ligand-induced PR conformations, we have determined, by using a mammalian two-hybrid assay, that the nuclear receptor corepressor (NCoR) and the silencing mediator for retinoid and thyroid hormone receptor (SMRT) differentially associate with PR depending upon the class of ligand bound to the receptor. Specifically, we observed that the corepressors preferentially associate with antagonist-occupied PR and that overexpression of these corepressors suppresses the partial agonist activity of antagonist-occupied PR. Binding studies performed in vitro, however, reveal that recombinant SMRT can interact with PR in a manner which is not influenced by the nature of the bound ligand. Thus, the inability of SMRT or NCoR to interact with agonist-activated PR when assayed in vivo may relate more to the increased affinity of PR for coactivators, with a subsequent displacement of corepressors, than to an inherent low affinity for the corepressor proteins. Previous work from other groups has shown that 8-bromo-cyclic AMP (8-bromo-cAMP) can convert the PR antagonist RU486 into an agonist and, additionally, can potentiate the transcriptional activity of agonist-bound PR. In this study, we show that exogenous expression of NCoR or SMRT suppresses all 8-bromo-cAMP-mediated potentiation of PR transcriptional activity. Further analysis revealed that 8-bromo-cAMP addition decreases the association of NCoR and SMRT with PR. Thus, we propose that 8-bromo-cAMP-mediated potentiation of PR transcriptional activity is due, at least in part, to a disruption of the interaction between PR and the corepressors NCoR and SMRT. Cumulatively, these results suggest that NCoR and SMRT expression may play a pivotal role in PR pharmacology.

Use of the probasin promoter ARR2PB to express Bax in androgen receptor-positive prostate cancer cells.

BACKGROUND: Adenovirus-mediated overexpression of the apoptosis-inducing protein Bax can induce apoptosis in prostate cancer cell lines. Constitutive overexpression of Bax could result in unwanted apoptosis in every site of accidental Bax accumulation in vivo. Therefore, we developed an adenoviral construct (Av-ARR2PB-Bax) in which the probasin promoter, modified to contain two androgen response elements, drives Bax expression. This promoter would be expected to limit expression of Bax to cells expressing the androgen receptor. METHODS: A variety of androgen receptor (AR)-positive and -negative cell lines of prostatic or nonprostatic origin were infected with Av-ARR2PB-Bax or a control virus, Av-ARR2PB-CAT, in which the same promoter drives expression of the chloramphenicol acetyl transferase-reporter gene. Bax expression and apoptosis in vitro were assessed by western blot analysis. Tumor size and apoptosis in vivo were assessed after four weekly injections of Av-ARR2PB-Bax or Av-ARR2PB-CAT into subcutaneous LNCaP xenografts growing in uncastrated male mice. All statistical tests were two-sided. RESULTS: Bax was overexpressed in an androgen-dependent way in AR-positive cell lines of prostatic origin but not in AR-positive cells of nonprostatic origin or in AR-negative cell lines of either prostatic or nonprostatic origin. The androgen dihydrotestosterone activated apoptosis in LNCaP cells infected with Av-ARR2PB-Bax but not in those infected with Av-ARR2PB-CAT. Av-ARR2PB-Bax-injected LNCaP xenograft tumors decreased in tumor size from 34.1 mm3 (95% confidence interval [CI] = 25.1 mm3 to 43.1 mm3) to 24.6 mm3 (95% CI = -2.5 mm3 to 51.7 mm3), but the difference was not statistically significant (P =.5). Tumors injected with Av-ARR2PB-CAT increased in size, from 28.9 mm3 (95% CI = 12.7 mm3 to 45.1 mm3) to 206 mm3 (95% CI = 122 mm3 to 290 mm3) (P =.002) and contained statistically significant more apoptotic cells (23.3% [95% CI = 21.1% to 25.6%] versus 9.5% [95% CI = 8.0% to 11.1]) (P<.001). CONCLUSIONS: Av-ARR2PB-Bax induces androgen-dependent therapeutic apoptosis in vitro and in vivo by activating apoptosis in AR-positive cells derived specifically from prostatic epithelium and does not affect nonprostatic cells.

A calcitriol analogue, EB1089, inhibits the growth of LNCaP tumors in nude mice.

Limited options for the treatment of prostate cancer have spurred the search for new therapies. One innovative approach is the use of 1alpha,25-dihydroxyvitamin D3 (calcitriol) analogues to inhibit cancer growth. We demonstrate here that the calcitriol analogue, EB1089, extensively inhibits the growth of LNCaP prostate cancer cells in culture and causes the cells to both accumulate in G0-G1 and undergo apoptosis. Importantly, we found that EB1089 inhibits the growth of LNCaP tumor xenografts in nude mice. Because of these antiproliferative properties in vivo, EB1089 is a potential new therapeutic agent for the treatment of prostate cancer.

Androgen receptor mutations in prostate cancer.

We analyzed the frequency and relevance of mutations in the coding region of the androgen receptor (AR) in genomic DNA extracted from 137 specimens of prostate cancer. The specimens were obtained from the primary tumors of patients affected by stage B disease [15 nonmicrodissected (group 1A) and 84 microdissected (group 1B)] and from the metastatic deposits of individuals with stage D1 disease [8 nonmicrodissected (group 2A) and 30 microdissected (group 2B)] who had not undergone androgen ablation therapy. The study was conducted by PCR-single strand conformational polymorphism (SSCP) analysis of exons 2-8 in the four groups and direct sequence analysis of exon 1 in group 1B. As positive and negative controls, we used genomic DNA extracted from genital skin fibroblasts of patients affected by various forms of androgen resistance with known mutations in the AR. To control for genetic instability, PCR-SSCP analysis of exon 2 of the human progesterone receptor was carried out on each specimen. The overall number of mutations detected was 11 (8%). No mutations were detected in any of the 99 patients with stage B disease. Eleven mutations were detected in exons 2-8 in 8 of the 38 patients with stage D1 disease (all in group 2B). Simultaneous analysis of exon 2 of the progesterone receptor was carried out, and no SSCP changes were identified. These data suggest that AR mutations are rare and presumably do not play a role in the initial phase of prostatic carcinogenesis. The presence of a significant number of AR mutations in metastatic disease indicates that mutations of this molecule may play a role in the most advanced phases of the natural history of this disease, either by facilitating growth or acquisition of the metastatic phenotype.

Ligand-independent activation of steroid hormone receptors.

In addition to the conventional hormone-dependent regulation of the activity of steroid/thyroid receptor family members, many studies have shown that there is substantial cross-talk between signal transduction pathways and steroid receptors. In a number of cases the modulation of kinase/phosphatase activity in cells leads to activation of steroid receptors in the absence of hormone. This novel mechanism may not be ubiquitous as the glucocorticoid receptor appears to be refractory to activation in the absence of hormone. However, estrogen receptors, progesterone receptors, androgen receptors, retinoic acid receptors, retinoid X receptors, and vitamin D receptors all exhibit ligand-independent activation under appropriate conditions. Whether a steroid receptor responds to a signal by inducing transcription of a target gene in the absence of hormone depends upon the cell type, promoter, and activator. The mechanism(s) by which ligand-independent activation is induced is currently a subject of great interest. Because the signals that activate receptors induce protein phosphorylation, altered phosphorylation of the receptors, and/or proteins that associate with the receptors are likely to be key to ligand-independent activation. In the case of the estrogen receptor there is good evidence that altered receptor phosphorylation plays a role in ligand-independent activation. Other likely targets are proteins in the heat shock protein complexes, corepressors, and/or coactivators of steroid receptors.

Identification of a hormone-dependent phosphorylation site adjacent to the DNA-binding domain of the chicken progesterone receptor.

We have previously identified three phosphorylation sites in chicken progesterone receptor (PR) isolated from the cytosol of chicken oviducts. Several lines of evidence suggested that there might be additional phosphorylations that occur only in the nuclear compartment. We have reexamined the phosphorylation of the PR using whole cell extracts and have identified a second hormone-dependent phosphorylation site (Ser367) which is located on the amino-terminal side of the DNA-binding domain. This site also contains a Ser-Pro consensus sequence, as do the three previously reported sites. There are four Ser-Pro sequences in chicken PR, and all four are phosphorylated. Two of the sites showed substantial phosphorylation in the absence of hormone, with some enhancement in response to hormone, whereas two of the sites showed very little phosphorylation in the absence of hormone. This suggests either that despite the Ser-Pro consensus sequences, the sites are phosphorylated by different enzymes, or that binding hormone changes the conformation of the protein sufficiently to expose the two hormone-dependent phosphorylation sites.

Phosphorylation of Ser530 facilitates hormone-dependent transcriptional activation of the chicken progesterone receptor.

Phosphorylation regulates the activities of many proteins, including transcription factors. However, the evidence for the significance of phosphorylation in steroid hormone receptor action is mainly indirect. In this study, one of the hormone-induced phosphorylation sites of chicken progesterone receptor, Ser530, was mutated to alanine, a nonphosphorylatable amino acid, and the transcriptional activity of the mutant receptor was compared with that of wild type in a transient cotransfection assay. The results showed that this mutation resulted in reduced transcriptional activity of chicken progesterone receptor at low hormone concentrations but did not affect the maximal activity of the receptor at saturating levels of hormone, suggesting that the phosphorylation at Ser530 influences the response of the receptor to its ligand. The decreased sensitivity of the mutant receptor is not due to a decrease in hormone-binding affinity, leading to our hypothesis that Ser530 phosphorylation stabilizes the receptor in its active state, perhaps by preventing its reassociation with heat shock proteins or by maintaining a conformation suitable for interaction with other transcription factors.

Effects of hormone and cellular modulators of protein phosphorylation on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors.

Human progesterone receptors (PR) in T47D breast cancer cells are synthesized as two different sized proteins, PR-A [94 kilodaltons (kDa)] and PR-B (120 kDa). Progestin addition to cells (in vivo) causes a 2-fold increase in total phosphorylation of PR and an increase in the apparent mol wt of both PR-A and PR-B on sodium dodecyl sulfate (SDS)-gels. Time-course experiments showed that increased PR phosphorylation that results from hormone addition is a multistep process and involves a rapid increase into total 32P labeling that takes place before the more slowly occurring phosphorylation(s) responsible for the change in electrophoretic mobility of PR on SDS-gels. As an approach to test whether phosphorylation is involved in regulating PR activity, we have examined the effects of cellular modulators of protein phosphorylation on PR-mediated target gene transcription in vivo using a T47D cloned cell line containing a stably transfected mouse mammary tumor virus-chloramphenicol acetyltransferase construct. Treatment with 8-bromo-cAMP (activator of cAMP-dependent protein kinases) or okadaic acid (protein phosphatase-1 and -2A inhibitor) did not stimulate target gene expression in the absence of progestin. When added together with progestin, either compound augmented PR-mediated target gene transcription by 3- to 4-fold. The cyclic nucleotide-dependent protein kinase inhibitor H8 completely blocked target gene responsiveness to hormone. Neither 8-bromo-cAMP, okadaic acid, nor H8 altered the hormone- or DNA-binding activities of PR, as measured in vitro or affected cellular concentrations of PR. These agents, therefore, appeared to selectively modulate PR transcriptional activity. Moreover, none of these compounds altered expression from a control reporter gene, pSV2CAT, indicating that these agents affect PR-mediated processes directly and are not acting through a general effect on transcription. Effects on PR phosphorylation were assessed by measuring 32P labeling of PR in vivo. None of these treatments had a substantial effect on the extent of total 32P labeling of immune isolated PR or on the phosphorylation(s) responsible for PR up-shifts on SDS-gels. This suggests that these agents modulate PR transcriptional activity either through phosphorylation of another protein intimately involved in PR-mediated transcription or through modification of a key site(s) not measurable as a change in total PR phosphorylation or electrophoretic mobility on SDS gels.

Multiple signaling pathways activate the chicken progesterone receptor.

We have previously reported that treatment of CV1 cells, transiently transfected with DNA encoding the chicken progesterone receptor (cPR) and a reporter plasmid PREtkCAT, with either 8-Br-cAMP or okadaic acid resulted in ligand-independent transcriptional activation of the cPR. The surprising finding that cPR can be activated in the absence of hormone has been followed by numerous studies from other laboratories examining the effects of modulators of kinases and phosphatases on the activity of other steroid receptors. These studies have yielded mixed results: ligand-independent activation has been observed in some cases, but not in others. In order to determine whether the ligand-independent activation of cPR was restricted to a specific reporter and cell type and to better characterize this phenomenon, the studies in this report were undertaken. Using both the original reporter, PREtkCAT, and a simpler reporter, GRE2E1bCAT, we found that ligand-independent activation of the cPR can be induced in both CV1 and HeLa cells. The magnitude of the response and the response of the reporter alone differed in the two cell types. Further analysis of the activation of cPR by inhibitors of protein phosphatases showed that inhibition of phosphatase 1 rather than phosphatase 2A was necessary for activation of cPR. Finally, treatment with vanadate, an inhibitor of phosphotyrosine phosphatases, or epidermal growth factor resulted in activation of cPR. These studies suggest that signals transduced through multiple signaling pathways can activate cPR.

Identification of a group of Ser-Pro motif hormone-inducible phosphorylation sites in the human progesterone receptor.

The human progesterone receptor (PR) is a member of the steroid/thyroid hormone superfamily of nuclear receptors. The receptor is expressed as two forms, PR-B and the shorter PR-A, which lacks the NH2-terminal 164 amino acids of PR-B; whereas PR-B seems to be predominantly a transcriptional activator, PR-A also functions as a repressor. Our previous studies of PR expressed in T47D breast cancer cells have shown that PR is a phosphoprotein whose phosphorylation is enhanced in response to hormone. There is an initial rapid (minutes) increase in phosphorylation followed by a slower, less substantial increase, which results in decreased mobility of the receptor on sodium dodecyl sulfate gels. We now report the identification of three phosphorylation sites, which are predominantly phosphorylated during the later phase of the response to hormone. These sites, Ser102, Ser294, and Ser345, are all found in Ser-Pro consensus sequences. Whereas Ser294 and Ser345 are common to PR-A and PR-B, Ser102 is unique to PR-B. Finally, we demonstrate that phosphorylation of Ser345 is associated with the altered mobility on sodium dodecyl sulfate gels.

Differential hormone-dependent phosphorylation of progesterone receptor A and B forms revealed by a phosphoserine site-specific monoclonal antibody.

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.

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.

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.

Chicken progesterone receptor is phosphorylated by a DNA-dependent protein kinase during in vitro transcription assays.

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.

Phosphorylation of human progesterone receptor by cyclin-dependent kinase 2 on three sites that are authentic basal phosphorylation sites in vivo.

The human progesterone receptor (hPR) in T47D breast cancer cells is phosphorylated on at least nine different serine residues. We have previously reported the identification of five sites; three are hormone inducible (Ser102, Ser294 and Ser345), and their phosphorylation correlates with the timing of the change in receptor mobility on gel electrophoresis in response to hormone treatment. The other two sites, Ser81 and Ser162, along with the remaining sites, are basally phosphorylated and exhibit a general increase in phosphorylation in response to hormone. With the exception of Ser81, all of these sites are in Ser-Pro motifs, suggesting that proline-directed kinases are responsible for their phosphorylation. We now report that cyclin A-cyclin-dependent kinase-2 complexes phosphorylate hPR-B in vitro with a high stoichiometry on three sites that are authentic basal sites in vivo. One of these is Ser162, which has been described previously. The other two sites are identified here as Ser190 and Ser400. The specificity and stoichiometry of the in vitro phosphorylation suggest that hPR phosphorylation may be regulated in a cell cycle-dependent manner in vivo.

Ligands induce conformational changes in the carboxyl-terminus of progesterone receptors which are detected by a site-directed antipeptide monoclonal antibody.

We have prepared a monoclonal antibody, C-262, to a synthetic peptide that contains the carboxy-terminal 14 amino acids from progesterone receptors (PR). This sequence is 100% conserved in all species of PRs that have been cloned to date, suggesting that this antibody will recognize all mammalian and avian PR. The C-262 antibody recognizes both native and denatured forms of the receptor. However, it does not recognize PR when they are bound to the hormone agonists progesterone or R5020. Surprisingly the antibody does recognize PR when they are bound to the steroid antagonist RU486. This suggests that progestin agonists induce a conformational change in the receptor that occludes the C-262 epitope in the carboxyl-terminus, whereas unliganded receptors and receptors bound with RU486 assume distinct conformations that leaves the C-terminal tail accessible to the C-262 antibody.

A C619Y mutation in the human androgen receptor causes inactivation and mislocalization of the receptor with concomitant sequestration of SRC-1 (steroid receptor coactivator 1)

Androgen ablation therapy is a primary treatment for advanced prostate cancer, but tumors become refractive to therapy. Consequently, the role of the androgen receptors (ARs) and of mutations in the AR in prostate cancer has been a subject of much concern. In the course of analyzing tumors for mutations, we identified a somatic mutation that substitutes tyrosine for a cysteine at amino acid 619 (C619Y), which is near the cysteines that coordinate zinc in the DNA binding domain in the AR. The mutation was re-created in a wild-type expression vector and functional analyses carried out using transfection assays with androgen-responsive reporters. The mutant is transcriptionally inactive and unable to bind DNA. In response to ligand treatment, AR619Y localizes abnormally in numerous, well circumscribed predominantly nuclear aggregates in the nucleus and cytoplasm. Interestingly, these aggregates also contain the bulk of the coexpressed steroid receptor coactivator SRC-1, suggesting, in analogy to AR in spinal bulbar muscular atrophy, that this mutant may alter cellular physiology through sequestration of critical proteins. Although many inactivating mutations have been identified in androgen insensitivity syndrome patients, to our knowledge, this is the first characterization of an inactivating mutation identified in human prostate cancer.